Targeted low potency il-12 FC fusion proteins and uses thereof

Abstract

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

Description

TargetedLow PotencyIL-12 Fc Fusion Proteins and Uses ThereofCross-reference to Related Applications[1] 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 to U.S. Provisional Application No. 63 / 711,820, filed October 25, 2024, each of which are entirely incorporated herein by reference.Sequence Listing[2] The instant application contains a Sequence Listing which is being submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on January 21, 2025, is named 51400_4004_WO_SL.xml and is 196,608 bytes in size.Background of the Invention[3] Interleukin-12 (IL-12) is a potent, pro-inflammatory cytokine that is produced by antigen presenting cells, such as, for example, dendritic cells, macrophages, and neutrophils. IL-12 belongs to the IL-12 family of cytokines. The IL-12 family of cytokines is unique in that they comprise heterodimeric cytokines. IL-12 is comprised of an alpha- (α-) subunit (encoded by the IL12A gene; also referred to herein as an “IL-12 p35 subunit”; Precursor Sequence – SEQ ID NO: 1 (as shown in Fig. 1); Mature Sequence – SEQ ID NO: 2 (as shown in Fig. 1)) and a beta- (β-) subunit (encoded by the IL12B gene; also referred to herein as an “IL-12 p40 subunit”; Precursor Sequence – SEQ ID NO: 3 (as shown in Fig. 1); Mature Sequence – SEQ ID NO: 4 (as shown in Fig. 1) that assemble to form a 70,000 Dalton (70 kDa) disulfide-linked heterodimer. Upon assembly of the IL-12 p35 and p40 subunits, a biologically active IL-12 heterodimer is formed. The receptor for IL-12, IL-12R, is a type I cytokine receptor comprising a beta-1 subunit (IL-12Rβ1 – SEQ ID NO: 5 (as shown in Fig. 2); Extracellular Domain – SEQ ID NO: 6 (as shown in Fig. 2)) and a beta-2 subunit (IL-12Rβ2 – SEQ ID NO: 7 (as shown in Fig. 2); Extracellular Domain – SEQ ID NO: 8 (as shown in Fig. 2)). The IL-12 p40 subunit has a binding affinity for IL-12Rβ1, and the IL-12 p35 subunit has a binding affinity for IL-12Rβ2.[4] The binding of IL-12 to IL-12R results in the phosphorylation of intracellular Signal Transducer And Activator Of Transcription 4 (STAT4) and triggers signaling pathways that: (i) induce TH1 cell differentiation, (ii) increase the activation and cytotoxic capacities of T- and natural killer (NK) cells, (iii) inhibit or reprogram immunosuppressive cells, such as, for example, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), and (iv) induce the production of large amounts of interferon gamma (IFNγ), which is cytostatic / cytotoxic, anti-angiogenic, and can upregulate major histocompatibility complex (MHC) class 1 and MHC class 2 molecules on tumor cells to enable immune recognition.[5] However, the targeting of wild-type IL-12 to a target cell using an antigen binding domain (ABD) has generally not been successful due to the high affinity of IL-12 for its receptor. Instead of trafficking the “immunocytokine” to an on-target cell expressing the antigen bound by the ABD, the immunocytokine is instead trafficked to an off-target cell that expresses high levels of IL-12R. As such, there remains an unmet need for the develop of a fusion protein comprising IL-12 and an ABD capable of directing IL-12 to an on-target cell expressing an antigen bound by the ABD.Brief Summary of the Invention[6] In one aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to 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-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 a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; (b) a second monomer comprising, from N-terminus to C-terminus: (i) a variable heavy (VH) domain, (ii) a constant heavy (CH1) domain, and (iii) a second Fc domain; and (c) a third monomer comprising, from N-terminus to C-terminus: (i) a variable light (VL) domain, and (ii) a constant light (CL) domain, wherein the VH domain and VL domain form an antigen binding domain (ABD) that binds a target antigen.[7] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.[8] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.[9] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[10] In a further embodiment and in accordance with any of the above, the ABD binds a target antigen selected from a group including: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7, and (7) human CCR8.

[11] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 101 and 102, respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 111 and 112, respectively. In still other embodiments, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In still other embodiments, the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In still other embodiments, the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 127 and 128, respectively. In still other embodiments, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 78 and 79, respectively. In still other embodiments, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[12] In a further embodiment and in accordance with any of the above, the ABD binds an extracellular domain (ECD) of the target antigen.

[13] In a further embodiment and in accordance with any of the above, the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[14] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a VH domain, (ii) a CH1 domain, and (iii) a first Fc domain; (b) a second monomer comprising, from N-terminus to C-terminus: (i) a VL domain, and (ii) a CL domain, wherein the VH domain and VL domain form an ABD that binds a first target antigen; (c) a third monomer comprising, from N-terminus to C-terminus: (i) a second Fc domain, (ii) a first domain linker, (iii) a first IL-12 subunit domain, (iv) a second domain linker, 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) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A.

[15] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[16] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[17] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[18] In a further embodiment and in accordance with any of the above, the ABD binds a target antigen selected from a group including: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7, and (7) human CCR8.

[19] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 101 and 102, respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 111 and 112, respectively. In still other embodiments, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In still other embodiments, the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In still other embodiments, the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 127 and 128, respectively. In still other embodiments, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 78 and 79, respectively. In still other embodiments, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[20] In a further embodiment and in accordance with any of the above, the ABD binds an extracellular domain (ECD) of the target antigen.

[21] In a further embodiment and in accordance with any of the above, the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[22] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain linker, and (iii) a first Fc domain; (b) a second monomer comprising, from N-terminus to C-terminus: (i) a second IL-12 subunit domain, (ii) a second domain linker, (iii) a second Fc domain, (iv) a third domain linker, (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) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; and (c) a third monomer comprising, from N-terminus to C-terminus: (i) a VL domain, and (ii) a CL domain, wherein the VH domain and VL domain form an ABD that binds a target antigen.

[23] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[24] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[25] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[26] In a further embodiment and in accordance with any of the above, the ABD binds a target antigen selected from a group including: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7, and (7) human CCR8.

[27] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 101 and 102, respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 111 and 112, respectively. In still other embodiments, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In still other embodiments, the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In still other embodiments, the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 127 and 128, respectively. In still other embodiments, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 78 and 79, respectively. In still other embodiments, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[28] In a further embodiment and in accordance with any of the above, the ABD binds an extracellular domain (ECD) of the target antigen.

[29] In a further embodiment and in accordance with any of the above, the first domain linker, the second domain linker, and / or the third domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, and / or the third domain linker comprises a covalent bond. In some other embodiments, at least one domain linker comprises any one of SEQ ID NOs: 31-40, and at least one domain linker comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[30] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a VH domain, (ii) a CH1 domain, (iii) a first Fc domain, (iv) a first domain linker, and (v) a first IL-12 subunit domain; and (b) a second monomer comprising, from N-terminus to C-terminus: (i) a VL domain, (ii) a CL domain, (iii) a second Fc domain, (iv) a second domain linker, 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) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (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 a target antigen.

[31] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[32] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[33] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[34] In a further embodiment and in accordance with any of the above, the ABD binds a target antigen selected from a group including: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7, and (7) human CCR8.

[35] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 101 and 102, respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 111 and 112, respectively. In still other embodiments, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In still other embodiments, the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In still other embodiments, the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 127 and 128, respectively. In still other embodiments, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 78 and 79, respectively. In still other embodiments, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[36] In a further embodiment and in accordance with any of the above, the ABD binds an extracellular domain (ECD) of the target antigen.

[37] In a further embodiment and in accordance with any of the above, the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[38] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a VH domain, (ii) a CH1 domain, and (iii) a first Fc domain; and (b) a second monomer comprising, from N-terminus to C-terminus: (i) a VL domain, (ii) a CL domain, (iii) a second Fc domain, (iv) a first domain linker, (v) a first IL-12 subunit domain, (vi) a second domain linker, 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 set of amino acid substitutions selected from a group including: (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 a target antigen.

[39] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[40] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[41] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[42] In a further embodiment and in accordance with any of the above, the ABD binds a target antigen selected from a group including: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7, and (7) human CCR8.

[43] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 101 and 102, respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 111 and 112, respectively. In still other embodiments, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In still other embodiments, the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In still other embodiments, the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 127 and 128, respectively. In still other embodiments, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 78 and 79, respectively. In still other embodiments, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[44] In a further embodiment and in accordance with any of the above, the ABD binds an extracellular domain (ECD) of the target antigen.

[45] In a further embodiment and in accordance with any of the above, the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[46] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising a first Fc domain; (b) a second monomer comprising, from N-terminus to C-terminus: (i) a VL domain, and (ii) a CL domain; and (c) a third monomer comprising, from N-terminus to 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, (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 an IL-12 p40 subunit domain, (2) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (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 a target antigen.

[47] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[48] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[49] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[50] In a further embodiment and in accordance with any of the above, the ABD binds a target antigen selected from a group including: (1) human OX40, (2) human LAG3, (3) human PD1, (4) human ICOS, (5) human 41BB, (6) human CCR7, and (7) human CCR8.

[51] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 101 and 102, respectively. In other embodiments, the target antigen comprises human LAG3, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 111 and 112, respectively. In still other embodiments, the target antigen comprises human OX40, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In still other embodiments, the target antigen comprises human ICOS, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In still other embodiments, the target antigen comprises human 41BB, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 127 and 128, respectively. In still other embodiments, the target antigen comprises human CCR7, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 78 and 79, respectively. In still other embodiments, the target antigen comprises human CCR8, and the ABD comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[52] In a further embodiment and in accordance with any of the above, the ABD binds an extracellular domain (ECD) of the target antigen.

[53] In a further embodiment and in accordance with any of the above, the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[54] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to 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 N-terminus to C-terminus: (i) a first VL (VL1) domain, and (ii) a first CL domain, wherein the VH1 domain and VL1 domain form a first ABD that binds a first target antigen; (c) a third monomer comprising, from N-terminus to C-terminus: (i) a second VH (VH2) domain, (ii) a second CH1 domain, (iii) a second Fc domain, (iv) a first domain linker, (v) a first IL-12 subunit domain, (vi) a second domain linker, 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, and (2) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; and (d) a fourth monomer comprising, from N-terminus to C-terminus: (i) a second VL (VL2) domain, and (ii) a second CL domain, wherein the VH2 domain and VL2 domain form a second ABD that binds a second target antigen.

[55] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[56] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[57] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

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

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

[60] In a further embodiment and in accordance with any of the above, the first ABD and the second ABD both bind a target antigen selected from a group including: (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 binds a first target antigen selected from a group including: (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 binds a second target antigen selected from a group including: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7, and (vii) human CCR8.

[61] In a further embodiment and in accordance with 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 a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. 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 a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[62] In a further embodiment and in accordance with any of the above, the first ABD binds an ECD of the first target antigen.

[63] In a further embodiment and in accordance with any of the above, the second ABD binds an ECD of the second target antigen.

[64] In a further embodiment and in accordance with any of the above, the first ABD binds an ECD of the first target antigen, and the second ABD binds an ECD of the second target antigen.

[65] In a further embodiment and in accordance with any of the above, the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[66] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a first VH (VH1) domain, (ii) a first CH1 domain, (iii) a first Fc domain, (iv) a first domain linker, and (v) a first IL-12 subunit domain; (b) a second monomer comprising, from N-terminus to C-terminus: (i) a first VL (VL1) domain, and (ii) a first CL domain, wherein the VH1 domain and VL1 domain form a first ABD that binds a first target antigen; (c) a third monomer comprising, from N-terminus to C-terminus: (i) a second VH (VH2) domain, (ii) a second CH1 domain, (iii) a second Fc domain, (iv) a second domain linker, 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) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; and (d) a fourth monomer comprising, from N-terminus to C-terminus: (i) a second VL (VL2) domain, and (ii) a second CL domain, wherein the VH2 domain and VL2 domain form a second ABD that binds a second target antigen.

[67] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[68] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[69] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 26-30, and the second Fc domain comprises any one of SEQ ID NOs: 26-30. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

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

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

[72] In a further embodiment and in accordance with any of the above, the first ABD and the second ABD both bind a target antigen selected from a group including: (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 binds a first target antigen selected from a group including: (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 binds a second target antigen selected from a group including: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7, and (vii) human CCR8.

[73] In a further embodiment and in accordance with 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 a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. 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 a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[74] In a further embodiment and in accordance with any of the above, the first ABD binds an ECD of the first target antigen.

[75] In a further embodiment and in accordance with any of the above, the second ABD binds an ECD of the second target antigen.

[76] In a further embodiment and in accordance with any of the above, the first ABD binds an ECD of the first target antigen, and the second ABD binds an ECD of the second target antigen.

[77] In a further embodiment and in accordance with any of the above, the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[78] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to 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 a set of amino acid substitutions selected from a group including: (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 linker, and a VL domain, (ii) a third domain linker, and (iii) a second Fc domain, wherein the anti-target antigen scFv is covalently attached to the third domain linker at the N-terminus of the second Fc domain.

[79] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[80] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

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

[82] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[83] In a further embodiment and in accordance with any of the above, the anti-target antigen scFv binds an extracellular domain (ECD) of the target antigen.

[84] In a further embodiment and in accordance with any of the above, the VH domain of the anti-target antigen scFv is covalently attached to the 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 attached to the third domain linker at the N-terminus of the second Fc domain.

[85] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[86] In a further embodiment and in accordance with any of the above, the second domain linker comprises any one of SEQ ID NOs: 31-40.

[87] In a further embodiment and in accordance with any of the above, the first domain linker and / or the third domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker and / or the third domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the third domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the third domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[88] In another aspect, the present 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 linker, and a VL domain, (ii) a second domain linker, and (iii) a first Fc domain, wherein the anti-target antigen scFv is covalently attached to the second domain linker at the N-terminus of the first Fc domain; and (b) a second monomer comprising, from N-terminus to C-terminus: (i) a second Fc domain, (ii) a third domain linker, (iii) a first IL-12 subunit domain, (iv) a fourth domain linker, 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) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A.

