Targeted low-potency IL-12Fc fusion protein and its applications

Abstract

The present invention relates to novel targeted low potency IL-12 Fc fusion proteins compositions, and methods of making and using such compositions.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 623,458, filed January 22, 2024; U.S. Provisional Application No. 63 / 560,957, filed March 4, 2024; and U.S. Provisional Application No. 63 / 711,820, filed October 25, 2024, each of which is incorporated herein by reference in its entirety.

[0002] sequence list This application contains a sequence list, which is submitted electronically in XML format and is hereby incorporated in its entirety by reference. The XML copy created on January 21, 2025, is named 51400_4004_WO_SL.xml and has a size of 196,608 bytes. Background Technology

[0003] Interleukin-12 (IL-12) is a potent pro-inflammatory cytokine produced by antigen-presenting cells, such as dendritic cells, macrophages, and neutrophils. IL-12 belongs to the IL-12 cytokine family. A unique feature of the IL-12 cytokine family is that they contain heterodimeric cytokines. IL-12 is composed of the alpha-(α-) subunit (encoded by the IL12A gene; also referred to herein as the "IL-12 p35 subunit"); precursor sequence - SEQ ID NO: 1 (e.g.) Figure 1 (as shown); mature sequence - SEQ ID NO: 2 (as shown) Figure 1 (as shown) and the beta-(β-) subunit (encoded by the IL12B gene; also referred to herein as the "IL-12 p40 subunit"); precursor sequence - SEQ ID NO: 3 (as shown) Figure 1 (as shown); mature sequence - SEQ ID NO: 4 (as shown) Figure 1 The components (shown) assemble to form a 70,000 Dalton (70 kDa) disulfide-linked heterodimer. After assembly, the IL-12 p35 and p40 subunits form the biologically active IL-12 heterodimer. The IL-12 receptor IL-12R is a type I cytokine receptor containing the β-1 subunit (IL-12Rβ1 - SEQ ID NO: 5). Figure 2 (as shown); Extracellular domain - SEQ ID NO: 6 (as shown) Figure 2 As shown)) and β-2 subunit (IL-12Rβ2 - SEQ ID NO: 7) Figure 2 (as shown); Extracellular domain - SEQ ID NO: 8 (as shown) Figure 2As shown in the figure), the IL-12 p40 subunit has a binding affinity for IL-12Rβ1, while the IL-12 p35 subunit has a binding affinity for IL-12Rβ2.

[0004] The binding of IL-12 to IL-12R leads to phosphorylation of intracellular signal transducers and activator of transcription 4 (STAT4) and triggers signal transduction pathways that: (i) induce TH1 cell differentiation, (ii) increase the activation and cytotoxicity of T cells and natural killer (NK) cells, (iii) suppress or reprogram immunosuppressive cells, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), and (iv) induce the production of large amounts of interferon-γ (IFNγ), which has cytosuppressive / cytotoxic, anti-angiogenic properties and can upregulate major histocompatibility complex (MHC) class 1 and MHC class 2 molecules on tumor cells for immune recognition.

[0005] However, due to the high affinity of IL-12 for its receptor, targeting wild-type IL-12 to target cells using the antigen-binding domain (ABD) is often unsuccessful. Instead of transporting "immunocytokines" to on-target cells expressing antigens bound by the ABD, the immune cytokines are often transported to off-target cells expressing high levels of IL-12R. Therefore, there remains an unmet need for the development of fusion proteins containing IL-12 and an ABD capable of directing IL-12 to on-target cells expressing antigens bound by the ABD. Summary of the Invention

[0006] In one aspect, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain linker, (iii) a second IL-12 subunit domain, (iv) a second domain linker, and (v) a first Fc domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, and (2) the variant IL-12 p35 subunit domain comprises an amino acid substitution selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a variable heavy chain (VH) domain, (ii) a constant heavy chain (CH1) domain, and (iii) a second Fc domain; and (c) a third monomer comprising, from the N-terminus to the C-terminus: (i) a variable light chain (VL) domain and (ii) a constant light chain (CL) domain, wherein the VH domain and the VL domain form an antigen-binding domain (ABD) that binds to the target antigen.

[0007] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0008] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0009] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0010] In a further embodiment and according to any of the above, ABD binds to target antigens selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0011] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In yet another embodiment, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In yet another embodiment, the target antigen comprises human ICOS, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In yet another embodiment, the target antigen comprises human 41BB, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In yet another embodiment, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In yet another embodiment, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0012] In a further implementation scheme and according to any of the above, ABD binds to the extracellular domain (ECD) of the target antigen.

[0013] In a further embodiment and according to any of the above, the first domain connector and / or the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0014] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a VH domain, (ii) a CH1 domain, and (iii) a first Fc domain; (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a VL domain, and (ii) a CL domain, wherein the VH domain and the VL domain form an ABD that binds to a first target antigen; and (c) a third monomer comprising, from the N-terminus to the C-terminus: (i) a second Fc domain, (ii) a first domain adapter, (iii) a first IL-12 subunit domain, (iv) a second domain adapter, and (v) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p40 subunit domain. The p35 subunit domain and the second IL-12 subunit domain include the IL-12 p40 subunit domain, and (2) the variant IL-12 p35 subunit domain includes a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

[0015] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0016] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0017] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0018] In a further embodiment and according to any of the above, ABD binds to target antigens selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0019] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In yet another embodiment, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In yet another embodiment, the target antigen comprises human ICOS, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In yet another embodiment, the target antigen comprises human 41BB, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In yet another embodiment, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In yet another embodiment, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0020] In a further implementation scheme and according to any of the above, ABD binds to the extracellular domain (ECD) of the target antigen.

[0021] In a further embodiment and according to any of the above, the first domain connector and / or the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0022] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain adapter, and (iii) a first Fc domain; (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a second IL-12 subunit domain, (ii) a second domain adapter, (iii) a second Fc domain, (iv) a third domain adapter, (v) a VH domain, and (vi) a CH1 domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, and (2) a variant IL-12 The p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; and (c) a third monomer that comprises, from the N-terminus to the C-terminus,: (i) a VL domain and (ii) a CL domain, wherein the VH domain and the VL domain form an ABD that binds to the target antigen.

[0023] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0024] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0025] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0026] In a further embodiment and according to any of the above, ABD binds to target antigens selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0027] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In yet another embodiment, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In yet another embodiment, the target antigen comprises human ICOS, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In yet another embodiment, the target antigen comprises human 41BB, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In yet another embodiment, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In yet another embodiment, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0028] In a further implementation scheme and according to any of the above, ABD binds to the extracellular domain (ECD) of the target antigen.

[0029] In a further embodiment and according to any of the above, the first domain connector, the second domain connector, and / or the third domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector, the second domain connector, and / or the third domain connector comprises a covalent bond. In some other embodiments, at least one domain connector comprises any of SEQ ID NO: 31-40, and at least one domain connector comprises a covalent bond. In some embodiments, at least one of the domain connectors comprises a hinge region of an Fc domain.

[0030] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a VH domain, (ii) a CH1 domain, (iii) a first Fc domain, (iv) a first domain adapter, and (v) a first IL-12 subunit domain; and (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a VL domain, (ii) a CL domain, (iii) a second Fc domain, (iv) a second domain adapter, and (v) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, (2) a variant IL-12 The p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) the VH domain and VL domain form an ABD that binds to the target antigen.

[0031] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0032] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0033] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0034] In a further embodiment and according to any of the above, ABD binds to target antigens selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0035] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In yet another embodiment, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In yet another embodiment, the target antigen comprises human ICOS, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In yet another embodiment, the target antigen comprises human 41BB, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In yet another embodiment, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In yet another embodiment, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0036] In a further implementation scheme and according to any of the above, ABD binds to the extracellular domain (ECD) of the target antigen.

[0037] In a further embodiment and according to any of the above, the first domain connector and / or the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0038] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a VH domain, (ii) a CH1 domain, and (iii) a first Fc domain; and (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a VL domain, (ii) a CL domain, (iii) a second Fc domain, (iv) a first domain adapter, (v) a first IL-12 subunit domain, (vi) a second domain adapter, and (vii) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises IL-12 p40 subunit domain. The p40 subunit domain, (2) the variant IL-12 p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) the VH and VL domains form an ABD that binds to the target antigen.

[0039] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0040] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0041] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0042] In a further embodiment and according to any of the above, ABD binds to target antigens selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0043] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In yet another embodiment, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In yet another embodiment, the target antigen comprises human ICOS, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In yet another embodiment, the target antigen comprises human 41BB, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In yet another embodiment, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In yet another embodiment, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0044] In a further implementation scheme and according to any of the above, ABD binds to the extracellular domain (ECD) of the target antigen.

[0045] In a further embodiment and according to any of the above, the first domain connector and / or the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0046] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising a first Fc domain; (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a VL domain and (ii) a CL domain; and (c) a third monomer comprising, from the N-terminus to the C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain adapter, (iii) a second IL-12 subunit domain, (iv) a second domain adapter, (v) a second Fc domain, (vi) a VH domain, and (vii) a CH1 domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises IL-12 p40 subunit domain. The p40 subunit domain, (2) the variant IL-12 p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) the VH and VL domains form an ABD that binds to the target antigen.

[0047] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0048] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0049] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0050] In a further embodiment and according to any of the above, ABD binds to target antigens selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0051] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In yet another embodiment, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In yet another embodiment, the target antigen comprises human ICOS, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In yet another embodiment, the target antigen comprises human 41BB, and the ABD comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In yet another embodiment, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In yet another embodiment, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0052] In a further implementation scheme and according to any of the above, ABD binds to the extracellular domain (ECD) of the target antigen.

[0053] In a further embodiment and according to any of the above, the first domain connector and / or the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0054] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a first VH (VH1) domain, (ii) a first CH1 domain, and (iii) a first Fc domain; (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a first VL (VL1) domain, and (ii) a first CL domain, wherein the VH1 domain and the VL1 domain form a first ABD binding to a first target antigen; and (c) a third monomer comprising, from the N-terminus to the C-terminus: (i) a second VH (VH2) domain, (ii) a second CH1 domain, (iii) a second Fc domain, (iv) a first domain adapter, (v) a first IL-12 subunit domain, (vi) a second domain adapter, and (vii) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain contains IL-12 The p40 subunit domain and the second IL-12 subunit domain include a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) the variant IL-12 p35 subunit domain includes a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (d) a fourth monomer that includes from the N-terminus to the C-terminus: (i) a second VL (VL2) domain and (ii) a second CL domain, wherein the VH2 domain and the VL2 domain form a second ABD that binds to the second target antigen.

[0055] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0056] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0057] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0058] In a further embodiment and according to any of the above, the first ABD binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0059] In a further embodiment and according to any of the above, the second ABD binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0060] In a further embodiment and according to any of the above, the combination of the first ABD and the second ABD is selected from target antigens comprising the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8. In other embodiments, the first ABD combination is selected from a first target antigen comprising the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8; and the second ABD combination is selected from a second target antigen comprising the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0061] In a further embodiment and according to any of the above, the first target antigen and / or the second target antigen comprises human PD1, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human LAG3, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63 respectively; (ii) SEQ ID NO: 103 and 104 respectively; (iii) SEQ ID NO: 105 and 106 respectively; (iv) SEQ ID NO: 107 and 108 respectively; (v) SEQ ID NO: 109 and 110 respectively; and (vi) SEQ ID NO: 111 and 112 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human OX40, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 160 and 148 respectively; 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the diagram. In other embodiments, the first target antigen and / or the second target antigen comprises human ICOS, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human 41BB, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR7, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR8, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0062] In a further implementation scheme and according to any of the above, the first ABD binds to the ECD of the first target antigen.

[0063] In a further implementation scheme and according to any of the above, the second ABD binds to the ECD of the second target antigen.

[0064] In a further embodiment and according to any of the above, the first ABD binds to the ECD of the first target antigen, and the second ABD binds to the ECD of the second target antigen.

[0065] In a further embodiment and according to any of the above, the first domain connector and / or the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0066] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a first VH (VH1) domain, (ii) a first CH1 domain, (iii) a first Fc domain, (iv) a first domain adapter, and (v) a first IL-12 subunit domain; (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a first VL (VL1) domain, and (ii) a first CL domain, wherein the VH1 domain and the VL1 domain form a first ABD binding to a first target antigen; and (c) a third monomer comprising, from the N-terminus to the C-terminus: (i) a second VH (VH2) domain, (ii) a second CH1 domain, (iii) a second Fc domain, (iv) a second domain adapter, and (v) a second IL-12 subunit domain, wherein: (i) the first IL-12 subunit domain contains IL-12 The p40 subunit domain and the second IL-12 subunit domain include a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) the variant IL-12 p35 subunit domain includes a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (d) a fourth monomer that includes from the N-terminus to the C-terminus: (i) a second VL (VL2) domain and (ii) a second CL domain, wherein the VH2 domain and the VL2 domain form a second ABD that binds to the second target antigen.

[0067] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0068] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0069] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 26-30, and the second Fc domain comprises any of SEQ ID NO: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0070] In a further embodiment and according to any of the above, the first ABD binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0071] In a further embodiment and according to any of the above, the second ABD binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0072] In a further embodiment and according to any of the above, the combination of the first ABD and the second ABD is selected from target antigens comprising the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8. In other embodiments, the first ABD combination is selected from a first target antigen comprising the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8; and the second ABD combination is selected from a second target antigen comprising the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0073] In a further embodiment and according to any of the above, the first target antigen and / or the second target antigen comprises human PD1, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human LAG3, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63 respectively; (ii) SEQ ID NO: 103 and 104 respectively; (iii) SEQ ID NO: 105 and 106 respectively; (iv) SEQ ID NO: 107 and 108 respectively; (v) SEQ ID NO: 109 and 110 respectively; and (vi) SEQ ID NO: 111 and 112 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human OX40, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 160 and 148 respectively; 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the diagram. In other embodiments, the first target antigen and / or the second target antigen comprises human ICOS, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human 41BB, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR7, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR8, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0074] In a further implementation scheme and according to any of the above, the first ABD binds to the ECD of the first target antigen.

[0075] In a further implementation scheme and according to any of the above, the second ABD binds to the ECD of the second target antigen.

[0076] In a further embodiment and according to any of the above, the first ABD binds to the ECD of the first target antigen, and the second ABD binds to the ECD of the second target antigen.

[0077] In a further embodiment and according to any of the above, the first domain connector and / or the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0078] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain linker, (iii) a second IL-12 subunit domain, and (iv) a first Fc domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, and (2) the variant IL-12 p35 subunit domain comprises an amino acid substitution selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) a second monomer comprising: (i) an anti-target antigen scFv, wherein the anti-target antigen scFv comprises a VH domain, a second domain adapter and a VL domain, (ii) a third domain adapter and (iii) a second Fc domain, wherein the anti-target antigen scFv is covalently linked to the third domain adapter at the N-terminus of the second Fc domain.

[0079] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0080] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0081] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0082] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In other embodiments, the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143, respectively; (ii) SEQ ID NO: 132 and 133, respectively; (iii) SEQ ID NO: 137 and 138, respectively; (iv) SEQ ID NO: 142 and 143, respectively; (v) SEQ ID NO: 147 and 148, respectively; (vi) SEQ ID NO: 130 and 131, respectively; (vii) SEQ ID NO: 154 and 143, respectively; (viii) SEQ ID NO: 156 and 143, respectively; (ix) SEQ ID NO: 158 and 148, respectively; (x) SEQ ID NO: 160 and 148, respectively; (xi) SEQ ID NO: 160 and 148, respectively; 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71 respectively; (ii) SEQ ID NO: 123 and 124 respectively; (iii) SEQ ID NO: 125 and 126 respectively; and (iv) SEQ ID NO: 127 and 128 respectively. In other embodiments, the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0083] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

[0084] In a further embodiment and according to any of the above, the VH domain of the anti-target antigen scFv is covalently linked to a third domain linker at the N-terminus of the second Fc domain. In other embodiments, the VL domain of the anti-target antigen scFv is covalently linked to a third domain linker at the N-terminus of the second Fc domain.

[0085] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0086] In a further embodiment and according to any of the above, the second domain connector comprises any of SEQ ID NO: 31-40.

[0087] In a further embodiment and according to any of the above, the first domain connector and / or the third domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector and / or the third domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the third domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the third domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0088] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising: (i) an anti-target antigen scFv, wherein the anti-target antigen scFv comprises a VH domain, a first domain adapter, and a VL domain; (ii) a second domain adapter; and (iii) a first Fc domain, wherein the anti-target antigen scFv is covalently linked to the second domain adapter at the N-terminus of the first Fc domain; and (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a second Fc domain; (ii) a third domain adapter; (iii) a first IL-12 subunit domain; (iv) a fourth domain adapter; and (v) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain. The p35 subunit domain and the second IL-12 subunit domain contain the IL-12 p40 subunit domain, and (2) the variant IL-12 p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

[0089] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0090] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0091] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0092] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In other embodiments, the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143, respectively; (ii) SEQ ID NO: 132 and 133, respectively; (iii) SEQ ID NO: 137 and 138, respectively; (iv) SEQ ID NO: 142 and 143, respectively; (v) SEQ ID NO: 147 and 148, respectively; (vi) SEQ ID NO: 130 and 131, respectively; (vii) SEQ ID NO: 154 and 143, respectively; (viii) SEQ ID NO: 156 and 143, respectively; (ix) SEQ ID NO: 158 and 148, respectively; (x) SEQ ID NO: 160 and 148, respectively; (xi) SEQ ID NO: 160 and 148, respectively; 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71 respectively; (ii) SEQ ID NO: 123 and 124 respectively; (iii) SEQ ID NO: 125 and 126 respectively; and (iv) SEQ ID NO: 127 and 128 respectively. In other embodiments, the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0093] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

[0094] In a further embodiment and according to any of the above, the VH domain of the anti-target antigen scFv is covalently linked to a second domain linker at the N-terminus of the first Fc domain. In other embodiments, the VL domain of the anti-target antigen scFv is covalently linked to a second domain linker at the N-terminus of the first Fc domain.

[0095] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0096] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40.

[0097] In a further embodiment and according to any of the above, the second, third, and / or fourth domain connectors comprise any of SEQ ID NO: 31-40. In some other embodiments, the second, third, and / or fourth domain connectors comprise covalent bonds. In some other embodiments, at least one domain connector comprises any of SEQ ID NO: 31-40, and at least one domain connector comprises covalent bonds. In some embodiments, at least one of the domain connectors comprises a hinge region of an Fc domain.

[0098] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain adapter, and (iii) a first Fc domain; and (b) a second monomer comprising: (i) a second IL-12 subunit domain, (ii) a second domain adapter, (iii) a second Fc domain, wherein the second IL-12 subunit domain is covalently linked to the second domain adapter at the N-terminus of the second Fc domain, (iv) a third domain adapter, and (v) an anti-target antigen scFv, wherein: (1) the second Fc domain is covalently linked to the third domain adapter at the N-terminus of the anti-target antigen scFv, (2) the anti-target antigen scFv comprises a VH domain, a fourth domain adapter, and a VL domain, and (3) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12. The p35 subunit domain, or the first IL-12 subunit domain contains the variant IL-12 p35 subunit domain and the second IL-12 subunit domain contains the IL-12 p40 subunit domain, and (4) the variant IL-12 p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

[0099] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0100] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0101] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0102] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In other embodiments, the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143, respectively; (ii) SEQ ID NO: 132 and 133, respectively; (iii) SEQ ID NO: 137 and 138, respectively; (iv) SEQ ID NO: 142 and 143, respectively; (v) SEQ ID NO: 147 and 148, respectively; (vi) SEQ ID NO: 130 and 131, respectively; (vii) SEQ ID NO: 154 and 143, respectively; (viii) SEQ ID NO: 156 and 143, respectively; (ix) SEQ ID NO: 158 and 148, respectively; (x) SEQ ID NO: 160 and 148, respectively; (xi) SEQ ID NO: 160 and 148, respectively; 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71 respectively; (ii) SEQ ID NO: 123 and 124 respectively; (iii) SEQ ID NO: 125 and 126 respectively; and (iv) SEQ ID NO: 127 and 128 respectively. In other embodiments, the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0103] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

[0104] In a further embodiment and according to any of the above, the VH domain of the anti-target antigen scFv is covalently linked to a third domain linker at the N-terminus of the second Fc domain. In other embodiments, the VL domain of the anti-target antigen scFv is covalently linked to a third domain linker at the N-terminus of the second Fc domain.

[0105] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0106] In a further embodiment and according to any of the above, the fourth domain connector comprises any of SEQ ID NO: 31-40.

[0107] In a further embodiment and according to any of the above, the first domain connector, the second domain connector, and / or the third domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector, the second domain connector, and / or the third domain connector comprises a covalent bond. In some other embodiments, at least one domain connector comprises any of SEQ ID NO: 31-40, and at least one domain connector comprises a covalent bond. In some embodiments, at least one of the domain connectors comprises a hinge region of an Fc domain.

[0108] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising: (i) a first IL-12 subunit domain, (ii) a first domain adapter, (iii) a second IL-12 subunit domain, wherein the first IL-12 subunit domain is covalently linked to the first domain adapter at the N-terminus of the second IL-12 subunit domain, (iv) a second domain adapter, (v) a first Fc domain, wherein the second IL-12 subunit domain is covalently linked to the second domain adapter at the N-terminus of the first Fc domain, (vi) a third domain adapter, and (vii) an anti-target antigen scFv, wherein: (1) the first Fc domain is covalently linked to the third domain adapter at the N-terminus of the anti-target antigen scFv, (2) the anti-target antigen scFv comprises a VH domain, a fourth domain adapter, and a VL domain, and (3) the first IL-12 subunit domain comprises IL-12 The p40 subunit domain and the second IL-12 subunit domain contain a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain contains a variant IL-12 p35 subunit domain and the second IL-12 subunit domain contains an IL-12 p40 subunit domain, and (4) the variant IL-12 p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) a second monomer containing a second Fc domain.

[0109] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0110] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0111] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

[0112] In a further embodiment and according to any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In other embodiments, the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143, respectively; (ii) SEQ ID NO: 132 and 133, respectively; (iii) SEQ ID NO: 137 and 138, respectively; (iv) SEQ ID NO: 142 and 143, respectively; (v) SEQ ID NO: 147 and 148, respectively; (vi) SEQ ID NO: 130 and 131, respectively; (vii) SEQ ID NO: 154 and 143, respectively; (viii) SEQ ID NO: 156 and 143, respectively; (ix) SEQ ID NO: 158 and 148, respectively; (x) SEQ ID NO: 160 and 148, respectively; (xi) SEQ ID NO: 160 and 148, respectively; 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as... Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises VH / VL domain pairs selected from the group consisting of: (i) SEQ ID NO: 70 and 71 respectively; (ii) SEQ ID NO: 123 and 124 respectively; (iii) SEQ ID NO: 125 and 126 respectively; and (iv) SEQ ID NO: 127 and 128 respectively. In other embodiments, the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0113] In a further embodiment and according to any of the above, the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

[0114] In a further embodiment and according to any of the above, the VH domain of the anti-target antigen scFv is covalently linked to a third domain linker at the N-terminus of the second Fc domain. In other embodiments, the VL domain of the anti-target antigen scFv is covalently linked to a third domain linker at the N-terminus of the second Fc domain.

[0115] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0116] In a further embodiment and according to any of the above, the fourth domain connector comprises any of SEQ ID NO: 31-40.

[0117] In a further embodiment and according to any of the above, the first domain connector, the second domain connector, and / or the third domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector, the second domain connector, and / or the third domain connector comprises a covalent bond. In some other embodiments, at least one domain connector comprises any of SEQ ID NO: 31-40, and at least one domain connector comprises a covalent bond. In some embodiments, at least one of the domain connectors comprises a hinge region of an Fc domain.

[0118] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising: (i) a first anti-target antigen scFv, wherein the first anti-target antigen scFv comprises a first VH (VH1) domain, a first domain adapter, and a first VL (VL1) domain; (ii) a second domain adapter; and (iii) a first Fc domain, wherein the first anti-target antigen scFv is covalently linked to the second domain adapter at the N-terminus of the first Fc domain; and (b) a second monomer comprising: (i) a second anti-target antigen scFv, wherein the second anti-target antigen scFv comprises a second VH (VH2) domain, a third domain adapter, and a second VL1 domain. (ii) a fourth domain linker, (iii) a second Fc domain, wherein the second anti-target antigen scFv is covalently linked to the fourth domain linker at the N-terminus of the second Fc domain, (iv) a fifth domain linker, (v) a first IL-12 subunit domain, (vi) a sixth domain linker, and (vii) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain contains an IL-12 p40 subunit domain and the second IL-12 subunit domain contains a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain contains a variant IL-12 p35 subunit domain and the second IL-12 subunit domain contains an IL-12 p40 subunit domain, (2) the variant IL-12 p35 subunit domain contains an amino acid substitution selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, (3) the second Fc structural domain is covalently connected to the fifth structural domain connector at the N end of the first IL-12 subunit structural domain, and (4) the first IL-12 subunit structural domain is covalently connected to the sixth structural domain connector at the N end of the second IL-12 subunit structural domain.

[0119] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0120] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0121] In a further embodiment and according to any of the above, the first anti-target antigen scFv binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0122] In a further embodiment and according to any of the above, the second anti-target antigen scFv binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0123] In a further embodiment and according to any of the above, the first anti-target antigen scFv and the second anti-target antigen scFv are both bound to target antigens selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8. In other embodiments, the first anti-target antigen scFv is bound to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8, and the second anti-target antigen scFv is bound to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0124] In a further embodiment and according to any of the above, the first target antigen and / or the second target antigen comprises human PD1, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human LAG3, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human OX40, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In other embodiments, the first target antigen and / or the second target antigen comprises human ICOS, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human 41BB, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR7, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR8, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0125] In a further embodiment and according to any of the above, the first anti-target antigen scFv binds to the ECD of the first target antigen.

[0126] In a further embodiment and according to any of the above, the second anti-target antigen scFv binds to the ECD of the second target antigen.

[0127] In a further embodiment and according to any of the above, the first anti-target antigen scFv binds to the ECD of the first target antigen, and the second anti-target antigen scFv binds to the ECD of the second target antigen.

[0128] In a further embodiment and according to any of the above, the VH1 domain of the first anti-target antigen scFv is covalently linked to a second domain linker at the N-terminus of the first Fc domain. In other embodiments, the VL1 domain of the anti-target antigen scFv is covalently linked to a second domain linker at the N-terminus of the first Fc domain.

[0129] In a further embodiment and according to any of the above, the VH2 domain of the second anti-target antigen scFv is covalently linked to a fourth domain linker at the N-terminus of the second Fc domain. In other embodiments, the VL2 domain of the second anti-target antigen scFv is covalently linked to a fourth domain linker at the N-terminus of the second Fc domain.

[0130] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0131] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, and the third domain connector comprises any of SEQ ID NO: 31-40.

[0132] In a further embodiment and according to any of the above, the second, fourth, fifth, and / or sixth domain connectors comprise any of SEQ ID NO: 31-40. In some other embodiments, the second, fourth, fifth, and / or sixth domain connectors comprise covalent bonds. In some other embodiments, at least one domain connector comprises any of SEQ ID NO: 31-40, and at least one domain connector comprises covalent bonds. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0133] In another aspect, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising: (i) a first anti-target antigen scFv, wherein the first anti-target antigen scFv comprises a first VH (VH1) domain, a first domain adapter, and a first VL (VL1) domain; (ii) a second domain adapter; (iii) a first Fc domain, wherein the first anti-target antigen scFv is covalently linked to the second domain adapter at the N-terminus of the first Fc domain; (iv) a third domain adapter; and (v) a first IL-12 subunit domain, wherein the first Fc domain is covalently linked to the third domain adapter at the N-terminus of the first IL-12 subunit domain; and (b) a second monomer comprising: (i) a second anti-target antigen scFv, wherein the second anti-target antigen scFv comprises a second VH (VH2) domain, a fourth domain adapter, and a second VL1 domain. (ii) a fifth domain linker, (iii) a second Fc domain, wherein the second anti-target antigen scFv is covalently linked to the fifth domain linker at the N-terminus of the second Fc domain, (iv) a sixth domain linker, and (v) a second IL-12 subunit domain, wherein: (1) the second Fc domain is covalently linked to the sixth domain linker at the N-terminus of the second IL-12 subunit domain, (2) the first IL-12 subunit domain contains an IL-12 p40 subunit domain and the second IL-12 subunit domain contains a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain contains a variant IL-12 p35 subunit domain and the second IL-12 subunit domain contains an IL-12 p40 subunit domain, and (3) the variant IL-12 p35 subunit domain contains an amino acid substitution selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

[0134] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0135] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0136] In a further embodiment and according to any of the above, the first anti-target antigen scFv binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0137] In a further embodiment and according to any of the above, the second anti-target antigen scFv binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0138] In a further embodiment and according to any of the above, the first anti-target antigen scFv and the second anti-target antigen scFv are both bound to target antigens selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8. In other embodiments, the first anti-target antigen scFv is bound to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8, and the second anti-target antigen scFv is bound to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

[0139] In a further embodiment and according to any of the above, the first target antigen and / or the second target antigen comprises human PD1, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human LAG3, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human OX40, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as Figure 13 Figures 75, 81, and 85 Figure 112As shown in the figure. In other embodiments, the first target antigen and / or the second target antigen comprises human ICOS, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human 41BB, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR7, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 78 and 79, respectively. In other embodiments, the first target antigen and / or the second target antigen comprises human CCR8, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

[0140] In a further embodiment and according to any of the above, the first anti-target antigen scFv binds to the ECD of the first target antigen.

[0141] In a further embodiment and according to any of the above, the second anti-target antigen scFv binds to the ECD of the second target antigen.

[0142] In a further embodiment and according to any of the above, the first anti-target antigen scFv binds to the ECD of the first target antigen, and the second anti-target antigen scFv binds to the ECD of the second target antigen.

[0143] In a further embodiment and according to any of the above, the VH1 domain of the first anti-target antigen scFv is covalently linked to a second domain linker at the N-terminus of the first Fc domain. In other embodiments, the VL1 domain of the anti-target antigen scFv is covalently linked to a second domain linker at the N-terminus of the first Fc domain.

[0144] In a further embodiment and according to any of the above, the VH2 domain of the second anti-target antigen scFv is covalently linked to the fifth domain linker at the N-terminus of the second Fc domain. In other embodiments, the VL2 domain of the second anti-target antigen scFv is covalently linked to the fifth domain linker at the N-terminus of the second Fc domain.

[0145] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0146] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, and the fourth domain connector comprises any of SEQ ID NO: 31-40.

[0147] In a further embodiment and according to any of the above, the second, third, fifth, and / or sixth domain connectors comprise any of SEQ ID NO: 31-40. In some other embodiments, the second, third, fifth, and / or sixth domain connectors comprise covalent bonds. In some other embodiments, at least one domain connector comprises any of SEQ ID NO: 31-40, and at least one domain connector comprises covalent bonds. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0148] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain linker, (iii) a second IL-12 subunit domain, and (iv) a first Fc domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, and (2) the variant IL-12 p35 subunit domain comprises an amino acid substitution selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a VHH domain, (ii) a second domain linker, and (iii) a second Fc domain, wherein the VHH domain binds to the target antigen.

[0149] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0150] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0151] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0152] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises any of SEQ ID NO: 31-40, and the second domain connector comprises a covalent bond. In some other embodiments, the first domain connector comprises a covalent bond, and the second domain connector comprises any of SEQ ID NO: 31-40. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0153] In further embodiments and according to any of the above, the VHH domain binds to target antigens selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[0154] In a further embodiment and according to any of the foregoing, the target antigen comprises PD1, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(g)).

[0155] In a further embodiment and according to any of the above, the target antigen comprises 41BB, and the VHH domain comprises: SEQ ID NO: 129. In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO: 86.

[0156] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a VHH domain, (ii) a first domain adapter, and (iii) a first Fc domain; and (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a second Fc domain, (ii) a second domain adapter, (iii) a first IL-12 subunit domain, (iv) a third domain adapter, and (v) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, and (2) a variant IL-12 The p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

[0157] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0158] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0159] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0160] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, the second domain connector comprises any of SEQ ID NO: 31-40, and the third domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector, the second domain connector, and / or the third domain connector comprises a covalent bond. In some other embodiments, at least one of the domain connectors comprises any of SEQ ID NO: 31-40, and at least one of the domain connectors comprises a covalent bond. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0161] In further embodiments and according to any of the above, the VHH domain binds to target antigens selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[0162] In a further embodiment and according to any of the foregoing, the target antigen comprises PD1, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(g)).

[0163] In a further embodiment and according to any of the above, the target antigen comprises 41BB, and the VHH domain comprises: SEQ ID NO: 129. In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO: 86.

[0164] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain adapter, and (iii) a first Fc domain; and (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a second IL-12 subunit domain, (ii) a second domain adapter, (iii) a second Fc domain, (iv) a third domain adapter, and (v) a VHH domain, wherein: (1) the VHH domain binds to the target antigen, (2) the first IL-12 subunit domain contains an IL-12 p40 subunit domain and the second IL-12 subunit domain contains a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain contains a variant IL-12 p35 subunit domain and the second IL-12 subunit domain contains an IL-12 p40 subunit domain, and (3) a variant IL-12 The p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

[0165] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0166] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0167] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0168] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, the second domain connector comprises any of SEQ ID NO: 31-40, and the third domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector, the second domain connector, and / or the third domain connector comprises a covalent bond. In some other embodiments, at least one of the domain connectors comprises any of SEQ ID NO: 31-40, and at least one of the domain connectors comprises a covalent bond. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0169] In further embodiments and according to any of the above, the VHH domain binds to target antigens selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[0170] In a further embodiment and according to any of the foregoing, the target antigen comprises PD1, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(g)).

[0171] In a further embodiment and according to any of the above, the target antigen comprises 41BB, and the VHH domain comprises: SEQ ID NO: 129. In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO: 86.

[0172] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain adapter, (iii) a second IL-12 subunit domain, (iv) a second domain adapter, (v) a first Fc domain, (vi) a third domain adapter, and (vii) a VHH domain, wherein: (1) the VHH domain binds to a target antigen, (2) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, and (3) the variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) a second monomer containing a second Fc domain.

[0173] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0174] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0175] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0176] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, the second domain connector comprises any of SEQ ID NO: 31-40, and the third domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first domain connector, the second domain connector, and / or the third domain connector comprises a covalent bond. In some other embodiments, at least one of the domain connectors comprises any of SEQ ID NO: 31-40, and at least one of the domain connectors comprises a covalent bond. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0177] In further embodiments and according to any of the above, the VHH domain binds to target antigens selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[0178] In a further embodiment and according to any of the foregoing, the target antigen comprises PD1, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(g)).

[0179] In a further embodiment and according to any of the above, the target antigen comprises 41BB, and the VHH domain comprises: SEQ ID NO: 129. In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO: 86.

[0180] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from its N-terminus to its C-terminus: (i) a first VHH domain, (ii) a first domain adapter, (iii) a first Fc domain, (iv) a second domain adapter, (v) a first IL-12 subunit domain, (vi) a third domain adapter, and (vii) a second IL-12 subunit domain, wherein: (1) the first IL-12 subunit domain comprises an IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain, (2) the variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) a first VHH domain that binds to a first target antigen; and (b) a second monomer that comprises, from the N-terminus to the C-terminus: (i) a second VHH domain, (ii) a fourth domain linker, and (iii) a second Fc domain, wherein the second VHH domain binds to a second target antigen.

[0181] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0182] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0183] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0184] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, the second domain connector comprises any of SEQ ID NO: 31-40, the third domain connector comprises any of SEQ ID NO: 31-40, and the fourth domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first, second, third, and / or fourth domain connectors comprise covalent bonds. In some other embodiments, at least one domain connector comprises any of SEQ ID NO: 31-40, and at least one domain connector comprises covalent bonds. In some embodiments, at least one of the domain connectors comprises a hinge region of an Fc domain.

[0185] In a further embodiment and according to any of the above, the first VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

[0186] In a further embodiment and according to any of the above, the second VHH domain binds to a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

[0187] In a further embodiment and according to any of the above, both the first VHH domain and the second VHH domain bind to a target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7, and (vii) CCR8. In other embodiments, the VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7, and (vii) CCR8, and the second VHH domain binds to a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7, and (vii) CCR8.

[0188] In a further embodiment and according to any of the foregoing, the first target antigen and / or the second target antigen comprises PD1, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(a)). In other embodiments, the first target antigen and / or the second target antigen comprises LAG3, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(b)). In other embodiments, the first target antigen and / or the second target antigen comprises OX40, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(c)). In other embodiments, the first target antigen and / or the second target antigen comprises 41BB, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(d)). In other embodiments, the first target antigen and / or the second target antigen comprises ICOS, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(e)). In other embodiments, the first target antigen and / or the second target antigen comprises CCR7, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(f)). In other embodiments, the first target antigen and / or the second target antigen comprises CCR8, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(g)).

[0189] In a further implementation and according to any of the above, the first VHH domain is humanized.

[0190] In a further implementation and according to any of the above, the second VHH domain is humanized.

[0191] In a further implementation and according to any of the above, the first VHH domain is humanized, and the second VHH domain is humanized.

[0192] In a further embodiment and according to any of the above, the first target antigen and / or the second target antigen comprises 41BB, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO: 129. In other embodiments, the first target antigen and / or the second target antigen comprises CCR8, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO: 86.

[0193] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer comprising, from the N-terminus to the C-terminus: (i) a first VHH domain, (ii) a first domain adapter, (iii) a first Fc domain, (iv) a second domain adapter, and (v) a first IL-12 subunit domain, wherein the first VHH domain binds a first target antigen; and (b) a second monomer comprising, from the N-terminus to the C-terminus: (i) a second VHH domain, (ii) a third domain adapter, (iii) a second Fc domain, (iv) a fourth domain adapter, and (v) a second IL-12 subunit domain, wherein: (1) the second VHH domain binds a second target antigen, (2) the first IL-12 subunit domain contains an IL-12 p40 subunit domain and the second IL-12 subunit domain contains a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain contains a variant IL-12 p35 subunit domain. The p35 subunit domain and the second IL-12 subunit domain contain the IL-12 p40 subunit domain, and (3) the variant IL-12 p35 subunit domain contains a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

[0194] In a further embodiment and as described above, the variant IL-12 p35 subunit domain includes any of SEQ ID NO: 17-21.

[0195] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises SEQ ID NO: 4. In other embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain. In some further embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, which comprises any of SEQ ID NO: 23-25. In still some further embodiments, the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25, and the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0196] In a further embodiment and according to any of the foregoing, the first Fc domain comprises any of SEQ ID NO: 29 or 30, and the second Fc domain comprises any of SEQ ID NO: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations including T366W / S354C, and the second Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C; while in other embodiments, the first Fc domain further comprises a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations including T366W / S354C.

[0197] In a further embodiment and according to any of the above, the first domain connector comprises any of SEQ ID NO: 31-40, the second domain connector comprises any of SEQ ID NO: 31-40, the third domain connector comprises any of SEQ ID NO: 31-40, and the fourth domain connector comprises any of SEQ ID NO: 31-40. In some other embodiments, the first, second, third, and / or fourth domain connectors comprise covalent bonds. In some other embodiments, at least one of the domain connectors comprises any of SEQ ID NO: 31-40, and at least one of the domain connectors comprises covalent bonds. In some embodiments, at least one of the domain connectors comprises a hinge region of the Fc domain.

[0198] In a further embodiment and according to any of the above, the first VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

[0199] In a further embodiment and according to any of the above, the second VHH domain binds to a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

[0200] In a further embodiment and according to any of the above, both the first VHH domain and the second VHH domain bind to a target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7, and (vii) CCR8. In other embodiments, the first VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7, and (vii) CCR8, and the second VHH domain binds to a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7, and (vii) CCR8.

[0201] In a further embodiment and according to any of the foregoing, the first target antigen and / or the second target antigen comprises PD1, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(a)). In other embodiments, the first target antigen and / or the second target antigen comprises LAG3, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(b)). In other embodiments, the first target antigen and / or the second target antigen comprises OX40, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(c)). In other embodiments, the first target antigen and / or the second target antigen comprises 41BB, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(d)). In other embodiments, the first target antigen and / or the second target antigen comprises ICOS, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(e)). In other embodiments, the first target antigen and / or the second target antigen comprises CCR7, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(f)). In other embodiments, the first target antigen and / or the second target antigen comprises CCR8, and the first VHH domain and / or the second VHH domain comprises the VHH domain described herein (see, for example, Section (III)(A)(2)(g)).

[0202] In a further implementation and according to any of the above, the first VHH domain is humanized.

[0203] In a further implementation and according to any of the above, the second VHH domain is humanized.

[0204] In a further implementation and according to any of the above, the first VHH domain is humanized, and the second VHH domain is humanized.

[0205] In a further embodiment and according to any of the above, the first target antigen and / or the second target antigen comprises 41BB, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO: 129. In other embodiments, the first target antigen and / or the second target antigen comprises CCR8, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO: 86.

[0206] On the other hand, this disclosure provides nucleic acid compositions comprising one or more nucleic acids encoding the IL-12 Fc fusion protein as described herein.

[0207] On the other hand, this disclosure provides an expression vector comprising one or more nucleic acids encoding the IL-12 Fc fusion protein as described herein.

[0208] On the other hand, this disclosure provides a host cell, wherein the host cell is transformed by an expression vector as described herein.

[0209] In another aspect, this disclosure provides a method for preparing an IL-12 Fc fusion protein, comprising: (a) culturing host cells transformed by an expression vector containing one or more nucleic acids encoding the IL-12 Fc fusion protein as described herein; and (b) isolating and / or purifying the IL-12 Fc fusion protein.

[0210] On the other hand, this disclosure provides an IL-12 Fc fusion protein comprising: (a) a first monomer, (b) a second monomer, and (c) a third monomer, wherein the first monomer, the second monomer, and the third monomer comprise: (i) respectively, SEQ ID NO: 49, 153, and 145; (ii) respectively, SEQ ID NO: 48, 146, and 145; (iii) respectively, SEQ ID NO: 48, 151, and 150; (iv) respectively, SEQ ID NO: 48, 153, and 145; (v) respectively, SEQ ID NO: 48, 155, and 145; (vi) respectively, SEQ ID NO: 48, 157, and 145; (vii) respectively, SEQ ID NO: 48, 159, and 150; (viii) respectively, SEQ ID NO: 48, 161, and 150; (ix) respectively, SEQ ID NO: 48, 163, and 150; (x) respectively, SEQ ID NO: 48, 165, and 150; (xi) respectively, SEQ ID NO: 48, 167, and 150; (xii) respectively, SEQ ID NO: 48, 169, and 150; (xiii) respectively, SEQ ID NO: 49, 141, and 140; (xiv) respectively, SEQ ID NO: 49, 146, and 145; (xv) respectively, SEQ ID NO: 49, 151, and 150; (xvi) respectively, SEQ ID NO: 48, 141, and 140; (xvii) respectively, SEQ ID NO: 49, 155, and 145; (xviii) respectively, SEQ ID NO: 49, 157, and 145; (xix) respectively, SEQ ID NO: 49, 159, and 150; (xx) respectively, SEQ ID NO: 49, 161, and 150; (xxi) respectively, SEQ ID NO: 49, 163, and 150; (xxii) respectively, SEQ ID NO: 49, 165, and 150; (xxiii) respectively, SEQ ID NO: 49, 167, and 150; (xxiv) respectively, SEQ ID NO: 49, 169, and 150; (xxv) respectively, SEQ ID NO: 50, 141, and 140; (xxvi) respectively, SEQ ID NO: 50, 146, and 145; (xxvii) respectively, SEQ ID NO: 50, 151, and 150; (xxviii) respectively, SEQ ID NO: 50, 153, and 145; (xxix) respectively, SEQ ID NO: 50, 155, and 145;(xxx) respectively, SEQ ID NO: 50, 157 and 145; (xxxi) respectively, SEQ ID NO: 50, 159 and 150; (xxxii) respectively, SEQ ID NO: 50, 161 and 150; (xxxiii) respectively, SEQ ID NO: 50, 163 and 150; (xxxiv) respectively, SEQ ID NO: 50, 165 and 150; (xxxv) respectively, SEQ ID NO: 50, 167 and 150; (xxxvi) respectively, SEQ ID NO: 50, 169 and 150; (xxxvii) respectively, SEQ ID NO: 51, 141 and 140; (xxxviii) respectively, SEQ ID NO: 51, 146 and 145; (xxxix) respectively, SEQ ID NO: 51, 151 and 150; (xl) respectively, SEQ ID NO: 51, 153, and 145; (xli) respectively, SEQ ID NO: 51, 155, and 145; (xlii) respectively, SEQ ID NO: 51, 157, and 145; (xliii) respectively, SEQ ID NO: 51, 159, and 150; (xliv) respectively, SEQ ID NO: 51, 161, and 150; (xlv) respectively, SEQ ID NO: 51, 163, and 150; (xlvi) respectively, SEQ ID NO: 51, 165, and 150; (xlvii) respectively, SEQ ID NO: 51, 167, and 150; (xlviii) respectively, SEQ ID NO: 51, 169, and 150; (xlix) respectively, SEQ ID NO: 52, 141, and 140; (l) respectively, SEQ ID NO: 52, 146, and 145; (li) respectively, SEQ ID NO: 52, 151, and 150; (lii) respectively, SEQ ID NO: 52, 153, and 145; (liii) respectively, SEQ ID NO: 52, 155, and 145; (liv) respectively, SEQ ID NO: 52, 157, and 145; (lv) respectively, SEQ ID NO: 52, 159, and 150; (lvi) respectively, SEQ ID NO: 52, 161, and 150; (lvii) respectively, SEQ ID NO: 52, 163, and 150; (lviii) respectively, SEQ ID NO: 52, 165, and 150; (lix) respectively, SEQ ID NO: 52, 167, and 150; (lx) respectively, SEQ ID NO: 52, 169, and 150;(lxi) respectively, SEQ ID NO: 48, 174 and 173; (lxii) respectively, SEQ ID NO: 49, 174 and 173; (lxiii) respectively, SEQ ID NO: 50, 174 and 173; (lxiv) respectively, SEQ ID NO: 51, 174 and 173; or (lxv) respectively, SEQ ID NO: 52, 174 and 173.

[0211] In a further embodiment and according to the foregoing, the first monomer comprises the amino acid sequence of SEQ ID NO: 49, the second monomer comprises the amino acid sequence of SEQ ID NO: 153, and the third monomer comprises the amino acid sequence of SEQ ID NO: 145. In some other further embodiments, the first monomer comprises the amino acid sequence of SEQ ID NO: 48, the second monomer comprises the amino acid sequence of SEQ ID NO: 174, and the third monomer comprises the amino acid sequence of SEQ ID NO: 173. In some other further embodiments, the first monomer comprises the amino acid sequence of SEQ ID NO: 49, the second monomer comprises the amino acid sequence of SEQ ID NO: 174, and the third monomer comprises the amino acid sequence of SEQ ID NO: 173. In some other further embodiments, the first monomer comprises the amino acid sequence of SEQ ID NO: 50, the second monomer comprises the amino acid sequence of SEQ ID NO: 174, and the third monomer comprises the amino acid sequence of SEQ ID NO: 173. In some further embodiments, the first monomer comprises the amino acid sequence of SEQ ID NO: 51, the second monomer comprises the amino acid sequence of SEQ ID NO: 174, and the third monomer comprises the amino acid sequence of SEQ ID NO: 173. In some further embodiments, the first monomer comprises the amino acid sequence of SEQ ID NO: 52, the second monomer comprises the amino acid sequence of SEQ ID NO: 174, and the third monomer comprises the amino acid sequence of SEQ ID NO: 173.

[0212] On the other hand, this disclosure provides a method for treating cancer in human subjects, comprising administering to human subjects in need an IL-12 Fc fusion protein as described herein, wherein the IL-12 Fc fusion protein binds to target cells.

[0213] In a further implementation scheme and as described above, the target cells express: (1) IL-12Rβ2, and (2) cell surface antigens selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7 and (g) human CCR8.

[0214] In a further embodiment and as described above, the cell surface antigen is human PD1, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human LAG3, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human OX40, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human ICOS, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human 41BB, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted NK cells; (iii) activated and / or exhausted CD8+ T cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human CCR7, and the target cells include: (i) naive CD4+ T cells; (ii) naive CD8+ T cells; or (iii) naive regulatory T cells. In other embodiments, the cell surface antigen is human CCR8, and the target cells include: (i) activated and / or exhausted CD4+ T cells; or (ii) activated and / or exhausted regulatory T cells.

[0215] On the other hand, this disclosure provides a method for activating target cells in a human subject, comprising administering to a human subject in need an IL-12 Fc fusion protein as described herein, wherein the IL-12 Fc fusion protein binds to the target cells.

[0216] In a further implementation scheme and as described above, the target cells express: (1) IL-12Rβ2, and (2) cell surface antigens selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7 and (g) human CCR8.

[0217] In a further embodiment and as described above, the cell surface antigen is human PD1, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human LAG3, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human OX40, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human ICOS, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human 41BB, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted NK cells; (iii) activated and / or exhausted CD8+ T cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human CCR7, and the target cells include: (i) naive CD4+ T cells; (ii) naive CD8+ T cells; or (iii) naive regulatory T cells. In other embodiments, the cell surface antigen is human CCR8, and the target cells include: (i) activated and / or exhausted CD4+ T cells; or (ii) activated and / or exhausted regulatory T cells.

[0218] In another aspect, this disclosure provides a method for stimulating IL-12 signaling in target cells, comprising administering an IL-12 Fc fusion protein as described herein to a human subject in need, wherein the IL-12 Fc fusion protein binds to the target cells.

[0219] In a further implementation scheme and as described above, the target cells express: (1) IL-12Rβ2, and (2) cell surface antigens selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7 and (g) human CCR8.

[0220] In a further embodiment and as described above, the cell surface antigen is human PD1, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human LAG3, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human OX40, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human ICOS, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human 41BB, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted NK cells; (iii) activated and / or exhausted CD8+ T cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human CCR7, and the target cells include: (i) naive CD4+ T cells; (ii) naive CD8+ T cells; or (iii) naive regulatory T cells. In other embodiments, the cell surface antigen is human CCR8, and the target cells include: (i) activated and / or exhausted CD4+ T cells; or (ii) activated and / or exhausted regulatory T cells.

[0221] In another aspect, this disclosure provides a method for inducing the production and / or secretion of one or more cytokines in target cells, comprising administering an IL-12 Fc fusion protein as described herein to a human subject in need, wherein the IL-12 Fc fusion protein binds to the target cells.

[0222] In a further implementation scheme and as described above, the target cells express: (1) IL-12Rβ2, and (2) cell surface antigens selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7 and (g) human CCR8.

[0223] In a further embodiment and as described above, the cell surface antigen is human PD1, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human LAG3, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human OX40, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human ICOS, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human 41BB, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted NK cells; (iii) activated and / or exhausted CD8+ T cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human CCR7, and the target cells include: (i) naive CD4+ T cells; (ii) naive CD8+ T cells; or (iii) naive regulatory T cells. In other embodiments, the cell surface antigen is human CCR8, and the target cells include: (i) activated and / or exhausted CD4+ T cells; or (ii) activated and / or exhausted regulatory T cells.

[0224] On the other hand, this disclosure provides a method for reprogramming and / or altering the differentiation state of target cells, comprising administering an IL-12 Fc fusion protein as described herein, wherein the IL-12 Fc fusion protein binds to the target cells.

[0225] In a further implementation scheme and as described above, the target cells express: (1) IL-12Rβ2, and (2) cell surface antigens selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7 and (g) human CCR8.

[0226] In a further embodiment and as described above, the cell surface antigen is human PD1, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human LAG3, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human OX40, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human ICOS, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human 41BB, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted NK cells; (iii) activated and / or exhausted CD8+ T cells; or (iv) activated and / or exhausted regulatory T cells. In other embodiments, the cell surface antigen is human CCR7, and the target cells include: (i) naive CD4+ T cells; (ii) naive CD8+ T cells; or (iii) naive regulatory T cells. In other embodiments, the cell surface antigen is human CCR8, and the target cells include: (i) activated and / or exhausted CD4+ T cells; or (ii) activated and / or exhausted regulatory T cells. Attached Figure Description

[0227] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments that utilize the principles of the invention, and in the drawings (also referred to herein as “Fig.”, “FIG.”, “Figure”, “Figures”, “Figs.”, and “FIGs.”): Figure 1 A sequence list of human wild-type IL-12 subunits α and β (precursor and mature form sequences) is shown.

[0228] Figure 2 A sequence list of human wild-type IL-12Rβ1 (+extracellular domain sequence) and IL-12Rβ2 (+extracellular domain sequence) is shown.

[0229] Figure 3 A sequence list of mouse wild-type IL-12 subunits α and β (precursor and mature forms) is shown.

[0230] Figure 4 A sequence list of mouse wild-type IL-12Rβ1 (+extracellular domain sequence) and IL-12Rβ2 (+extracellular domain sequence) is shown.

[0231] Figure 5 A list of variant IL-12 p35 subunit sequences (both human and mouse) containing one or more substitution mutations is shown.

[0232] Figure 6 A list of variant IL-12 p40 subunit sequences is shown.

[0233] Figure 7 An exemplary sequence of the human IgG1 Fc domain (G1m allotype and Fc domain containing one or more substitutions) is shown.

[0234] Figure 8 A list of exemplary domain connector sequences is shown.

[0235] Figure 9 A list of exemplary antigen-binding domain (ABD) amino acid sequences of chicken egg white lysozyme is shown, along with exemplary heavy and light chain amino acid sequences (mouse and human) for the control IL-12 fusion protein.

[0236] Figure 10 ( Figures 10A-10D The diagram shows a list of amino acid sequences of exemplary IL-12 Fc fusion protein monomers for use in the IL-12 Fc fusion protein of the present invention.

[0237] Figure 11 ( Figure 11A and Figure 11B This section shows a list of exemplary antigen-binding domain (ABD) sequences for PD1 (human and mouse), as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of this invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0238] Figure 12 ( Figure 12A and Figure 12B This section shows a list of exemplary ABD sequences that bind to LAG3 (human / mouse cross-reactivity and human), as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of this invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0239] Figure 13 A list of exemplary ABD sequences binding to OX40 (human and mouse) is shown, along with exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of this invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0240] Figure 14 ( Figure 14A and Figure 14B The diagram shows a list of exemplary ABD sequences that bind to 41BB (human / mouse cross-reactivity and human), as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of the present invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0241] Figure 15 Figure 15A and Figure 15B This section shows a list of exemplary ABD sequences that bind to ICOS (human / mouse cross-reactivity and human), as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of this invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0242] Figure 16 A list of exemplary ABD sequences that bind to CCR7 (human) is shown, as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of the present invention.

[0243] Figure 17 ( Figure 17A and Figure 17BThis section shows a list of exemplary ABD sequences that bind to CCR8 (human / mouse cross-reactivity), as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of this invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0244] Figure 18 ( Figures 18A-18HThe diagram illustrates an exemplary form of the IL-12-ABD fusion protein. In some exemplary configurations, the IL-12-ABD fusion protein of the present invention is configured such that it comprises one antigen-binding domain (i.e., a monovalent monospecific form). In other exemplary configurations, the IL-12-ABD fusion protein of the present invention is configured such that it comprises two antigen-binding domains. In such configurations, the IL-12-ABD fusion protein of the present invention may be configured such that the ABDs bind to the same target antigen (i.e., a bivalent monospecific form), while in other configurations, the IL-12-ABD fusion protein of the present invention is configured such that the first ABD and the second ABD bind to different target antigens (i.e., a monovalent bispecific form). As will be understood by those skilled in the art, it is clearly understood that one or more ABDs depicted herein may take different configurations. Suitable ABD forms include, but are not limited to, Fab forms, scFv forms, and nanobody forms (VHH domains). Furthermore, in some exemplary configurations, the IL-12-ABD fusion protein of the present invention is configured such that the first IL-12 subunit domain and the second IL-12 subunit domain are located on the same monomer of the IL-12 fusion protein of the present invention (typically covalently linked to each other using domain linkers), while in other exemplary configurations, the first IL-12 subunit domain and the second IL-12 subunit domain are located on different monomers constituting the IL-12 fusion protein of the present invention. Additionally, although the schematic diagram generally shows a single-stranded IL-12 arranged from the N-terminus to the C-terminus as (variant) IL-12 p40 subunit domain - domain linker - (variant) IL-12 p35 subunit domain, it is also contemplated that the IL-12 subunit domains may be arranged from the N-terminus to the C-terminus as variant IL-12 p35 subunit domain - domain linker - (variant) IL-12 p40 subunit domain. Those skilled in the art will understand that, while all exemplary configurations show a first IL-12 subunit domain and a second IL-12 subunit domain covalently linked to a monomer of the IL-12-ABD fusion protein of the present invention, or covalently linked to a first and a second monomer of the fusion protein, it is also contemplated that the IL-12-ABD fusion protein of the present invention may be configured such that: only one of the IL-12 subunit domains (e.g., the variant IL-12 p35 subunit domain, or (variant) IL-12 p40 subunit domain) is covalently linked to a monomer of the IL-12-ABD fusion protein of the present invention, and the other IL-12 subunit domain (e.g., (variant) IL-12 p40 subunit domain, or variant IL-12 p35 subunit domain) is expressed in a soluble protein form that is not covalently linked to any of the monomers of the IL-12-ABD fusion protein of the present invention.