[89] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[90] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

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

[92] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[93] In a further embodiment and in accordance with any of the above, the anti-target antigen scFv binds an extracellular domain (ECD) of the target antigen.

[94] In a further embodiment and in accordance with any of the above, the VH domain of the anti-target antigen scFv is covalently attached to the 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 attached to the second domain linker at the N-terminus of the first Fc domain.

[95] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[96] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40.

[97] In a further embodiment and in accordance with any of the above, the second domain linker, the third domain linker, and / or the fourth domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the second domain linker, the third domain linker, and / or the fourth domain linker comprises a covalent bond. In some other embodiments, at least one domain linker comprises any one of SEQ ID NOs: 31-40, and at least one domain linker comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[98] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain linker, 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 attached 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 attached to the third domain linker at the N-terminus of the anti-target antigen scFv, (2) the anti-target antigen scFv comprises a VH domain, a fourth domain linker, and a VL domain, (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 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 (4) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A.

[99] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[100] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

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

[102] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[103] In a further embodiment and in accordance with any of the above, the anti-target antigen scFv binds an extracellular domain (ECD) of the target antigen.

[104] In a further embodiment and in accordance with any of the above, the VH domain of the anti-target antigen scFv is covalently attached to the 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 attached to the third domain linker at the N-terminus of the second Fc domain.

[105] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[106] In a further embodiment and in accordance with any of the above, the fourth domain linker comprises any one of SEQ ID NOs: 31-40.

[107] In a further embodiment and in accordance with any of the above, the first domain linker, the second domain linker, and / or the third domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, and / or the third domain linker comprises a covalent bond. In some other embodiments, at least one domain linker comprises any one of SEQ ID NOs: 31-40, and at least one domain linker comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[108] In another aspect, the present 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 linker, (iii) a second IL-12 subunit domain, wherein the first IL-12 subunit domain is covalently attached to the first domain linker at the N-terminus of the second IL-12 subunit domain, (iv) a second domain linker, (v) a first Fc domain, wherein the second IL-12 subunit domain is covalently attached to the second domain linker at the N-terminus of the first Fc domain, (vi) a third domain linker, and (vii) an anti-target antigen scFv, wherein: (1) the first Fc domain is covalently attached to the third domain linker at the N-terminus of the anti-target antigen scFv, (2) the anti-target antigen scFv comprises a VH domain, a fourth domain linker, and a VL domain, (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 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 (4) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; and (b) a second monomer comprising a second Fc domain.

[109] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[110] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

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

[112] In a further embodiment and in accordance with any of the above, the target antigen comprises human PD1, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. In other embodiments, the target antigen comprises human ICOS, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 121 and 122, respectively. In other embodiments, the target antigen comprises human 41BB, and the anti-target antigen scFv comprises a VH / VL domain pair selected from a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[113] In a further embodiment and in accordance with any of the above, the anti-target antigen scFv binds an extracellular domain (ECD) of the target antigen.

[114] In a further embodiment and in accordance with any of the above, the VH domain of the anti-target antigen scFv is covalently attached to the 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 attached to the third domain linker at the N-terminus of the second Fc domain.

[115] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[116] In a further embodiment and in accordance with any of the above, the fourth domain linker comprises any one of SEQ ID NOs: 31-40.

[117] In a further embodiment and in accordance with any of the above, the first domain linker, the second domain linker, and / or the third domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, and / or the third domain linker comprises a covalent bond. In some other embodiments, at least one domain linker comprises any one of SEQ ID NOs: 31-40, and at least one domain linker comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[118] In another aspect, the present 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 linker, and a first VL (VL1) domain, (ii) a second domain linker, and (iii) a first Fc domain, wherein the first anti-target antigen scFv is covalently attached to the second domain linker 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 attached 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 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 set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, (3) the second Fc domain is covalently attached to the fifth domain linker at the N-terminus of the first IL-12 subunit domain, and (4) the first IL-12 subunit domain is covalently attached to the sixth domain linker at the N-terminus of the second IL-12 subunit domain.

[119] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[120] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

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

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

[123] In a further embodiment and in accordance with any of the above, the first anti-target antigen scFv and the second anti-target antigen scFv both bind a target antigen selected from a group including: (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 binds a first target antigen selected from a group including: (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 binds a second target antigen selected from a group including: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7, and (vii) human CCR8.

[124] In a further embodiment and in accordance with 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 a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. 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 a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[125] In a further embodiment and in accordance with any of the above, the first anti-target antigen scFv binds an ECD of the first target antigen.

[126] In a further embodiment and in accordance with any of the above, the second anti-target antigen scFv binds an ECD of the second target antigen.

[127] In a further embodiment and in accordance with any of the above, the first anti-target antigen scFv binds an ECD of the first target antigen, and the second anti-target antigen scFv binds an ECD of the second target antigen.

[128] In a further embodiment and in accordance with any of the above, the VH1 domain of the first anti-target antigen scFv is covalently attached to the 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 attached to the second domain linker at the N-terminus of the first Fc domain.

[129] In a further embodiment and in accordance with any of the above, the VH2 domain of the second anti-target antigen scFv is covalently attached to the 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 attached to the fourth domain linker at the N-terminus of the second Fc domain.

[130] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[131] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the third domain linker comprises any one of SEQ ID NOs: 31-40.

[132] In a further embodiment and in accordance with any of the above, the second domain linker, the fourth domain linker, the fifth domain linker, and / or the sixth domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the second domain linker, the fourth domain linker, the fifth domain linker, and / or the sixth domain linker comprises a covalent bond. In some other embodiments, at least one domain linker comprises any one of SEQ ID NOs: 31-40, and at least one domain linker comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[133] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: the IL-12 Fc fusion protein comprises: (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 linker, and a first VL (VL1) domain, (ii) a second domain linker, (iii) a first Fc domain, wherein the first anti-target antigen scFv is covalently attached to the second domain linker at the N-terminus of the first Fc domain, (iv) a third domain linker, and (v) a first IL-12 subunit domain, wherein the first Fc domain is covalently attached to the third domain linker 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 attached 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 attached to the sixth domain linker at the N-terminus of the second IL-12 subunit domain, (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 set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A.

[134] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[135] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

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

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

[138] In a further embodiment and in accordance with any of the above, the first anti-target antigen scFv and the second anti-target antigen scFv both bind a target antigen selected from a group including: (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 binds a first target antigen selected from a group including: (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 binds a second target antigen selected from a group including: (i) human OX40, (ii) human LAG3, (iii) human PD1, (iv) human ICOS, (v) human 41BB, (vi) human CCR7, and (vii) human CCR8.

[139] In a further embodiment and in accordance with 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 a group including: (i) SEQ ID NOs: 54 and 55, respectively, (ii) SEQ ID NOs: 93 and 94, respectively, (iii) SEQ ID NOs: 95 and 96, respectively, (iv) SEQ ID NOs: 97 and 98, respectively, (v) SEQ ID NOs: 99 and 100, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 62 and 63, respectively, (ii) SEQ ID NOs: 103 and 104, respectively, (iii) SEQ ID NOs: 105 and 106, respectively, (iv) SEQ ID NOs: 107 and 108, respectively, (v) SEQ ID NOs: 109 and 110, respectively, and (vi) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively, as shown in Figs. 13, 75, 81, 85, and 112. 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 a group including: (i) SEQ ID NOs: 74 and 75, respectively, (ii) SEQ ID NOs: 117 and 118, respectively, (iii) SEQ ID NOs: 119 and 120, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 70 and 71, respectively, (ii) SEQ ID NOs: 123 and 124, respectively, (iii) SEQ ID NOs: 125 and 126, respectively, and (iv) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 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 a group including: (i) SEQ ID NOs: 82 and 83, respectively, (ii) SEQ ID NOs: 113 and 114, respectively, and (iii) SEQ ID NOs: 115 and 116, respectively.

[140] In a further embodiment and in accordance with any of the above, the first anti-target antigen scFv binds an ECD of the first target antigen.

[141] In a further embodiment and in accordance with any of the above, the second anti-target antigen scFv binds an ECD of the second target antigen.

[142] In a further embodiment and in accordance with any of the above, the first anti-target antigen scFv binds an ECD of the first target antigen, and the second anti-target antigen scFv binds an ECD of the second target antigen.

[143] In a further embodiment and in accordance with any of the above, the VH1 domain of the first anti-target antigen scFv is covalently attached to the 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 attached to the second domain linker at the N-terminus of the first Fc domain.

[144] In a further embodiment and in accordance with any of the above, the VH2 domain of the second anti-target antigen scFv is covalently attached 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 attached to the fifth domain linker at the N-terminus of the second Fc domain.

[145] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[146] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the fourth domain linker comprises any one of SEQ ID NOs: 31-40.

[147] In a further embodiment and in accordance with any of the above, the second domain linker, the third domain linker, the fifth domain linker, and / or the sixth domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the second domain linker, the third domain linker, the fifth domain linker, and / or the sixth domain linker comprises a covalent bond. In some other embodiments, at least one domain linker comprises any one of SEQ ID NOs: 31-40, and at least one domain linker comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[148] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to 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 a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; and (b) a second monomer comprising, from N-terminus to C-terminus: (i) a VHH domain, (ii) a second domain linker, and (iii) a second Fc domain, wherein the VHH domain binds a target antigen.

[149] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[150] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[151] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[152] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises any one of SEQ ID NOs: 31-40, and the second domain linker comprises a covalent bond. In some other embodiments, the first domain linker comprises a covalent bond, and the second domain linker comprises any one of SEQ ID NOs: 31-40. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[153] In a further embodiment and in accordance with any of the above, the VHH domain binds a target antigen selected from a group including: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[154] In a further embodiment and in accordance with any of the above, the target antigen comprises PD1, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(g)).

[155] In a further embodiment and in accordance with 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.

[156] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a VHH domain, (ii) a first domain linker, and (iii) a first Fc domain; and (b) a second monomer comprising, from N-terminus to C-terminus: (i) a second Fc domain, (ii) a second domain linker, (iii) a first IL-12 subunit domain, (iv) a third domain linker, 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) the variant IL-12 p35 subunit domain comprises a set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A.

[157] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[158] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[159] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[160] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, the second domain linker comprises any one of SEQ ID NOs: 31-40, and the third domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, and / or the third domain linker comprises a covalent bond. In some other embodiments, at least one of the domain linkers comprises any one of SEQ ID NOs: 31-40, and at least one of the domain linkers comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[161] In a further embodiment and in accordance with any of the above, the VHH domain binds a target antigen selected from a group including: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[162] In a further embodiment and in accordance with any of the above, the target antigen comprises PD1, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(g)).

[163] In a further embodiment and in accordance with 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.

[164] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a first IL-12 subunit domain, (ii) a first domain linker, and (iii) a first Fc domain; and (b) a second monomer comprising, from N-terminus to C-terminus: (i) a second IL-12 subunit domain, (ii) a second domain linker, (iii) a second Fc domain, (iv) a third domain linker, and (v) a VHH domain, wherein: (1) the VHH domain binds 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 set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A.

[165] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[166] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[167] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[168] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, the second domain linker comprises any one of SEQ ID NOs: 31-40, and the third domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, and / or the third domain linker comprises a covalent bond. In some other embodiments, at least one of the domain linkers comprises any one of SEQ ID NOs: 31-40, and at least one of the domain linkers comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[169] In a further embodiment and in accordance with any of the above, the VHH domain binds a target antigen selected from a group including: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[170] In a further embodiment and in accordance with any of the above, the target antigen comprises PD1, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(g)).

[171] In a further embodiment and in accordance with 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.

[172] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to 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, (v) a first Fc domain, (vi) a third domain linker, and (vii) a VHH domain, wherein: (1) the VHH domain binds 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 set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A; and (b) a second monomer comprising a second Fc domain.

[173] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[174] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[175] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[176] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, the second domain linker comprises any one of SEQ ID NOs: 31-40, and the third domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, and / or the third domain linker comprises a covalent bond. In some other embodiments, at least one of the domain linkers comprises any one of SEQ ID NOs: 31-40, and at least one of the domain linkers comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

[177] In a further embodiment and in accordance with any of the above, the VHH domain binds a target antigen selected from a group including: (1) OX40, (2) LAG3, (3) PD1, (4) ICOS, (5) 41BB, (6) CCR7, and (7) CCR8. In some further embodiments, the VHH domain is humanized.

[178] In a further embodiment and in accordance with any of the above, the target antigen comprises PD1, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(a)). In other embodiments, the target antigen comprises LAG3, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(b)). In other embodiments, the target antigen comprises OX40, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(c)). In other embodiments, the target antigen comprises 41BB, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(d)). In other embodiments, the target antigen comprises ICOS, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(e)). In other embodiments, the target antigen comprises CCR7, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(f)). In other embodiments, the target antigen comprises CCR8, and the VHH domain comprises a VHH domain described herein (see, e.g., Section (III)(A)(2)(g)).

[179] In a further embodiment and in accordance with 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.

[180] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a first VHH domain, (ii) a first domain linker, (iii) a first Fc domain, (iv) a second domain linker, (v) a first IL-12 subunit domain, (vi) a third domain linker, 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 set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, and (3) the first VHH domain binds a first target antigen; and (b) a second monomer comprising, from N-terminus to C-terminus: (i) a second VHH domain, (ii) a fourth domain linker, and (iii) a second Fc domain, wherein the second VHH domain binds a second target antigen.

[181] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[182] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[183] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[184] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, the second domain linker comprises any one of SEQ ID NOs: 31-40, the third domain linker comprises any one of SEQ ID NOs: 31-40, and the fourth domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, the third domain linker, and / or the fourth domain linker comprises a covalent bond. In some other embodiments, at least one domain linker comprises any one of SEQ ID NOs: 31-40, and at least one domain linker comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

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

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

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

[188] In a further embodiment and in accordance with any of the above, the first target antigen and / or the second target antigen comprises PD1, and the first VHH domain and / or the second VHH domain comprises a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., Section (III)(A)(2)(g)).

[189] In a further embodiment and in accordance with any of the above, the first VHH domain is humanized.