[0245] Figure 19A list of exemplary IL-12 Fc fusion proteins is shown, including ABD specificity (human), IL-12 p35 subunit domain mutations, and amino acid sequence identifiers of such exemplary IL-12 Fc fusion proteins, as well as exemplary control fusion proteins (ABD specificity to chicken oocyte lysozyme, or lacking ABD).

[0246] Figure 20 Figure 20A and Figure 20B The diagram shows SDS-PAGE analysis of an exemplary purified IL-12-ABD fusion protein in both reduced (+DTT) and non-reduced (-DTT) states.

[0247] Figure 21 Representative flow cytometry analysis of PD-1 expression on purified naïve human NK cells is shown.

[0248] Figure 22 ( Figure 22A and Figure 22B The results of alpha-LISA measurements of IFNγ in naïve NK cells incubated with commercially available recombinant IL-12 (rIL-12) or the IL-12-ABD fusion protein shown are presented. The sign and error bar indicate the mean and SD, respectively.

[0249] Figure 23 Flow cytometry analysis of PD-1 on human PBMCs from healthy donors activated with 600 U / mL IL-2 and 32 ng / mL OKT3 is shown.

[0250] Figure 24 ( Figure 24A and Figure 24B The results of alpha-LISA measurements of IFNγ in IL-2 / OKT3-activated human PBMCs in the presence of 50 U / mL human IL-2, in culture medium incubated with commercially available recombinant IL-12 (rIL-12) or the IL-12-ABD fusion protein shown, are presented. The symbols and error bars indicate the mean and SD, respectively.

[0251] Figure 25 shows the comparisons displayed in the listed datasets. Figures 25A-25I Selected volcano plots of differential gene expression within the range (log-transformation fold change (logFC) on the x-axis; adjusted p-values ​​of negative log-transformation (-log(adj)) on the y-axis). p ))), and a summary table with the upregulated rank of each gene within each dataset and the rank of each gene after multiplying the rank products of the listed datasets and comparisons ( Figure 25J ).

[0252] Figure 26This shows each listed cell type that was positive for each cell surface target in a range of tissue and tumor samples (defined in the legend). x The percentage of (axis). For RNA / transcription abundance data, a cell is considered positive if at least one transcript is detected. For ADT / cell surface antibody markers, a cell is considered positive if the abundance of transformation is greater than 1 (an arbitrary threshold selected to exclude 'background staining' on various markers in the relevant dataset).

[0253] Figure 27 This shows a list of cell type clusters from a range of tissues in the Human Protein Atlas. x Selected genes in the axis y The average transcript abundance (as measured by normalized transcript per kilobase million normalized transcripts (nTPM)) is categorized by axis. Individual cell types have been clustered into relevant groups. At the bottom ).

[0254] Figure 28 shows different cell types ( x (axis) and a series of structures (see legend) CCR8 Percentage of positive cells ( Figure 28A ); a list of cell type clusters from a series of tissues in the human protein atlas ( x In the axis, the average transcript abundance, as measured by normalized transcript per kilobase million normalized transcripts (nTPM), is used to cluster cell types into related groups. At the bottom ) ( Figure 28B ); and FOXP3, a Treg marker for T cell clusters within the human protein atlas ( x (axis) and CCR8 ( y The average transcript abundance of the axis ( Figure 28C ).

[0255] Figure 29 shows the comparisons displayed in the listed datasets. Figures 29A-29F Selected volcano plots of differential gene expression within the range (log-transformation fold change (logFC) on the x-axis; adjusted p-values ​​of negative log-transformation (log(adj)) on the y-axis). p ))). Figure 29A , Figure 29B and Figure 29C Includes a comparison between tumor-resident naïve CD8 T cells and other tumor-resident CD8 T cell subsets. Figure 29D , Figure 29E and Figure 29F Includes a comparison between tumor-resident CD8 T cells and CD8 T cells from matched healthy tissue. Figure 29G This shows CD8 T cells from peripheral blood or tumors of colorectal cancer patients. CCR7 Normalized transcript abundance, cell subpopulation by tissue origin and TCF7 and / or PDCD1 Co-expression to identify initial ( TCF7 + / PDCD1 - T cells and putative central memory T cells (T cells) scm ; TCF7 + / PDCD1 + ).

[0256] Figure 30 ( Figure 30A and Figure 30B This paper summarizes the flow cytometry analysis of immune cells harvested from different organs of C57 / BL6 mice carrying MC38 14 days after IL-12 treatment.

[0257] Figure 31 ( Figure 31 A- Figure 31 C) shows a flow cytometry plot analyzing the expression of potential ABD targets on a subset of mouse immune cells harvested from tumors of C57 / BL6 mice carrying MC38 14 days after IL-12 treatment.

[0258] Figure 32 ( Figure 32 A- Figure 32 C) shows a flow cytometry plot analyzing the expression of potential ABD targets on a subset of mouse immune cells harvested from the spleen of C57 / BL6 mice carrying MC38 14 days after IL-12 treatment.

[0259] Figure 33 A list of exemplary IL-12 Fc fusion proteins is shown, including ABD-specific (mouse), IL-12 p35 subunit domain mutations, and amino acid sequence identifiers of such exemplary IL-12 Fc fusion proteins, as well as exemplary control fusion proteins (ABD-specific to chicken oocyte lysozyme, or lacking ABD).

[0260] Figure 34 The reduced (+DTT) and non-reduced (-DTT) SDS-PAGE analysis of an exemplary purified IL-12-ABD fusion protein is shown.

[0261] Figure 35 ( Figure 35 A and Figure 35 B) shows flow cytometry analysis of purified C57 / BL6 mouse T cells activated with 1 μg / mL anti-CD3 and 600 U / mL human IL-2.

[0262] Figure 36 ( Figure 36A and Figure 36B The results of alpha-LISA measurements of IFNγ in culture medium containing purified mouse T cells incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the IL-12-ABD fusion protein shown are presented in the presence of 50 U / mL human IL-2. The sign and error bar indicate the mean and SD, respectively.

[0263] Figure 37 Flow cytometry analysis of LAG-3 on human PBMCs from healthy donors activated with 600 U / mL IL-2 and 32 ng / mL OKT3 and amplified in 600 U / mL IL-2 for 5 days is shown.

[0264] Figure 38 AlphaLISA measurements of IFNγ in activated / amplified human PBMCs in the presence of 50 U / mL human IL-2 in medium incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the IL-12-ABD fusion protein shown are presented. The sign and error bar indicate the mean and SD, respectively.

[0265] Figure 39 Histograms are shown representing CCR8 expression as determined by flow cytometry in HEK-Blue IL-12 reporter cells (untransduced) and CCR8-HEK-Blue IL-12 reporter cells (transduced).

[0266] Figure 40 SEAP measurements (expressed as absorbance at 650 nm) of CCR8-HEK-Blue IL-12 reporter cells incubated with commercially available recombinant IL-12 (rIL-12) or the IL-12 Fc or IL-12-ABD fusion protein shown are presented. The symbols and error bars indicate the mean and SD, respectively.

[0267] Figure 41 Flow cytometry analysis of CCR7 on human PBMCs from healthy donors activated with 600 U / mL IL-2 and 32 ng / mL OKT3 is shown.

[0268] Figure 42 AlphaLISA measurements of IFNγ in IL-2 / OKT3-activated human PBMCs in the presence of 50 U / mL human IL-2, in culture medium incubated with commercially available recombinant IL-12 (rIL-12) or the IL-12-ABD fusion protein shown. The symbols and error bars indicate the mean and SD, respectively.

[0269] Figure 43 Flow cytometry analysis of CCR7 on purified naïve human NK cells is shown.

[0270] Figure 44 The results of alpha-LISA measurements of IFNγ in naïve NK cells incubated with commercially available recombinant IL-12 (rIL-12) or the IL-12-ABD fusion protein shown are presented. The symbols and error bars indicate the mean and SD, respectively.

[0271] Figure 45 The amino acid sequence of the full-length human CCR8 protein is shown.

[0272] Figure 46 Flow cytometry analysis of OX40 on purified C57 / BL6 mouse T cells activated with 0.5 ug / mL anti-CD3, 0.5 ug / mL anti-CD28, and 600 U / mL human IL-2 is shown.

[0273] Figure 47 The results of alpha-LISA measurements of IFNγ in culture medium containing purified mouse T cells activated with anti-CD3 / anti-CD28 / IL-2 and incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the IL-12-ABD fusion protein shown are presented in the presence of 50 U / mL human IL-2. The sign and error bar indicate the mean and SD, respectively.

[0274] Figure 48 The growth of MC38 tumor cells after subcutaneous implantation in C57 / BL6 mice is shown. Unless otherwise specified, mice were treated with the test substance shown on days 0, 7, and 14 at a dose equivalent to 0.03 mg / kg IL-12 Fc Y36E. Mice euthanized due to ulceration are indicated by 'x'.

[0275] Figure 49 ( Figures 49A-49C The results show the plasma IFNγ measurements in C57 / BL6 mice carrying MC38 treated with PBS, IL-12 Fc Y36E, or the indicated IL-12 ABD on days 0, 7, and 14. Measurements were taken 3 days after each treatment (day 3...). Figure 49A Day 10 Figure 49B ) and the 17th day ( Figure 49C The plasma samples were measured. Symbols indicate the measurement results for each mouse, and the horizontal line indicates the median for each treatment group.

[0276] Figure 50 Figures 50A-50CThe results show the measurement of IL-12-Fc / IL-12 ABD fusion protein in the plasma of C57 / BL6 mice carrying MC38 treated with PBS, IL-12 Fc Y36E, or the indicated IL-12 ABD on days 0, 7, and 14. Measurements were taken 3 days after each treatment (day 3...). Figure 50A Day 10 Figure 50B ) and the 17th day ( Figure 50C The plasma samples were measured. Symbols represent the measurement results for individual mice, and the horizontal line indicates the median for each treatment group. Data points below the detection limit were designated as any value of 1 pg / mL.

[0277] Figure 51 The weights of MC38 tumors harvested from C57BL / 6 mice 5 days after treatment with PBS, 0.03 mg / kg wild-type (WT) IL-12 Fc, or an equimolar dose of the indicated IL-12ABD fusion protein are shown. '10X' indicates mice treated with 10 times the dose of the indicated test substance. Symbols represent individual mouse measurements, and the horizontal line indicates the median for each treatment group.

[0278] Figure 52 The following figures show the weight of spleens harvested from C57BL / 6 mice carrying MC38 tumors 5 days after treatment with PBS, 0.03 mg / kg wild-type (WT) IL-12 Fc, or an equimolar dose of the indicated IL-12ABD fusion protein. '10X' indicates mice treated with 10 times the dose of the indicated test substance. Symbols represent individual mouse measurements, and the horizontal line indicates the median for each treatment group.

[0279] Figure 53 The CD45 concentrations in spleens harvested from C57BL / 6 mice carrying MC38 tumors were shown 5 days after treatment with PBS, 0.03 mg / kg wild-type (WT) IL-12 Fc, or an equimolar dose of the indicated IL-12ABD fusion protein (n = 6 / group). + Cell frequency. '10X' indicates mice treated with 10 times the dose of the test substance shown. Data are plotted in box-and-whisker form, where whiskers represent the minimum and maximum values ​​in each treatment group.

[0280] Figure 54 Figures 54A-54EThe data show the quantification of immune cell types (as shown) in MC38 tumors harvested from C57BL / 6 mice 5 days after treatment with PBS, 0.03 mg / kg wild-type (WT) IL-12 Fc, or an equimolar dose of the indicated IL-12 ABD fusion protein (n = 6 / group). '10X' indicates mice treated with a 10-fold higher dose of the indicated test substance. Data are plotted in box-and-whisker form, where whiskers represent the minimum and maximum values ​​in each treatment group.

[0281] Figure 55 The quantification of IFNγ in plasma, spleen, and MC38 tumor tissue of C57BL / 6 mice 5 days after treatment with PBS, 0.03 mg / kg wild-type (WT) IL-12 Fc, or an equimolar dose of the indicated IL-12ABD fusion protein (n = 6 / group) is shown. '10X' indicates mice treated with a 10-fold higher dose of the indicated test substance. Symbols represent individual mouse measurements, and the horizontal line indicates the median for each treatment group.

[0282] Figure 56 The ratio of IFNγ in MC38 tumors to spleen in C57BL / 6 mice treated 5 days after administration of 0.03 mg / kg wild-type (WT) IL-12 Fc or an equimolar dose of the indicated IL-12 ABD fusion protein is shown. '10X' indicates mice treated with 10 times the dose of the indicated test substance. The tumor:spleen IFNγ ratio is calculated by dividing the mean tumor IFNγ value by the mean spleen IFNγ value (from...). Figure 55 The calculation is based on the data depicted in the text.

[0283] Figure 57 The growth of A20 tumor cells following subcutaneous implantation in BALB / c mice is shown. Unless otherwise specified, mice were treated on days 0, 7, and 14 with 0.05 mg / kg wild-type (WT) IL-12 Fc or a molar equivalent of 0.03 mg / kg IL-12 Fc of the IL-12 ABD fusion protein. '10X' indicates mice treated with 10 times the dose of the test substance shown (molar equivalent of 0.3 mg / kg IL-12 Fc). 'Single' indicates the group of mice treated once (on day 0).

[0284] Figure 58 Figures 58A-58CThe results show the plasma IFNγ measurements in A20-carrying BALB / c mice treated with 0.05 mg / kg wild-type (WT) IL-12 Fc or a molar equivalent of 0.03 mg / kg IL-12 Fc by the IL-12 ABD fusion protein on days 0, 7, and 14. '10X' indicates mice treated with 10 times the dose of the test substance shown (molar equivalent of 0.3 mg / kg IL-12 Fc). 'Single' indicates the group of mice treated once (on day 0). On day 3 (D3; Figure 58A Day 10; Figure 58B ) and day 17 (D17; Figure 58C Plasma IFNγ levels were measured. Symbols indicate individual mouse measurements, and horizontal lines indicate the median for each treatment group. The lower limit of quantitation (LLoQ) is indicated by a dashed line.

[0285] Figure 59 A welfare scoring system was described for assessing the toxicity of IL-12-based treatments in mice. In addition... Figure 59 In addition to the parameters outlined, mice may undergo unplanned euthanasia due to tumor size and / or ulceration of the tumor / the skin covering the tumor.

[0286] Figure 60 The growth of MC38 tumor cells in C57BL / 6 mice treated with PBS or the indicated dose of IL-12 ABD fusion protein on days 0, 7, and 14 is shown.

[0287] Figure 61 ( Figure 61A and Figure 61B This shows a heatmap depicting the welfare scores of C57BL / 6 mice carrying MC38 tumors treated with PBS or the indicated dose of IL-12 ABD fusion protein on days 0, 7, and 14. Individual mice are represented in separate rows. Each 'block' is colored according to the welfare score received by each mouse on a given day (as shown in the example). *Indicates mice euthanized due to treatment-related toxicity.

[0288] Figure 62 The quantification of welfare scores in C57BL / 6 mice carrying MC38 tumors treated with IL-12 ABD fusion protein on days 0, 7, and 14 is shown. Symbols denote the mean welfare score (measured by quantification of the area under the curve (AUC), and error bars indicate the standard deviation of the measurements. The mean welfare score of mice treated with PBS is indicated by a horizontal solid line.

[0289] Figure 63 ( Figures 63A-63CThe results show the plasma IFNγ measurements in C57BL / 6 mice carrying MC38 treated with PBS or the IL-12 ABD fusion protein shown on days 0, 7, and 14. The numbers in parentheses indicate the dose of each test substance in mg / kg. Plasma IFNγ levels were measured on day 3 (D3). Figure 63A Day 10; Figure 63B ) and day 17 (D17; Figure 63C Measurements. Symbols represent the measurement results for individual mice, and horizontal lines indicate the median for each treatment group.

[0290] Figure 64 The quantification of plasma IFNγ levels in C57BL / 6 mice carrying MC38 three days after treatment with PBS or IL-12 ABD fusion protein is shown (n = 6 per group). The symbols and error bars represent the mean and standard deviation, respectively.

[0291] Figure 65 Figure 65A and Figure 65B The growth of AT3 tumor cells in C57BL / 6 mice treated subcutaneously with PBS, anti-PD-1 and / or IL-12-based test subjects as shown is illustrated.

[0292] Figure 66 ( Figure 66A and Figure 66B The cumulative welfare scores of C57BL / 6 mice carrying AT3 tumors, treated with PBS, anti-PD-1 and / or IL-12-based test subjects as shown, are displayed. Solid lines indicate the cumulative welfare scores of individual mice (according to...). Figure 59 (See the table below). The horizontal dashed line represents the extrapolation of the welfare curve from the point where the mice were euthanized. 'Mean AUC' indicates the mean area under the curve for each treatment group.

[0293] Figure 67 The results show the plasma IFNγ measurements in C57BL / 6 mice carrying AT3 four days after treatment with PBS, anti-PD-1 and / or IL-12-based test agents as shown. The numbers in parentheses indicate the dose of each test agent in mg / kg. Symbols represent individual mouse measurements, and the horizontal line indicates the median for each treatment group.

[0294] Figure 68 The results show the plasma IFNγ measurements in healthy C57BL / 6 mice (tumor-free) or C57BL / 6 mice (tumor-bearing) treated with a single dose of mono-Y36E-OX86 (as shown in the example) on day 0. The symbols and error bars represent the mean and standard deviation of measurements from 3 mice in each group. The horizontal dashed line indicates the lower limit of quantitation (LLoQ).

[0295] Figure 69 The results of plasma mono-Y36E-OX86 measurements in healthy C57BL / 6 mice (tumor-free) or C57BL / 6 mice (tumor-bearing) treated with a single dose of mono-Y36E-OX86 (as shown in the example) on day 0 are presented. The symbols and error bars represent the mean and standard deviation of measurements from 3 mice in each group. The horizontal dashed line indicates the lower limit of quantitation (LLoQ).

[0296] Figure 70 A summary of the pharmacokinetic characteristics of mono-Y36E-OX86 in C57BL / 6 mice with subcutaneous injection of MC-38 tumors is presented. Using Figure 69 The data shown are used to model pharmacokinetic properties in PK Solver software.

[0297] Figure 71 Histograms showing the expression of mouse OX40 (mOX40; top), human IL-12Rb1 (hIL-12Rb1; middle), and human IL-12Rb2 (hIL-12Rb2; bottom) in Jurkat NF-κB reporter cells and mOX40 / hIL-12R / Jurkat NF-κB reporter cells are shown.

[0298] Figure 72 The percentage of mOX40 / hIL-12R / Jurkat NF-κB reporter cells expressing GFP after incubation with anti-OX40 mAb (OX86) or IL-12 ABD fusion protein is shown in the presence or absence of A20 cells expressing mouse FcγRI.

[0299] Figure 73 It shows that in the presence of 40 nM OVA 323-339 Flow cytometry analysis of OT-II spleen cells activated for three days with peptides and 100 U / mL recombinant human IL-2. FMO (fluorescence reduced by one; top right panel) indicates staining with all antibodies in the figure (complete staining; bottom right panel), except for recognition of OX40 (anti-mouse CD134 BV605, BD Biosciences).

[0300] Figure 74 The OVA incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the IL-12-ABD fusion protein shown in the presence of 50 U / mL human IL-2 is illustrated. 323-339AlphaLISA measurements of IFNγ in culture medium of peptide / IL-2 activated OT-II splenocytes. The sign and error bar indicate the mean and SD, respectively.

[0301] Figure 75 Figures 75A-75C This section shows a list of exemplary ABD sequences that bind to human OX40, as well as exemplary heavy and light chain amino acid sequences for anti-OX40 mAb or the IL-12 Fc fusion protein of the present invention. For selected VH / VLABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0302] Figure 76 A list of exemplary conventional bivalent IgG1 anti-human OX40 antibodies is shown, including amino acid sequence identifiers.

[0303] Figure 77 A list of exemplary IL-12 Fc fusion proteins is shown, including ABD-specific (human), IL-12 p35 subunit domain mutations, and amino acid sequence identifiers of such exemplary IL-12 Fc fusion proteins.

[0304] Figure 78 The binding of the conventional bivalent form of anti-human OX40 antibody (solid symbol) and the OX40-targeted IL-12 Fc fusion protein (hollow symbol) to hOX40-Jurkat-NF-κB cells, as determined by flow cytometry, is shown.

[0305] Figure 79 The binding of the conventional bivalent form of anti-human OX40 antibody (solid symbol) in the form of IgG1 and the OX40-targeted IL-12 Fc fusion protein (hollow symbol) to cOX40-Jurkat-NF-κB cells, as determined by flow cytometry, is shown.

[0306] Figure 80 The EC50 values ​​of the conventional bivalent form of anti-human OX40 antibody and the OX40-targeted IL-12 Fc fusion protein, as determined by flow cytometry, are shown for binding to hOX40-Jurkat-NF-κB cells and cOX40-Jurkat-NF-κB cells.

[0307] Figure 81 ( Figure 81A and Figure 81B This section shows a list of exemplary ABD sequences that bind to human OX40, as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of the present invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0308] Figure 82 A list of exemplary IL-12 Fc fusion proteins is shown, including ABD-specific (human), IL-12 p35 subunit domain mutations, and amino acid sequence identifiers of such exemplary IL-12 Fc fusion proteins.

[0309] Figure 83 The binding of the OX40-targeted IL-12 Fc fusion protein to hOX40-Jurkat-NF-κB cells, as determined by flow cytometry, is shown.

[0310] Figure 84 The EC50 values ​​of the OX40-targeted IL-12 Fc fusion protein bound to hOX40-Jurkat-NF-κB cells, as determined by flow cytometry, are shown.

[0311] Figure 85 Figures 85A-85C This section shows a list of exemplary ABD sequences that bind to human OX40, as well as exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of the present invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0312] Figure 86 A list of exemplary IL-12 Fc fusion proteins is shown, including ABD specificity, IL-12 p35 subunit domain mutations, and amino acid sequence identifiers of such exemplary IL-12 Fc fusion proteins.

[0313] Figure 87 The binding of the conventional bivalent form of anti-human OX40 antibody and the OX40-targeted IL-12 Fc fusion protein to hOX40-Jurkat-NF-κB cells, as determined by flow cytometry, is shown.

[0314] Figure 88 The EC50 values ​​of the conventional bivalent form of anti-human OX40 antibody and the OX40-targeted IL-12 Fc fusion protein bound to hOX40-Jurkat-NF-κB cells are shown, as determined by flow cytometry.

[0315] Figure 89 This demonstrates the binding of OX40L-Fc to OX40-positive hOX40-Jurkat-NF-κB cells in the presence of conventional bivalent IgG1 anti-human OX40 antibody or OX40-targeted IL-12 Fc fusion protein.

[0316] Figure 90 ( Figure 90A and Figure 90BThe following figures show the expression of OX40 (on the left y-axis) and GFP after incubation with 10 nM OX40L-Fc and / or various concentrations of the indicated IL-12ABD fusion protein for 1 hour (1 h) or overnight (o / n). + Percentage of hOX40-Jurkat-NF-κB cells (on the right y-axis). GFP measurements were performed only after overnight incubation. The horizontal dashed line indicates GFP levels after incubation with OX40L-Fc alone. + Percentage of cells. Horizontal dotted lines indicate OX40 MFI after overnight incubation with individual OX40L-Fc cells.

[0317] Figure 91 Figure 91A and Figure 91B The following figures show the expression of OX40 (on the left y-axis) and GFP after incubation with 10 nM OX40L-Fc and / or various concentrations of the indicated IL-12ABD fusion protein for 1 hour (1 h) or overnight (o / n). + Percentage of hOX40-Jurkat-NF-κB cells (on the right y-axis). GFP measurements were performed only after overnight incubation. Cells expressing only high levels of OX40 were included in the analysis. The horizontal dashed line indicates GFP levels after incubation with OX40L-Fc alone. + Percentage of cells.

[0318] Figure 92 ( Figure 92A and Figure 92B The results show two healthy donors (B113) activated with 600 U / mL IL-2 and 32 ng / mL OKT3. Figure 92A ) and B121 ( Figure 92B Flow cytometry analysis of PBMCs. OX40 positive cells are indicated by horizontal lines.

[0319] Figure 93 AlphaLISA measurements of IFNγ in IL-2 / OKT3-activated human PBMCs in culture medium in the presence of 50 U / mL human IL-2, incubated with wild-type IL-12 Fc (WT IL-12 Fc) or the IL-12-ABD fusion protein shown. Data for two PBMC donors (B113 (top panel) and B121 (bottom panel)) are shown. Symbols and error bars indicate mean and SD, respectively.

[0320] Figure 94 A graph showing EC50 values ​​of wild-type IL-12 Fc (WT IL-12 Fc) and various IL-12-ABD fusion proteins, determined by PBMC IFNγ release assays, is presented. Symbols represent individual measurements, and horizontal lines indicate the median for each treatment group.

[0321] Figure 95 A summary of EC50 values ​​for wild-type IL-12 Fc (WT IL-12 Fc) and various IL-12-ABD fusion proteins, as determined by PBMC IFNγ release assay, is presented.

[0322] Figure 96 Figures 96A-96C The results showed that in the presence of 50 U / mL IL2 (for T cells) and 1 ng / mL IL-15 (for NK cells), the levels of IL-15 in cells from three different PBMC donors (B130, ... Figure 96A B134 Figure 96B ) and B177 ( Figure 96C Purified CD4 + T cells (apical row), CD8 + AlphaLISA measurements of IFNγ in culture medium for T cells (middle row) and NK cells (bottom row), which were freshly isolated (D0, left column), activated for 2 days (D2, middle column), or maintained in culture for 5 days after activation (D7, right column) and treated with WT IL-12 Fc or the indicated IL-12 ABD fusion protein. The sign and error bar indicate the mean and SD, respectively.

[0323] Figure 97 The figures show OX40 (left column of each figure), IL-12Rb1 (middle column of each figure), and IL-12Rb2 (right column of each figure) in purified CD4 from three different PBMC donors (B130 (top left), B134 (top right), and B177 (bottom)). + T cells, CD8 + Expression on T cells and NK cells, which were freshly isolated (D0; top row of each figure), activated for 2 days (D2; middle row of each figure), or maintained in culture for 5 days after activation (D7; bottom row of each figure).

[0324] Figure 98 Figure 98A and Figure 98B ) shows IL-12Rb2 ( Figure 98A ) and OX40 ( Figure 98B In purified NK cells (left column), CD4 + T cells (middle column) and CD8 + Expression on T cells (right column), which were freshly isolated (D0, top row), activated for 2 days (D2, middle row), or maintained in culture for 5 days after activation (D7, bottom row).

[0325] Figure 99The results showed that purified CD4 cells were effective in the presence of 50 U / mL IL-2 (for T cells) and 1 ng / mL IL-15 (for NK cells). + T cells (CD4, apical row), CD8 + AlphaLISA measurements of IFNγ in culture medium for T cells (CD8, middle row) and NK cells (NK, bottom row), which were freshly isolated (D0, left column), activated for 2 days (D2, middle column), or maintained in culture for 5 days after activation (D7, right column) and treated with WT IL-12 Fc or the indicated IL-12 ABD fusion protein. The sign and error bar indicate the mean and SD, respectively.

[0326] Figure 100 The purified CD4 was shown. + T cells, CD8 + T cells and NK cells (as shown) respond to WT IL-12Fc (as shown) Figure 99 The maximum amount of IFNγ produced by the treatment (described in the text) was determined. Cells were freshly isolated (D0), activated for 2 days (D2), or maintained in culture for 5 days after activation (D7). At D0, CD4... + T cells and CD8 + The IFNγ measurement result of T cells was below the limit of quantitation and was specified as an arbitrary value of 0 pg / ml.

[0327] Figure 101 Freshly isolated and purified CD4 cells were shown after treatment with WT IL-12 Fc or the IL-12 ABD fusion protein as shown in the absence of IL-15. + AlphaLISA results of IFNγ in T cell culture medium. The sign and error bar indicate the mean and SD, respectively. The horizontal dotted line indicates the lower limit of quantitation.

[0328] Figure 102 This shows that conventional CD4 has been maintained in culture for 7 days after CD3 / 28 activation. + Phenotypes of cells (ConCD4; top and third rows) and induced Treg cells (Treg; second and fourth rows) that have been activated / differentiated for 10 days in the presence of CD3 / 28 Dynabeads, 500 U / mL human IL-2, 5 ng / mL TGF-b and 10 nM retinoic acid.

[0329] Figure 103The figures show the conventional CD4 counts from two different PBMC donors (B363 (top left and bottom left) and B364 (top right and bottom right)) in the absence or presence of 5 ng / mL TGF-b and 10 nM retinoic acid, respectively. + T cells (CD4) + conv AlphaLISA measurements of IFNγ in the culture medium of induced Treg cells (top left and bottom left) and induced Treg cells (top right and bottom right), which were activated with CD3 / 28 Dynabeads and maintained in culture for 10 days. The symbols and error bars indicate the mean and SD of two replicates, respectively.

[0330] Figure 104 This demonstrates the use of conventional CD4 from two different PBMC donors (B363 and B364). + T cells (CD4) + conv Summary of EC50 values ​​and fold changes (relative to WT IL-12 Fc) of wild-type IL-12 Fc (WT IL-12 Fc) and various IL-12-ABD fusion proteins determined by IFNγ release assays in induced Treg cells (Treg) and induced Treg cells (Treg).

[0331] Figure 105 The results showed a positive result for phosphorylated STAT4 (pStat4) after incubation with wild-type IL-12 Fc (WT IL-12 Fc) or IL-12-ABD fusion protein for 30 minutes. + The OX40 is derived from two independent PBMC donors (B130 (top left and top right images) and B323 (bottom left and bottom right images)). + / CD4 + Cells (top left and bottom left images) and OX40 - / CD4 + Percentage of cells (top right and bottom right images).

[0332] Figure 106 The purified CD4 was shown to have been activated for 2 days with CD3 / 28 Dynabeads and 100 U / mL IL-2 and maintained in culture for 5 days after activation. + T cells (including regular CD4) + Flow cytometry analysis of both T cells (ConCD4) and Treg cells (as depicted in the left figure). The middle and right figures show conventional CD4 cells, respectively. + OX40 expression on T cells (ConCD4) and Treg cells.

[0333] Figure 107 The results showed a positive result for phosphorylated STAT4 (pStat4) after incubation with wild-type IL-12 Fc (WT IL-12 Fc) or IL-12-ABD fusion protein for 30 minutes. + ) of OX40 + / Treg cells (top left image), OX40 - / Treg cells (bottom left image), OX40 + / Conventional CD4 cells (top right image) and OX40 - / Percentage of regular CD4 cells (bottom right figure).

[0334] Figure 108 Histograms showing the expression of human OX40 (top), human IL-12Rb1 (middle), and human IL-12Rb2 (bottom) in Jurkat NF-κB reporter cells (parental Jurkat) and hOX40 / hIL-12R / Jurkat NF-κB reporter cells (hIL-12Rb1 / hIL-12Rb2 / hOX40.Jurkat) are presented.

[0335] Figure 109 ( Figure 109A and Figure 109B ) shows that in the existence ( Figure 109A ) or does not exist ( Figure 109B The percentage of GFP-positive hOX40 / hIL-12R / Jurkat NF-κB reporter cells in A20 cells expressing human FcγRI (A20-FcγRI) after overnight incubation with a conventional bivalent form of anti-human OX40 antibody and an OX40-targeted IL-12 Fc fusion protein. Data were normalized so that unstimulated cells were assigned a value of 0% GFP positivity.

[0336] Figure 110 The results of IFNγ measurements by AlphaLISA are shown in activated PBMCs stimulated with plate-bound anti-CD3 and plate-bound conventional IgG1 bivalent form of anti-human OX40 (hG1-BGB-A445 (PB)) or plate-bound anti-CD3 and WT IL-12 Fc or IL-12 ABD fusion protein. Prior to stimulation, genes encoding IL-12Rb2 (top right), STAT4 (bottom left), or OX40 (bottom right) were knocked out using CRISPR / Cas9 genome editing, as shown. AAVS1 (top left) indicates CRISPR / Cas9-edited cells where the targeting ribonucleoprotein is specific to the gene desert (used as a negative control). Symbols and error bars indicate mean and SD, respectively.

[0337] Figure 111The growth of MC38 tumor cells is shown after subcutaneous implantation in naïve C57BL / 6 mice (untreated; top panel) and previously treated C57BL / 6 mice (previously treated; bottom panel) inoculated with MC38 cells and treated with a single dose of 0.143 mg / kg mono-Y36E-OX86. Solid lines indicate tumor growth in each mouse (n=5 per group).

[0338] Figure 112 A list of exemplary ABD sequences binding to human OX40 is shown, along with exemplary heavy and light chain amino acid sequences for the IL-12 Fc fusion protein of the present invention. For selected VH / VL ABD pairs, the vhCDR1-3 and vlCDR1-3 sequences are underlined.

[0339] Figure 113 The binding of the conventional bivalent form of anti-human OX40 antibody to hOX40-Jurkat-NF-κB cells, as determined by flow cytometry, is shown.

[0340] Figure 114 The binding of the conventional bivalent form of anti-human OX40 antibody to cOX40-Jurkat-NF-κB cells, as determined by flow cytometry, is shown.

[0341] Figure 115 The EC50 values ​​of the conventional bivalent form of anti-human OX40 antibody and the OX40-targeted IL-12 Fc fusion protein bound to hOX40-Jurkat-NF-κB cells are shown, as determined by flow cytometry.

[0342] Figure 116 The binding of OX40L-Fc to OX40-positive hOX40-Jurkat-NF-κB cells was demonstrated in the presence of anti-human OX40 antibody in the conventional bivalent form of IgG1.

[0343] Figure 117 The graph shows OX40 expression (on the left y-axis) and the percentage of GFP+ hOX40-Jurkat-NF-κB cells (on the right y-axis) after incubation with 10 nM OX40L-Fc and / or various concentrations of the indicated anti-OX40 antibody for 10 min or overnight. GFP measurements were performed only after overnight incubation. The horizontal dashed line indicates the percentage of GFP+ cells after incubation with OX40L-Fc alone. The horizontal dotted line indicates the OX40 MFI after overnight incubation with OX40L-Fc alone.

[0344] Figure 118 ( Figure 118A and Figure 118B ) shows that in the existence ( Figure 118A ) or does not exist ( Figure 118B The percentage of GFP-positive hOX40-Jurkat NF-κB reporter cells in A20 cells expressing human FcγRI (A20-FcγRI) after overnight incubation with anti-human OX40 antibody in the conventional bivalent form of IgG1. Data were normalized so that unstimulated cells were assigned to a value of 0% GFP positivity. Detailed Implementation

[0345] The specification is presented to enable those skilled in the art to make and use the invention, and is provided in the context of the patent application and its claims. Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter. While various embodiments of the invention disclosed herein have been shown and described, such embodiments are provided by way of example only and will be apparent to those skilled in the art. Many modifications, variations, and substitutions will be readily apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice any of the inventions set forth herein.

[0346] All patents, published patent applications, other publications, sequences from GenBank, sequences from UniProt (including but not limited to UniProt accessions: P43431, P43432, Q60837, and P97378), and other databases mentioned herein are incorporated in their entirety by reference to the relevant technologies.

[0347] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For the purposes of this disclosure, the following terms are defined. The definitions provided are intended to apply to the given terms as well as other derivative language restates and grammatical equivalents of said terms.

[0348] As used herein, the term "protein" refers to at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. When a "biologically functional molecule" contains two or more proteins, such as, for example, IL-12 (which contains IL-12 p35 polypeptide and IL-12 p40 polypeptide), the individual proteins containing two or more proteins may each be referred to as a "subunit," "monomer," or "domain," and the biologically functional molecule may be referred to as a "complex." In some embodiments, the two or more proteins of the functional complex are non-covalently linked. In some embodiments, the two or more proteins of the functional complex are covalently linked, such as, for example, through disulfide bonds.

[0349] As used herein, the term "cytokine" refers to a large class of proteins, such as chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, which are secreted by the first cell and exert their effects on one or more cells by binding to receptors on those cells. Cytokines can participate in autocrine, paracrine, procrine, and / or endocrine signaling.

[0350] As used herein, the term "immunocytokine" generally refers to a fusion protein that contains a cytokine (e.g., IL-12) and at least one antibody domain (e.g., a variable heavy chain domain, a variable light chain domain, a constant heavy chain domain, a constant light chain domain, a VHH domain, etc.).

[0351] As used herein, the term "wildtype" refers to an amino acid or nucleotide sequence found in nature, including allelic variations. Wild-type proteins have an unmodified amino acid sequence (or the nucleotide sequence encoding that amino acid sequence).

[0352] As used herein, unless the context clearly indicates otherwise, the term "IL-12 p35 subunit" refers to the human wild-type IL-12 p35 polypeptide, whether natural or recombinant. Therefore, IL-12 p35 subunit refers to recombinant IL-12 p35 polypeptides, synthetic IL-12 p35 polypeptides, and IL-12 p35 extracted from cells or tissues. Figure 1 The amino acid sequence of the human wild-type IL-12 p35 subunit was depicted (precursor: SEQ ID NO: 1; mature: SEQ ID NO: 2). Figure 3The amino acid sequence of the mouse wild-type IL-12 p35 subunit is depicted (precursor: SEQ ID NO: 9; mature: SEQ ID NO: 10). In the context of fusion proteins, the IL-12 p35 subunit may also be referred to as the “IL-12 p35 subunit domain,” where the IL-12 p35 subunit domain contains at least a portion of the amino acid sequence encoding the IL-12 p35 subunit. In many of these cases, the IL-12 p35 subunit domain contains one or more mutations in the “parental protein” of the IL-12 p35 subunit. In such cases, the IL-12 p35 subunit domain is referred to herein as the “variant IL-12 p35 subunit domain.”

[0353] As used herein, unless the context clearly indicates otherwise, the term "IL-12 p40 subunit" refers to the human wild-type IL-12 p40 polypeptide, whether natural or recombinant. Therefore, IL-12 p40 subunit refers to recombinant IL-12 p40 polypeptides, synthetic IL-12 p40 polypeptides, and IL-12 p40 extracted from cells or tissues. Figure 1 The amino acid sequence of the human wild-type IL-12 p40 subunit was depicted (precursor: SEQ ID NO: 3; mature: SEQ ID NO: 4). Figure 3 The amino acid sequence of the human wild-type IL-12 p40 subunit is depicted (precursor: SEQ ID NO: 11; mature: SEQ ID NO: 12). In the context of fusion proteins, the IL-12 p40 subunit may also be referred to as the "IL-12 p40 subunit domain," wherein the IL-12 p40 subunit domain contains at least a portion of the amino acid sequence encoding the IL-12 p40 subunit. In some of these cases, the IL-12 p40 subunit domain contains one or more mutations in the "parental sequence" of the IL-12 p40 subunit. In such cases, the IL-12 p40 subunit domain is referred to herein as the "variant IL-12 p40 subunit domain." For the sake of brevity, as used herein, the phrase “(variant) IL-12 p40 subunit” is used to disclose embodiments in which the IL-12 p40 subunit (or, in the case of a fusion protein, the IL-12 p40 subunit domain) may comprise a wild-type IL-12 p40 polypeptide or a variant IL-12 p40 subunit (domain), as if each embodiment had been described separately.

[0354] As used herein, the term "single-chain" refers to a molecule comprising two or more protein domains linearly linked by peptide bonds. In some embodiments, the biologically functional IL-12 is a single-chain IL-12 complex (sc-IL-12) (i.e., the IL-12 p35 subunit (or IL-12 p35 subunit domain) and the IL-12 p40 subunit (or IL-12 p40 subunit domain) are fused to form a single peptide chain). In a further embodiment, the C-terminus of the IL-12 p40 subunit (domain) is attached to the N-terminus of the IL-12 p35 subunit (domain) (sc-IL-12(p35 / p40)). In yet another embodiment, sc-IL-12(p35 / p40) further comprises a "linker" wherein the C-terminus of the IL-12 p35 subunit is attached to the N-terminus of the linker, and the C-terminus of the linker is attached to the N-terminus of the IL-12 p40 subunit. In other embodiments, the C-terminus of the IL-12 p40 subunit is connected to the N-terminus of the IL-12 p35 subunit (sc-IL-12(p40 / p35)). In yet another embodiment, sc-IL-12(p40 / p35) further includes a connector, wherein the C-terminus of the IL-12 p40 subunit is connected to the N-terminus of the connector, and the C-terminus of the connector is connected to the N-terminus of the IL-12 p35 subunit. In many of these embodiments, the IL-12 p35 subunit (domain) includes a variant of the IL-12 p35 subunit (domain). Furthermore, in some of these embodiments, the IL-12 p40 subunit (domain) includes a variant of the IL-12 p40 subunit (domain).

[0355] As used herein, the interchangeable terms “linker” and “domain linker” generally refer to a structure (such as, for example, a polymer (e.g., containing any of the amino acid sequences of SEQ ID NO: 31-40)) or a bond (such as, for example, a covalent bond) that “links” a first domain to a second domain (such as, for example, a CH1 domain and an Fc domain, a VH domain and a VL domain, a VL domain and a CL domain, a subunit of a cytokine (e.g., the p35 subunit domain of IL-12, the p40 subunit domain of IL-12, etc.) and a subunit of an antibody (e.g., the VH domain, the VL domain, the CH1 domain, the Fc domain, etc.), a first subunit of a cytokine, and a second subunit of a cytokine). Such a linking can be “direct” (i.e., where the domain linker contains a covalent bond) or “indirect” (i.e., where the domain linker contains a structure (e.g., any of the amino acid sequences of SEQ ID NO: 31-40)). In many embodiments, multiple domain linkers are incorporated into the "low-potency IL-12 Fc fusion protein" of the present invention, as further described in detail herein. In some cases, the low-potency IL-12 Fc fusion protein of the present invention may comprise one or more domain linkers. In some further cases, the one or more domain linkers comprise one or more of the amino acid sequences of SEQ ID NO: 31-40, and may optionally comprise one or more covalent bonds. In some other further cases, the one or more domain linkers comprise one or more covalent bonds, and may optionally comprise one or more of the amino acid sequences of SEQ ID NO: 31-40. In some cases, at least one domain linker comprises a hinge region of an Fc domain.

[0356] As used herein, the term "residue" refers to a position in a protein and its associated amino acid identity. For example, cysteine ​​252 (also known as Cys252 or C252) is a residue at position 252.

[0357] As used herein, the term "parental protein" refers to a "reference" protein, the amino acid sequence encoding the reference protein, or a DNA sequence encoding the amino acid sequence of a reference protein. In some embodiments, the reference protein includes a wild-type protein, the amino acid sequence encoding the wild-type protein, and / or a nucleic acid sequence encoding the amino acid sequence of a wild-type protein. In some embodiments, the reference protein includes a human wild-type protein, the amino acid sequence encoding the human wild-type protein, and / or a nucleic acid sequence encoding the human wild-type protein. In some embodiments, the reference protein includes a (human) wild-type protein conjugated to an Fc domain and / or an antigen-binding domain (such as, for example, an ABD comprising a VH / VL domain pair, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 41 and the VL domain comprises the amino acid sequence of SEQ ID NO: 42).

[0358] As used herein, the terms "variant protein," "protein variant," or "variant" refer to a protein that differs from the parent protein due to at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more modifications. The term may refer to the protein itself, a composition comprising the protein, the amino acid sequence encoding the protein, or the DNA sequence encoding the amino acid sequence. In some embodiments, the parent protein refers to the wild-type sequence. In some embodiments, the parent protein refers to the human wild-type sequence. Therefore, a “variant” of the IL-12p35 subunit (or a “variant” of the IL-12p35 subunit domain) refers to a polypeptide with one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid substitutions, deletions and / or insertions compared to the amino acid sequence of a reference IL-12p35 subunit (such as, for example, the (human) wild-type IL-12p35 subunit). Furthermore, a “variant” of the IL-12 p40 subunit (or a “variant” of the IL-12 p40 subunit domain) refers to a polypeptide with one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid substitutions, deletions and / or insertions compared to the amino acid sequence of a reference IL-12 p40 subunit (such as, for example, the (human) wild-type IL-12 p40 subunit).

[0359] As used herein, the term "modification" refers to the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence, or to any alteration of the portion of the polypeptide that is chemically linked to the protein. As used herein, the term "amino acid modification" refers to the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence. There are no particular restrictions on the location of amino acid modifications or the number of amino acids that can be modified in the amino acid sequence.

[0360] Those skilled in the art will understand that the modifications to the IL-12 p35 subunit (domain) described throughout refer to modifications to the mature form of the sequence (SEQ ID NO: 2) and / or variations thereof, and not modifications to the precursor sequence (SEQ ID NO: 1). The precursor sequence of the IL-12 p35 subunit contains an additional 22 amino acid residues at the N-terminus, comprising the sequence: MCPARSLLLVATLVLLDHLSLA (SEQ ID NO: 91). However, the precursor form of the IL-12 p35 subunit may be modified to the mature form of the IL-12 p35 subunit as listed, after adjusting for the additional leading amino acid residues in the precursor sequence. For example, the substitution modification Y40A is one of many potential modifications to the mature sequence of the IL-12 p35 subunit, but it may also refer to a substitution modification of the IL-12 p35 subunit precursor sequence containing Y62A.

[0361] Similarly, the modifications to the IL-12 p40 subunit (domain) described throughout this document are intended to refer to modifications of the mature form of the sequence (SEQ ID NO: 4) and / or its variants, and not modifications to the precursor sequence (SEQ ID NO: 3). The precursor sequence of the IL-12 p40 subunit contains an additional 22 amino acid residues at the N-terminus, comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 92). However, it will also be understood that the precursor form of the IL-12 p40 subunit may be modified to the mature form of the IL-12 p40 subunit as listed, after adjusting for the additional leading amino acid residues in the precursor sequence. For example, the substitution modification C177S is disclosed herein as a potential modification to the mature sequence of the IL-12 p40 subunit, but may also refer to a substitution modification of the IL-12 p40 subunit precursor sequence containing C199S.

[0362] As used herein, the term "amino acid substitution" or "substitution" refers to replacing an amino acid at a specific position in the parent polypeptide sequence with a different amino acid. For example, Y40A indicates that tyrosine at position 40 is replaced by alanine at the same position. In some embodiments, the substitution is made of an amino acid that is not naturally present at the specific position, meaning that the amino acid is not naturally present in the organism or any organism. For clarity, a protein engineered to alter the nucleic acid coding sequence but not the resulting amino acid (e.g., exchanging CCU (encoding proline) for CCC (still encoding proline)) is not an "amino acid substitution." In other words, although a new gene encoding the same protein is generated, if the protein has the same amino acid at the specific position as its starting position, this is not an amino acid substitution.

[0363] As used herein, the term "amino acid insertion" or "insertion" refers to the addition of an amino acid residue or sequence at a specific position in the parent polypeptide sequence. For example, -40A indicates the insertion of an alanine residue after position 40 and before position 41. As a separate example, D8EPKSS or -8EPKSS indicates the insertion of the sequence Glu-Pro-Lys-Ser-Ser after position 8 and before position 9.

[0364] As used herein, the term "amino acid deletion" or "deletion" refers to the removal of an amino acid or sequence at a specific position in the parent polypeptide sequence. For example, Y40-, Y40#, Y40(), or Y40del indicates the deletion of a tyrosine residue at position 40. As a separate example, EPKSS8-, EPKSS8#, and EPKSS8del indicate the deletion of the sequence Glu-Pro-Lys-Ser-Ser starting at position 8.

[0365] As used herein, the term "non-naturally occurring protein" or "non-naturally occurring protein variant" refers to a variant protein that differs from its parent protein due to at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more non-isomorphic modifications. For example, because the IL-12 p35 subunit of IL-12 does not contain an alanine residue at position 40, the substitution Y40A is considered a non-naturally occurring IL-12p35 variant (or more generally, a non-naturally occurring IL-12 variant). Non-isomorphic modifications to a protein may be referred to as "non-naturally occurring modifications."

[0366] As used herein, the terms “percentage (%) identity” and “percentage (%) sequence identity” when used in the context of two or more proteins or nucleic acids refer to the percentage of amino acid residues (or nucleic acids encoding amino acid residues) in a candidate sequence that are identical to amino acid residues (or nucleic acids encoding amino acid residues) in a particular sequence (such as, for example, the amino acid sequence of a parent protein), after sequence alignment and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity, without considering any conserved substitutions as part of sequence identity. Alignments for determining percentage sequence identity can be performed in various ways within the scope of the art, such as, for example, using publicly available computer software (e.g., BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. In some embodiments, two or more amino acid sequences are at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical.

[0367] As used herein, the term "half-life" can refer to the time required for an agent to lose half of its pharmacological, physiological, or other activity relative to the time at which such activity is administered to a organism or subject's serum or tissue, or relative to any other defined time point. "Half-life" can also refer to the time required for the amount or concentration of an agent to decrease to half the initial amount administered to a organism or subject's serum or tissue, relative to the time at which such amount or concentration is administered to a organism or subject's serum or tissue, or relative to any other defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0368] As used herein, the terms "host cell" and "recombinant cell" refer to an individual cell or cell culture that can be or has become a recipient of any recombinant vector or isolated polynucleotide. A host cell can be a transfected, transformed, transduced, or infected cell from any source, including but not limited to prokaryotic cells, eukaryotic cells, mammalian cells, avian cells, insect cells, plant cells, or bacterial cells, or can be any source from which the nucleic acids described herein can be propagated. Host cells include cells transfected or infected in vivo or in vitro with the recombinant vector or polynucleotide of the present invention. A host cell containing the recombinant vector of the present invention may be referred to as a "recombinant host cell."

[0369] Host cells may include, but are not limited to, cells of mammals, plants, insects, fungi, and bacteria. Bacterial cells include, but are not limited to, cells of Gram-positive bacteria (such as species of Bacillus, Streptomyce, and Staphylococcus), and cells of Gram-negative bacteria (such as cells of Escherichia and Pseudomonas). Fungal cells may include, but are not limited to, yeast cells, such as Saccharomyces, Pichia pastoris, and Hansenula polymorpha. Insect cells may include, but are not limited to, cells of Drosophila and Sf9 cells. Plant cells include, but are not limited to, cells derived from crops, medicinal or ornamental plants, or bulbs. Mammalian cells suitable for use in this invention include, but are not limited to, epithelial cell lines (e.g., porcine epithelial cells), osteosarcoma cell lines, neuroblastoma cell lines, epithelial carcinoma, glial cells, hepatocyte lines, Chinese hamster ovary (CHO) cells, COS cells, BHK cells, HeLa cells, mouse embryonic stem cell (mESC) line D3 cells, human embryonic stem cells (e.g., HS293 cells and BG01V cells), NIH 3T3 cells, human embryonic kidney (HEK) 293T cells, and human mesenchymal stem cells (hMSCS).

[0370] As used herein, the terms “cell,” “cell culture,” “cell line,” and “host cell” refer not only to a specific cell, cell culture, cell line, or host cell, but also to the offspring or potential offspring of such a cell, cell culture, cell line, or host cell, regardless of the number of transfers or passages in the culture. It should be understood that not all progeny are identical to the parent cell. This is because certain modifications can occur in the offspring due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), making the progeny practically different from the parent cell, but still included in the terminology used herein, as long as the progeny retains the same or substantially the same function as the original cell, cell culture, cell line, or host cell.

[0371] As used herein, the term "medium" or "culture medium" includes any culture medium, solution, solid, semi-solid, or rigid support that can support or contain any host cell, including but not limited to bacterial host cells, yeast or fungal host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, Escherichia coli (E. coli) host cells, Pseudomonas host cells, etc., and cell contents. Therefore, the term may cover culture media for host cell growth (e.g., media containing secreted peptides, including media before, during, or after the proliferation step). The term may also cover buffer solutions or other reagents containing host cell lysates, such as in cases where polynucleotides are produced intracellularly and the host cell is lysed or destroyed to release peptides.

[0372] As used herein, the term "fusion protein" refers to the covalently coupled binding of at least two proteins or protein domains. Fusion proteins may contain artificial sequences, such as, for example, domain linkers, Fc domains, variant Fc domains, variant IL-12p35 subunit domains, (variant) IL-12p40 subunit domains, etc., as described herein. As used herein, the term "Fc fusion protein" refers to a protein containing a (variant) Fc domain, which is typically linked (optionally via a domain linker, as described herein) to one or more different protein domains. In some embodiments, the C-terminus of the Fc domain is linked to the N-terminus of one or more different protein domains (optionally via a domain linker, wherein the C-terminus of the Fc domain is linked to the N-terminus of the domain linker, and the C-terminus of the domain linker is linked to the N-terminus of one or more different protein domains). In some implementations, the N-terminus of the Fc domain is connected to the C-terminus of one or more different protein domains (optionally via a domain linker, wherein the C-terminus of one or more protein domains is connected to the N-terminus of the domain linker, and the C-terminus of the domain linker is connected to the N-terminus of the Fc domain).

[0373] As used herein, the interchangeable terms “low-potency IL-12 Fc fusion protein,” “IL-12-ABD fusion protein,” “IL-12 Fc fusion protein,” or “targeted IL-12 Fc fusion protein” generally refer to a fusion protein comprising: (1) a first IL-12 subunit domain and a second IL-12 subunit domain, wherein (i) the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises a (variant) IL-12 p40 subunit domain, or the first IL-12 subunit domain comprises a (variant) IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, and (ii) the variant IL-12 p35 subunit domain comprises a reduced amount of IL-12 compared to a reference IL-12. The Fc fusion protein has a set of amino acid substitutions that increase its binding affinity to IL-12Rβ2, wherein the set of amino acid substitutions is selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; (2) at least one antigen-binding domain (ABD; i.e., one ABD, two ABDs, three ABDs, etc.), wherein the ABD binds to a target antigen selected from the group consisting of: (a) OX40, (b) LAG3, (c) PD1, (d) ICOS, (e) 41BB, (f) CCR7 and (g) CCR8; (3) one or more domain linkers; and (4) a first Fc domain and a second Fc domain. Those skilled in the art will understand that the IL-12 Fc fusion protein of the present invention can present several different conformations (including, but not limited to, those described herein and the figures (see, for example, ...). Figures 18A-18HThe IL-12 Fc fusion protein of the present invention may be configured such that the ABD binds to a human target antigen, a mouse target antigen, a camelid target antigen, a cynomolgus monkey target antigen, or the ABD is a humanized ABD. In some embodiments, the target antigen includes a human target antigen (e.g., human PD1, human LAG3, human OX40, human ICOS, human 41BB, human CCR7, or human CCR8). In some embodiments, the target antigen includes a mouse target antigen. In some embodiments, the target antigen includes a camelid target antigen. In some embodiments, the target antigen includes a cynomolgus monkey target antigen. In some further embodiments, the ABD is a humanized ABD. As another example, the IL-12 Fc fusion protein of the present invention can be configured such that ABD is in Fab form, scFv form, nanobody form, and other forms known in the art (including but not limited to: bivalent antibody form, triantibody form, microantibody form, single-chain Fab (scFab) form, F(ab)2 form, scFv-Fc form, BiTE form, intracellular antibody form, tandem scFv form, DART form, etc.). In some cases, the IL-12 Fc fusion protein is configured such that ABD is in Fab form (i.e., the IL-12 Fc fusion protein includes a variable heavy chain (VH) domain, a variable light chain (VL) domain, a constant heavy chain (CH1) domain, and a constant light chain (CL) domain). In some other cases, the IL-12 Fc fusion protein is configured such that the ABD is in scFv form (i.e., the IL-12 Fc fusion protein contains a VH domain, a linker, and a VL domain from the N-terminus to the C-terminus; or conversely, wherein the IL-12 Fc fusion protein contains a VL domain, a linker, and a VH domain from the N-terminus to the C-terminus). In still other cases, the IL-12 Fc fusion protein is configured such that the ABD is in nanobody form (i.e., the IL-12 Fc fusion protein contains a VHH domain, wherein the VHH domain contains complementarity-determining regions (CDRs) 1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 bind to the target antigen). As another example, the IL-12 Fc fusion protein of the present invention may be configured such that the fusion protein contains one ABD (i.e., a monovalent monospecific form), two ABDs (e.g., a bivalent monospecific form or a monovalent bispecific form), or three or more ABDs (e.g., a monovalent multispecific form, a multivalent monospecific form, a multivalent multispecific form, etc.). In the case where the IL-12 Fc fusion protein of the present invention contains two ABDs, the first ABD and the second ABD can bind to the same target antigen (i.e., a bivalent monospecific form), or the first ABD can bind to a first target antigen and the second ABD can bind to a different second target antigen (i.e., a monovalent bispecific form).Those skilled in the art will understand that the IL-12 Fc fusion protein of the present invention, comprising three or more ABDs, can be constructed in a similar manner according to the total number of ABDs and the specificity of such ABDs (e.g., monovalent multispecific form; multivalent monospecific form; multivalent multispecific format; etc.). As another example, the IL-12 Fc fusion protein of the present invention can be constructed such that the first Fc domain and / or the second Fc domain comprises a wild-type Fc domain or comprises one or more Fc domains with substitution mutations. In some embodiments, at least one of the first Fc domain and the second Fc domain comprises a variant Fc domain (e.g., the first Fc domain comprises a variant Fc domain and the second Fc domain comprises an Fc domain or a variant Fc domain, or the second Fc domain comprises a variant Fc domain and the first Fc domain comprises an Fc domain or a variant Fc domain). In some embodiments, the first Fc domain and / or the second Fc domain comprises modifications that promote heterodimerization of the first Fc domain and the second Fc domain. In some embodiments, the first Fc domain and / or the second Fc domain includes one or more modifications that alter Fc binding. In some embodiments, the first Fc domain and / or the second Fc domain includes one or more modifications that alter the Fc half-life. In some embodiments, the first Fc domain and / or the second Fc domain includes one or more modifications that alter the binding of the first Fc domain and / or the second Fc domain to the neonatal Fc receptor (FcRn). In some further embodiments, the first Fc domain and / or the second Fc domain includes one or more modifications that increase the half-life and / or binding to FcRn. As another example, the IL-12 Fc fusion protein of the present invention can be configured such that: (1) the first IL-12 subunit domain and the second IL-12 subunit domain are located on the same monomer of the IL-12 Fc fusion protein of the present invention (in several embodiments, domain linkers are used for covalent connection (e.g., from the N-terminus to the C-terminus, variant IL-12 p35 subunit domain - domain linker - (variant) IL-12 p40 subunit domain or (variant) IL-12 p40 subunit domain - domain linker - variant IL-12 p35 subunit domain)), (2) the first IL-12 subunit domain and the second IL-12 subunit domain are located on different monomers of the IL-12 Fc fusion protein of the present invention (e.g., the first monomer and the second monomer, respectively, or vice versa), or (3) the first IL-12 subunit domain is covalently linked to the IL-12 Fc fusion protein of the present invention. The monomer of the Fc fusion protein, and the second IL-12 subunit domain is expressed in the form of a soluble protein, wherein the soluble IL-12 subunit interacts with / binds to the IL-12 subunit of the monomer of the IL-12 Fc fusion protein of the present invention.An exemplary conformation in which one IL-12 subunit is covalently linked to the IL-12 Fc fusion protein of the present invention and one IL-12 subunit is expressed as a soluble fragment is described in PCT Publication No. WO 2023 / 242769 A1, which is incorporated herein by reference in its entirety. In some embodiments, the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, and the second IL-12 subunit domain comprises an IL-12 p40 subunit domain or a variant IL-12 p40 subunit domain. In some embodiments, the first IL-12 subunit domain comprises an IL-12 p40 subunit domain or a variant IL-12 p40 subunit domain, and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain. In some embodiments, the C-terminus of the first IL-12 subunit domain is connected to the N-terminus of the first Fc domain (optionally via a domain connector), and the C-terminus of the second IL-12 subunit domain is connected to the N-terminus of the second Fc domain (optionally via a domain connector).