[190] In a further embodiment and in accordance with any of the above, the second VHH domain is humanized.

[191] In a further embodiment and in accordance with any of the above, the first VHH domain is humanized, and the second VHH domain is humanized.

[192] In a further embodiment and in accordance with 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.

[193] In another aspect, the present disclosure provides an IL-12 Fc fusion protein, comprising: (a) a first monomer comprising, from N-terminus to C-terminus: (i) a first VHH domain, (ii) a first domain linker, (iii) a first Fc domain, (iv) a second domain linker, 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 N-terminus to C-terminus: (i) a second VHH domain, (ii) a third domain linker, (iii) a second Fc domain, (iv) a fourth domain linker, 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 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 set of amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A.

[194] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17-21.

[195] In a further embodiment and in accordance with 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 wherein the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 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.

[196] In a further embodiment and in accordance with any of the above, the first Fc domain comprises any one of SEQ ID NOs: 29 or 30, and the second Fc domain comprises any one of SEQ ID NOs: 30 or 29. In some further embodiments, the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, whereas in other embodiments the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

[197] In a further embodiment and in accordance with any of the above, the first domain linker comprises any one of SEQ ID NOs: 31-40, the second domain linker comprises any one of SEQ ID NOs: 31-40, the third domain linker comprises any one of SEQ ID NOs: 31-40, and the fourth domain linker comprises any one of SEQ ID NOs: 31-40. In some other embodiments, the first domain linker, the second domain linker, the third domain linker, and / or the fourth domain linker comprises a covalent bond. In some other embodiments, at least one of the domain linkers comprises any one of SEQ ID NOs: 31-40, and at least one of the domain linkers comprises a covalent bond. In some embodiments, at least one of the domain linkers comprises a hinge region of a Fc domain.

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

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

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

[201] In a further embodiment and in accordance with any of the above, the first target antigen and / or the second target antigen comprises PD1, and the first VHH domain and / or the second VHH domain comprises a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., 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 a VHH domain described herein (see, e.g., Section (III)(A)(2)(g)).

[202] In a further embodiment and in accordance with any of the above, the first VHH domain is humanized.

[203] In a further embodiment and in accordance with any of the above, the second VHH domain is humanized.

[204] In a further embodiment and in accordance with any of the above, the first VHH domain is humanized, and the second VHH domain is humanized.

[205] In a further embodiment and in accordance with 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.

[206] In another aspect, the present disclosure provides a nucleic acid composition, comprising: one or more nucleic acids encoding an IL-12 Fc fusion protein, as described herein.

[207] In another aspect, the present disclosure provides an expression vector, comprising: one or more nucleic acids encoding an IL-12 Fc fusion protein, as described herein.

[208] In another aspect, the present disclosure provides a host cell, wherein the host cell is transformed with an expression vector, as described herein.

[209] In another aspect, the present disclosure provides a method of making an IL-12 Fc fusion protein, comprising: (a) culturing a host cell transformed with an expression vector comprising one or more nucleic acids encoding an IL-12 Fc fusion protein, as described herein; and (b) isolating and / or purifying the IL-12 Fc fusion protein.

[210] In another aspect, the present 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) SEQ ID NOs: 49, 153, and 145, respectively, (ii) SEQ ID NOs: 48, 146, and 145, respectively, (iii) SEQ ID NOs: 48, 151, and 150, respectively, (iv) SEQ ID NOs: 48, 153, and 145, respectively, (v) SEQ ID NOs: 48, 155, and 145, respectively, (vi) SEQ ID NOs: 48, 157, and 145, respectively, (vii) SEQ ID NOs: 48, 159, and 150, respectively, (viii) SEQ ID NOs: 48, 161, and 150, respectively, (ix) SEQ ID NOs: 48, 163, and 150, respectively, (x) SEQ ID NOs: 48, 165, and 150, respectively, (xi) SEQ ID NOs: 48, 167, and 150, respectively, (xii) SEQ ID NOs: 48, 169, and 150, respectively, (xiii) SEQ ID NOs: 49, 141, and 140, respectively, (xiv) SEQ ID NOs: 49, 146, and 145, respectively, (xv) SEQ ID NOs: 49, 151, and 150, respectively, (xvi) SEQ ID NOs: 48, 141, and 140, respectively, (xvii) SEQ ID NOs: 49, 155, and 145, respectively, (xviii) SEQ ID NOs: 49, 157, and 145, respectively, (xix) SEQ ID NOs: 49, 159, and 150, respectively, (xx) SEQ ID NOs: 49, 161, and 150, respectively, (xxi) SEQ ID NOs: 49, 163, and 150, respectively, (xxii) SEQ ID NOs: 49, 165, and 150, respectively, (xxiii) SEQ ID NOs: 49, 167, and 150, respectively, (xxiv) SEQ ID NOs: 49, 169, and 150, respectively, (xxv) SEQ ID NOs: 50, 141, and 140, respectively, (xxvi) SEQ ID NOs: 50, 146, and 145, respectively, (xxvii) SEQ ID NOs: 50, 151, and 150, respectively, (xxviii) SEQ ID NOs: 50, 153, and 145, respectively, (xxix) SEQ ID NOs: 50, 155, and 145, respectively, (xxx) SEQ ID NOs: 50, 157, and 145, respectively, (xxxi) SEQ ID NOs: 50, 159, and 150, respectively, (xxxii) SEQ ID NOs: 50, 161, and 150, respectively, (xxxiii) SEQ ID NOs: 50, 163, and 150, respectively, (xxxiv) SEQ ID NOs: 50, 165, and 150, respectively, (xxxv) SEQ ID NOs: 50, 167, and 150, respectively, (xxxvi) SEQ ID NOs: 50, 169, and 150, respectively, (xxxvii) SEQ ID NOs: 51, 141, and 140, respectively, (xxxviii) SEQ ID NOs: 51, 146, and 145, respectively, (xxxix) SEQ ID NOs: 51, 151, and 150, respectively, (xl) SEQ ID NOs: 51, 153, and 145, respectively, (xli) SEQ ID NOs: 51, 155, and 145, respectively, (xlii) SEQ ID NOs: 51, 157, and 145, respectively, (xliii) SEQ ID NOs: 51, 159, and 150, respectively, (xliv) SEQ ID NOs: 51, 161, and 150, respectively, (xlv) SEQ ID NOs: 51, 163, and 150, respectively, (xlvi) SEQ ID NOs: 51, 165, and 150, respectively, (xlvii) SEQ ID NOs: 51, 167, and 150, respectively, (xlviii) SEQ ID NOs: 51, 169, and 150, respectively, (xlix) SEQ ID NOs: 52, 141, and 140, respectively, (l) SEQ ID NOs: 52, 146, and 145, respectively, (li) SEQ ID NOs: 52, 151, and 150, respectively, (lii) SEQ ID NOs: 52, 153, and 145, respectively, (liii) SEQ ID NOs: 52, 155, and 145, respectively, (liv) SEQ ID NOs: 52, 157, and 145, respectively, (lv) SEQ ID NOs: 52, 159, and 150, respectively, (lvi) SEQ ID NOs: 52, 161, and 150, respectively, (lvii) SEQ ID NOs: 52, 163, and 150, respectively, (lviii) SEQ ID NOs: 52, 165, and 150, respectively, (lix) SEQ ID NOs: 52, 167, and 150, respectively, (lx) SEQ ID NOs: 52, 169, and 150, respectively, (lxi) SEQ ID NOs: 48, 174, and 173, respectively, (lxii) SEQ ID NOs: 49, 174, and 173, respectively, (lxiii) SEQ ID NOs: 50, 174, and 173, respectively, (lxiv) SEQ ID NOs: 51, 174, and 173, respectively, or (lxv) SEQ ID NOs: 52, 174, and 173, respectively.

[211] In a further embodiment and in accordance with the above, 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 other 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 other 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

[212] In another aspect, the present disclosure provides a method of treating a cancer in a human subject, comprising administering an IL-12 Fc fusion protein, as described herein, to a human subject in need thereof, wherein the IL-12 Fc fusion protein binds to a target cell.

[213] In a further embodiment and in accordance with the above, the target cell expresses: (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.

[214] In a further embodiment and in accordance with the above, the cell surface antigen is human PD1, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human LAG3, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human OX40, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human ICOS, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human 41BB, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted NK cell(s), (iii) activated and / or exhausted CD8+ T cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR7, and the target cell comprises: (i) naïve CD4+ T cell(s), (ii) naïve CD8+ T cell(s), or (iii) naïve regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR8, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), or (ii) activated and / or exhausted regulatory T cell(s).

[215] In another aspect, the present disclosure provides a method of activating a target cell in a human subject, comprising administering an IL-12 Fc fusion protein, as described herein, to a human subject in need thereof, wherein the IL-12 Fc fusion protein binds to the target cell.

[216] In a further embodiment and in accordance with the above, the target cell expresses: (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.

[217] In a further embodiment and in accordance with the above, the cell surface antigen is human PD1, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human LAG3, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human OX40, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human ICOS, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human 41BB, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted NK cell(s), (iii) activated and / or exhausted CD8+ T cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR7, and the target cell comprises: (i) naïve CD4+ T cell(s), (ii) naïve CD8+ T cell(s), or (iii) naïve regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR8, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), or (ii) activated and / or exhausted regulatory T cell(s).

[218] In another aspect, the present disclosure provides a method of stimulating IL-12 signaling in a target cell comprising administering an IL-12 Fc fusion protein, as described herein, to a human subject in need thereof, wherein the IL-12 Fc fusion protein binds to the target cell.

[219] In a further embodiment and in accordance with the above, the target cell expresses: (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.

[220] In a further embodiment and in accordance with the above, the cell surface antigen is human PD1, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human LAG3, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human OX40, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human ICOS, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human 41BB, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted NK cell(s), (iii) activated and / or exhausted CD8+ T cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR7, and the target cell comprises: (i) naïve CD4+ T cell(s), (ii) naïve CD8+ T cell(s), or (iii) naïve regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR8, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), or (ii) activated and / or exhausted regulatory T cell(s).

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

[222] In a further embodiment and in accordance with the above, the target cell expresses: (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.

[223] In a further embodiment and in accordance with the above, the cell surface antigen is human PD1, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human LAG3, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human OX40, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human ICOS, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human 41BB, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted NK cell(s), (iii) activated and / or exhausted CD8+ T cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR7, and the target cell comprises: (i) naïve CD4+ T cell(s), (ii) naïve CD8+ T cell(s), or (iii) naïve regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR8, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), or (ii) activated and / or exhausted regulatory T cell(s).

[224] In another aspect, the present disclosure provides a method of reprogramming and / or altering a differentiation state of a target cell comprising administering an IL-12 Fc fusion protein, as described herein, wherein the IL-12 Fc fusion protein binds to the target cell.

[225] In a further embodiment and in accordance with the above, the target cell expresses: (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.

[226] In a further embodiment and in accordance with the above, the cell surface antigen is human PD1, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human LAG3, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human OX40, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human ICOS, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human 41BB, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted NK cell(s), (iii) activated and / or exhausted CD8+ T cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR7, and the target cell comprises: (i) naïve CD4+ T cell(s), (ii) naïve CD8+ T cell(s), or (iii) naïve regulatory T cell(s). In other embodiments, the cell surface antigen is human CCR8, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), or (ii) activated and / or exhausted regulatory T cell(s).Brief Description of the Drawings

[227] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Fig.,” “FIG.,” “Figure,” “Figures,” “Figs.,” and “FIGs.” herein) of which:

[228] Fig. 1 shows a list of sequences for human, wild-type IL-12 subunits alpha and beta (precursor and mature form sequences).

[229] Fig. 2 shows a list of sequences for human, wild-type IL-12Rβ1 (+ the sequence for the extracellular domain) and IL-12Rβ2 (+ the sequence for the extracellular domain).

[230] Fig. 3 shows a list of sequences for mouse, wild-type IL-12 subunits alpha and beta (precursor and mature form sequences).

[231] Fig. 4 shows a list of sequences for mouse, wild-type IL-12Rβ1 (+ the sequence for the extracellular domain) and IL-12Rβ2 (+ the sequence for the extracellular domain).

[232] Fig. 5 shows a list of variant IL-12 p35 subunit sequences (both human and mouse) comprising one or more substitution mutations.

[233] Fig. 6 shows a list of variant IL-12 p40 subunit sequences.

[234] Fig. 7 shows exemplary sequences for human IgG1 Fc domains (G1m allotype, as well as Fc domains comprising one or more substitutions).

[235] Fig. 8 shows a list of exemplary domain linker sequences.

[236] Fig. 9 shows a list of amino acid sequences for exemplary antigen binding domains (ABDs) for hen egg-white lysozyme, as well as exemplary heavy chain and light chain amino acid sequences (mouse and human) for use in control IL-12 fusion proteins.

[237] Fig. 10 (Figs. 10A-10D) shows a list of amino acid sequences for exemplary IL-12 Fc fusion protein monomers for use in subject IL-12 Fc fusion proteins.

[238] Fig. 11 (Figs. 11A and 11B) shows a list of sequences for exemplary antigen binding domains (ABDs) for PD1 (human and mouse), as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[239] Fig. 12 (Figs. 12A and 12B) shows a list of sequences for exemplary ABDs that bind to LAG3 (human / mouse cross-reactive and human), as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[240] Fig. 13 shows a list of sequences for exemplary ABDs that bind to OX40 (human and mouse), as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[241] Fig. 14 (Figs. 14A and 14B) shows a list of sequences for exemplary ABDs that bind to 41BB (human / mouse cross-reactive and human), as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[242] Fig. 15 (Figs. 15A and 15B) shows a list of sequences for exemplary ABDs that bind to ICOS (human / mouse cross-reactive and human), as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[243] Fig. 16 shows a list of sequences for exemplary ABDs that bind to CCR7 (human), as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein.