[0374] As used herein, the term "isolated" in describing the various polypeptides disclosed herein refers to a polypeptide that has been identified and isolated and / or recovered from the cells (e.g., host cells and / or cell lines) or cell cultures in which it is expressed. Typically, isolated polypeptides are prepared by at least one purification step. As used herein, the term "isolated protein" refers to a protein that is substantially free of other proteins (such as, for example, host cell proteins) from the cell culture.

[0375] As used herein, the interchangeable terms “Fc,” “Fc region,” or “Fc domain” refer to a polypeptide containing the CH2-CH3 domain of an immunoglobulin G (IgG) molecule, and in some cases include all or part of the hinge and its variants. In the EU designation of human IgG1, the CH2-CH3 domain contains amino acids 231 to 447, and the hinge is 216 to 230. Therefore, the definition of an “Fc domain” includes amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3) or fragments thereof. In this context, an “Fc fragment” may contain fewer amino acids from either or both of the N-terminus and C-terminus, but still retains the ability to form a dimer with another Fc domain or Fc fragment, which can be detected using standard methods, typically based on size (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). In this document, unless otherwise specified, “Fc domain” generally refers to the CH2-CH3 domain (and optionally all or part of the hinge) of human IgG1. Various human IgG1 Fc domains are described below, at least some of which contain one or more modifications. Unless the context clearly indicates otherwise, amino acid position numbering relative to Fc domains (e.g., first Fc domain, second Fc domain, etc.) is based on the EU index. The interchangeable terms “EU index” or “EU index in Kabat” or “EU numbering” refer to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). For clarity, although modifications are discussed primarily in the context of human IgG1 Fc domains for brevity, mutations in other immunoglobulins (such as, for example, IgG2, IgG3, IgG4, IgA, IgM, and IgE) may occur at residue positions corresponding to the mutations described in human IgG1 herein. Those skilled in the art will be able to easily determine the residue positions in other immunoglobulins corresponding to the human IgG1 modification described herein.

[0376] As used herein, the term "vector" refers to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid molecule is typically linked to (e.g., inserted into) a vector nucleic acid molecule. Typically, a vector is capable of replication when associated with appropriate control elements. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integration vectors. An "expression vector" is a vector containing a regulatory region that enables the expression of DNA sequences and fragments thereof in vitro and / or in vivo. Vectors may include sequences that direct autonomous replication in cells or sequences sufficient to allow integration into the host cell's DNA. Useful vectors include, but are not limited to, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, granules, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, but are not limited to, replication-deficient retroviruses and lentiviruses. In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery medium to transfer genes into cells.

[0377] As used herein, with respect to nucleic acid molecules, the term "recombinant" refers to genomic, cDNA, viral, semi-synthetic and / or synthetically derived polynucleotides that, due to their own origin or manipulation, are not wholly or partially related to naturally occurring polynucleotides of the same origin. With respect to proteins or polypeptides, the term "recombinant" refers to polypeptides produced through the expression of recombinant polynucleotides. With respect to host cells, the term "recombinant" refers to host cells in which recombinant polynucleotides or vectors containing recombinant polynucleotides have been introduced.

[0378] As used herein, the term "operably linked" refers to a physical or functional connection between two or more elements (e.g., a polypeptide sequence or a polynucleotide sequence) that allows them to operate in their intended manner. For example, an operably linked connection between a polynucleotide of interest and a regulatory sequence (such as, for example, a promoter) is a functional connection that enables the polynucleotide of interest to be expressed. In this sense, the term "operably linked" refers to the localization of a regulatory region and a coding sequence to be transcribed such that the regulatory region effectively regulates the transcription or translation of the coding sequence of interest. Thus, if a promoter mediates the transcription of a nucleic acid sequence, it is operably linked to the nucleic acid sequence. It should be understood that the elements operably linked can be continuous or discontinuous. In the context of polypeptides, "operably linked" refers to a physical connection (e.g., a direct or indirect link) between amino acid sequences (e.g., different segments, modules, or domains) to provide the said activity of the polypeptide.

[0379] As used herein, the term "binding affinity" refers to the "strength" of the binding of a given molecule (such as, for example, the p35 subunit domain of variant IL-12, or the antigen-binding domain) to its ligands (such as, for example, IL-12Rβ2, PD1, LAG3, OX40, ICOS, 41BB, CCR7, CCR8, etc.) and / or the rate of association and / or dissociation of the molecule with its ligands. Binding affinity is typically expressed as a dissociation constant (K0). D The binding activity of the IL-12 Fc fusion protein of this disclosure can be determined by any suitable method known in the art, such as, for example, surface plasmon resonance (SPR) assay, enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Biacore assay, KinExA assay, etc. Since the IL-12 Fc fusion protein binds to at most one target (e.g., IL-12Rβ2 and at least one target antigen (PD1, LAG3, OX40, ICOS, 41BB, CCR7, CCR8)), such IL-12 Fc fusion proteins will exhibit different binding affinities for IL-12Rβ2 and at least one target antigen. Therefore, the term "binding affinity" is intended to refer to the "strength" of binding to IL-12Rβ2 when referring to the "IL-12 binding arm" of the IL-12 Fc fusion protein of this invention, or the "strength" of binding to a target antigen when referring to the ABD of the IL-12 Fc fusion protein of this invention. Furthermore, some conformations of the IL-12 Fc fusion protein described herein contain more than one ABD, and are therefore capable of binding more than one target antigen. Therefore, in such embodiments, the term "binding affinity" is intended to refer to the "strength" of the binding of the first ABD to the first target antigen (e.g., the strength of the binding of an ABD "specifically binding" to PD1 to PD1) or the "strength" of the binding of the second ABD to the second target antigen (e.g., the strength of the binding of an ABD "specifically binding" to LAG3 to LAG3).

[0380] As used herein, the interchangeable terms “specific binding,” “specific binding to” a specific antigen or “epitope,” or “to” a specific antigen or “epitope,” and “having specificity” mean a binding that is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule to a control molecule, typically a structurally similar but non-binding molecule. For example, specific binding can be determined by competition with a control molecule similar to the target.

[0381] As used herein, the term "epitaph" refers to a determinant cluster, also known as a complementary site, that interacts with a specific antigen-binding site in the variable region of an antibody molecule. Epitopes are groups of molecules, such as amino acids or sugar side chains, that possess specific structural and charge characteristics. A single antigen may have more than one epitope.

[0382] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) to elicit a response at a given dose or concentration in a given biological system or experimental setting. The potency of the IL-12 Fc fusion protein of this disclosure can be determined by any suitable method known in the art, such as, for example, the IL-12 HEK reporter gene assay (e.g., InvivoGen's IL-12 HEK reporter gene assay (catalog number hkb-il12)), ligand binding assays (e.g., ELISA or flow cytometry), and / or functional assays (e.g., AlphaLISA). Typically, changes in potency can be graphically represented as a leftward or rightward shift of the response curve compared to a control. A rightward shift of the response curve generally indicates decreased potency, while a leftward shift generally indicates increased potency.

[0383] As used herein, the term "activity" refers to a specific response elicited by a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) at a given dose or concentration in a specific biological system or experimental setting. The activity of the IL-12 Fc fusion protein of this disclosure can be determined by any suitable method known in the art, such as, for example, ligand binding assays and / or functional assays. Typically, changes in activity can be graphically represented as an upward or downward shift of the response curve compared to a control. An upward shift of the response curve generally indicates increased activity, while a downward shift of the response curve generally indicates decreased activity. As described in further detail below, changes in activity may or may not be related to and / or caused by changes in potency.

[0384] As used herein, the term “manufacturability” refers to any property that can affect the production and / or storage of a given protein, cytokine, fusion protein, antibody, etc., on a scale and in quantity sufficient for individual administration. Examples of properties that affect manufacturability include, but are not limited to, the stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein).

[0385] As used herein, the term "stability" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) to retain the same properties and characteristics as it had at the time of manufacture within specific limits and / or storage and / or use parameters. The stability of the IL-12 Fc fusion protein of this disclosure can be determined by any suitable method known in the art, such as, for example, ELISA, Western blot, Biacore assay, SDS-PAGE, size exclusion chromatography, dynamic light scattering, differential scanning calorimetry, and differential scanning fluorescence.

[0386] As used herein, the term "expression yield" refers to the amount or quantity of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, etc., is well known in the relevant fields. Expression yield can be quantified or determined using any suitable method, such as, for example, UV absorbance measurements, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorescence assays.

[0387] As used herein, the terms “subject” or “individual” for therapeutic purposes may generally refer to any animal classified as a mammal (including, but not limited to, humans, primates and / or non-human primates), livestock, farm animal, zoo animal, research animal, sporting animal and / or pet animal, such as dogs, horses, cats, cattle, etc. However, unless the context clearly indicates otherwise, references to “subject” are intended to refer to human subjects.

[0388] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include multiple indicators. Thus, for example, reference to “an antigen” includes a mixture of antigens; reference to “a pharmaceutically acceptable carrier” includes a mixture of two or more such carriers, etc. Therefore, the terms “a” (or “an”), “one or more”, and “at least one” are used interchangeably herein.

[0389] Furthermore, when used herein, “and / or” should be considered as specifically disclosing each of two specified features or components and the presence or absence of the other. Thus, as used in the phrase, the term “and / or,” such as “A and / or B,” is intended to include “A and B,” “A or B,” “A (alone),” and “B (alone).”

[0390] As used herein, the use of ordinal terms (such as, for example, "first," "second," "third," "fourth," "fifth," etc.) to modify elements of this disclosure does not in itself imply any priority, order of precedence, or sequence of events of one element relative to another, or the chronological order in which the actions of the method are performed, but is merely used as a marker to distinguish one element having a certain name (e.g., assay, domain connector, etc.) from another element having the same name but using ordinal terms used to distinguish elements (e.g., "first assay," "second assay," "third assay," "fourth assay," "fifth assay," etc.). For example, a "first assay" may be performed to detect changes in the potency of the IL-12 Fc fusion protein, and a "second assay" may be performed to detect changes in the binding affinity to IL-12Rβ2. In such instances, the terms "first assay" and "second assay" are not used to describe any priority, order of precedence, or sequence of events of the assays performed, but rather to indicate that, apart from the use of the ordinal terms "first" and "second," the elements are different despite having the same name. As a standalone example, a "third measurement" can be performed to detect changes in the ability to stimulate IFNγ without performing a "first measurement" or a "second measurement." Alternatively, it is also considered that the "first measurement" may be performed before or after the "third measurement," and that in the example, the "second measurement" may be omitted, performed before or after the "first measurement," or before or after the "third measurement." Although the examples described above utilize specific ordinal terms, the concepts presented above should be considered. Make the necessary changes This applies to other instances where any number of ordinal terms are used in various configurations.

[0391] As used herein, the term "about" means ±10% of a specified value. When referring to a range of values ​​(or parameters), the term "about" means +10% of the upper limit and -10% of the lower limit of the specified range. When providing ranges of values, it should be understood that each intermediate value between the upper and lower limits of the range, as well as any other specified or intermediate values ​​within the range, are included within the scope of this disclosure. Where a specified range includes the upper and / or lower limits, ranges excluding one of those included limits are also included in this disclosure.

[0392] It should be understood that certain features of this disclosure described in the context of individual embodiments for clarity may also be provided in combination with individual embodiments. Conversely, various features of this disclosure described in the context of individual embodiments for simplicity may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to this disclosure are specifically covered in this disclosure and disclosed herein as if each and every combination were individually and explicitly disclosed herein. In addition, all sub-combinations of various embodiments and their elements are also specifically covered in this disclosure and disclosed herein as if each and every such sub-combination were individually and explicitly disclosed herein.

[0393] II. Overview This application generally relates to compositions and methods for modulating IL-12-mediated signal transduction. Specifically, this disclosure provides a low-potency IL-12 Fc fusion protein comprising: (1) a variant IL-12 p35 subunit domain with reduced binding affinity to interleukin-12 receptor β2 (IL-12Rβ2), (2) an IL-12 p40 subunit domain, and (3) an antigen-binding domain (ABD) facilitating the transport of the fusion protein to target cells of interest, and methods for preparing and using the thereof. In some preferred embodiments, the ABD binds to OX40. In some further preferred embodiments, the ABD binds to the extracellular domain (ECD) of OX40. In still some further preferred embodiments, the ABD binds to the ECD of human OX40.

[0394] As described above, IL-12 is a potent pro-inflammatory cytokine with high affinity for its receptors (the p35 subunit of IL-12Rβ2; the p40 subunit of IL-12Rβ1). Due to this high affinity, IL-12 therapies formulated to target various cell populations using a combination of wild-type IL-12 and ABD are often unsuccessful because the affinity and / or potency of the "IL-12 binding arm" of the therapy is usually greater than the binding affinity / potency of the "ABD arm." Fusions of reduced-potency IL-12 variants with ABD targeting have the potential to overcome this limitation. However, the properties of the resulting fusion protein are heavily influenced by the identity of the ABD target. For example, the type and frequency of immune cells expressing the ABD target can affect the efficacy, pharmacokinetics, and tolerability of IL-12 ABD fusion proteins. As demonstrated in this paper, targeting low-potency IL-12 with ABDs specific to Lag-3, CCR7, or CCR8 does not enhance the antitumor activity of IL-12. While some other targeting arms (e.g., PD-1 targeting, ICOS targeting, or 41BB targeting) enhance the antitumor activity of IL-12, the resulting IL-12 ABD fusion proteins exhibit less desirable pharmacokinetic characteristics (e.g., faster clearance) and / or poorer safety profiles, likely due to the relatively widespread expression of these targets. This application especially This paper describes a reduced-potency IL-12 fusion protein targeting OX40 that exhibits enhanced properties relative to other targeted IL-12 therapeutics described herein. The reduced-potency IL-12 fusion protein targeting OX40 demonstrates potent antitumor activity and, importantly, affects pharmacokinetic characteristics and tolerability to a minimal degree. Therefore, OX40 targeting produces the greatest improvement in the therapeutic index of any of the molecules tested. Correspondingly, co-administration of the reduced-potency IL-12 fusion protein targeting OX40 with an anti-PD-1 antibody further enhances antitumor activity without significantly affecting pharmacokinetic characteristics and tolerability, while targeting additional cell types with the reduced-potency IL-12 fusion protein targeting PD-1 results in increased toxicity (as described further in detail herein).

[0395] In some embodiments, the compositions and methods described herein facilitate the targeting of target cells with the IL-12 Fc fusion protein, wherein the target cells express: (1) IL-12Rβ2, and (2) a cell surface antigen selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7 or (g) human CCR8.

[0396] In some embodiments, the compositions and methods described herein facilitate the delivery of the IL-12 Fc fusion protein to target cells expressing: (1) IL-12Rβ2, and (2) a cell surface antigen selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7, (g) human CCR8, or any combination thereof. In still some further embodiments, the cell surface antigen is PD1, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other further embodiments, the cell surface antigen is LAG3, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other further embodiments, the cell surface antigen is OX40, and the target cells include: (i) activated and / or exhausted CD8+ T cells; (ii) activated and / or exhausted CD4+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other further embodiments, the cell surface antigen is ICOS, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted CD8+ T cells; (iii) activated and / or exhausted NK cells; or (iv) activated and / or exhausted regulatory T cells. In other further embodiments, the cell surface antigen is 41BB, and the target cells include: (i) activated and / or exhausted CD4+ T cells; (ii) activated and / or exhausted NK cells; (iii) activated and / or exhausted CD8+ T cells; or (iv) activated and / or exhausted regulatory T cells. In other further embodiments, the cell surface antigen is CCR7, and the target cells include: (i) naive CD4+ T cells; (ii) naive CD8+ T cells; or (iii) naive regulatory T cells. In other further embodiments, the cell surface antigen is CCR8, and the target cells include: (i) activated and / or depleted CD4+ T cells; or (ii) activated and / or depleted regulatory T cells.

[0397] III. Composition Those skilled in the art will understand that any aspect of the compositions described herein and any of the embodiments thereof may be used in any aspect of the manufacturing methods described below and / or in any of the embodiments thereof, or also in the methods of use described below.

[0398] A. Low-potency interleukin-12 (IL-12) Fc fusion protein This disclosure provides several aspects and embodiments of a low-potency IL-12 Fc fusion protein (sometimes referred to herein as "IL-12 Fc fusion protein" for brevity). The IL-12 Fc fusion protein described herein comprises: (1) a first IL-12 subunit domain and a second IL-12 subunit domain, wherein (i) the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises a (variant) IL-12 p40 subunit domain, or the first IL-12 subunit domain comprises a (variant) IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, and (ii) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions that reduce the binding affinity of the resulting IL-12 Fc fusion protein to IL-12Rβ2 compared to a reference IL-12, wherein the set of amino acid substitutions is selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; (2) at least one antigen-binding domain (ABD), wherein the ABD binds to a target antigen selected from the group consisting of: (a) OX40, (b) LAG3, (c) PD1, (d) ICOS, (e) 41BB, (f) CCR7, and (g) CCR8; (3) one or more domain linkers; and (4) a first Fc domain and a second Fc domain. In some embodiments, the target antigen includes a human target antigen (e.g., human PD1, human LAG3, human OX40, human ICOS, human 41BB, human CCR7, or human CCR8). In some embodiments, the target antigen includes a mouse target antigen. In some embodiments, the ABD includes a humanized ABD.

[0399] Those skilled in the art will understand that the IL-12 Fc fusion protein of the present invention can present one of several different conformations. In some cases, the IL-12 Fc fusion protein is configured such that ABD is in the Fab form (i.e., the IL-12 Fc fusion protein includes a variable heavy chain (VH) domain, a variable light chain (VL) domain, a constant heavy chain (CH1) domain, and a constant light chain (CL) domain). In other cases, the IL-12 Fc fusion protein is configured such that ABD is in the scFv form (i.e., the IL-12 Fc fusion protein includes a VH domain, a domain linker, and a VL domain from the N-terminus to the C-terminus; or conversely, wherein the IL-12 Fc fusion protein includes a VL domain, a domain linker, and a VH domain from the N-terminus to the C-terminus). In other cases, the IL-12Fc fusion protein is configured such that ABD is in the form of a nanobody (i.e., the IL-12 Fc fusion protein contains a VHH domain, wherein the VHH domain contains complementarity-determining regions (CDRs) 1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 bind to the target antigen).

[0400] Due to the modular nature of the conformation of the IL-12 Fc fusion protein, the following provides first a discussion of exemplary components comprising the IL-12 Fc fusion protein of the present invention (such as, for example, variant IL-12 p35 subunit domain, (variant) IL-12 p40 subunit domain, ABD, VH domain, VL domain, Fc domain, domain linker, etc.), followed by a discussion of exemplary IL-12 Fc fusion protein conformations that have found utility in some embodiments described herein.

[0401] 1. Interleukin-12 : This disclosure provides an IL-12 Fc fusion protein comprising: (1) a first IL-12 subunit domain and a second IL-12 subunit domain, wherein (i) the first IL-12 subunit domain comprises a variant IL-12 p35 subunit domain and the second IL-12 subunit domain comprises a (variant) IL-12 p40 subunit domain, or the first IL-12 subunit domain comprises a (variant) IL-12 p40 subunit domain and the second IL-12 subunit domain comprises a variant IL-12 p35 subunit domain, and (ii) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions that reduce the binding affinity of the resulting IL-12 Fc fusion protein to IL-12Rβ2 compared to a reference IL-12, wherein the set of amino acid substitutions is selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; (2) at least one antigen-binding domain (ABD), wherein the ABD binds to a target antigen selected from the group consisting of (a) OX40, (b) LAG3, (c) PD1, (d) ICOS, (e) 41BB, (f) CCR7 and (g) CCR8; (3) one or more domain linkers; and (4) a first Fc domain and a second Fc domain.

[0402] In some embodiments, the first IL-12 subunit domain and the second IL-12 subunit domain are configured such that the first IL-12 subunit domain and the second IL-12 subunit domain are located on different monomers of the IL-12 Fc fusion protein (e.g., the first monomer of the IL-12 Fc fusion protein contains the first IL-12 subunit domain and the second monomer contains the second IL-12 subunit domain, or vice versa, wherein the first monomer contains the second IL-12 subunit domain and the second monomer contains the first IL-12 subunit domain). In other embodiments, the first IL-12 subunit domain and the second IL-12 subunit domain are configured such that they are located on the same monomer of the IL-12 Fc fusion protein, typically in a single-stranded form linked by domain linkers (e.g., the first monomer contains the first IL-12 subunit domain and the second IL-12 subunit domain and the second monomer does not contain the IL-12 subunit domain; the second monomer contains the first IL-12 subunit domain and the second IL-12 subunit domain and the first monomer does not contain the IL-12 subunit domain).

[0403] In some cases where the first IL-12 subunit domain and the second IL-12 subunit domain are located on different monomers of the IL-12 Fc fusion protein, the first IL-12 subunit domain and the second IL-12 subunit domain may be configured such that they are located at the N-terminus of their respective monomers, or they may be configured such that they are located at the C-terminus of their respective monomers. In cases where the first IL-12 subunit domain and the second IL-12 subunit domain are located on a single monomer of the IL-12 Fc fusion protein, the single-stranded IL-12 containing the first IL-12 subunit domain, the domain linker, and the second IL-12 subunit domain may be configured such that the single-stranded IL-12 is located at the N-terminus of the monomer or is configured such that the single-stranded IL-12 is located at the C-terminus of the monomer. In either configuration, the variant p35 subunit domain may be located at the N-terminus of a single-stranded IL-12 and the (variant) p40 subunit domain may be located at the C-terminus of a single-stranded IL-12, or the (variant) p40 subunit domain may be located at the N-terminus of a single-stranded IL-12 and the variant p35 subunit domain may be located at the C-terminus of a single-stranded IL-12.

[0404] While exemplary IL-12 Fc fusion proteins are typically described such that: (1) the first IL-12 subunit domain and the second IL-12 subunit domain are located on the same monomer of the IL-12 Fc fusion protein of the present invention (in several embodiments, domain linkers are covalently linked (e.g., from the N-terminus to the C-terminus, variant IL-12 p35 subunit domain - domain linker - (variant) IL-12 p40 subunit domain or (variant) IL-12 p40 subunit domain - domain linker - variant IL-12 p35 subunit domain)), or (2) the first IL-12 subunit domain and the second IL-12 subunit domain are located on different monomers of the IL-12 Fc fusion protein of the present invention (e.g., the first monomer and the second monomer, respectively, or vice versa), it is also expressly contemplated that the IL-12 Fc fusion protein of the present invention may be configured such that the first IL-12 The Fc subunit domain is covalently linked to a monomer of the IL-12Fc fusion protein of the present invention, and the second IL-12 subunit domain is expressed in a soluble protein form, wherein the soluble IL-12 subunit interacts with / binds to the IL-12 subunit of the monomer covalently linked to the IL-12 Fc fusion protein of the present invention (or conversely, wherein the first IL-12 subunit domain is expressed in a soluble protein form, and the second IL-12 subunit domain is covalently linked to a monomer of the IL-12 Fc fusion protein of the present invention). An exemplary configuration in which one IL-12 subunit is covalently linked to the IL-12 Fc fusion protein of the present invention and one IL-12 subunit is expressed in a soluble fragment form is described, for example, in PCT Publication No. WO2023 / 242769 A1, which is incorporated herein by reference in its entirety.

[0405] The binding affinity to IL-12Rβ2 can be assessed using any number of assays known to those skilled in the art. Non-limiting examples of suitable assays for evaluating binding affinity include: surface plasmon resonance (SPR) assay, enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Biacore assay, KinExA assay, flow cytometry, etc.

[0406] a. p35 subunit domain : In some embodiments, the p35 subunit domain of variant IL-12 comprises one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A. In some embodiments, one or more amino acid substitutions include Y40P / K168A. In other embodiments, one or more amino acid substitutions include Y40S / K170T. In other embodiments, one or more amino acid substitutions include Y40S / K170A. In other embodiments, one or more amino acid substitutions include Y40P / K170A. In other embodiments, one or more amino acid substitutions include Y40E / K170A.

[0407] In some embodiments, the p35 subunit domain of the variant IL-12 comprises an amino acid sequence selected from the group consisting of any of the following: (i) SEQ ID NO: 17 (Y40P / K168A), (ii) SEQ ID NO: 18 (Y40P / K170A), (iii) SEQ ID NO: 19 (Y40S / K170T), (iv) SEQ ID NO: 20 (Y40S / K170A), and (v) SEQ ID NO: 21 (Y40E / K170A), as shown below. Figure 5 As shown in the image.

[0408] In some embodiments, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence selected from the group consisting of any of the following: SEQ ID NO: 17-21 (e.g.) Figure 5 (As shown).

[0409] In some embodiments, the variant IL-12 p35 subunit domain contains a first substitution mutation comprising Y40P, and further comprises a second substitution mutation selected from the group consisting of K168A and K170A. In some embodiments, the variant IL-12 p35 subunit domain contains either SEQ ID NO: 17 or 18, such as... Figure 5 As shown.

[0410] In some embodiments, the variant IL-12 p35 subunit domain contains a first substitution mutation, including Y40S, and also contains a second substitution mutation selected from the group consisting of K170T and K170A. In some embodiments, the variant IL-12 p35 subunit domain contains either SEQ ID NO: 19 or 20, such as... Figure 5 As shown.

[0411] In some embodiments, the variant IL-12 p35 subunit domain contains a first substitution mutation, including Y40E, and also contains a second substitution mutation selected from the group consisting of K170A. In some embodiments, the variant IL-12 p35 subunit domain contains SEQ ID NO: 21, as shown in the image. Figure 5 As shown.

[0412] In some embodiments, compared to SEQ ID NO: 2, the variant IL-12 p35 subunit domain also contains a C74S substitution mutation (particularly useful in embodiments where the IL-12 Fc fusion protein of the present invention contains a variant IL-12 p40 subunit domain with a C177S or C177S / C252S substitution mutation).

[0413] In addition to the aforementioned IL-12 Fc fusion protein, further modifications to various residues are known in the field. Non-limiting examples of modifiable residues include: Q20, N21, Q35, L37, E38, F39, P41, T43, S44, E45, E46, I47, H49, E50, I52, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, T69, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, M98, A99, L123, L124, and M12. 5. D126, K128, Q130, I131, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, P164, D165, F166, Y167, T169, I171, K172, L173, I175, R181, I182, R183, V185, T186, D188, R189, V190, M191, S192, Y193, N195, A196, and S197. In some embodiments, this document or figures (see, for example, Figure 1 , 3The (variant) IL-12 p35 subunit domain described in (and 5) contains one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more additional amino acid substitutions. In some embodiments, the present document or figures (see, for example, Figure 1 , 3 The (variant) IL-12 p35 subunit domain described in (and 5) contains about 1 to about 5, about 6 to about 10, about 11 to about 15, about 16 to about 20, about 21 to about 25, about 26 to about 30, about 31 to about 35, about 36 to about 40, about 1 to about 10, about 11 to about 20, about 21 to about 30, about 31 to about 40, about 1 to about 20, or about 21 to about 40 additional amino acid substitutions.