[244] Fig. 17 (Figs. 17A and 17B) shows a list of sequences for exemplary ABDs that bind to CCR8 (human and human / mouse cross-reactive), as well as exemplary heavy chain and light chain amino acid sequences for use in subject IL-12 Fc fusion proteins. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[245] Fig. 18 (Figs. 18A-18H) shows schematic representations of exemplary IL-12-ABD fusion protein formats. In some exemplary configurations, the subject IL-12-ABD fusion protein is configured such that it comprises one antigen binding domain (i.e., a monovalent, monospecific format). In other exemplary configurations, the subject IL-12-ABD fusion protein is configured such that it comprises two antigen binding domains. In such configurations, the subject IL-12-ABD fusion protein may be configured such that the ABD’s bind the same target antigen (i.e., a bivalent, monospecific format), whereas in other configurations the subject IL-12-ABD fusion protein is configured such that the first ABD and the second ABD bind distinct target antigens (i.e., a monovalent, bispecific format). It is expressly contemplated that the one or more ABD depicted herein can take on different configurations, as appreciated by those skilled in the art. Suitable ABD formats include, but are not limited to, a Fab format, a scFv format, and a nanobody format (VHH domain). Further, in some exemplary configurations, the subject IL-12-ABD fusion protein is configured such that the first IL-12 subunit domain and the second IL-12 subunit domain are on the same monomer of a subject IL-12 fusion protein (generally, covalently linked to each other using a domain linker), whereas in other exemplary configurations the first IL-12 subunit domain and the second IL-12 subunit domain are on separate monomers comprising a subject IL-12 fusion protein. Further, while the schematics generally show single chain IL-12 arranged, from N-terminus to 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 can be arranged, from N-terminus to C-terminus, as variant IL-12 p35 subunit domain-domain linker-(variant) IL-12 p40 subunit domain. As will be appreciated by those skilled in the relevant art, while all exemplary configurations show the first IL-12 subunit domain and the second IL-12 subunit domain covalently linked to one monomer or covalently linked to a first monomer and a second monomer of a subject IL-12-ABD fusion protein, it is also contemplated that a subject IL-12-ABD fusion protein can be configured such that only one of the IL-12 subunit domains (e.g., the variant IL-12 p35 subunit domain, or the (variant) IL-12 p40 subunit domain) is covalently attached to a monomer of the subject IL-12-ABD fusion protein and the other IL-12 subunit domain (e.g., the (variant) IL-12 p40 subunit domain, or the variant IL-12 p35 subunit domain) is expressed as a soluble protein that is not covalently attached to the monomers of the subject IL-12-ABD fusion protein.

[246] Fig. 19 shows a list of exemplary IL-12 Fc fusion proteins, including ABD specificity (human), IL-12 p35 subunit domain mutations, and the amino acid sequence identifiers for such exemplary IL-12 Fc fusion proteins, as well as exemplary control fusion proteins (ABD specificity for hen egg-white lysozyme, or lacking an ABD).

[247] Fig. 20 (Figs. 20A and 20B) shows reducing (+DTT) and non-reducing (-DTT) SDS-PAGE analysis of exemplary purified IL-12-ABD fusion proteins.

[248] Fig. 21 shows a representative flow cytometry analysis of PD-1 expression on purified naïve human NK cells.

[249] Fig. 22 (Figs. 22A and 22B) shows AlphaLISA measurements of IFNγ in the medium of naïve NK cells that were incubated with commercially available recombinant IL-12 (rIL-12) or the indicated IL-12-ABD fusion proteins. Symbols and error bars indicate the mean and SD, respectively.

[250] Fig. 23 shows flow cytometry analysis of PD-1 on healthy donor-derived human PBMC that were activated with 600 U / mL IL-2 and 32 ng / mL OKT3.

[251] Fig. 24 (Figs. 24A and 24B) shows AlphaLISA measurements of IFNγ in the medium of IL-2 / OKT3 activated human PBMC that were incubated with commercially available recombinant IL-12 (rIL-12) or the indicated IL-12-ABD fusion proteins, in the presence of 50 U / mL human IL-2. Symbols and error bars indicate the mean and SD, respectively.

[252] Fig. 25 shows selected volcano plots that display differential gene expression (logtransformed fold change (logFC) on the x-axis; negative log-transformed adjusted pvalue (log(adj. p)) on the y-axis) within the listed data sets and comparisons (Figs. 25A-25I), as well as a summary table (Fig. 25J) with the upregulation rank of each gene within each data set, and the rank of each gene after taking a product-of-ranks across the listed data sets and comparisons.

[253] Fig. 26 shows the percentage of each listed cell type (x-axis) that is positive for each cell surface target across a range of tissues and tumor samples (defined in legend). For RNA / transcript abundance data, a cell was considered positive if at least 1 transcript was detected. For ADT / cell surface antibody labelling, a cell was considered positive if the transformed abundance is above 1 (an arbitrary threshold that was selected to exclude ‘background staining’ across a variety of markers within the associated data set).

[254] Fig. 27 (Figs. 27A-27L) shows the average transcript abundance, as measured by normalized transcript per kilobase million (nTPM), for selected genes (y-axis) across the listed cell type clusters (xaxis) from a range of tissues in the human protein atlas. Individual cell types have been clustered in related groups (at bottom).

[255] Fig. 28 shows the percentage of CCR8-positive cells across different cell types (x-axis) and a range of tissues (see legend) (Fig. 28A); the average transcript abundance, as measured by normalized transcript per kilobase million (nTPM), across the listed cell type clusters (x-axis) from a range of tissues in the human protein atlas, with individual cell types clustered into related groups (at bottom) (Fig. 28B); and the average transcript abundance for the Treg marker FOXP3 (x-axis) and CCR8 (y-axis) for T cell clusters within the human protein atlas (Fig. 28C).

[256] Fig. 29 shows selected volcano plots that display differential gene expression (log-transformed fold change (logFC) on the x-axis; negative log-transformed adjusted p-value (log(adj. p)) on the y-axis) within the listed data sets and comparisons (Figs. 29A – 29F). Figs. 29A, 29B, and 29C contain comparisons between tumor-resident naïve CD8 T cells and other tumor-resident CD8 T cell subsets. Figs. 29D, 29E, and 29F contain comparisons between tumor-resident CD8 T cells and CD8 T cells from matched heathy tissue. Fig. 29G shows the normalized transcript abundance for CCR7 within CD8 T cells from the peripheral blood or tumor of colorectal cancer patients, with cells subset by their tissue source and co-expression of TCF7 and / or PDCD1 to identify naïve (TCF7+ / PDCD1-) T cells and putative central memory T cells (Tscm; TCF7+ / PDCD1+).

[257] Fig. 30 (Figs. 30A and 30B) shows a summary of flow cytometry analysis performed on immune cells that were harvested from different organs of MC38-bearing C57 / BL6 mice 14 days following IL-12 treatment.

[258] Fig. 31 (Figs. 31A-31C) shows flow cytometry plots analyzing the expression of potential ABD targets on mouse immune cell subsets that were harvested from tumors of MC38-bearing C57 / BL6 mice 14 days following IL-12 treatment.

[259] Fig. 32 (Figs. 32A-32C) shows flow cytometry plots analyzing the expression of potential ABD targets on mouse immune cell subsets that were harvested from spleens of MC38-bearing C57 / BL6 mice 14 days following IL-12 treatment.

[260] Fig. 33 shows a list of exemplary IL-12 Fc fusion proteins, including ABD specificity (mouse), IL-12 p35 subunit domain mutations, and the amino acid sequence identifiers for such exemplary IL-12 Fc fusion proteins, as well as exemplary control fusion proteins (ABD specificity for hen egg-white lysozyme, or lacking an ABD).

[261] Fig. 34 shows reducing (+DTT) and non-reducing (-DTT) SDS-PAGE analysis of exemplary purified IL-12-ABD fusion proteins.

[262] Fig. 35 (Figs. 35A and 35B) shows flow cytometry analysis of purified C57 / BL6 mouse T cells that were activated with 1 ug / mL anti-CD3 and 600 U / mL human IL-2.

[263] Fig. 36 (Figs. 36A and 36B) show AlphaLISA measurements of IFNγ in the medium of anti-CD3 / IL-2 activated purified mouse T cells that were incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the indicated IL-12-ABD fusion proteins, in the presence of 50 U / mL human IL-2. Symbols and error bars indicate the mean and SD, respectively.

[264] Fig. 37 shows flow cytometry analysis of LAG-3 on healthy donor-derived human PBMC that were activated with 600 U / mL IL-2 and 32 ng / mL OKT3 and expanded in for 5 days in 600 U / mL IL-2.

[265] Fig. 38 shows AlphaLISA measurements of IFNγ in the medium of activated / expanded human PBMC that were incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the indicated IL-12-ABD fusion proteins, in the presence of 50 U / mL human IL-2. Symbols and error bars indicate the mean and SD, respectively.

[266] Fig. 39 shows a histogram representing CCR8 expression as determined by flow cytometry on HEK-Blue IL-12 reporter cells (untransduced) and CCR8-HEK-Blue IL-12 reporter cells (transduced).

[267] Fig. 40 shows SEAP measurements (represented as absorbance at 650 nm) of CCR8-HEK-Blue IL-12 reporter cells that were incubated with commercially available recombinant IL-12 (rIL-12) or the indicated IL-12 Fc or IL-12-ABD fusion proteins. Symbols and error bars indicate the mean and SD, respectively.

[268] Fig. 41 shows flow cytometry analysis of CCR7 on healthy donor-derived human PBMC that were activated with 600 U / mL IL-2 and 32 ng / mL OKT3.

[269] Fig. 42 shows AlphaLISA measurements of IFNγ in the medium of IL-2 / OKT3 activated human PBMC that were incubated with commercially available recombinant IL-12 (rIL-12) or the indicated IL-12-ABD fusion proteins, in the presence of 50 U / mL human IL-2. Symbols and error bars indicate the mean and SD, respectively.

[270] Fig. 43 shows flow cytometry analysis of CCR7 on purified naïve human NK cells.

[271] Fig. 44 shows AlphaLISA measurements of IFNγ in the medium of naïve NK cells that were incubated with commercially available recombinant IL-12 (rIL-12) or the indicated IL-12-ABD fusion proteins. Symbols and error bars indicate the mean and SD, respectively.

[272] Fig. 45 shows the amino acid sequence of full-length, human CCR8 protein.

[273] Fig. 46 shows flow cytometry analysis of OX40 on purified C57 / BL6 mouse T cells that were activated with 0.5 ug / mL anti-CD3, 0.5 ug / mL anti-CD28, and 600 U / mL human IL-2.

[274] Fig. 47 shows AlphaLISA measurements of IFNγ in the medium of anti-CD3 / anti-CD28 / IL-2 activated purified mouse T cells that were incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the indicated IL-12-ABD fusion proteins, in the presence of 50 U / mL human IL-2. Symbols and error bars indicate the mean and SD, respectively.

[275] Fig. 48 (Figs. 48A and 48B) shows the growth of MC38 tumor cells following subcutaneous implantation into C57 / BL6 mice. Mice were treated on days 0, 7 and 14 with the indicated test articles at a dose that was the molar equivalent to 0.03 mg / kg of IL-12 Fc Y36E unless otherwise indicated. Mice that were euthanized due to ulceration are indicated by an ‘x’.

[276] Fig. 49 (Figs. 49A-49C) shows measurements of IFNγ in the plasma of MC38-bearing C57 / BL6 mice that were treated with either PBS, IL-12 Fc Y36E, or the indicated IL-12 ABD on days 0, 7, and 14. Measurements were performed on plasma that was sampled 3 days after each treatment (days 3 (Fig. 49A), 10 (Fig. 49B), and 17 (Fig. 49C)). Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group.

[277] Fig. 50 (Figs. 50A-50C) shows measurements of IL-12-Fc / IL-12 ABD fusion proteins in the plasma of MC38-bearing C57 / BL6 mice that were treated with either PBS, IL-12 Fc Y36E, or the indicated IL-12 ABD on days 0, 7, and 14. Measurements were performed on plasma that was sampled 3 days after each treatment (days 3 (Fig. 50A), 10 (Fig. 50B), and 17 (Fig. 50C)). Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group. Data points that were below the detection limit of the assay were assigned an arbitrary value of 1 pg / mL.

[278] Fig. 51 shows the weight of MC38 tumors that were harvested from C57BL / 6 mice 5 days after treatment with either PBS, 0.03 mg / kg of wild type (WT) IL-12 Fc or an equimolar dose of the indicated IL-12 ABD fusion proteins. ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article. Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group.

[279] Fig. 52 shows the weight of spleens harvested from MC38 tumor bearing C57BL / 6 mice 5 days after treatment with either PBS, 0.03 mg / kg of wild type (WT) IL-12 Fc or an equimolar dose of the indicated IL-12 ABD fusion proteins. ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article. Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group.

[280] Fig. 53 shows the frequency of CD45+ cells in spleens that were harvested from MC38 tumor bearing C57BL / 6 mice 5 days after treatment with either PBS, 0.03 mg / kg of wild type (WT) IL-12 Fc or an equimolar dose of the indicated IL-12 ABD fusion proteins (n = 6 / group). ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article. Data is plotted in box-and-whisker format, where the whiskers represent the minimum and maximum values in each treatment group.

[281] Fig. 54 (Figs. 54A-54E) shows quantification of immune cell types (as indicated) in MC38 tumors that were harvested from C57BL / 6 mice 5 days after treatment with either PBS, 0.03 mg / kg of wild type (WT) IL-12 Fc or an equimolar dose of the indicated IL-12 ABD fusion proteins (n = 6 / group). ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article. Data is plotted in box-and-whisker format, where the whiskers represent the minimum and maximum values in each treatment group.

[282] Fig. 55 shows quantification of IFNγ in plasma, spleen and MC38 tumor tissue of C57BL / 6 mice 5 days after treatment with either PBS, 0.03 mg / kg of wild type (WT) IL-12 Fc or an equimolar dose of the indicated IL-12 ABD fusion proteins (n = 6 / group). ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article. Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group.

[283] Fig. 56 shows the ratio of IFNγ in MC38 tumors relative to spleens of C57BL / 6 mice 5 days after treatment with either 0.03 mg / kg of wild type (WT) IL-12 Fc or an equimolar dose of the indicated IL-12 ABD fusion proteins. ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article. Tumor:Spleen IFNγ ratio was calculated by dividing the mean tumor IFNγ value by the mean spleen IFNγ value (from data depicted in Figure 55).

[284] Fig. 57 (Figs. 57A-57C) shows the growth of A20 tumor cells following subcutaneous implantation into BALB / C mice. Unless otherwise indicated, mice were treated on days, 0, 7 and 14 with 0.05 mg / kg wild type (WT) IL-12 Fc, or IL-12 ABD fusion protein at a dose that was molar equivalent to 0.03 mg / kg of IL-12 Fc. ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article (molar equivalent to 0.3 mg / kg of IL-12 Fc). ‘Single’ indicates groups of mice that were treated once (on day 0).

[285] Fig. 58 (Figs. 58A-58C) shows measurements of IFNγ in the plasma of A20-bearing BALB / C mice that were treated on days, 0, 7 and 14 with 0.05 mg / kg wild type (WT) IL-12 Fc, or IL-12 ABD fusion protein at a dose that was molar equivalent to 0.03 mg / kg of IL-12 Fc. ‘10X’ indicates where mice were treated with a 10-fold higher dose of the indicated test article (molar equivalent to 0.3 mg / kg of IL-12 Fc). ‘Single’ indicates groups of mice that were treated once (on day 0). Plasma IFNγ levels were measured on day 3 (D3; Fig. 58A), day 10 (D10; Fig. 58B) and day 17 (D17; Fig. 58C). Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group. The lower limit of quantification (LLoQ) is indicated by a dashed line.

[286] Fig. 59 (Figs. 59A-59C) depicts the welfare scoring system employed for assessing toxicity in mice receiving IL-12 based treatments. In addition to the parameters outlined in Fig. 59, mice could undergo unscheduled euthanasia due to tumor size and / or ulceration of the tumor / skin overlying the tumor.

[287] Fig. 60 (Figs. 60A and 60B) shows the growth of MC38 tumor cells following subcutaneous implantation into C57BL / 6 mice treated on days, 0, 7 and 14 with PBS or the indicated dosage of IL-12 ABD fusion protein.

[288] Fig. 61 (Figs. 61A and 61B) shows heatmaps depicting welfare scores for MC38 tumor-bearing C57BL / 6 mice that were treated on days, 0, 7 and 14 with PBS or the indicated dosage of IL-12 ABD fusion protein. Individual mice are represented in separate rows. Each ‘block’ is colored according to the welfare score received by each mouse (as indicated in the legend) on that given day. * indicates mice that were euthanized due to treatment related toxicity.

[289] Fig. 62 shows quantification of welfare scores for MC38 tumor-bearing C57BL / 6 mice that were treated on days, 0, 7 and 14 with IL-12 ABD fusion protein. Symbols represent mean welfare score (measured by quantification of area-under-the-curve (AUC)), and error bars indicate the standard deviation of the measurements. The mean welfares score for mice that received PBS treatment is indicated by a solid horizontal line.

[290] Fig. 63 (Figs. 63A-63C) shows measurements of IFNγ in the plasma of MC38-bearing C57BL / 6 mice that were treated on days, 0, 7 and 14 with PBS or the indicated IL-12 ABD fusion protein. Numbers in parathesis indicate the dose of each test article in units of mg / kg. Plasma IFNγ levels were measured on day 3 (D3; Fig. 63A), day 10 (D10; Fig. 63B) and day 17 (D17; Fig. 63C). Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group.

[291] Fig. 64 shows quantification of IFNγ levels in the plasma of MC38-bearing C57BL / 6 mice 3 days after treatment with PBS or IL-12 ABD fusion protein (n = 6 per group). Symbols and error bars represent the mean and standard deviation, respectively.

[292] Fig. 65 (Figs. 65A and 65B) shows the growth of AT3 tumor cells following subcutaneous implantation into C57BL / 6 mice that were treated with PBS, anti-PD-1 and / or IL-12-based test articles as indicated.

[293] Fig. 66 (Figs. 66A and 66B) shows the cumulative welfare score for AT3 tumor-bearing C57BL / 6 mice that were treated with PBS, anti-PD-1 and / or IL-12-based test articles as indicated. Solid lines indicate the cumulative welfare score (according to the table in Figure 59) for individual mice. Horizontal dashed lines represent extrapolation of welfare curves from the point at which mice were euthanized. ‘Avg AUC’ indicates the average area under the curve for each treatment group.

[294] Fig. 67 shows measurements of IFNγ in the plasma of AT3-bearing C57BL / 6 mice four days after treatment with PBS, anti-PD-1 and / or IL-12-based test articles as indicated. Numbers in parathesis indicate the dose of each test article in units of mg / kg. Symbols represent measurements for individual mice and horizontal lines indicate the median for each treatment group.

[295] Fig. 68 shows measurement of IFNγ in the plasma of healthy C57BL / 6 mice (No tumor) or C57BL / 6 mice that were subcutaneously injected with MC-38 tumors (Tumor) after treatment with a single dose of mono-Y36E-OX86 (as indicated in the legend) on day 0. Symbols and error bars represent the mean and standard deviation of measurements from 3 mice per group. The horizonal dashed line indicated the lower limit of quantification (LLoQ) of the assay.

[296] Fig. 69 shows measurement of mono-Y36E-OX86 in the plasma of healthy C57BL / 6 mice (No tumor) or C57BL / 6 mice that were subcutaneously injected with MC-38 tumors (Tumor) after treatment with a single dose of mono-Y36E-OX86 (as indicated in the legend) on day 0. Symbols and error bars represent the mean and standard deviation of measurements from 3 mice per group. The horizonal dashed line indicated the lower limit of quantification (LLoQ) of the assay.

[297] Fig. 70 shows a summary of pharmacokinetic properties of mono-Y36E-OX86 in C57BL / 6 mice that were subcutaneously injected with MC-38 tumors. Pharmacokinetic properties were modelled in PK Solver software using the data shown in Fig. 69.

[298] Fig. 71 shows histograms representing the expression of mouse OX40 (mOX40; top panel), human IL-12Rb1 (hIL-12Rb1; middle panel) and human IL-12Rb2 (hIL-12Rb2; bottom panel) on Jurkat NF-κB reporter cells and mOX40 / hIL-12R / Jurkat NF-κB reporter cells.

[299] Fig. 72 shows the percentage of mOX40 / hIL-12R / Jurkat NF-κB reporter cells that expressed GFPfollowing incubation with anti-OX40 mAb (OX86) or IL-12 ABD fusion proteins in the presence or absence of A20 cells that expressed mouse FcγRI.

[300] Fig. 73 shows flow cytometry analysis of OT-II splenocytes that were activated for three days in the presence of 40 nM OVA323-339 peptide and 100 U / mL recombinant human IL-2. FMO (fluorescence minus one; top right panel) indicates staining with all antibodies in the panel (full stain; bottom right panel) except that recognizing OX40 (anti-mouse CD134 BV605, BD Biosciences).

[301] Fig. 74 shows AlphaLISA measurements of IFNγ in the medium of OVA323-339 peptide / IL-2 activated OT-II splenocytes that were incubated with commercially available recombinant mouse IL-12 (rmIL-12) or the indicated IL-12-ABD fusion proteins, in the presence of 50 U / mL human IL-2. Symbols and error bars indicate the mean and SD, respectively.

[302] Fig. 75 (Figs. 75A-75C) shows a list of sequences for exemplary ABDs that bind to human OX40, as well as exemplary heavy chain and light chain amino acid sequences for use in anti-OX40 mAbs or subject IL-12 Fc fusion proteins. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[303] Fig. 76 shows a list of exemplary anti-human OX40 antibodies in traditional IgG1 bivalent format, including amino acid sequence identifiers.

[304] Fig. 77 shows a list of exemplary IL-12 Fc fusion proteins, including ABD specificity (human), IL-12 p35 subunit domain mutations, and the amino acid sequence identifiers for such exemplary IL-12 Fc fusion proteins.

[305] Fig. 78 shows the binding of anti-human OX40 antibodies in traditional IgG1 bivalent format (solid symbols) and OX40-targeted IL-12 Fc fusion proteins (open symbols) to hOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[306] Fig. 79 shows the binding of anti-human OX40 antibodies in traditional IgG1 bivalent format (solid symbols) and OX40-targeted IL-12 Fc fusion proteins (open symbols) to cOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[307] Fig. 80 shows EC50 values for the binding of anti-human OX40 antibodies in traditional IgG1 bivalent format and OX40-targeted IL-12 Fc fusion proteins to hOX40-Jurkat-NF-κB cells and cOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[308] Fig. 81 (Figs. 81A and 81B) shows a list of sequences for exemplary ABDs that bind to human OX40, as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[309] Fig. 82 shows a list of exemplary IL-12 Fc fusion proteins, including ABD specificity (human), IL-12 p35 subunit domain mutations, and the amino acid sequence identifiers for such exemplary IL-12 Fc fusion proteins.

[310] Fig. 83 shows the binding of OX40-targeted IL-12 Fc fusion proteins to hOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[311] Fig. 84 shows EC50 values for the binding of OX40-targeted IL-12 Fc fusion proteins to hOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[312] Fig. 85 (Figs. 85A-85C) shows a list of sequences for exemplary ABDs that bind to human OX40, as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences underlined for select VH / VL ABD pairs.

[313] Fig. 86 shows a list of exemplary IL-12 Fc fusion proteins, including ABD specificity, IL-12 p35 subunit domain mutations, and the amino acid sequence identifiers for such exemplary IL-12 Fc fusion proteins.

[314] Fig. 87 shows the binding of an anti-human OX40 antibodies in traditional IgG1 bivalent format and OX40-targeted IL-12 Fc fusion proteins to hOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[315] Fig. 88 shows EC50 values for the binding of an anti-human OX40 antibody in traditional IgG1 bivalent format and OX40-targeted IL-12 Fc fusion proteins to hOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[316] Fig. 89 shows binding of OX40L-Fc to OX40 positive hOX40-Jurkat-NF-κB cells in the presence of anti-human OX40 antibodies in traditional IgG1 bivalent format or OX40-targeted IL-12 Fc fusion proteins.

[317] Fig. 90 (Figs. 90A and 90B) shows expression of OX40 (on the left y-axis) and the percentage of GFP+ hOX40-Jurkat-NF-κB cells (on the right y-axis) following incubation with 10 nM OX40L-Fc and / or various concentrations of the indicated IL-12 ABD fusion protein for either 1 hour (1h) or overnight (o / n). GFP measurements were only performed following overnight incubation. Horizonal dashed lines indicate the percentage of GFP+ cells following incubation with OX40L-Fc alone. Horizonal dotted lines indicate the OX40 MFI following overnight incubation with OX40L-Fc alone.

[318] Fig. 91 (Figs. 91A and 91B) shows expression of OX40 (on the left y-axis) and the percentage of GFP+ hOX40-Jurkat-NF-κB cells (on the right y-axis) following incubation with 10 nM OX40L-Fc and / or various concentrations of the indicated IL-12 ABD fusion protein for either 1 hour (1h) or overnight (o / n). GFP measurements were only performed following overnight incubation. Only cells that expressed high levels of OX40 were included in the analysis. Horizonal dashed lines indicate the percentage of GFP+ cells following incubation with OX40L-Fc alone.

[319] Fig. 92 (Figs. 92A and 92B) shows flow cytometry analysis of PBMC derived from two healthy donors (B113 (Fig. 92A) and B121 (Fig. 92B)) that were activated with 600 U / mL IL-2 and 32 ng / mL OKT3. OX40 positive cells are indicated by horizontal lines.

[320] Fig. 93 shows AlphaLISA measurements of IFNγ in the medium of IL-2 / OKT3 activated human PBMC that were incubated with wild type IL-12 Fc (WT IL-12 Fc) or the indicated IL-12-ABD fusion proteins, in the presence of 50 U / mL human IL-2. Data is shown for two PBMC donors (B113 (top panel) and B121 (bottom panel)). Symbols and error bars indicate the mean and SD, respectively.

[321] Fig. 94 shows a graph of EC50 values for wild type IL-12 Fc (WT IL-12 Fc) and various IL-12-ABD fusion proteins determined from a PBMC IFNγ release assay. Symbols represent individual measurements and horizontal lines indicate the median value for each treatment group.

[322] Fig. 95 shows a summary of EC50 values for wild type IL-12 Fc (WT IL-12 Fc) and various IL-12-ABD fusion proteins determined by PBMC IFNγ release assays.

[323] Fig. 96 (Figs. 96A-96C) shows AlphaLISA measurements of IFNγ in the medium of purified CD4+ T cells (top row), CD8+ T cells (middle row), and NK cells (bottom row) from 3 different PBMC donors (B130 (Fig. 96A), B134 (Fig. 96B), and B177 (Fig. 96C)) that were either freshly isolated (D0, left column), activated for 2 days (D2, middle column), or maintained in culture for 5 days post activation (D7, right column) and treated with WT IL-12 Fc or the indicated IL-12 ABD fusion protein in the presence of 50 U / mL IL2 (for T cells) and 1 ng / mL IL-15 (for NK cells). Symbols and error bars indicate the mean and SD, respectively.

[324] Fig. 97 shows the expression of OX40 (left column for each panel), IL-12Rb1 (middle column for each panel), and IL-12Rb2 (right column for each panel) on purified CD4+ T cells, CD8+ T cells and NK cells from 3 different PBMC donors (B130 (top left panel), B134 (top right panel), and B177 (bottom panel)) that were either freshly isolated (D0; top row for each panel), activated for 2 days (D2; middle row for each panel), or maintained in culture for 5 days post activation (D7; bottom row for each panel).

[325] Fig. 98 (Figs. 98A and 98B) shows expression of IL-12Rb2 (Fig. 98A) and OX40 (Fig. 98B) on purified NK cells (left column), CD4+ T cells (middle column) and CD8+ T cells (right column) that were either freshly isolated (D0, top row), activated for 2 days (D2, middle row) or maintained in culture for 5 days post activation (D7, bottom row).