[0414] Mutations in the p35 subunit domain of the variant IL-12 can lead to changes in one or more of the following parameters: (i) binding affinity, (ii) potency, (iii) activity, (iv) manufacturability, or (v) stability; however, changes in one or more of the foregoing parameters (such as, for example, binding affinity) are not necessarily related to changes in one or more of the other foregoing parameters (such as, for example, potency or activity). In some embodiments, substitutions of one or more amino acids in the p35 subunit domain of the variant IL-12 result in an altered binding affinity for IL-12Rβ2 compared to the binding affinity of reference IL-12. In some embodiments, substitutions of one or more amino acids reduce the binding affinity of the p35 subunit domain of the variant IL-12 to IL-12Rβ2 compared to reference IL-12. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein that does not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof. In other embodiments, one or more amino acid substitutions in the p35 subunit of the variant IL-12 do not affect binding affinity but may result in changes in one or more of the following parameters: (i) potency, (ii) activity, (iii) manufacturability, (iv) stability, or (v) any combination thereof.

[0415] Various assays can be used to select IL-12 Fc fusion proteins (e.g., IL-12Rβ2 and / or IL-12Rβ1) that bind to ligands of interest. Non-limiting examples include: solid-phase ELISA immunoassay, immunoprecipitation, Biacore assay, KinExA assay, fluorescence activated cell sorting (FACS), Octet assay, Western blot analysis, etc. The binding activity of the IL-12 Fc fusion protein disclosed herein can be determined by any suitable method known in the art, such as, for example, surface plasmon resonance (SPR) assay, enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Biacore assay, KinExA assay, etc.

[0416] Those skilled in the art will understand that binding affinity can also be used as a measure of the “strength” of the non-covalent interaction between two binding partners (e.g., the p35 subunit domain of variant IL-12 and IL-12Rβ2). The binding affinity between two molecules can be determined by determining the dissociation constant (K0). D ) can be quantified. Conversely, K can be determined by measuring the kinetics of complex formation and dissociation using suitable assays known in the art (such as, for example, the SPR assay). D The rate constants corresponding to the association and dissociation of monovalent complexes are called the association rate constants k and k, respectively. a (or k) on and dissociation rate constant k d (or k) off K D Through the following equation and k a and k d Related: K D = k d / k a The value of the dissociation constant can be determined directly using well-known methods.

[0417] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) to elicit a response at a given dose or concentration in a given biological system or experimental setting. The potency of the IL-12 Fc fusion protein of this disclosure can be determined by any suitable method known in the art, such as, for example, the IL-12 HEK reporter gene assay (e.g., InvivoGen's IL-12 HEK reporter gene assay (catalog number hkb-il12)), ligand binding assays (e.g., ELISA or flow cytometry), and / or functional assays (e.g., AlphaLISA). Typically, changes in potency can be graphically represented as a leftward or rightward shift of the response curve compared to a control. A rightward shift of the response curve generally indicates decreased potency, while a leftward shift generally indicates increased potency. As used herein, the term "activity" refers to the specific response elicited by a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) at a given dose or concentration in a specific biological system or experimental setting. The activity of the IL-12 Fc fusion protein disclosed herein can be determined by any suitable method known in the art, such as, for example, ligand binding assays and / or functional assays. Typically, changes in activity can be graphically represented as an upward or downward shift of the response curve compared to a control. An upward shift of the response curve generally indicates increased activity, while a downward shift of the response curve generally indicates decreased activity.

[0418] As used herein, the term "manufacturability" refers to any property that can affect the production and / or storage of a given protein, cytokine, fusion protein, antibody, etc., on a scale and in quantity sufficient for individual administration. Examples of properties affecting manufacturability include, but are not limited to, the stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein). As used herein, the term "stability" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) to retain the same properties and characteristics as it had at the time of manufacture within certain limits and / or storage and / or use parameters. The stability of the IL-12 Fc fusion protein of this disclosure can be determined by any suitable method known in the art, such as, for example, ELISA, Western blot, Biacore assay, SDS-PAGE, size exclusion chromatography, dynamic light scattering, differential scanning calorimetry, and differential scanning fluorescence. As used herein, the term "expression yield" refers to the amount or quantity of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, the IL-12 Fc fusion protein) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, etc., is well known in the relevant fields. Expression yield can be quantified or determined using any suitable method, such as, for example, UV absorbance measurements, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorescence assays.

[0419] In some embodiments, the binding affinity of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to IL-12Rβ2 is reduced by at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% or more, as determined by an assay. In some embodiments, the assay includes an SPR assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0420] In some embodiments, the binding affinity of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to IL-12Rβ2 is reduced by about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% or higher compared to the binding affinity of reference IL-12, as determined by an assay. In some embodiments, the assay includes an SPR assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0421] In some implementations, the binding affinity of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to IL-12Rβ2 is reduced by at least approximately 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, and 3.5-fold, respectively, compared to the binding affinity of reference IL-12. 0.9 times, approximately 4.0 times, approximately 4.1 times, approximately 4.2 times, approximately 4.3 times, approximately 4.4 times, approximately 4.5 times, approximately 4.6 times, approximately 4.7 times, approximately 4.8 times, approximately 4.9 times, approximately 5.0 times, approximately 5.1 times, approximately 5.2 times, approximately 5.3 times, approximately 5.4 times, approximately 5.5 times, approximately 6.0 times, approximately 7.0 times, approximately 8.0 times, approximately 9.0 times, approximately 10.0 times, approximately 20.0 times, approximately 30.0 times, approximately 40.0 times, approximately 50.0 times, approximately 100.0 times, approximately 150.0 times, approximately 200.0 times, approximately 250.0 times. Approximately 300.0 times, approximately 350.0 times, approximately 400.0 times, approximately 450.0 times, approximately 500.0 times, approximately 550.0 times, approximately 600.0 times, approximately 650.0 times, approximately 700.0 times, approximately 750.0 times, approximately 800.0 times, approximately 850.0 times, approximately 900.0 times, approximately 950.0 times, approximately 1,000.0 times, approximately 2,000.0 times, approximately 3,000.0 times, approximately 4,000.0 times, approximately 5,000.0 times, approximately 6,000.0 times, approximately 7,000.0 times, approximately 8,000.0 times. 0.0 times, approximately 9,000.0 times, approximately 10,000.0 times, approximately 20,000.0 times, approximately 30,000.0 times, approximately 40,000.0 times, approximately 50,000.0 times, approximately 100,000.0 times, approximately 150,000.0 times, approximately 200,000.0 times, approximately 250,000.0 times, approximately 300,000.0 times, approximately 350,000.0 times, approximately 400,000.0 times, approximately 450,000.0 times, approximately 500,000.0 times or more, as determined by measurement. In some embodiments, the measurement includes an SPR measurement. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0422] In some embodiments, the binding affinity of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to IL-12Rβ2 is reduced by approximately 1.5-fold to approximately 2.0-fold, approximately 2.0-fold to approximately 3.0-fold, approximately 3.0-fold to approximately 4.0-fold, approximately 4.0-fold to approximately 5.0-fold, approximately 5.0-fold to approximately 10.0-fold, approximately 10.0-fold to approximately 20.0-fold, approximately 20.0-fold to approximately 30.0-fold, approximately 30.0-fold to approximately 40.0-fold, approximately 40.0-fold to approximately 50.0-fold, approximately 50.0-fold to approximately 100.0-fold, approximately 100.0-fold to approximately 150.0-fold, approximately 150.0-fold to approximately 200.0-fold, approximately 200.0-fold to approximately 250.0-fold, approximately 250.0-fold to approximately 300.0-fold, approximately 3... 0.0 times to about 350.0 times, about 350.0 times to about 400.0 times, about 400.0 times to about 450.0 times, about 450.0 times to about 500.0 times, about 500.0 times to about 550.0 times, about 550.0 times to about 600.0 times, about 600.0 times to about 650.0 times, about 650.0 times to about 700.0 times, about 700.0 times to about 750.0 times, about 750.0 times to about 800.0 times, about 800.0 times to about 850.0 times, about 850.0 times to about 900.0 times, about 900.0 times to about 950.0 times, about 950.0 times to about 1,000.0 times, about 1,000.0 times to about 2,000.0 times 0.0 times, approximately 2,000.0 times to approximately 3,000.0 times, approximately 3,000.0 times to approximately 4,000.0 times, approximately 4,000.0 times to approximately 5,000.0 times, approximately 5,000.0 times to approximately 6,000.0 times, approximately 6,000.0 times to approximately 7,000.0 times, approximately 7,000.0 times to approximately 8,000.0 times, approximately 8,000.0 times to approximately 9,000.0 times, approximately 9,000.0 times to approximately 10,000.0 times, approximately 10,000.0 times to approximately 20,000.0 times, approximately 20,000.0 times to approximately 30,000.0 times, approximately 30,000.0 times to approximately 40,000.0 times, approximately 40,000.0 times. From approximately 50,000.0 times to approximately 100,000.0 times, from approximately 100,000.0 times to approximately 150,000.0 times, from approximately 150,000.0 times to approximately 200,000.0 times, from approximately 200,000.0 times to approximately 250,000.0 times, from approximately 250,000.0 times to approximately 300,000.0 times, from approximately 300,000.0 times to approximately 350,000.0 times, from approximately 350,000.0 times to approximately 400,000.0 times, from approximately 400,000.0 times to approximately 450,000.0 times, from approximately 450,000.0 times to approximately 500,000.0 times or higher, as determined by measurement.In some embodiments, the assay includes an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein that does not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0423] In some embodiments, the binding affinity of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to IL-12Rβ2 is lower than the measured lower detectable level, and the binding affinity of the reference IL-12 to IL-12Rβ2 is between or equivalent to the measured lower or higher detectable level (i.e., the binding affinity of the reference IL-12 may be equal to the lower detectable level, the higher detectable level, or a value between the lower and higher detectable levels; in other words, the binding affinity is "detectable" for the reference IL-12 and "undetectable" for the IL-12 Fc fusion protein). In some embodiments, the assay includes an SPR assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0424] In some implementations, the potency of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain is reduced by at least about 50.0-fold, about 100.0-fold, about 150.0-fold, about 200.0-fold, about 250.0-fold, about 300.0-fold, about 350.0-fold, about 400.0-fold, about 450.0-fold, about 500.0-fold, about 550.0-fold, about 600.0-fold, about 650.0-fold, about 700.0-fold, about 750.0-fold, about 800.0-fold, about 850.0-fold, about 900.0-fold, about 950.0-fold, or about 1,000-fold compared to the potency of reference IL-12. 0.0 times, approximately 2,000.0 times, approximately 3,000.0 times, approximately 4,000.0 times, approximately 5,000.0 times, approximately 6,000.0 times, approximately 7,000.0 times, approximately 8,000.0 times, approximately 9,000.0 times, approximately 10,000.0 times, approximately 20,000.0 times, approximately 30,000.0 times, approximately 40,000.0 times, approximately 50,000.0 times, approximately 100,000.0 times, approximately 200,000 times, or approximately 300,000 times or higher, as determined by assay. In some embodiments, the assay includes an IL-12 HEK reporter gene assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0425] In some embodiments, the potency of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain is reduced by at least 50.0-fold to about 100.0-fold, about 100.0-fold to about 150.0-fold, about 150.0-fold to about 200.0-fold, about 200.0-fold to about 250.0-fold, about 250.0-fold to about 300.0-fold, about 300.0-fold to about 350.0-fold, about 350.0-fold to about 400.0-fold, about 400.0-fold to about 450.0-fold, about 450.0-fold to about 500.0-fold, about 500.0-fold to about 550.0-fold, and about 550.0-fold. From approximately 600.0 times, from approximately 600.0 times to approximately 650.0 times, from approximately 650.0 times to approximately 700.0 times, from approximately 700.0 times to approximately 750.0 times, from approximately 750.0 times to approximately 800.0 times, from approximately 800.0 times to approximately 850.0 times, from approximately 850.0 times to approximately 900.0 times, from approximately 900.0 times to approximately 950.0 times, from approximately 950.0 times to approximately 1,000.0 times, from approximately 1,000.0 times to approximately 2,000.0 times, from approximately 2,000.0 times to approximately 3,000.0 times, from approximately 3,000.0 times to approximately 4,000.0 times. 0.0 times, approximately 4,000.0 times to approximately 5,000.0 times, approximately 5,000.0 times to approximately 6,000.0 times, approximately 6,000.0 times to approximately 7,000.0 times, approximately 7,000.0 times to approximately 8,000.0 times, approximately 8,000.0 times to approximately 9,000.0 times, approximately 9,000.0 times to approximately 10,000.0 times, approximately 10,000.0 times to approximately 20,000.0 times, approximately 20,000.0 times to approximately 30,000.0 times, approximately 30,000.0 times to approximately 40,000.0 times, Approximately 40,000.0 times to approximately 50,000.0 times, approximately 50,000.0 times to approximately 60,000.0 times, approximately 60,000.0 times to approximately 70,000.0 times, approximately 70,000.0 times to approximately 80,000.0 times, approximately 80,000.0 times to approximately 90,000.0 times, approximately 90,000.0 times to approximately 100,000.0 times, approximately 100,000.0 times to approximately 200,000.0 times, or approximately 200,000.0 times to approximately 300,000.0 times or higher, as determined by assay. In some embodiments, the assay includes an IL-12 HEK reporter gene assay.In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0426] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain exhibit potency too weak to be accurately determined, and the potency of the reference IL-12 is between or equivalent to the measured lower or higher detectable levels (i.e., the potency of the reference IL-12 may be equal to the lower detectable level, the higher detectable level, or a value between the lower and higher detectable levels; in other words, the potency is "detectable" for the reference IL-12 and "undetectable" for the IL-12 Fc fusion protein). In some embodiments, the assay includes an IL-12 HEK reporter gene assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0427] In some implementations, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain have a reduced ability to stimulate STAT4 signaling compared to reference IL-12, as determined by assays. Reduced ability to stimulate STAT4 signaling may refer to a decrease in the observed maximum response and / or EC. 50The value shift. In some embodiments, the ability of the IL-12 Fc fusion protein to stimulate STAT4 signaling is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% or more compared to a reference IL-12, as determined by assay. In some embodiments, the assay includes an IL-12 HEK reporter gene assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0428] In some embodiments, the ability of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to stimulate STAT4 signaling is reduced by approximately 10% to approximately 100%, approximately 10% to approximately 50%, approximately 20% to approximately 70%, approximately 30% to approximately 80%, approximately 40% to approximately 90%, approximately 50% to approximately 100%, approximately 20% to approximately 50%, approximately 40% to approximately 70%, approximately 30% to approximately 60%, approximately 40% to approximately 100%, approximately 20% to approximately 80%, or approximately 10% to approximately 90% compared to reference IL-12, as determined by assay. In some embodiments, the assay includes an IL-12 HEK reporter gene assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0429] In some implementations, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain have a reduced ability to stimulate IFNγ production compared to reference IL-12, as determined by assays. Reduced ability to stimulate IFNγ production may refer to a decrease in the observed maximum response and / or EC. 50Value shift. In some implementations, the ability of the IL-12 Fc fusion protein and / or variant IL-12 p35 subunit domain to stimulate IFNγ production is reduced by approximately 50.0-fold, 100.0-fold, 150.0-fold, 200.0-fold, 250.0-fold, 300.0-fold, 350.0-fold, 400.0-fold, 450.0-fold, 500.0-fold, 550.0-fold, 600.0-fold, 650.0-fold, 700.0-fold, 750.0-fold, 800.0-fold, 850.0-fold, 900.0-fold, 950.0-fold, and approximately 1,000.0-fold compared to reference IL-12. Approximately 0.0 times, about 2,000.0 times, about 3,000.0 times, about 4,000.0 times, about 5,000.0 times, about 6,000.0 times, about 7,000.0 times, about 8,000.0 times, about 9,000.0 times, about 10,000.0 times, about 20,000.0 times, about 30,000.0 times, about 40,000.0 times, about 50,000.0 times, about 100,000.0 times, about 200,000 times, or about 300,000 times or higher, as determined by assay. In some embodiments, the assay includes an AlphaLISA assay, an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA, or an ELISpot assay. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0430] In some embodiments, the ability of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to stimulate IFNγ production is reduced by at least 50.0-fold to about 100.0-fold, about 100.0-fold to about 150.0-fold, about 150.0-fold to about 200.0-fold, about 200.0-fold to about 250.0-fold, about 250.0-fold to about 300.0-fold, about 300.0-fold to about 350.0-fold, about 350.0-fold to about 400.0-fold, about 400.0-fold to about 450.0-fold, about 450.0-fold to about 500.0-fold, about 500.0-fold to about 550.0-fold, about 550.0-fold, and about 550.0-fold compared to reference IL-12. 0.0 times to about 600.0 times, about 600.0 times to about 650.0 times, about 650.0 times to about 700.0 times, about 700.0 times to about 750.0 times, about 750.0 times to about 800.0 times, about 800.0 times to about 850.0 times, about 850.0 times to about 900.0 times, about 900.0 times to about 950.0 times, about 950.0 times to about 1,000.0 times, about 1,000.0 times to about 2,000.0 times, about 2,000.0 times to about 3,000.0 times, about 3,000.0 times to about 4 Approximately 4,000.0 times, approximately 5,000.0 times, approximately 6,000.0 times, approximately 6,000.0 times, approximately 7,000.0 times, approximately 7,000.0 times, approximately 8,000.0 times, approximately 8,000.0 times, approximately 9,000.0 times, approximately 10,000.0 times, approximately 10,000.0 times, approximately 20,000.0 times, approximately 30,000.0 times, approximately 30,000.0 times, approximately 40,000.0 times. Approximately 40,000.0 times to approximately 50,000.0 times, approximately 50,000.0 times to approximately 60,000.0 times, approximately 60,000.0 times to approximately 70,000.0 times, approximately 70,000.0 times to approximately 80,000.0 times, approximately 80,000.0 times to approximately 90,000.0 times, approximately 90,000.0 times to approximately 100,000.0 times, approximately 100,000.0 times to approximately 200,000.0 times, or approximately 200,000.0 times to approximately 300,000.0 times or higher, as determined by assay. In some embodiments, the assay includes an AlphaLISA assay, an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA, or an ELISpot assay.In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0431] In some implementations, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain exhibit an ability to stimulate IFNγ production that is too weak to be accurately determined, and the ability of the reference IL-12 to stimulate IFNγ production is between or equivalent to a measured lower or higher detectable level (i.e., the potency of the reference IL-12 may be equal to a lower detectable level, a higher detectable level, or a value between a lower and higher detectable level; in other words, the ability to stimulate IFNγ production is "detectable" for the reference IL-12, and the ability to stimulate IFNγ production is "detectable" for the IL-12). The Fc fusion protein is "undetectable". In some embodiments, the assay includes an AlphaLISA assay, an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA, or an ELISpot assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein that does not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0432] In some embodiments, an IL-12 Fc fusion protein and / or a variant IL-12 p35 subunit domain are provided, wherein one or more amino acid substitutions in the variant IL-12 p35 subunit domain improve the half-life compared to that of a reference IL-12. In some embodiments, the fusion of the variant IL-12 p35 subunit domain and / or (variant) IL-12 p40 subunit domain improves the half-life compared to a reference. In some implementations, the half-life of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain is reduced or increased by about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, about 6.0-fold, about 7.0-fold, about 8.0-fold, about 9.0-fold, about 10.0-fold, about 15.0-fold, about 20.0-fold, about 30.0-fold, about 40.0-fold, about 50.0-fold, or about 100.0-fold or more compared to the half-life of the reference IL-12. In some implementations, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, and IL-12 Fc fusion proteins that do not contain one or more amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[0433] In some embodiments, an IL-12 Fc fusion protein and / or a variant IL-12 p35 subunit domain are provided, wherein one or more amino acid substitutions in the variant IL-12 p35 subunit domain improve the half-life compared to that of a reference IL-12. In some embodiments, the fusion of the variant IL-12 p35 subunit domain and / or (variant) IL-12 p40 subunit domain improves the half-life compared to a reference. In some embodiments, the half-life of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain is reduced or increased by about 1.5-fold to about 2.0-fold, about 2.0-fold to about 3.0-fold, about 3.0-fold to about 4.0-fold, about 4.0-fold to about 5.0-fold, about 5.0-fold to about 6.0-fold, about 6.0-fold to about 7.0-fold, about 7.0-...

Claims

1. An IL-12 Fc fusion protein comprising: (a) A first monomer, comprising from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, (iii) a second IL-12 subunit domain, (iv) a second domain connector, and (v) a first Fc domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a variable heavy chain (VH) domain, (ii) a constant heavy chain (CH1) domain, and (iii) a second Fc domain; and (c) A third monomer comprising, from the N-terminus to the C-terminus: (i) a variable light chain (VL) domain and (ii) a constant light chain (CL) domain, wherein the VL domain and the VH domain form an antigen-binding domain (ABD) for binding the target antigen.

2. The IL-12 Fc fusion protein according to claim 1, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

3. The IL-12 Fc fusion protein according to claim 1 or 2, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

4. The IL-12 Fc fusion protein according to claim 1 or 2, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

5. The IL-12 Fc fusion protein according to claim 4, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

6. The IL-12 Fc fusion protein according to claim 5, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

7. The IL-12 Fc fusion protein according to any one of claims 1 to 6, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

8. The IL-12 Fc fusion protein according to claim 7, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

9. The IL-12 Fc fusion protein according to any one of claims 1 to 8, wherein the ABD binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

10. The IL-12 Fc fusion protein of claim 9, wherein the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 160 and 148 respectively; NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

11. The IL-12 Fc fusion protein of claim 9, wherein the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

12. The IL-12 Fc fusion protein of claim 9, wherein the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

13. The IL-12 Fc fusion protein of claim 9, wherein the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

14. The IL-12 Fc fusion protein of claim 9, wherein the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

15. The IL-12 Fc fusion protein of claim 9, wherein the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

16. The IL-12 Fc fusion protein of claim 9, wherein the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

17. The IL-12 Fc fusion protein according to any one of claims 1 to 16, wherein the ABD binds to the extracellular domain (ECD) of the target antigen.

18. The IL-12 Fc fusion protein according to any one of claims 1 to 17, wherein the first domain adapter and / or the second domain adapter comprises any one of SEQ ID NO: 31-40.

19. An IL-12 Fc fusion protein comprising: (a) A first monomer, which comprises from the N-terminus to the C-terminus: (i) a first VH (VH1) domain, (ii) a first CH1 domain, and (iii) a first Fc domain; (b) A second monomer comprising, from the N-terminus to the C-terminus: (i) a first VL (VL1) domain and (ii) a first CL domain, wherein the VH1 domain and the VL1 domain form a first ABD that binds to the first target antigen; (c) A third monomer, comprising from its N-terminus to its C-terminus: (i) a second VH (VH2) domain, (ii) a second CH1 domain, (iii) a second Fc domain, (iv) a first domain connector, (v) a first IL-12 subunit domain, (vi) a second domain connector, and (vii) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (d) A fourth monomer comprising, from the N-terminus to the C-terminus: (i) a second VL (VL2) domain and (ii) a second CL domain, wherein the VH2 domain and the VL2 domain form a second ABD that binds to the second target antigen.

20. The IL-12 Fc fusion protein of claim 19, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

21. The IL-12 Fc fusion protein according to claim 19 or 20, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

22. The IL-12 Fc fusion protein of claim 19 or 20, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

23. The IL-12 Fc fusion protein of claim 22, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

24. The IL-12 Fc fusion protein according to claim 23, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

25. The IL-12 Fc fusion protein according to any one of claims 19 to 24, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

26. The IL-12 Fc fusion protein according to claim 25, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

27. The IL-12 Fc fusion protein according to any one of claims 19 to 26, wherein the first ABD binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

28. The IL-12 Fc fusion protein according to any one of claims 19 to 27, wherein the second ABD binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

29. The IL-12 Fc fusion protein according to any one of claims 19 to 28, wherein both the first ABD and the second ABD bind to a target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

30. The IL-12 Fc fusion protein according to any one of claims 19 to 28, wherein: (1) The first ABD binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8; and (2) The second ABD binds to the second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

31. The IL-12 Fc fusion protein according to any one of claims 27 to 30, wherein the first target antigen and / or the second target antigen comprises human OX40, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

32. The IL-12 Fc fusion protein according to any one of claims 27 to 30, wherein the first target antigen and / or the second target antigen comprises human LAG3, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 62 and 63; (ii) respectively, SEQ ID NO: 103 and 104; (iii) respectively, SEQ ID NO: 105 and 106; (iv) respectively, SEQ ID NO: 107 and 108; (v) respectively, SEQ ID NO: 109 and 110; and (vi) respectively, SEQ ID NO: 111 and 112.

33. The IL-12 Fc fusion protein according to any one of claims 27 to 30, wherein the first target antigen and / or the second target antigen comprises human PD1, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively.

34. The IL-12 Fc fusion protein according to any one of claims 27 to 30, wherein the first target antigen and / or the second target antigen comprises human ICOS, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively.

35. The IL-12 Fc fusion protein according to any one of claims 27 to 30, wherein the first target antigen and / or the second target antigen comprises human 41BB, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

36. The IL-12 Fc fusion protein according to any one of claims 27 to 30, wherein the first target antigen and / or the second target antigen comprises human CCR7, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

37. The IL-12 Fc fusion protein according to any one of claims 27 to 30, wherein the first target antigen and / or the second target antigen comprises human CCR8, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

38. The IL-12 Fc fusion protein according to any one of claims 19 to 37, wherein the first ABD binds to the ECD of the first target antigen.

39. The IL-12 Fc fusion protein according to any one of claims 19 to 38, wherein the second ABD binds to the ECD of the second target antigen.

40. The IL-12 Fc fusion protein according to any one of claims 19 to 39, wherein the first domain adapter and / or the second domain adapter comprises any one of SEQ ID NO: 31-40.

41. An IL-12 Fc fusion protein comprising: (a) A first monomer, which comprises from the N-terminus to the C-terminus: (i) a VH domain, (ii) a CH1 domain, and (iii) a first Fc domain; (b) A second monomer comprising, from its N-terminus to its C-terminus: (i) a VL domain and (ii) a CL domain, wherein the VH domain and the VL domain form an ABD that binds to the first target antigen; and (c) A third monomer, comprising from its N-terminus to its C-terminus: (i) a second Fc domain, (ii) a first domain connector, (iii) a first IL-12 subunit domain, (iv) a second domain connector, and (v) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

42. The IL-12 Fc fusion protein of claim 41, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

43. The IL-12 Fc fusion protein according to claim 41 or 42, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

44. The IL-12 Fc fusion protein according to claim 41 or 42, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

45. The IL-12 Fc fusion protein of claim 44, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

46. ​​The IL-12 Fc fusion protein according to claim 45, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

47. The IL-12 Fc fusion protein according to any one of claims 41 to 46, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

48. The IL-12 Fc fusion protein according to claim 47, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

49. The IL-12 Fc fusion protein according to any one of claims 41 to 48, wherein the ABD binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

50. The IL-12 Fc fusion protein of claim 49, wherein the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 160 and 148 respectively; NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

51. The IL-12 Fc fusion protein of claim 49, wherein the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

52. The IL-12 Fc fusion protein of claim 49, wherein the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

53. The IL-12 Fc fusion protein of claim 49, wherein the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

54. The IL-12 Fc fusion protein of claim 49, wherein the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

55. The IL-12 Fc fusion protein of claim 49, wherein the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

56. The IL-12 Fc fusion protein of claim 49, wherein the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

57. The IL-12 Fc fusion protein according to any one of claims 41 to 56, wherein the ABD binds to the extracellular domain (ECD) of the target antigen.

58. The IL-12 Fc fusion protein according to any one of claims 41 to 57, wherein the first domain adapter and / or the second domain adapter comprises any one of SEQ ID NO: 31-40.

59. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising, from the N-terminus to the C-terminus: (i) a first VH (VH1) domain, (ii) a first CH1 domain, (iii) a first Fc domain, (iv) a first domain connector, and (v) a first IL-12 subunit domain. (b) A second monomer comprising, from the N-terminus to the C-terminus: (i) a first VL (VL1) domain and (ii) a first CL domain, wherein the VH1 domain and the VL1 domain form a first ABD that binds to the first target antigen; (c) A third monomer, comprising from the N-terminus to the C-terminus: (i) a second VH (VH2) domain, (ii) a second CH1 domain, (iii) a second Fc domain, (iv) a second domain connector, and (v) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (d) A fourth monomer comprising, from the N-terminus to the C-terminus: (i) a second VL (VL2) domain and (ii) a second CL domain, wherein the VH2 domain and the VL2 domain form a second ABD that binds to the second target antigen.