[326] Fig. 99 (Figs. 99A and 99B) shows AlphaLISA measurements of IFNγ in the medium of purified CD4+ T cells (CD4, top row), CD8+ T cells (CD8, middle row) and NK cells (NK, bottom row) that were either freshly isolated (D0, left column), activated for 2 days (D2, middle column) or maintained in culture for 5 days post activation (D7, right column) and treated with WT IL-12 Fc or the indicated IL-12 ABD fusion protein in the presence of 50 U / mL IL2 (for T cells) and 1 ng / mL IL-15 (for NK cells). Symbols and error bars indicate the mean and SD, respectively.

[327] Fig. 100 shows the maximum amount of IFNγ produced by purified CD4+ T cells, CD8+ T cells and NK cells (as indicated) in response to treatment with WT IL-12 Fc (as depicted in Fig. 99). Cells were either freshly isolated (D0), activated for 2 days (D2) or maintained in culture for 5 days post activation (D7). IFNγ measurements for CD4+ T cells and CD8+ T cells on D0 were below the lower limit of quantification of the assay and were assigned an arbitrary value of 0 pg / ml.

[328] Fig. 101 shows AlphaLISA measurements of IFNγ in the medium of freshly isolated purified CD4+ T cells that were treated with WT IL-12 Fc or the indicated IL-12 ABD fusion protein in the absence of IL-15. Symbols and error bars indicate the mean and SD, respectively. The horizontal dotted line indicated the lower limit of quantification of the assay.

[329] Fig. 102 (Figs. 102A-102D) shows the phenotype of conventional CD4+ cells (ConCD4; top row and third row) that had been maintained in culture for 7 days post CD3 / 28 activation, and induced Treg cells (Treg; second row and fourth row) that had been activated / differentiated for 10 days in the presence of CD3 / 28 Dynabeads, 500 U / mL of human IL-2, 5 ng / mL TGF-b and 10 nM retinoic acid.

[330] Fig. 103 shows AlphaLISA measurements of IFNγ in the medium of conventional CD4+ T cells (CD4+conv; top left panel and bottom left panel) and induced Treg cells (Treg; top right panel and bottom right panel) from 2 different PBMC donors (B363 (top left panel and bottom left panel) and B364 (top right panel and bottom right panel)) that were activated with CD3 / 28 Dynabeads and maintained in culture for 10 days in the absence or presence of 5 ng / mL TGF-b and 10 nM retinoic acid, respectively. Symbols and error bars indicate the mean and SD of 2 replicates, respectively.

[331] Fig. 104 shows a summary of EC50 values and fold change (relative to WT IL-12 Fc) for wild type IL-12 Fc (WT IL-12 Fc) and various IL-12-ABD fusion proteins determined by IFNγ release assays performed in conventional CD4+ T cells (CD4+conv) and induced Treg cells (Treg) from 2 different PBMC donors (B363 and B364).

[332] Fig. 105 shows the percentage of OX40+ / CD4+ cells (top left panel and bottom left panel) and OX40- / CD4+ cells (top right panel and bottom right panel) derived from two independent PBMC donors (B130 (top left panel and top right panel) and B323 (bottom left panel and bottom right panel)) that were positive for phosphorylated STAT4 (pStat4+) after a 30-minute incubation with wild type IL-12 Fc (WT IL-12 Fc) or IL-12-ABD fusion proteins.

[333] Fig. 106 shows flow cytometry analysis of purified CD4+ T cells (containing both conventional CD4+ T cells (ConCD4) and Treg cells, as depicted in the left panel) that were activated for 2 days with CD3 / 28 Dynabeads and 100 U / mL IL-2 and maintained in culture for 5 days post activation. The middle and right panels show OX40 expression on conventional CD4+ T cells (ConCD4) and Treg cells, respectively.

[334] Fig. 107 shows the percentage of OX40+ / Treg cells (top left panel), OX40- / Treg cells (bottom left panel), OX40+ / conventional CD4 cells (top right panel) and OX40- / conventional CD4 cells (bottom right panel) that were positive for phosphorylated STAT4 (pStat4+) after a 30-minute incubation with wild type IL-12 Fc (WT IL-12 Fc) or IL-12-ABD fusion proteins.

[335] Fig. 108 shows histograms representing the expression of human OX40 (top panel), human IL-12Rb1 (middle panel), and human IL-12Rb2 (bottom panel) on Jurkat NF-κB reporter cells (parental Jurkat) and hOX40 / hIL-12R / Jurkat NF-κB reporter cells (hIL-12Rb1 / hIL-12Rb2 / hOX40.Jurkat).

[336] Fig. 109 (Figs. 109A and 109B) shows the percentage of GFP positive hOX40 / hIL-12R / Jurkat NF-κB reporter cells following overnight incubation with anti-human OX40 antibodies in traditional IgG1 bivalent format and OX40-targeted IL-12 Fc fusion proteins in the presence (Fig. 109A) or absence (Fig. 109B) of A20 cells that expressed human FcγRI (A20-FcγRI). Data were normalized such that unstimulated cells were attributed a value of 0 % GFP positive.

[337] Fig. 110 shows AlphaLISA measurements of IFNγ in the medium of activated PBMC that were stimulated with plate bound anti-CD3 and plate bound anti-human OX40 in traditional IgG1 bivalent format (hG1-BGB-A445 (PB)), or plate bound anti-CD3 and either WT IL-12 Fc or IL-12 ABD fusion proteins. Prior to stimulation, genes encoding IL-12Rb2 (top right panel), STAT4 (bottom left panel), or OX40 (bottom right panel) were knocked out using CRISPR / Cas9 genome editing, as indicated. AAVS1 (top left panel) indicates CRISPR / Cas9 edited cells whereby the targeting ribonucleoproteins were specific for a gene desert (used as a negative control). Symbols and error bars indicate the mean and SD, respectively.

[338] Fig. 111 shows the growth of MC38 tumor cells following subcutaneous implantation into naïve C57BL / 6 mice (treatment naïve; top panel) and C57BL / 6 mice that had previously been inoculated with MC38 cells and treated with a single dose of 0.143 mg / kg mono-Y36E-OX86 (previously treated; bottom panel). Solid lines indicate tumor growth in individual mice (n=5 per group).

[339] Fig. 112 shows a list of sequences for exemplary ABDs that bind to human OX40, as well as exemplary heavy chain and light chain amino acid sequences for use in a subject IL-12 Fc fusion protein. vhCDR1-3 and vlCDR1-3 sequences are underlined for select VH / VL ABD pairs.

[340] Fig. 113 shows the binding of anti-human OX40 antibodies in traditional IgG1 bivalent format to hOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[341] Fig. 114 shows the binding of anti-human OX40 antibodies in traditional IgG1 bivalent format to cOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[342] Fig. 115 shows EC50 values for the binding of an anti-human OX40 antibody in traditional IgG1 bivalent format and OX40-targeted IL-12 Fc fusion proteins to hOX40-Jurkat-NF-κB cells as determined by flow cytometry.

[343] Fig. 116 shows binding of OX40L-Fc to OX40 positive hOX40-Jurkat-NF-κB cells in the presence of anti-human OX40 antibodies in traditional IgG1 bivalent format.

[344] Fig. 117 (Figs. 117A-117C) shows expression of OX40 (on the left y-axis) and the percentage of GFP+ hOX40-Jurkat-NF-κB cells (on the right y-axis) following incubation with 10 nM OX40L-Fc and / or various concentrations of the indicated anti-OX40 antibodies for either 10 minutes (10 min) or overnight (o / n). GFP measurements were only performed following overnight incubation. Horizonal dashed lines indicate the percentage of GFP+ cells following incubation with OX40L-Fc alone. Horizonal dotted lines indicate the OX40 MFI following overnight incubation with OX40L-Fc alone.

[345] Fig. 118 (Fig. 118A and Fig. 118B) shows the percentage of GFP positive hOX40-Jurkat NF-κB reporter cells following overnight incubation with anti-human OX40 antibodies in traditional IgG1 bivalent format in the presence (Fig. 118A) or absence (Fig. 118B) of A20 cells that expressed human FcγRI (A20-FcγRI). Data were normalized such that unstimulated cells were attributed a value of 0 % GFP positive.Detailed Description of the Invention

[346] The description is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its requirements. The section headings used herein are for organization purposes only and are not to be construed as limiting the subject matter described. While various embodiments of the invention(s) of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention(s). It should be understood that various alternatives to the embodiments of the invention(s) described herein may be employed in practicing any one of the inventions(s) set forth herein.

[347] All patents, published patent applications, other publications, sequences from GenBank, sequences from UniProt (including, but not limited to, UniProt accession numbers: P43431, P43432, Q60837, and P97378) and other databases referred to herein are incorporated by reference in their entirety respective to the related technology.I. Definitions

[348] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For purposes of the present disclosure, the following terms are defined below. The definitions provided are intended to apply to a given term, as well as other derivative linguistic re-phrasings and grammatical equivalents of the term.

[349] 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 attached amino acids, which includes proteins, polypeptides, oligopeptides, and peptides. When a “biologically functional molecule” comprises two or more proteins, such as, for example, IL-12 (which comprises an IL-12 p35 polypeptide and an IL-12 p40 polypeptide), the individual proteins comprising the 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 a functional complex are non-covalently attached. In some embodiments, the two or more proteins of a functional complex are covalently attached, such as, for example, through a disulfide bond.

[350] As used herein, the term “cytokine” refers to a broad category of proteins, such as, for example, chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and the like, that are secreted by a first cell to cause an effect on one or more cells through a binding of the secreted cytokine to a receptor on the one or more cells. Cytokines may be involved in autocrine, paracrine, juxtacrine, and / or endocrine signaling.

[351] As used herein, the term “immunocytokine” generally refers to a fusion protein comprising a cytokine (e.g., IL-12) and at least one antibody domain (e.g., a variable heavy domain, a variable light domain, a constant heavy domain, a constant light domain, a VHH domain, and the like).

[352] As used herein, the term “wild-type” refers to an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A wild-type protein has an amino acid sequence (or a nucleotide sequence encoding the amino acid sequence) that has not been intentionally modified.

[353] As used herein, the term “IL-12 p35 subunit” refers to the human wild-type IL-12 p35 polypeptide, whether native or recombinant, unless the context clearly dictates otherwise. As such, an IL-12 p35 subunit refers to recombinantly produced IL-12 p35 polypeptide, synthetically produced IL-12 p35 polypeptide, as well as IL-12 p35 extracted from cells or tissues. An amino acid sequence of human wild-type IL-12 p35 subunit is depicted in Fig. 1 (Precursor: SEQ ID NO: 1; Mature: SEQ ID NO: 2). An amino acid sequence of mouse wild-type IL-12 p35 subunit is depicted in Fig. 3 (Precursor: SEQ ID NO: 9; Mature: SEQ ID NO: 10). In the context of a fusion protein, the IL-12 p35 subunit may also be referred to as an “IL-12 p35 subunit domain,” wherein the IL-12 p35 subunit domain comprises at least part of the amino acid sequence encoding the IL-12 p35 subunit. In many of these instances, the IL-12 p35 subunit domain comprises one or more mutations to the IL-12 p35 subunit “parent protein.” In such instances, the IL-12 p35 subunit domain is referred to herein as a “variant IL-12 p35 subunit domain.”

[354] As used herein, the term “IL-12 p40 subunit” refers to the human wild-type IL-12 p40 polypeptide, whether native or recombinant, unless the context clearly dictates otherwise. As such, an IL-12 p40 subunit refers to recombinantly produced IL-12 p40 polypeptide, synthetically produced IL-12 p40 polypeptide, as well as IL-12 p40 extracted from cells or tissues. An amino acid sequence of human wild-type IL-12 p40 subunit is depicted in Fig. 1 (Precursor: SEQ ID NO: 3; Mature: SEQ ID NO: 4). An amino acid sequence of mouse wild-type IL-12 p40 subunit is depicted in Fig. 3 (Precursor: SEQ ID NO: 11; Mature: SEQ ID NO: 12). In the context of a fusion protein, the IL-12 p40 subunit may also be referred to as an “IL-12 p40 subunit domain,” wherein the IL-12 p40 subunit domain comprises at least part of the amino acid sequence encoding the IL-12 p40 subunit. In some of these instances, the IL-12 p40 subunit domain comprises one or more mutations to the IL-12 p40 subunit “parent sequence.” In such instances, the IL-12 p40 subunit domain is referred to herein as a “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 wherein the IL-12 p40 subunit (or, in the case of fusion proteins, IL-12 p40 subunit domain) may comprise a wildtype IL-12 p40 polypeptide or a variant IL-12 p40 subunit (domain) as if each embodiment had been set forth individually.

[355] 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 further embodiments, the C-terminus of the IL-12 p35 subunit (domain) is connected to the N-terminus of the IL-12 p40 subunit (domain) (sc-IL-12(p35 / p40)). In yet further embodiments, the sc-IL-12(p35 / p40) further comprises a “linker,” wherein the C-terminus of the IL-12 p35 subunit is linked to the N-terminus of the linker and the C-terminus of the linker is linked 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 further embodiments, the sc-IL-12(p40 / p35) further comprises a linker, wherein the C-terminus of the IL-12 p40 subunit is linked to the N-terminus of the linker and the C-terminus of the linker is linked to the N-terminus of the IL-12 p35 subunit. In many of these embodiments, the IL-12 p35 subunit (domain) comprises a variant IL-12 p35 subunit (domain). Further, in some of these embodiments, the IL-12 p40 subunit (domain) comprises a variant IL-12 p40 subunit (domain).

[356] As used herein, the terms “linker” and “domain linker,” which can be used interchangeably, generally refer to a structure (such as, for example, a polymer (e.g., any one of the amino acid sequences comprising SEQ ID NOs: 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 a 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., a VH domain, a VL domain, a CH1 domain, a Fc domain, etc.), a first subunit of a cytokine and a second subunit of the cytokine, and the like). Such a linkage may be “direct” (i.e., in instances where the domain linker comprises a covalent bond) or “indirect” (i.e., in instances where the domain linker comprises a structure, such as, for example, any one of the amino acid sequences of SEQ ID NOs: 31-40). In many embodiments, a plurality of domain linkers is incorporated into a subject “low potency IL-12 Fc fusion proteins,” as is described in further detail herein. In some instances, a subject low potency IL-12 Fc fusion protein may comprise one or more domain linkers. In some further instances, the one or more domain linkers comprise one or more of the amino acid sequences of SEQ ID NOs: 31-40, and may optionally comprise one or more covalent bonds. In some other further instances, 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 NOs: 31-40. In some instances, at least one domain linker comprises a hinge region of a Fc domain.

[357] As used herein, the term “residue” refers to a position in a protein and its associated amino acid identity. For example, Cysteine 252 (also referred to as Cys252 or C252) is a residue at position 252.

[358] As used herein, the term “parent protein” refers to a “reference” protein, the amino acid sequence that encodes the reference protein, or the DNA sequence that encodes the amino acid sequence that encodes the reference protein. In some embodiments, the reference protein comprises a wild-type protein, the amino acid sequence encoding the wild-type protein, and / or the nucleic acid sequence encoding the amino acid sequence encoding the wild-type protein. In some embodiments, the reference protein comprises a human wild-type protein, the amino acid sequence encoding the human wild-type protein, and / or the nucleic acid sequence encoding the amino acid sequence encoding the human wild-type protein. In some embodiments, the reference protein comprises 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).

[359] As used herein, the terms “variant protein,” “protein variant,” or “variant” refer to a protein that differs from that of a parent protein by virtue of 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 terms may refer to the protein itself, a composition comprising the protein, the amino acid sequence that encodes it, or the DNA sequence that encodes it. In some embodiments, the parent protein refers to a wild-type sequence. In some embodiments, the parent protein refers to a human wild-type sequence. Thus, a “variant” of an IL-12 p35 subunit (or “variant” of an IL-12 p35 subunit domain) refers to a polypeptide in which 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 are present as compared to the amino acid sequence of a reference IL-12 p35 subunit, such as, for example, a (human) wild-type IL-12 p35 subunit. Further, a “variant” of an IL-12 p40 subunit (or “variant” of an IL-12 p40 subunit domain) refers to a polypeptide in which 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 are present as compared to the amino acid sequence of a reference IL-12 p40 subunit, such as, for example, a (human) wild-type IL-12 p40 subunit.

[360] As used herein, the term “modification” refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein. As used herein, the term “amino acid modification” refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. The position(s) where the amino acid(s) are modified and the number of amino acid(s) that may be modified in the amino acid sequence are not particularly limited.

[361] As will be appreciated by those skilled in the relevant art, the modifications to the IL-12 p35 subunit (domain) described throughout refer to modifications made to the mature form of the sequence (SEQ ID NO: 2) and / or variants thereof, as opposed to the precursor sequence (SEQ ID NO: 1). The precursor sequence for the IL-12 p35 subunit comprises an additional 22 amino acid residues on the N-terminus comprising the sequence: MCPARSLLLVATLVLLDHLSLA (SEQ ID NO: 91). However, the listed modifications to the mature form of the IL-12 p35 subunit can be made to the precursor form of the IL-12 p35 subunit after correcting the position of the stated modification in view of the additional leading amino acid residues in the precursor sequence. For example, the substitution modification Y40A is one of many potential modifications to the IL-12 p35 subunit mature sequence but can also refer to a substitution modification of the IL-12 p35 subunit precursor sequence comprising Y62A.

[362] Similarly, modifications to the IL-12 p40 subunit (domain) described throughout are intended to refer to modifications made to the mature form of the sequence (SEQ ID NO: 4) and / or variants thereof, as opposed to the precursor sequence (SEQ ID NO: 3). The precursor sequence for the IL-12 p40 subunit comprises an additional 22 amino acid residues on the N-terminus comprising the sequence: MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 92). However, it will also be appreciated that the listed modifications to the mature form of the IL-12 p40 subunit can be made to the precursor form of the IL-12 p40 subunit after correcting the position of the stated modification in view of 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 IL-12 p40 subunit mature sequence but can also refer to a substitution modification of the IL-12 p40 subunit precursor sequence comprising C199S.

[363] As used herein, the terms “amino acid substitution” or “substitution” refer to the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. For example, Y40A designates a substitution of tyrosine at position 40 with an alanine at the same position. In some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For clarity, a protein that has been engineered to change the nucleic acid coding sequence but not to change the resulting amino acid (for example, exchanging CCU (encoding proline) to CCC (still encoding proline)) is not an “amino acid substitution.” Phrased differently, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.

[364] As used herein, the terms “amino acid insertion” or “insertion” refer to the addition of an amino acid residue or sequence at a particular position in a parent polypeptide sequence. For example, -40A designates an insertion of alanine after position 40 and before position 41. As a separate example, D8EPKSS or -8EPKSS designates an insertion of the sequence Glu-Pro-Lys-Ser-Ser after position 8 and before position 9.

[365] As used herein, the terms “amino acid deletion” or “deletion” refer to the removal of an amino acid or sequence at a particular position in a parent polypeptide sequence. For example, Y40-, Y40#, Y40( ), or Y40del designates a deletion of tyrosine at position 40. As a separate example, EPKSS8-, EPKSS8#, EPKSS8del designates a deletion of the sequence Glu-Pro-Lys-Ser-Ser that begins at position 8.

[366] As used herein, the terms “non-naturally occurring protein” or “non-naturally occurring protein variant” refer to a variant protein that differs from that of a parent protein by virtue of 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 that are not isotypic. For example, because the IL-12 p35 subunit of IL-12 does not comprise an alanine at position 40, the substitution Y40A is considered a non-naturally occurring IL-12 p35 variant (or, more generally, a non-naturally occurring IL-12 variant). Modifications to a protein that are not isotypic can be referred to as “non-naturally occurring modifications.”

[367] As used herein, the terms “percent (%) identity” and “percent (%) sequence identity,” when used in the context of two or more proteins or nucleic acids, refer to a percentage of amino acid residues (or nucleic acids encoding the amino acid residues) in a candidate sequence that are identical with the amino acid residues (or nucleic acids encoding the amino acid residues) in a specific sequence, such as, for example, the amino acid sequence of a parent protein, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill 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 needed to achieve maximal alignment over 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.

[368] As used herein, the term “half-life” of an agent can refer to the time it takes for the agent to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism or subject, or relative to any other defined time-point. “Half-life” can also refer to the time it takes for the amount or concentration of an agent to be reduced by half of a starting amount administered into the serum or tissue of an organism or subject, relative to such amount or concentration at the time of administration into the serum or tissue of an organism or subject, or to any other defined time-point. The half-life can be measured in serum and / or any one or more selected tissues.

[369] As used herein, the terms “host cell” and “recombinant cell” refer to an individual cell or a cell culture that can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide(s). A host cell can be a transfected, transformed, transduced, or infected cell of any origin, including, but not limited to, prokaryotic, eukaryotic, mammalian, avian, insect, plant, or bacteria cells, or it can be of any origin that can be used to propagate a nucleic acid described herein. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell that comprises a recombinant vector of the invention may be called a “recombinant host cell.”

[370] Host cells can include, without limitation, the cells of mammals, plants, insects, fungi, and bacteria. Bacterial cells include, without limitation, the cells of Gram-positive bacteria, such as, for example, species of the genus Bacillus, Streptomyces, and Staphylococcus, and cells of Gram-negative bacteria, such as, for example, cells of the genus Escherichia and Pseudomonas. Fungal cells can include, without limitation, yeast cells, such as, for example, Saccharomyces, Pichia pastoris, and Hansenula polymorpha. Insect cells can include, without limitation, cells of Drosophila and Sf9 cells. Plant cells include, without limitations, cells from crop plants, medicinal or ornamental plants or bulbs. Suitable mammal cells for the present invention include, but are not limited to, epithelial cell lines (e.g., porcine epithelial cells), osteosarcoma cell lines, neuroblastoma cell lines, epithelial carcinomas, glial cells, liver cell lines, Chinese hamster ovary (CHO) cells, COS cells, BHK cells, HeLa cells, D3 cells of the line of murine embryonic stem cells (mESCs), human embryonic stem cells (e.g., HS293 cells and BG01V cells), NIH 3T3 cells, human embryonic kidney (HEK) 293T cells, human mesenchymal stem cells (hMSCSs), and the like.

[371] As used herein, the terms “cell,” “cell culture,” “cell line,” and “host cell” refer not only to the particular cell, cell culture, cell line, or host cell, but also to the progeny or potential progeny of such a cell, cell culture, cell line, or host cell, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the terms as used herein, so long as the progeny retain the same functionality or substantially the same functionality as that of the original cell, cell culture, cell line, or host cell.

[372] As used herein, the terms “medium” or “culture medium” include any culture medium, solution, solid, semi-solid, or rigid support that may support or contain any host cells, 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, E. coli host cells, Pseudomonas host cells, and the like, and cell contents. Thus, the term may encompass medium in which the host cell has been grown, (e.g., medium into which a polypeptide has been secreted, including medium either before, during, or after a proliferation step). The term also may encompass buffers or other reagents that contain host cell lysates, such as, for example, in the case where a polynucleotide is produced intracellularly, and the host cells are lysed or disrupted to release the polypeptide.

[373] As used herein, the term “fusion protein” refers to a covalent joining of at least two proteins or protein domains. Fusion proteins may comprise artificial sequences, such as, for example, a domain linker, Fc domains, variant Fc domains, a variant IL-12 p35 subunit domain, a(n) (variant) IL-12 p40 subunit domain, and the like, as described herein. As used herein, the term “Fc fusion protein” refers to a protein comprising a (variant) Fc domain, generally linked (optionally through 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 through 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 the one or more different protein domains). In some embodiments, the N-terminus of the Fc domain is linked to the C-terminus of one or more different protein domains (optionally through a domain linker, wherein the C-terminus of the one or more protein domains 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 the Fc domain).

[374] As used herein, the terms “low potency IL-12 Fc fusion proteins,” “IL-12-ABD fusion proteins,” “IL-12 Fc fusion proteins,” or “targeted IL-12 Fc fusion proteins,” which can be used interchangeably, 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 set of amino acid substitutions that reduces binding affinity of the resultant IL-12 Fc fusion protein for IL-12Rβ2 as compared to a reference IL-12, wherein the set of amino acid substitutions is selected from a group including: (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 a target antigen selected from a group including: (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. As will be appreciated by those skilled in the art, a subject IL-12 Fc fusion protein can take on one of several different configurations (including, but not limited to, those described herein and shown in the figures (see, e.g., Figs. 18A-18H)). By way of a first example, a subject IL-12 Fc fusion protein can be configured such that the ABD binds 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 comprises 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 comprises a mouse target antigen. In some embodiments, the target antigen comprises a camelid target antigen. In some embodiments, the target antigen comprises a cynomolgus monkey target antigen. In some further embodiments, the ABD is a humanized ABD. As another example, a subject IL-12 Fc fusion protein can be configured such that the ABD is in a Fab format, a scFv format, a nanobody format, as well as other formats known in the relevant art (including, but not limited to: a diabody format, a triabody format, a minibody format, a single-chain Fab (scFab) format, a F(ab)2 format, a scFv-Fc format, a BiTE format, an intrabody format, a tandem scFv format, a DART format, and the like). In some instances, the IL-12 Fc fusion protein is configured such that the ABD is in a Fab format (i.e., the IL-12 Fc fusion protein comprises a variable heavy (VH) domain, a variable light (VL) domain, a constant heavy (CH1) domain, and a constant light (CL) domain). In some other instances, the IL-12 Fc fusion protein is configured such that the ABD is in an scFv format (i.e., the IL-12 Fc fusion protein comprises, from N-terminus to C-terminus, a VH domain, a linker, and a VL domain; or the reverse where the IL-12 Fc fusion protein comprises, from N-terminus to C-terminus, a VL domain, a linker, and a VH domain). In still other instances, the IL-12 Fc fusion protein is configured such that the ABD is in a nanobody format (i.e., the IL-12 Fc fusion protein comprises a VHH domain, wherein the VHH domain comprises a complementarity determining region (CDR) 1, a CDR2, and a CDR3, wherein the CDR1, CDR2, and CDR3 bind to a target antigen). As another example, a subject IL-12 Fc fusion protein can be configured such that the fusion protein comprises one ABD (i.e., a monovalent, monospecific format), two ABDs (e.g., a bivalent, monospecific format, or a monovalent, bispecific format), or three or more ABDs (e.g., a monovalent, multispecific format, a multivalent, monospecific format, a multivalent, multispecific format, and the like). In instances where the subject IL-12 Fc fusion protein comprises two ABDs, the first ABD and the second ABD may bind the same target antigen (i.e., a bivalent, monospecific format), or the first ABD may bind a first target antigen and the second ABD may bind a second, distinct target antigen (i.e., a monovalent, bispecific format). As will be appreciated by those skilled in the relevant art, a subject IL-12 Fc fusion protein comprising three or more ABDs can be configured in similar manners depending on the total number of ABDs and the specificity of such ABDs (e.g., monovalent, multispecific formats; multivalent, monospecific formats; multivalent, multispecific formats; and the like). As another example, a subject IL-12 Fc fusion protein can be configured such that the first Fc domain and / or the second Fc domain comprises a wild-type Fc domain or a Fc domain that comprises one or more substitution mutations. In some embodiments, at least one of the first Fc domain and second Fc domain comprises a variant Fc domain (e.g., the first Fc domain comprises a variant Fc domain and the second Fc domain either comprises a Fc domain or a variant Fc domain, the second Fc domain comprises a variant Fc domain and the first Fc domain either comprises a Fc domain or a variant Fc domain). In some embodiments, the first Fc domain and / or the second Fc domain comprise modifications that promote heterodimerization of the first and second Fc domains. In some embodiments, the first Fc domain and / or the second Fc domain comprises one or more modifications that alter Fc binding. In some embodiments, the first Fc domain and / or the second Fc domain comprises one or more modifications that alter Fc half-life. In some embodiments, the first Fc domain and / or the second Fc domain comprises 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 certain further embodiments, the first Fc domain and / or the second Fc domain comprise one or more modifications that increase half-life and / or binding to FcRn. As another example, a subject IL-12 Fc fusion protein can be configured such that: (1) the first IL-12 subunit domain and the second IL-12 subunit domain are on the same monomer of the subject IL-12 Fc fusion protein (covalently linked using a domain linker in several embodiments (e.g., from N-terminus to 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 on separate monomers of the subject IL-12 Fc fusion protein (e.g., a first monomer and a second monomer, respectively, or the reverse), or (3) the first IL-12 subunit domain is covalently attached to a monomer of the subject IL-12 Fc fusion protein and the second IL-12 subunit domain is expressed as a soluble protein, wherein the soluble IL-12 subunit interacts with / binds the IL-12 subunit covalently attached to a monomer of the subject IL-12 Fc fusion protein. Exemplary configurations wherein one IL-12 subunit is covalently attached to a subject IL-12 Fc fusion protein and one IL-12 subunit is expressed as a soluble fragment is described in PCT Publication No. WO 2023 / 242769 A1, which is incorporated 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 linked to the N-terminus of the first Fc domain (optionally through a domain linker), and the C-terminus of the second IL-12 subunit domain is linked to the N-terminus of the second Fc domain (optionally through a domain linker). In some embodiments, the C-terminus of the first Fc domain is linked to the N-terminus of the first IL-12 subunit domain (optionally through a domain linker), and the C-terminus of the second Fc domain is linked to the N-terminus of the second IL-12 subunit domain (optionally through a domain linker).