60. The IL-12 Fc fusion protein of claim 59, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

61. The IL-12 Fc fusion protein according to claim 59 or 60, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

62. The IL-12 Fc fusion protein according to claim 59 or 60, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

63. The IL-12 Fc fusion protein of claim 62, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

64. The IL-12 Fc fusion protein according to claim 63, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

65. The IL-12 Fc fusion protein according to any one of claims 59 to 64, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

66. The IL-12 Fc fusion protein according to claim 65, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

67. The IL-12 Fc fusion protein according to any one of claims 59 to 66, wherein the first ABD binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

68. The IL-12 Fc fusion protein according to any one of claims 59 to 67, wherein the second ABD binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

69. The IL-12 Fc fusion protein according to any one of claims 59 to 68, wherein both the first ABD and the second ABD bind to a target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

70. The IL-12 Fc fusion protein according to any one of claims 59 to 68, wherein: (1) The first ABD binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8; and (2) The second ABD binds to the second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

71. The IL-12 Fc fusion protein according to any one of claims 67 to 70, wherein the first target antigen and / or the second target antigen comprises human OX40, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

72. The IL-12 Fc fusion protein according to any one of claims 67 to 70, wherein the first target antigen and / or the second target antigen comprises human LAG3, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 62 and 63; (ii) respectively, SEQ ID NO: 103 and 104; (iii) respectively, SEQ ID NO: 105 and 106; (iv) respectively, SEQ ID NO: 107 and 108; (v) respectively, SEQ ID NO: 109 and 110; and (vi) respectively, SEQ ID NO: 111 and 112.

73. The IL-12 Fc fusion protein according to any one of claims 67 to 70, wherein the first target antigen and / or the second target antigen comprises human PD1, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55 respectively; (ii) SEQ ID NO: 93 and 94 respectively; (iii) SEQ ID NO: 95 and 96 respectively; (iv) SEQ ID NO: 97 and 98 respectively; (v) SEQ ID NO: 99 and 100 respectively; and (vi) SEQ ID NO: 101 and 102 respectively.

74. The IL-12 Fc fusion protein according to any one of claims 67 to 70, wherein the first target antigen and / or the second target antigen comprises human ICOS, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively.

75. The IL-12 Fc fusion protein according to any one of claims 67 to 70, wherein the first target antigen and / or the second target antigen comprises human 41BB, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

76. The IL-12 Fc fusion protein according to any one of claims 67 to 70, wherein the first target antigen and / or the second target antigen comprises human CCR7, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

77. The IL-12 Fc fusion protein according to any one of claims 67 to 70, wherein the first target antigen and / or the second target antigen comprises human CCR8, and the first ABD and / or the second ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

78. The IL-12 Fc fusion protein according to any one of claims 59 to 77, wherein the first ABD binds to the ECD of the first target antigen.

79. The IL-12 Fc fusion protein according to any one of claims 59 to 78, wherein the second ABD binds to the ECD of the second target antigen.

80. The IL-12 Fc fusion protein according to any one of claims 59 to 79, wherein the first domain adapter and / or the second domain adapter comprises any one of SEQ ID NO: 31-40.

81. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising, from the N-terminus to the C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, and (iii) a first Fc domain; (b) A second monomer, comprising from the N-terminus to the C-terminus: (i) a second IL-12 subunit domain, (ii) a second domain connector, (iii) a second Fc domain, (iv) a third domain connector, (v) a VH domain, and (vi) a CH1 domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (c) A third monomer comprising, from the N-terminus to the C-terminus: (i) a VL domain and (ii) a CL domain, wherein the VH domain and the VL domain form an ABD that binds to the target antigen.

82. The IL-12 Fc fusion protein of claim 81, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

83. The IL-12 Fc fusion protein according to claim 81 or 82, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

84. The IL-12 Fc fusion protein according to claim 81 or 82, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

85. The IL-12 Fc fusion protein of claim 84, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

86. The IL-12 Fc fusion protein according to claim 85, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

87. The IL-12 Fc fusion protein according to any one of claims 81 to 86, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

88. The IL-12 Fc fusion protein according to claim 87, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

89. The IL-12 Fc fusion protein according to any one of claims 81 to 88, wherein the ABD binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

90. The IL-12 Fc fusion protein of claim 89, wherein the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148 respectively; (xi) SEQ ID NO: 160 and 148 respectively; NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

91. The IL-12 Fc fusion protein of claim 89, wherein the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

92. The IL-12 Fc fusion protein of claim 89, wherein the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

93. The IL-12 Fc fusion protein of claim 89, wherein the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

94. The IL-12 Fc fusion protein of claim 89, wherein the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

95. The IL-12 Fc fusion protein of claim 89, wherein the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

96. The IL-12 Fc fusion protein of claim 89, wherein the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

97. The IL-12 Fc fusion protein according to any one of claims 81 to 96, wherein the ABD binds to the extracellular domain (ECD) of the target antigen.

98. The IL-12 Fc fusion protein according to any one of claims 81 to 97, wherein the first domain adapter, the second domain adapter and / or the third domain adapter comprises any one of SEQ ID NO: 31-40.

99. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising, from its N-terminus to its C-terminus: (i) a VH domain, (ii) a CH1 domain, (iii) a first Fc domain, (iv) a first domain connector, and (v) a first IL-12 subunit domain; and (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a VL domain, (ii) a CL domain, (iii) a second Fc domain, (iv) a second domain connector, and (v) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain. (2) The p35 subunit domain of the variant IL-12 comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) The VH domain and the VL domain form an ABD that binds to the target antigen.

100. The IL-12 Fc fusion protein of claim 99, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

101. The IL-12 Fc fusion protein according to claim 99 or 100, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

102. The IL-12 Fc fusion protein according to claim 99 or 100, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

103. The IL-12 Fc fusion protein of claim 102, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

104. The IL-12 Fc fusion protein according to claim 103, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

105. The IL-12 Fc fusion protein according to any one of claims 99 to 104, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

106. The IL-12 Fc fusion protein according to claim 105, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

107. The IL-12 Fc fusion protein according to any one of claims 99 to 106, wherein the ABD binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

108. The IL-12 Fc fusion protein of claim 107, wherein the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

109. The IL-12 Fc fusion protein of claim 107, wherein the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

110. The IL-12 Fc fusion protein of claim 107, wherein the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

111. The IL-12 Fc fusion protein of claim 107, wherein the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

112. The IL-12 Fc fusion protein of claim 107, wherein the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

113. The IL-12 Fc fusion protein of claim 107, wherein the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

114. The IL-12 Fc fusion protein of claim 107, wherein the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

115. The IL-12 Fc fusion protein according to any one of claims 99 to 114, wherein the ABD binds to the extracellular domain (ECD) of the target antigen.

116. The IL-12 Fc fusion protein according to any one of claims 99 to 115, wherein the first domain adapter and / or the second domain adapter comprises any one of SEQ ID NO: 31-40.

117. An IL-12 Fc fusion protein comprising: (a) A first monomer, comprising from its N-terminus to its C-terminus: (i) a VH domain, (ii) a CH1 domain, and (iii) a first Fc domain; and (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a VL domain, (ii) a CL domain, (iii) a second Fc domain, (iv) a first domain connector, (v) a first IL-12 subunit domain, (vi) a second domain connector, and (vii) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain. (2) The p35 subunit domain of the variant IL-12 comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) The VH domain and the VL domain form an ABD that binds to the target antigen.

118. The IL-12 Fc fusion protein of claim 117, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

119. The IL-12 Fc fusion protein according to claim 117 or 118, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

120. The IL-12 Fc fusion protein of claim 117 or 118, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

121. The IL-12 Fc fusion protein of claim 120, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

122. The IL-12 Fc fusion protein according to claim 121, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

123. The IL-12 Fc fusion protein according to any one of claims 117 to 122, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

124. The IL-12 Fc fusion protein according to claim 123, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

125. The IL-12 Fc fusion protein according to any one of claims 117 to 124, wherein the ABD binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

126. The IL-12 Fc fusion protein of claim 125, wherein the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

127. The IL-12 Fc fusion protein of claim 125, wherein the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

128. The IL-12 Fc fusion protein of claim 125, wherein the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

129. The IL-12 Fc fusion protein of claim 125, wherein the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

130. The IL-12 Fc fusion protein of claim 125, wherein the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

131. The IL-12 Fc fusion protein of claim 125, wherein the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

132. The IL-12 Fc fusion protein of claim 125, wherein the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

133. The IL-12 Fc fusion protein according to any one of claims 117 to 132, wherein the ABD binds to the extracellular domain (ECD) of the target antigen.

134. The IL-12 Fc fusion protein according to any one of claims 117 to 133, wherein the first domain adapter and / or the second domain adapter comprises any one of SEQ ID NO: 31-40.

135. An IL-12 Fc fusion protein comprising: (a) A first monomer containing a first Fc domain; (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a VL domain, and (ii) a CL domain; and (c) A third monomer, comprising from the N-terminus to the C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, (iii) a second IL-12 subunit domain, (iv) a second domain connector, (v) a second Fc domain, (vi) a VH domain, and (vii) a CH1 domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain. (2) The p35 subunit domain of the variant IL-12 comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) The VH domain and the VL domain form an ABD that binds to the target antigen.

136. The IL-12 Fc fusion protein of claim 135, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

137. The IL-12 Fc fusion protein according to claim 135 or 136, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

138. The IL-12 Fc fusion protein according to claim 135 or 136, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

139. The IL-12 Fc fusion protein of claim 138, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

140. The IL-12 Fc fusion protein according to claim 139, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

141. The IL-12 Fc fusion protein according to any one of claims 135 to 140, wherein: (i) The first Fc domain comprises any one of SEQ ID NO: 26-30; and (ii) The second Fc domain contains any of SEQ ID NO: 26-30.

142. The IL-12 Fc fusion protein according to claim 141, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

143. The IL-12 Fc fusion protein according to any one of claims 135 to 142, wherein the ABD binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

144. The IL-12 Fc fusion protein of claim 143, wherein the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

145. The IL-12 Fc fusion protein of claim 143, wherein the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

146. The IL-12 Fc fusion protein of claim 143, wherein the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

147. The IL-12 Fc fusion protein of claim 143, wherein the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

148. The IL-12 Fc fusion protein of claim 143, wherein the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

149. The IL-12 Fc fusion protein of claim 143, wherein the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

150. The IL-12 Fc fusion protein of claim 143, wherein the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

151. The IL-12 Fc fusion protein according to any one of claims 135 to 150, wherein the ABD binds to the extracellular domain (ECD) of the target antigen.

152. The IL-12 Fc fusion protein according to any one of claims 135 to 151, wherein the first domain adapter and / or the second domain adapter comprises any one of SEQ ID NO: 31-40.

153. An IL-12 Fc fusion protein comprising: (a) A first monomer, comprising from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, (iii) a second IL-12 subunit domain, and (iv) a first Fc domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) A second monomer comprising: (i) an anti-target antigen scFv, wherein the anti-target antigen scFv comprises a VH domain, a second domain adapter and a VL domain, (ii) a third domain adapter and (iii) a second Fc domain, wherein the anti-target antigen scFv is covalently linked to the third domain adapter at the N-terminus of the second Fc domain.

154. The IL-12 Fc fusion protein of claim 153, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

155. The IL-12 Fc fusion protein according to claim 153 or 154, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

156. The IL-12 Fc fusion protein according to claim 153 or 154, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

157. The IL-12 Fc fusion protein of claim 156, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

158. The IL-12 Fc fusion protein according to claim 157, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

159. The IL-12 Fc fusion protein according to any one of claims 153 to 158, wherein the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

160. The IL-12 Fc fusion protein of claim 159, wherein the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

161. The IL-12 Fc fusion protein of claim 159, wherein the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

162. The IL-12 Fc fusion protein of claim 159, wherein the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

163. The IL-12 Fc fusion protein of claim 159, wherein the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

164. The IL-12 Fc fusion protein of claim 159, wherein the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

165. The IL-12 Fc fusion protein of claim 159, wherein the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

166. The IL-12 Fc fusion protein of claim 159, wherein the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

167. The IL-12 Fc fusion protein according to any one of claims 153 to 166, wherein the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

168. The IL-12 Fc fusion protein according to any one of claims 153 to 167, wherein the VH domain of the anti-target antigen scFv is covalently linked to the third domain linker at the N-terminus of the second Fc domain.

169. The IL-12 Fc fusion protein according to any one of claims 153 to 167, wherein the VL domain of the anti-target antigen scFv is covalently linked to the third domain linker at the N-terminus of the second Fc domain.

170. The IL-12 Fc fusion protein according to any one of claims 153 to 169, wherein: (i) The first Fc domain contains either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

171. The IL-12 Fc fusion protein according to claim 170, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

172. The IL-12 Fc fusion protein according to any one of claims 153 to 171, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; and (iii) The third structural domain connector includes any one of SEQ ID NO: 31-40.

173. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising: (i) a first anti-target antigen scFv, wherein the first anti-target antigen scFv comprises a first VH (VH1) domain, a first domain adapter, and a first VL (VL1) domain; (ii) a second domain adapter; and (iii) a first Fc domain, wherein the first anti-target antigen scFv is covalently linked to the second domain adapter at the N-terminus of the first Fc domain; and (b) A second monomer comprising: (i) a second anti-target antigen scFv, wherein the second anti-target antigen scFv comprises a second VH (VH2) domain, a third domain linker, and a second VL (VL2) domain, (ii) a fourth domain linker, (iii) a second Fc domain, wherein the second anti-target antigen scFv is covalently linked to the fourth domain linker at the N-terminus of the second Fc domain, (iv) a fifth domain linker, (v) a first IL-12 subunit domain, (vi) a sixth domain linker, and (vii) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain. (2) The p35 subunit domain of the variant IL-12 comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A. (3) The second Fc domain is covalently connected to the fifth domain connector at the N-terminus of the first IL-12 subunit domain, and (4) The first IL-12 subunit structural domain is covalently connected to the sixth structural domain connector at the N end of the second IL-12 subunit structural domain.

174. The IL-12 Fc fusion protein of claim 173, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

175. The IL-12 Fc fusion protein according to claim 173 or 174, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

176. The IL-12 Fc fusion protein according to claim 173 or 174, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

177. The IL-12 Fc fusion protein of claim 176, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

178. The IL-12 Fc fusion protein according to claim 177, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

179. The IL-12 Fc fusion protein according to any one of claims 173 to 178, wherein the first anti-target antigen scFv binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

180. The IL-12 Fc fusion protein according to any one of claims 173 to 179, wherein the second anti-target antigen scFv binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

181. The IL-12 Fc fusion protein according to any one of claims 173 to 180, wherein the first anti-target antigen scFv and the second anti-target antigen scFv both bind to a target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

182. The IL-12 Fc fusion protein according to any one of claims 173 to 180, wherein: (1) The first anti-target antigen scFv binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8; and (2) The second anti-target antigen scFv binds to the second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

183. The IL-12 Fc fusion protein according to any one of claims 179 to 182, wherein the first target antigen and / or the second target antigen comprises human OX40, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 152 and 143; (ii) respectively, SEQ ID NO: 132 and 133; (iii) respectively, SEQ ID NO: 137 and 138; (iv) respectively, SEQ ID NO: 142 and 143; (v) respectively, SEQ ID NO: 147 and 148; (vi) respectively, SEQ ID NO: 130 and 131; (vii) respectively, SEQ ID NO: 154 and 143; (viii) respectively, SEQ ID NO: 156 and 143; (ix) respectively, SEQ ID NO: 158 and 148; (x) respectively, SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

184. The IL-12 Fc fusion protein according to any one of claims 179 to 182, wherein the first target antigen and / or the second target antigen comprises human LAG3, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 62 and 63; (ii) respectively, SEQ ID NO: 103 and 104; (iii) respectively, SEQ ID NO: 105 and 106; (iv) respectively, SEQ ID NO: 107 and 108; (v) respectively, SEQ ID NO: 109 and 110; and (vi) respectively, SEQ ID NO: 111 and 112.

185. The IL-12 Fc fusion protein according to any one of claims 179 to 182, wherein the first target antigen and / or the second target antigen comprises human PD1, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 54 and 55; (ii) respectively, SEQ ID NO: 93 and 94; (iii) respectively, SEQ ID NO: 95 and 96; (iv) respectively, SEQ ID NO: 97 and 98; (v) respectively, SEQ ID NO: 99 and 100; and (vi) respectively, SEQ ID NO: 101 and 102.

186. The IL-12 Fc fusion protein according to any one of claims 179 to 182, wherein the first target antigen and / or the second target antigen comprises human ICOS, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 74 and 75; (ii) respectively, SEQ ID NO: 117 and 118; (iii) respectively, SEQ ID NO: 119 and 120; and (iv) respectively, SEQ ID NO: 121 and 122.

187. The IL-12 Fc fusion protein according to any one of claims 179 to 182, wherein the first target antigen and / or the second target antigen comprises human 41BB, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

188. The IL-12 Fc fusion protein according to any one of claims 179 to 182, wherein the first target antigen and / or the second target antigen comprises human CCR7, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

189. The IL-12 Fc fusion protein according to any one of claims 179 to 182, wherein the first target antigen and / or the second target antigen comprises human CCR8, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83 respectively; (ii) SEQ ID NO: 113 and 114 respectively; and (iii) SEQ ID NO: 115 and 116 respectively.

190. The IL-12 Fc fusion protein according to any one of claims 173 to 189, wherein the first ABD binds to the ECD of the first target antigen.

191. The IL-12 Fc fusion protein according to any one of claims 173 to 190, wherein the second ABD binds to the ECD of the second target antigen.

192. The IL-12 Fc fusion protein according to any one of claims 173 to 191, wherein the VH1 domain of the first anti-target antigen scFv is covalently linked to the second domain linker at the N-terminus of the first Fc domain.

193. The IL-12 Fc fusion protein according to any one of claims 173 to 191, wherein the VL1 domain of the anti-target antigen scFv is covalently linked to the second domain linker at the N-terminus of the first Fc domain.

194. The IL-12 Fc fusion protein according to any one of claims 173 to 193, wherein the VH2 domain of the second anti-target antigen scFv is covalently linked to the fourth domain linker at the N-terminus of the second Fc domain.

195. The IL-12 Fc fusion protein according to any one of claims 173 to 193, wherein the VL2 domain of the second anti-target antigen scFv is covalently linked to the fourth domain linker at the N-terminus of the second Fc domain.

196. The IL-12 Fc fusion protein according to any one of claims 173 to 195, wherein: (i) The first Fc domain comprises either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

197. The IL-12 Fc fusion protein according to claim 196, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

198. The IL-12 Fc fusion protein according to any one of claims 173 to 197, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; (iii) The third structural domain connector comprises any one of SEQ ID NO: 31-40; (iv) The fourth structural domain connector comprises any one of SEQ ID NO: 31-40; (v) The fifth structural domain connector comprises any one of SEQ ID NO: 31-40; and (vi) The sixth structural domain connector includes any one of SEQ ID NO: 31-40.

199. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising: (i) an anti-target antigen scFv, wherein the anti-target antigen scFv comprises a VH domain, a first domain adapter, and a VL domain; (ii) a second domain adapter; and (iii) a first Fc domain, wherein the anti-target antigen scFv is covalently linked to the second domain adapter at the N-terminus of the first Fc domain; and (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a second Fc domain, (ii) a third domain connector, (iii) a first IL-12 subunit domain, (iv) a fourth domain connector, and (v) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

200. The IL-12 Fc fusion protein of claim 199, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

201. The IL-12 Fc fusion protein according to claim 199 or 200, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

202. The IL-12 Fc fusion protein according to claim 199 or 200, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

203. The IL-12 Fc fusion protein of claim 202, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

204. The IL-12 Fc fusion protein according to claim 203, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

205. The IL-12 Fc fusion protein according to any one of claims 199 to 204, wherein the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

206. The IL-12 Fc fusion protein of claim 205, wherein the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

207. The IL-12 Fc fusion protein of claim 205, wherein the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

208. The IL-12 Fc fusion protein of claim 205, wherein the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

209. The IL-12 Fc fusion protein of claim 205, wherein the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

210. The IL-12 Fc fusion protein of claim 205, wherein the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

211. The IL-12 Fc fusion protein of claim 205, wherein the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

212. The IL-12 Fc fusion protein of claim 205, wherein the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

213. The IL-12 Fc fusion protein according to any one of claims 199 to 212, wherein the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

214. The IL-12 Fc fusion protein according to any one of claims 199 to 213, wherein the VH domain of the anti-target antigen scFv is covalently linked to the second domain linker at the N-terminus of the first Fc domain.

215. The IL-12 Fc fusion protein according to any one of claims 199 to 213, wherein the VL domain of the anti-target antigen scFv is covalently linked to the second domain linker at the N-terminus of the first Fc domain.

216. The IL-12 Fc fusion protein according to any one of claims 199 to 215, wherein: (i) The first Fc domain comprises either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

217. The IL-12 Fc fusion protein according to claim 216, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

218. The IL-12 Fc fusion protein according to any one of claims 199 to 217, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; (iii) The third structural domain connector comprises any one of SEQ ID NO: 31-40; and (iv) The fourth structural domain connector comprises any one of SEQ ID NO: 31-40.

219. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising: (i) a first anti-target antigen scFv, wherein the first anti-target antigen scFv comprises a first VH (VH1) domain, a first domain adapter, and a first VL (VL1) domain; (ii) a second domain adapter; (iii) a first Fc domain, wherein the first anti-target antigen scFv is covalently linked to the second domain adapter at the N-terminus of the first Fc domain; (iv) a third domain adapter; and (v) a first IL-12 subunit domain, wherein the first Fc domain is covalently linked to the third domain adapter at the N-terminus of the first IL-12 subunit domain; and (b) A second monomer comprising: (i) a second anti-target antigen scFv, wherein the second anti-target antigen scFv comprises a second VH (VH2) domain, a fourth domain linker, and a second VL (VL2) domain, (ii) a fifth domain linker, (iii) a second Fc domain, wherein the second anti-target antigen scFv is covalently linked to the fifth domain linker at the N-terminus of the second Fc domain, (iv) a sixth domain linker, and (v) a second IL-12 subunit domain, wherein: (1) The second Fc domain is covalently connected to the sixth domain connector at the N-terminus of the second IL-12 subunit domain. (2) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (3) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

220. The IL-12 Fc fusion protein of claim 219, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

221. The IL-12 Fc fusion protein according to claim 219 or 220, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

222. The IL-12 Fc fusion protein of claim 219 or 220, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

223. The IL-12 Fc fusion protein of claim 222, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

224. The IL-12 Fc fusion protein according to claim 223, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

225. The IL-12 Fc fusion protein according to any one of claims 219 to 224, wherein the first anti-target antigen scFv binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

226. The IL-12 Fc fusion protein according to any one of claims 219 to 225, wherein the second anti-target antigen scFv binds to a second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

227. The IL-12 Fc fusion protein according to any one of claims 219 to 226, wherein the first anti-target antigen scFv and the second anti-target antigen scFv both bind to a target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

228. The IL-12 Fc fusion protein according to any one of claims 219 to 226, wherein: (1) The first anti-target antigen scFv binds to a first target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8; and (2) The second anti-target antigen scFv binds to the second target antigen selected from the group consisting of: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7 and (vii) human CCR8.

229. The IL-12 Fc fusion protein according to any one of claims 225 to 228, wherein the first target antigen and / or the second target antigen comprises human OX40, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 152 and 143; (ii) respectively, SEQ ID NO: 132 and 133; (iii) respectively, SEQ ID NO: 137 and 138; (iv) respectively, SEQ ID NO: 142 and 143; (v) respectively, SEQ ID NO: 147 and 148; (vi) respectively, SEQ ID NO: 130 and 131; (vii) respectively, SEQ ID NO: 154 and 143; (viii) respectively, SEQ ID NO: 156 and 143; (ix) respectively, SEQ ID NO: 158 and 148; (x) respectively, SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

230. The IL-12 Fc fusion protein according to any one of claims 225 to 228, wherein the first target antigen and / or the second target antigen comprises human LAG3, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 62 and 63; (ii) respectively, SEQ ID NO: 103 and 104; (iii) respectively, SEQ ID NO: 105 and 106; (iv) respectively, SEQ ID NO: 107 and 108; (v) respectively, SEQ ID NO: 109 and 110; and (vi) respectively, SEQ ID NO: 111 and 112.

231. The IL-12 Fc fusion protein according to any one of claims 225 to 228, wherein the first target antigen and / or the second target antigen comprises human PD1, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 54 and 55; (ii) respectively, SEQ ID NO: 93 and 94; (iii) respectively, SEQ ID NO: 95 and 96; (iv) respectively, SEQ ID NO: 97 and 98; (v) respectively, SEQ ID NO: 99 and 100; and (vi) respectively, SEQ ID NO: 101 and 102.

232. The IL-12 Fc fusion protein according to any one of claims 225 to 228, wherein the first target antigen and / or the second target antigen comprises human ICOS, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75 respectively; (ii) SEQ ID NO: 117 and 118 respectively; (iii) SEQ ID NO: 119 and 120 respectively; and (iv) SEQ ID NO: 121 and 122 respectively.

233. The IL-12 Fc fusion protein according to any one of claims 225 to 228, wherein the first target antigen and / or the second target antigen comprises human 41BB, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

234. The IL-12 Fc fusion protein according to any one of claims 225 to 228, wherein the first target antigen and / or the second target antigen comprises human CCR7, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

235. The IL-12 Fc fusion protein according to any one of claims 225 to 228, wherein the first target antigen and / or the second target antigen comprises human CCR8, and the first anti-target antigen scFv and / or the second anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

236. The IL-12 Fc fusion protein according to any one of claims 219 to 235, wherein the first ABD binds to the ECD of the first target antigen.

237. The IL-12 Fc fusion protein according to any one of claims 219 to 236, wherein the second ABD binds to the ECD of the second target antigen.

238. The IL-12 Fc fusion protein according to any one of claims 219 to 237, wherein the VH1 domain of the first anti-target antigen scFv is covalently linked to the second domain linker at the N-terminus of the first Fc domain.

239. The IL-12 Fc fusion protein according to any one of claims 219 to 237, wherein the VL1 domain of the anti-target antigen scFv is covalently linked to the second domain linker at the N-terminus of the first Fc domain.

240. The IL-12 Fc fusion protein according to any one of claims 219 to 239, wherein the VH2 domain of the second anti-target antigen scFv is covalently linked to the fifth domain linker at the N-terminus of the second Fc domain.

241. The IL-12 Fc fusion protein according to any one of claims 219 to 239, wherein the VL2 domain of the second anti-target antigen scFv is covalently linked to the fifth domain linker at the N-terminus of the second Fc domain.

242. The IL-12 Fc fusion protein according to any one of claims 219 to 241, wherein: (i) The first Fc domain contains either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

243. The IL-12 Fc fusion protein according to claim 242, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

244. The IL-12 Fc fusion protein according to any one of claims 219 to 243, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; (iii) The third structural domain connector comprises any one of SEQ ID NO: 31-40; (iv) The fourth structural domain connector comprises any one of SEQ ID NO: 31-40; (v) The fifth structural domain connector comprises any one of SEQ ID NO: 31-40; and (vi) The sixth structural domain connector includes any one of SEQ ID NO: 31-40.

245. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising, from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, and (iii) a first Fc domain; and (b) A second monomer comprising: (i) a second IL-12 subunit domain, (ii) a second domain linker, (iii) a second Fc domain, wherein the second IL-12 subunit domain is covalently linked to the second domain linker at the N-terminus of the second Fc domain, (iv) a third domain linker, and (v) an anti-target antigen scFv, wherein: (1) The second Fc domain is covalently linked to the third domain linker at the N-terminus of the anti-target antigen scFv. (2) The anti-target antigen scFv contains a VH domain, a fourth domain linker, and a VL domain. (3) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (4) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

246. The IL-12 Fc fusion protein of claim 245, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

247. The IL-12 Fc fusion protein according to claim 245 or 246, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

248. The IL-12 Fc fusion protein of claim 245 or 246, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

249. The IL-12 Fc fusion protein of claim 248, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

250. The IL-12 Fc fusion protein according to claim 249, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

251. The IL-12 Fc fusion protein according to any one of claims 245 to 250, wherein the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

252. The IL-12 Fc fusion protein of claim 251, wherein the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

253. The IL-12 Fc fusion protein of claim 251, wherein the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

254. The IL-12 Fc fusion protein of claim 251, wherein the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

255. The IL-12 Fc fusion protein of claim 251, wherein the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

256. The IL-12 Fc fusion protein of claim 251, wherein the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

257. The IL-12 Fc fusion protein of claim 251, wherein the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

258. The IL-12 Fc fusion protein of claim 251, wherein the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

259. The IL-12 Fc fusion protein according to any one of claims 245 to 258, wherein the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

260. The IL-12 Fc fusion protein according to any one of claims 245 to 259, wherein the VH domain of the anti-target antigen scFv is covalently linked to the third domain linker at the C-terminus of the second Fc domain.

261. The IL-12 Fc fusion protein according to any one of claims 245 to 259, wherein the VL domain of the anti-target antigen scFv is covalently linked to the third domain linker at the C-terminus of the second Fc domain.

262. The IL-12 Fc fusion protein according to any one of claims 245 to 261, wherein: (i) The first Fc domain comprises either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

263. The IL-12 Fc fusion protein according to claim 262, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

264. The IL-12 Fc fusion protein according to any one of claims 245 to 263, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; (iii) The third structural domain connector comprises any one of SEQ ID NO: 31-40; and (iv) The fourth structural domain connector comprises any one of SEQ ID NO: 31-40.

265. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising: (i) a first IL-12 subunit domain, (ii) a first domain connector, (iii) a second IL-12 subunit domain, wherein the first IL-12 subunit domain is covalently connected to the first domain connector at the N-terminus of the second IL-12 subunit domain, (iv) a second domain connector, (v) a first Fc domain, wherein the second IL-12 subunit domain is covalently connected to the second domain connector at the N-terminus of the first Fc domain, (vi) a third domain connector, and (vii) an anti-target antigen scFv, wherein: (1) The first Fc domain is covalently linked to the third domain linker at the N-terminus of the anti-target antigen scFv. (2) The anti-target antigen scFv contains a VH domain, a fourth domain linker, and a VL domain. (3) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (4) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) A second monomer containing a second Fc domain.

266. The IL-12 Fc fusion protein of claim 265, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

267. The IL-12 Fc fusion protein according to claim 265 or 266, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

268. The IL-12 Fc fusion protein according to claim 265 or 266, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

269. The IL-12 Fc fusion protein of claim 268, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

270. The IL-12 Fc fusion protein according to claim 269, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

271. The IL-12 Fc fusion protein according to any one of claims 265 to 270, wherein the anti-target antigen scFv binds to a target antigen selected from the group consisting of: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7 and (7) human CCR8.

272. The IL-12 Fc fusion protein of claim 271, wherein the target antigen comprises human OX40, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 152 and 143 respectively; (ii) SEQ ID NO: 132 and 133 respectively; (iii) SEQ ID NO: 137 and 138 respectively; (iv) SEQ ID NO: 142 and 143 respectively; (v) SEQ ID NO: 147 and 148 respectively; (vi) SEQ ID NO: 130 and 131 respectively; (vii) SEQ ID NO: 154 and 143 respectively; (viii) SEQ ID NO: 156 and 143 respectively; (ix) SEQ ID NO: 158 and 148 respectively; (x) SEQ ID NO: 160 and 148; (xi) respectively, SEQ ID NO: 162 and 148; (xii) respectively, SEQ ID NO: 164 and 148; (xiii) respectively, SEQ ID NO: 166 and 148; (xiv) respectively, SEQ ID NO: 168 and 148; and (xv) respectively, SEQ ID NO: 170 and 171, as shown in Figures 13, 75, 81, 85 and 112.

273. The IL-12 Fc fusion protein of claim 271, wherein the target antigen comprises human LAG3, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 62 and 63, respectively; (ii) SEQ ID NO: 103 and 104, respectively; (iii) SEQ ID NO: 105 and 106, respectively; (iv) SEQ ID NO: 107 and 108, respectively; (v) SEQ ID NO: 109 and 110, respectively; and (vi) SEQ ID NO: 111 and 112, respectively.

274. The IL-12 Fc fusion protein of claim 271, wherein the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 54 and 55, respectively; (ii) SEQ ID NO: 93 and 94, respectively; (iii) SEQ ID NO: 95 and 96, respectively; (iv) SEQ ID NO: 97 and 98, respectively; (v) SEQ ID NO: 99 and 100, respectively; and (vi) SEQ ID NO: 101 and 102, respectively.

275. The IL-12 Fc fusion protein of claim 271, wherein the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 74 and 75, respectively; (ii) SEQ ID NO: 117 and 118, respectively; (iii) SEQ ID NO: 119 and 120, respectively; and (iv) SEQ ID NO: 121 and 122, respectively.

276. The IL-12 Fc fusion protein of claim 271, wherein the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 70 and 71, respectively; (ii) SEQ ID NO: 123 and 124, respectively; (iii) SEQ ID NO: 125 and 126, respectively; and (iv) SEQ ID NO: 127 and 128, respectively.

277. The IL-12 Fc fusion protein of claim 271, wherein the target antigen comprises human CCR7, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) respectively, SEQ ID NO: 78 and 79.

278. The IL-12 Fc fusion protein of claim 271, wherein the target antigen comprises human CCR8, and the anti-target antigen scFv comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NO: 82 and 83, respectively; (ii) SEQ ID NO: 113 and 114, respectively; and (iii) SEQ ID NO: 115 and 116, respectively.

279. The IL-12 Fc fusion protein according to any one of claims 265 to 278, wherein the anti-target antigen scFv binds to the extracellular domain (ECD) of the target antigen.

280. The IL-12 Fc fusion protein according to any one of claims 265 to 279, wherein the VH domain of the anti-target antigen scFv is covalently linked to the third domain linker at the C-terminus of the first Fc domain.

281. The IL-12 Fc fusion protein according to any one of claims 265 to 279, wherein the VL domain of the anti-target antigen scFv is covalently linked to the third domain linker at the C-terminus of the first Fc domain.

282. The IL-12 Fc fusion protein according to any one of claims 265 to 281, wherein: (i) The first Fc domain comprises either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

283. The IL-12 Fc fusion protein according to claim 282, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

284. The IL-12 Fc fusion protein according to any one of claims 265 to 283, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; (iii) The third structural domain connector comprises any one of SEQ ID NO: 31-40; and (iv) The fourth structural domain connector comprises any one of SEQ ID NO: 31-40.

285. An IL-12 Fc fusion protein comprising: (a) A first monomer, comprising from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, (iii) a second IL-12 subunit domain, and (iv) a first Fc domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) A second monomer comprising, from the N-terminus to the C-terminus: (i) a VHH domain, (ii) a second domain linker, and (iii) a second Fc domain, wherein the VHH domain binds to the target antigen.

286. The IL-12 Fc fusion protein of claim 285, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

287. The IL-12 Fc fusion protein according to claim 285 or 286, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

288. The IL-12 Fc fusion protein according to claim 285 or 286, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

289. The IL-12 Fc fusion protein of claim 288, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

290. The IL-12 Fc fusion protein according to claim 289, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

291. The IL-12 Fc fusion protein according to any one of claims 285 to 290, wherein: (i) The first Fc domain contains either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

292. The IL-12 Fc fusion protein according to claim 291, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

293. The IL-12 Fc fusion protein according to any one of claims 285 to 292, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; and (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40.

294. The IL-12 Fc fusion protein according to any one of claims 285 to 293, wherein the VHH domain binds to a target antigen selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7 and (7) CCR8.

295. The IL-12 Fc fusion protein of claim 294, wherein the target antigen comprises OX40.

296. The IL-12 Fc fusion protein of claim 294, wherein the target antigen comprises LAG3.

297. The IL-12 Fc fusion protein of claim 294, wherein the target antigen comprises PD1.

298. The IL-12 Fc fusion protein of claim 294, wherein the target antigen comprises ICOS.

299. The IL-12 Fc fusion protein of claim 294, wherein the target antigen comprises 41BB, and the VHH domain comprises: SEQ ID NO:

129.

300. The IL-12 Fc fusion protein of claim 294, wherein the target antigen comprises CCR7.

301. The IL-12 Fc fusion protein of claim 294, wherein the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO:

86.

302. The IL-12 Fc fusion protein according to any one of claims 285 to 301, wherein the VHH domain is humanized.

303. An IL-12 Fc fusion protein comprising: (a) A first monomer, comprising from its N-terminus to its C-terminus: (i) a first VHH domain, (ii) a first domain connector, (iii) a first Fc domain, (iv) a second domain connector, (v) a first IL-12 subunit domain, (vi) a third domain connector, and (vii) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain. (2) The p35 subunit domain of the variant IL-12 comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A, and (3) The first VHH domain binds to the first target antigen; and (b) A second monomer comprising, from the N-terminus to the C-terminus: (i) a second VHH domain, (ii) a fourth domain linker, and (iii) a second Fc domain, wherein the second VHH domain binds to the second target antigen.

304. The IL-12 Fc fusion protein of claim 303, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

305. The IL-12 Fc fusion protein according to claim 303 or 304, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

306. The IL-12 Fc fusion protein according to claim 303 or 304, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

307. The IL-12 Fc fusion protein of claim 306, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

308. The IL-12 Fc fusion protein according to claim 307, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

309. The IL-12 Fc fusion protein according to any one of claims 303 to 308, wherein: (i) The first Fc domain contains either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

310. The IL-12 Fc fusion protein according to claim 309, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

311. The IL-12 Fc fusion protein according to any one of claims 303 to 310, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; (iii) The third structural domain connector comprises any one of SEQ ID NO: 31-40; and (iv) The fourth structural domain connector comprises any one of SEQ ID NO: 31-40.

312. The IL-12 Fc fusion protein according to any one of claims 303 to 311, wherein the first VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

313. The IL-12 Fc fusion protein according to any one of claims 303 to 312, wherein the second VHH domain binds a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

314. The IL-12 Fc fusion protein according to any one of claims 303 to 313, wherein both the first VHH domain and the second VHH domain bind to a target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

315. The IL-12 Fc fusion protein according to any one of claims 303 to 313, wherein: (1) The first VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8; and (2) The second VHH domain binds to a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

316. The IL-12 Fc fusion protein according to any one of claims 312 to 315, wherein the first target antigen and / or the second target antigen comprises OX40.

317. The IL-12 Fc fusion protein according to any one of claims 312 to 315, wherein the first target antigen and / or the second target antigen comprises LAG3.

318. The IL-12 Fc fusion protein according to any one of claims 312 to 315, wherein the first target antigen and / or the second target antigen comprises PD1.

319. The IL-12 Fc fusion protein according to any one of claims 312 to 315, wherein the first target antigen and / or the second target antigen comprises ICOS.

320. The IL-12 Fc fusion protein according to any one of claims 312 to 315, wherein the first target antigen and / or the second target antigen comprises 41BB, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO:

129.

321. The IL-12 Fc fusion protein according to any one of claims 312 to 315, wherein the first target antigen and / or the second target antigen comprises CCR7.

322. The IL-12 Fc fusion protein according to any one of claims 312 to 315, wherein the first target antigen and / or the second target antigen comprises CCR8, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO:

86.

323. The IL-12 Fc fusion protein according to any one of claims 303 to 322, wherein the first VHH domain is humanized.

324. The IL-12 Fc fusion protein according to any one of claims 303 to 323, wherein the second VHH domain is humanized.

325. An IL-12 Fc fusion protein comprising: (a) A first monomer, which comprises from the N-terminus to the C-terminus: (i) a VHH domain, (ii) a first domain connector, and (iii) a first Fc domain; (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a second Fc domain, (ii) a second domain connector, (iii) a first IL-12 subunit domain, (iv) a third domain connector, and (v) a second IL-12 subunit domain, wherein: (1) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (2) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

326. The IL-12 Fc fusion protein of claim 325, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

327. The IL-12 Fc fusion protein according to claim 325 or 326, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

328. The IL-12 Fc fusion protein of claim 325 or 326, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

329. The IL-12 Fc fusion protein of claim 328, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

330. The IL-12 Fc fusion protein according to claim 329, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

331. The IL-12 Fc fusion protein according to any one of claims 325 to 330, wherein: (i) The first Fc domain contains either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

332. The IL-12 Fc fusion protein according to claim 331, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

333. The IL-12 Fc fusion protein according to any one of claims 325 to 332, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; and (iii) The third structural domain connector includes any one of SEQ ID NO: 31-40.

334. The IL-12 Fc fusion protein according to any one of claims 325 to 333, wherein the VHH domain binds to a target antigen selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7 and (7) CCR8.

335. The IL-12 Fc fusion protein of claim 334, wherein the target antigen comprises OX40.

336. The IL-12 Fc fusion protein of claim 334, wherein the target antigen comprises LAG3.

337. The IL-12 Fc fusion protein of claim 334, wherein the target antigen comprises PD1.

338. The IL-12 Fc fusion protein of claim 334, wherein the target antigen comprises ICOS.

339. The IL-12 Fc fusion protein of claim 334, wherein the target antigen comprises 41BB, and the VHH domain comprises: SEQ ID NO:

129.

340. The IL-12 Fc fusion protein of claim 334, wherein the target antigen comprises CCR7.

341. The IL-12 Fc fusion protein of claim 334, wherein the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO:

86.

342. The IL-12 Fc fusion protein according to any one of claims 325 to 341, wherein the VHH binds to the extracellular domain (ECD) of the target antigen.

343. The IL-12 Fc fusion protein according to any one of claims 325 to 342, wherein the VHH domain is humanized.

344. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising, from its N-terminus to its C-terminus: (i) a first VHH domain, (ii) a first domain adapter, (iii) a first Fc domain, (iv) a second domain adapter, and (v) a first IL-12 subunit domain, wherein the first VHH domain binds to a first target antigen; and (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a second VHH domain, (ii) a third domain connector, (iii) a second Fc domain, (iv) a fourth domain connector, and (v) a second IL-12 subunit domain, wherein: (1) The second VHH domain binds to the second target antigen. (2) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (3) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

345. The IL-12 Fc fusion protein of claim 344, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

346. The IL-12 Fc fusion protein according to claim 344 or 345, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

347. The IL-12 Fc fusion protein according to claim 344 or 345, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

348. The IL-12 Fc fusion protein of claim 347, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

349. The IL-12 Fc fusion protein according to claim 348, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

350. The IL-12 Fc fusion protein according to any one of claims 344 to 349, wherein: (i) The first Fc domain contains either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

351. The IL-12 Fc fusion protein according to claim 350, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

352. The IL-12 Fc fusion protein according to any one of claims 344 to 351, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; (iii) The third structural domain connector comprises any one of SEQ ID NO: 31-40; and (iv) The fourth structural domain connector comprises any one of SEQ ID NO: 31-40.

353. The IL-12 Fc fusion protein according to any one of claims 344 to 352, wherein the first VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

354. The IL-12 Fc fusion protein according to any one of claims 344 to 353, wherein the second VHH domain binds a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

355. The IL-12 Fc fusion protein according to any one of claims 344 to 354, wherein both the first VHH domain and the second VHH domain bind to a target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

356. The IL-12 Fc fusion protein according to any one of claims 344 to 354, wherein: (1) The first VHH domain binds to a first target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8; and (2) The second VHH domain binds to a second target antigen selected from the group consisting of: (i) OX40, (ii) LAG3, (iii) PD1, (iv) ICOS, (v) 41BB, (vi) CCR7 and (vii) CCR8.

357. The IL-12 Fc fusion protein according to any one of claims 353 to 356, wherein the first target antigen and / or the second target antigen comprises OX40.

358. The IL-12 Fc fusion protein according to any one of claims 353 to 356, wherein the first target antigen and / or the second target antigen comprises LAG3.

359. The IL-12 Fc fusion protein according to any one of claims 353 to 356, wherein the first target antigen and / or the second target antigen comprises PD1.

360. The IL-12 Fc fusion protein according to any one of claims 353 to 356, wherein the first target antigen and / or the second target antigen comprises ICOS.

361. The IL-12 Fc fusion protein according to any one of claims 353 to 356, wherein the first target antigen and / or the second target antigen comprises 41BB, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO:

129.

362. The IL-12 Fc fusion protein according to any one of claims 353 to 356, wherein the first target antigen and / or the second target antigen comprises CCR7.

363. The IL-12 Fc fusion protein according to any one of claims 353 to 356, wherein the first target antigen and / or the second target antigen comprises CCR8, and the first VHH domain and / or the second VHH domain comprises: SEQ ID NO:

86.

364. The IL-12 Fc fusion protein according to any one of claims 344 to 363, wherein the first VHH domain is humanized.

365. The IL-12 Fc fusion protein according to any one of claims 344 to 364, wherein the second VHH domain is humanized.

366. An IL-12 Fc fusion protein comprising: (a) A first monomer comprising, from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, and (iii) a first Fc domain; and (b) A second monomer, comprising from its N-terminus to its C-terminus: (i) a second IL-12 subunit domain, (ii) a second domain connector, (iii) a second Fc domain, (iv) a third domain connector, and (v) a VHH domain, wherein: (1) The VHH domain binds to the target antigen. (2) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (3) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A.

367. The IL-12 Fc fusion protein of claim 366, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

368. The IL-12 Fc fusion protein according to claim 366 or 367, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

369. The IL-12 Fc fusion protein according to claim 366 or 367, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

370. The IL-12 Fc fusion protein of claim 369, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

371. The IL-12 Fc fusion protein according to claim 370, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

372. The IL-12 Fc fusion protein according to any one of claims 366 to 371, wherein: (i) The first Fc domain comprises either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

373. The IL-12 Fc fusion protein according to claim 372, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

374. The IL-12 Fc fusion protein according to any one of claims 366 to 373, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; and (iii) The third structural domain connector includes any one of SEQ ID NO: 31-40.

375. The IL-12 Fc fusion protein according to any one of claims 366 to 374, wherein the VHH domain binds to a target antigen selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7 and (7) CCR8.

376. The IL-12 Fc fusion protein of claim 375, wherein the target antigen comprises OX40.

377. The IL-12 Fc fusion protein of claim 375, wherein the target antigen comprises LAG3.

378. The IL-12 Fc fusion protein of claim 375, wherein the target antigen comprises PD1.

379. The IL-12 Fc fusion protein of claim 375, wherein the target antigen comprises ICOS.

380. The IL-12 Fc fusion protein of claim 375, wherein the target antigen comprises 41BB and the VHH domain comprises: SEQ ID NO:

129.

381. The IL-12 Fc fusion protein of claim 375, wherein the target antigen comprises CCR7.

382. The IL-12 Fc fusion protein of claim 375, wherein the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO:

86.

383. The IL-12 Fc fusion protein according to any one of claims 366 to 382, ​​wherein the VHH domain is humanized.

384. An IL-12 Fc fusion protein comprising: (a) A first monomer, comprising from its N-terminus to its C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain connector, (iii) a second IL-12 subunit domain, (iv) a second domain connector, (v) a first Fc domain, (vi) a third domain connector, and (vii) a VHH domain, wherein: (1) The VHH domain binds to the target antigen. (2) The first IL-12 subunit domain includes an IL-12 p40 subunit domain and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain, or the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain and the second IL-12 subunit domain includes an IL-12 p40 subunit domain, and (3) The variant IL-12 p35 subunit domain comprises a group of amino acid substitutions selected from the group consisting of: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A and (e) Y40E / K170A; and (b) A second monomer containing a second Fc domain.

385. The IL-12 Fc fusion protein of claim 384, wherein the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 17-21.

386. The IL-12 Fc fusion protein according to claim 384 or 385, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

4.

387. The IL-12 Fc fusion protein according to claim 384 or 385, wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

388. The IL-12 Fc fusion protein of claim 387, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NO: 23-25.

389. The IL-12 Fc fusion protein according to claim 388, wherein: (i) The variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (ii) The variant IL-12 p35 subunit domain also contains a C74S substitution mutation.

390. The IL-12 Fc fusion protein according to any one of claims 384 to 389, wherein: (i) The first Fc domain contains either SEQ ID NO: 29 or 30; and (ii) The second Fc domain contains either SEQ ID NO: 30 or 29.

391. The IL-12 Fc fusion protein according to claim 390, wherein: (i) The first Fc domain further includes a set of substitution mutations including T366W / S354C, and the second Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C; or (ii) The first Fc domain further includes a set of substitution mutations including T366S / L368A / Y407V / Y349C, and the second Fc domain further includes a set of substitution mutations including T366W / S354C.

392. The IL-12 Fc fusion protein according to any one of claims 384 to 391, wherein: (i) The first structural domain connector comprises any one of SEQ ID NO: 31-40; (ii) The second structural domain connector comprises any one of SEQ ID NO: 31-40; and (iii) The third structural domain connector includes any one of SEQ ID NO: 31-40.

393. The IL-12 Fc fusion protein according to any one of claims 384 to 392, wherein the VHH domain binds to a target antigen selected from the group consisting of: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7 and (7) CCR8.

394. The IL-12 Fc fusion protein of claim 393, wherein the target antigen comprises OX40.

395. The IL-12 Fc fusion protein of claim 393, wherein the target antigen comprises LAG3.

396. The IL-12 Fc fusion protein of claim 393, wherein the target antigen comprises PD1.

397. The IL-12 Fc fusion protein of claim 393, wherein the target antigen comprises ICOS.

398. The IL-12 Fc fusion protein of claim 393, wherein the target antigen comprises 41BB, and the VHH domain comprises: SEQ ID NO:

129.

399. The IL-12 Fc fusion protein of claim 393, wherein the target antigen comprises CCR7.

400. The IL-12 Fc fusion protein of claim 393, wherein the target antigen comprises CCR8, and the VHH domain comprises: SEQ ID NO:

86.

401. The IL-12 Fc fusion protein according to any one of claims 384 to 400, wherein the VHH domain is humanized.

402. An IL-12 Fc fusion protein comprising: (a) First monomer; (b) the second monomer; and (c) a third monomer, wherein the first monomer, the second monomer, and the third monomer comprise: (i) Specifically, SEQ ID NO: 49, 153 and 145, (ii) Specifically, SEQ ID NO: 48, 146, and 145, (iii) Specifically, SEQ ID NO: 48, 151, and 150, (iv) Specifically, SEQ ID NO: 48, 153 and 145, (v) Specifically, SEQ ID NO: 48, 155, and 145, (vi) Specifically, SEQ ID NO: 48, 157 and 145, (vii) Specifically, SEQ ID NO: 48, 159 and 150, (viii) Specifically, SEQ ID NO: 48, 161, and 150, (ix) Specifically, SEQ ID NO: 48, 163 and 150, (x) Specifically, SEQ ID NO: 48, 165, and 150, (xi) Specifically, SEQ ID NO: 48, 167 and 150, (xii) Specifically, SEQ ID NO: 48, 169 and 150, (xiii) Specifically, SEQ ID NO: 49, 141 and 140, (xiv) Specifically, SEQ ID NO: 49, 146, and 145, (xv) Specifically, SEQ ID NO: 49, 151, and 150, (xvi) Specifically, SEQ ID NO: 48, 141 and 140, (xvii) Specifically, SEQ ID NO: 49, 155 and 145, (xviii) Specifically, SEQ ID NO: 49, 157 and 145, (xix) Specifically, SEQ ID NO: 49, 159 and 150, (xx) Specifically, SEQ ID NO: 49, 161 and 150, (xxi) respectively, SEQ ID NO: 49, 163 and 150, (xxii) Specifically, SEQ ID NO: 49, 165 and 150, (xxiii) Specifically, SEQ ID NO: 49, 167 and 150, (xxiv) Specifically, SEQ ID NO: 49, 169 and 150, (xxv) respectively, SEQ ID NO: 50, 141 and 140, (xxvi) Specifically, SEQ ID NO: 50, 146, and 145, (xxvii) Specifically, SEQ ID NO: 50, 151 and 150, (xxviii) Specifically, SEQ ID NO: 50, 153, and 145, (xxix) respectively, SEQ ID NO: 50, 155 and 145, (xxx) respectively, SEQ ID NO: 50, 157 and 145, (xxxi) respectively, SEQ ID NO: 50, 159 and 150, (xxxii) Specifically, SEQ ID NO: 50, 161 and 150, (xxxiii) Specifically, SEQ ID NO: 50, 163 and 150, (xxxiv) Specifically, SEQ ID NO: 50, 165, and 150, (xxxv) respectively, SEQ ID NO: 50, 167 and 150, (xxxvi) Specifically, SEQ ID NO: 50, 169 and 150, (xxxvii) Specifically, SEQ ID NO: 51, 141 and 140, (xxxviii) Specifically, SEQ ID NO: 51, 146, and 145, (xxxix) respectively, SEQ ID NO: 51, 151 and 150, (xl) Specifically, SEQ ID NO: 51, 153 and 145, (xli) respectively, SEQ ID NO: 51, 155 and 145, (xlii) respectively, SEQ ID NO: 51, 157 and 145, (xliii) respectively, SEQ ID NO: 51, 159 and 150, (xliv) respectively, SEQ ID NO: 51, 161 and 150, (xlv) respectively, SEQ ID NO: 51, 163 and 150, (xlvi) Specifically, SEQ ID NO: 51, 165 and 150, (xlvii) respectively, SEQ ID NO: 51, 167 and 150, (xlviii) Specifically, SEQ ID NO: 51, 169, and 150, (xlix) respectively, SEQ ID NO: 52, 141 and 140, (l) Specifically, SEQ ID NO: 52, 146, and 145, (li) Specifically, SEQ ID NO: 52, 151 and 150, (lii) Specifically, SEQ ID NO: 52, 153 and 145, (liii) respectively, SEQ ID NO: 52, 155 and 145, (liv) respectively, SEQ ID NO: 52, 157 and 145, (lv) Specifically, SEQ ID NO: 52, 159 and 150, (lvi) Specifically, SEQ ID NO: 52, 161 and 150, (lvii) respectively, SEQ ID NO: 52, 163 and 150, (lviii) Specifically, SEQ ID NO: 52, 165, and 150, (lix) Specifically, SEQ ID NO: 52, 167 and 150, (lx) respectively, SEQ ID NO: 52, 169 and 150, (lxi) respectively, SEQ ID NO: 48, 174 and 173, (lxii) respectively, SEQ ID NO: 49, 174 and 173, (lxiii) Specifically, SEQ ID NO: 50, 174 and 173, (lxiv) respectively, SEQ ID NO: 51, 174 and 173, or (lxv) respectively, SEQ ID NO: 52, 174 and 173.

403. A nucleic acid composition comprising one or more nucleic acids encoding the IL-12 Fc fusion protein according to any one of claims 1 to 402.

404. An expression vector comprising one or more nucleic acids according to claim 403.

405. A host cell transformed by the expression vector according to claim 404.

406. A method for preparing IL-12 Fc fusion protein, comprising: (a) Culture the host cells according to claim 405 under conditions expressing the IL-12 Fc fusion protein; as well as (b) Isolate and / or purify the IL-12 Fc fusion protein.

407. A method of treating cancer in a human subject, comprising administering to a human subject in need an IL-12 Fc fusion protein according to any one of claims 1 to 402, wherein the IL-12 Fc fusion protein binds to target cells.

408. A method of activating target cells in a human subject, comprising administering to a human subject in need an IL-12 Fc fusion protein according to any one of claims 1 to 402, wherein the IL-12 Fc fusion protein binds to the target cells.

409. A method for stimulating IL-12 signaling in target cells, comprising administering to a human subject in need an IL-12 Fc fusion protein according to any one of claims 1 to 402, wherein the IL-12 Fc fusion protein binds to the target cells.

410. A method for inducing the production and / or secretion of one or more cytokines in target cells, comprising administering to a human subject in need an IL-12 Fc fusion protein according to any one of claims 1 to 402, wherein the IL-12 Fc fusion protein binds to the target cells.

411. A method for reprogramming and / or altering the differentiation state of target cells, comprising administering to a human subject in need an IL-12 Fc fusion protein according to any one of claims 1 to 402, wherein the IL-12 Fc fusion protein binds to the target cells.

412. The method according to any one of claims 407 to 411, wherein the target cells express: (1) IL-12Rβ2, and (2) a cell surface antigen selected from the group consisting of: (a) human OX40, (b) human LAG3, (c) human PD1, (d) human ICOS, (e) human 41BB, (f) human CCR7 and (g) human CCR8.

413. The method of claim 412, wherein the cell surface antigen is human PD1, and the target cells comprise: (i) Activated and / or depleted CD4+ T cells; (ii) Activated and / or depleted CD8+ T cells; (iii) Activated and / or depleted NK cells; or (iv) Activated and / or depleted regulatory T cells.

414. The method of claim 412, wherein the cell surface antigen is human LAG3, and the target cells comprise: (i) Activated and / or depleted CD8+ T cells; (ii) Activated and / or depleted CD4+ T cells; (iii) Activated and / or depleted NK cells; or (iv) Activated and / or depleted regulatory T cells.

415. The method of claim 412, wherein the cell surface antigen is human OX40, and the target cells comprise: (i) Activated and / or depleted CD8+ T cells; (ii) Activated and / or depleted CD4+ T cells; (iii) Activated and / or depleted NK cells; or (iv) Activated and / or depleted regulatory T cells.

416. The method of claim 412, wherein the cell surface antigen is human ICOS, and the target cell comprises: (i) Activated and / or depleted CD4+ T cells; (ii) Activated and / or depleted CD8+ T cells; (iii) Activated and / or depleted NK cells; or (iv) Activated and / or depleted regulatory T cells.

417. The method of claim 412, wherein the cell surface antigen is human 41BB, and the target cell comprises: (i) Activated and / or depleted CD4+ T cells; (ii) Activated and / or depleted NK cells; (iii) Activated and / or depleted CD8+ T cells; or (iv) Activated and / or depleted regulatory T cells.

418. The method of claim 412, wherein the cell surface antigen is human CCR7, and the target cell comprises: (i) Naïve CD4+ T cells; (ii) Naïve CD8+ T cells; Or (iii) initial regulatory T cells.

419. The method of claim 412, wherein the cell surface antigen is human CCR8, and the target cell comprises: (i) Activated and / or depleted CD4+ T cells; Or (ii) activated and / or depleted regulatory T cells.

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