[375] As used herein, the term “isolated,” when used to describe the various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from a cell (e.g., a host cell and / or a cell line) or cell culture from which it was expressed. Generally, an isolated polypeptide will be prepared by at least one purification step. As used herein, the term “isolated protein” refers to a protein which is substantially free of other proteins from a cell culture, such as, for example, host cell proteins.

[376] As used herein, the terms “Fc,” “Fc region,” or “Fc domain,” which can be used interchangeably, refer to the polypeptide comprising the CH2-CH3 domains of an immunoglobulin G (IgG) molecule, and in some cases, inclusive of all or part of the hinge, as well as variants thereof. In EU numbering for human IgG1, the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Thus, the definition of “Fc domain” includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. A “Fc fragment” in this context may contain fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another Fc domain or Fc fragment as can be detected using standard methods, generally based on size (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). Herein, unless specifically outlined, a “Fc domain” generally refers to the CH2-CH3 domains (and optionally all or part of the hinge) of human IgG1. Described below are various human IgG1 Fc domains, at least some of which comprise one or more modifications. Unless the context clearly indicates otherwise, amino acid position numbering with respect to a Fc domain (e.g., a first Fc domain, a second Fc domain, etc.) is according to the EU index. The “EU index” or “EU index as in Kabat” or “EU numbering” scheme, which can be used interchangeably, refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference). For clarity, while the modifications are discussed herein primarily in the context of human IgG1 Fc domains for the sake of brevity, it is expressly contemplated that mutations to other immunoglobulins, such as, for example, IgG2, IgG3, IgG4, IgA, IgM, and IgE, can be made at residue positions corresponding to the mutations in human IgG1 that are described herein. Those skilled in the relevant art will readily be able to determine residue locations in other immunoglobulins that correspond with the modifications to human IgG1 described herein.

[377] 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 generally linked to (e.g., inserted into) the vector nucleic acid molecule. Generally, a vector is capable of replication when associated with the proper control elements. The term “vector” includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region, thereby capable of expressing DNA sequences and fragments thereof in vitro and / or in vivo. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, but are not limited to, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, but are not limited to, replication defective retroviruses and lentiviruses. In some embodiments, a vector is a gene delivery vector. In some embodiments, a vector is used as a gene delivery vehicle to transfer a gene into a cell.

[378] As used herein, the term “recombinant,” with respect to a nucleic acid molecule, refers to a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin which, by virtue of its own origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant,” as used with respect to a protein or polypeptide, refers to a polypeptide produced by expression of a recombinant polynucleotide. The term “recombinant,” as used with respect to a host cell, refers to a host cell into which a recombinant polynucleotide or vector comprising a recombinant polynucleotide has been introduced.

[379] As used herein, the term “operably linked” refers to a physical or functional linkage between two or more elements (e.g., polypeptide sequences or polynucleotide sequences) that permits them to operate in their intended fashion. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (such as, for example, a promoter) is a functional link that allows for expression of the polynucleotide of interest. In this sense, the term “operably linked” refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. Thus, a promoter is in an operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence. It should be understood that elements that are operably linked may be contiguous or non-contiguous. In the context of a polypeptide, “operably linked” refers to a physical linkage (e.g., directly, or indirectly linked) between amino acid sequences (e.g., different segments, modules, or domains) to provide for a described activity of the polypeptide.

[380] As used herein, the term “binding affinity” refers to the “strength” of binding of a given molecule (such as, for example, a variant IL-12 p35 subunit domain, or an antigen binding domain) to its ligand (such as, for example, IL-12Rβ2, PD1, LAG3, OX40, ICOS, 41BB, CCR7, CCR8, etc.) and / or the rate at which the molecule associates with and / or dissociates from its ligand. Binding affinity is often expressed in terms of the dissociation constant (KD). The binding activity of the IL-12 Fc fusion proteins of the disclosure can be assayed by any suitable method known in the art, such as, for example, a surface plasmon resonance (SPR) assay, an enzyme-linked immunosorbent assay (ELISA), an ELISpot assay, Biacore assays, KinExA assays, and the like. As the IL-12 Fc fusion proteins bind to more than 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 separate binding affinities for IL-12Rβ2 and the at least one target antigen. As such, 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 a subject IL-12 Fc fusion protein, or to the “strength” of binding to the target antigen when referring to the ABD of a subject IL-12 Fc fusion protein. Further, some configurations of IL-12 Fc fusion proteins described herein comprise more than one ABD and are therefore capable of binding more than one target antigen. As such, in such embodiments, the term “binding affinity” is intended to refer to the “strength” of binding to a first target antigen by a first ABD (e.g., the strength of binding to PD1 by an ABD that “specifically binds” to PD1) or the “strength” of binding to a second target antigen by a second ABD (e.g., the strength of binding to LAG3 by an ABD that “specifically binds” to LAG3).

[381] As used herein, the terms “specific binding,” “specifically binds to,” or is “specific for” a particular antigen or an “epitope,” which can be used interchangeably, means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[382] As used herein, the term “epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.

[383] As used herein, the term “potency” refers to the ability of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) to elicit a response at a certain dose or concentration in a given biological system or experimental setting. The potency of the IL-12 Fc fusion proteins of the disclosure can be assayed by any suitable method known in the art, such as, for example, an IL-12 HEK reporter assay (e.g., InvivoGen’s IL-12 HEK reporter assay (Catalog No. hkb-il12)), a ligand-binding assay (e.g., ELISA or flow cytometry), and / or a functional assay (e.g., AlphaLISA). Generally, changes in potency may be demonstrated graphically as a leftward or rightward shift in a response curve compared to a control. A rightward shift of the response curve is generally indicative of a reduction in potency, whereas as a leftward shift of the response curve is generally indicative of an increase in potency.

[384] As used herein, the term “activity” refers to the specific response of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) elicited in a particular biological system or experimental setting at a given dose or concentration. The activity of the IL-12 Fc fusion proteins of the disclosure can be assayed by any suitable method known in the art, such as, for example, a ligand-binding assay and / or a functional assay. Generally, changes in activity may be demonstrated graphically as an upward or downward shift in a response curve compared to a control. An upward shift of the response curve is generally indicative of an increase in activity, whereas a downward shift of the response curve is generally indicative of a decrease in activity. As described in further detail below, a change in activity may or may not be related to and / or caused by a change in potency.

[385] As used herein, the term “manufacturability” refers to any property that may impact the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, and the like at a scale and quantity sufficient for administration to an individual. Examples of properties that impact manufacturability include, but are not limited to, the stability, purity, aggregation levels, and / or yield of expression of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein).

[386] As used herein, the term “stability” refers to the ability of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) to retain the same properties and characteristics that it possessed at the time of its manufacture within specified limits and / or storage and / or use parameters. The stability of the IL-12 Fc fusion proteins of the disclosure can be assayed 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 fluorimetry.

[387] As used herein, the term “yield of expression” refers to the amount or quantity of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, and the like are well-known in relevant arts. Any suitable method for quantitating or determining a yield of expression may be employed, such as, for example, UV absorption measurements, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorometric assays.

[388] As used herein, the terms “subject” or an “individual” for purposes of treatment can generally refer to any animal classified as a mammal (including but not limited to humans, primates, and / or non-human primates), domestic animals, farm animals, zoo animals, research animals, sports animals, and / or pet animals, such as dogs, horses, cats, cows, etc. However, unless the context clearly dictates otherwise, references to a “subject” are intended to refer to a human subject.

[389] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antigen” includes mixtures of antigens; reference to “a pharmaceutically acceptable carrier” includes mixtures of two or more such carriers, and the like. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[390] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A (alone),” and “B (alone).”

[391] As used herein, the use of ordinal terms, such as, for example, “first,” “second,” “third,” “fourth”, “fifth,” etc., to modify an element of the disclosure does not by itself connote any priority, precedence, or order of one element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one element having a certain name (e.g., an assay, a domain linker, etc.) from another element having a same name but for the use of the ordinal term to distinguish the elements (e.g., “a first assay,” “a second assay,” “a third assay,” “a fourth assay,” “a fifth assay,” etc.). For example, a “first assay” may be performed to detect changes in potency of an IL-12 Fc fusion protein and a “second assay” may be performed to detect changes in binding affinity for IL-12Rβ2. In such an example, the terms “first assay” and “second assay” are not used to describe any priority, precedence, or order in which the assays are performed, but rather to denote that the elements are distinct despite having the same name except for the use of the ordinal terms “first” and “second.” As a separate example, a “third assay” may be performed to detect changes in capability to stimulate IFNγ without a “first assay” or a “second assay” having been performed. Alternatively, it is also contemplated that a “first assay” could be performed before or after the “third assay,” and that the “second assay” of the example may not be performed, may be performed before or after the “first assay,” or before or after the “third assay.” While the examples set forth above utilize specific ordinal terms, it is contemplated that the concepts described above apply mutatis mutandis to other examples using any number of ordinal terms in various configurations.

[392] As used herein, the term “about” a value (or parameter) refers to ±10% of a stated value. When referring to a range of values (or parameters), the term “about” refers to +10% of the upper limit and -10% of the lower limit of a stated range of values. When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the scope of the present disclosure. Where the stated range includes upper and / or lower limits, ranges excluding either of those included limits are also included in the present disclosure.

[393] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.II. Overview

[394] The present application generally relates to compositions and methods for modulating signal transduction mediated by IL-12. In particular, the disclosure provides low potency IL-12 Fc fusion proteins comprising: (1) a variant IL-12 p35 subunit domain that reduces binding affinity to interleukin 12 receptor beta 2 (IL-12Rβ2), (2) an IL-12 p40 subunit domain, and (3) an antigen binding domain (ABD) to facilitate trafficking of the fusion protein to a target cell of interest, as well as methods of making and using the same. 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.

[395] As noted above, IL-12 is a potent, pro-inflammatory cytokine with a high affinity for its receptors (p35 subunit for IL-12Rβ2; p40 subunit for IL-12Rβ1). Due to this high affinity, IL-12 therapies configured to target various cell populations utilizing wild-type IL-12 in combination with an ABD have generally not been successful, as the affinity and / or potency of the “IL-12 binding arm” of the therapy is generally greater than the binding affinity / potency of the “ABD arm” of the therapy. Fusion of reduced potency IL-12 variants to targeting ABD has the potential to overcome this limitation. However, the properties of the resultant fusion protein are heavily influenced by the identity of the ABD target. For example, the type and frequency of immune cells that express the ABD target can impact the efficacy, pharmacokinetics, and tolerability of the IL-12 ABD fusion protein. As demonstrated herein, targeting of low potency IL-12 using ABD specific for Lag-3, CCR7, or CCR8 did not enhance IL-12 anti-tumor activity. While some other targeting arms (for example, PD-1 targeting, ICOS-targeting, or 41BB-targeting) enhanced IL-12 anti-tumor activity, the resultant IL-12 ABD fusion proteins exhibited a less preferred pharmacokinetic profile (e.g., faster clearance) and / or a worse safety profile, potentially due to the relatively broad expression of these targets. The present application describes, inter alia, OX40-targeted reduced potency IL-12 fusion proteins that exhibit enhanced properties relative to other targeted IL-12 therapeutics described herein. OX40-targeted reduced potency IL-12 fusion proteins demonstrated potent anti-tumor activity and importantly, only minimally impacted pharmacokinetic profile and tolerability. Accordingly, OX40 targeting resulted in the largest improvement of therapeutic index of any of the molecules tested. Relatedly, coadministration of an OX40-targeted reduced potency IL-12 fusion protein with an anti-PD-1 antibody further enhanced the anti-tumor activity without significantly affecting the pharmacokinetic profile and tolerability, whereas the PD-1-targeted reduced potency IL-12 fusion protein targeted additional cell types resulting in an increase in toxicity (as is described in further detail herein).

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

[397] In some embodiments, the compositions and methods described herein facilitate the trafficking of an IL-12 Fc fusion protein to a target cell expressing: (1) IL-12Rβ2, and (2) a cell surface antigen selected from a group that includes: (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 cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other further embodiments, the cell surface antigen is LAG3, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other further embodiments, the cell surface antigen is OX40, and the target cell comprises: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other further embodiments, the cell surface antigen is ICOS, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted CD8+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other further embodiments, the cell surface antigen is 41BB, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), (ii) activated and / or exhausted NK cell(s), (iii) activated and / or exhausted CD8+ T cell(s), or (iv) activated and / or exhausted regulatory T cell(s). In other further embodiments, the cell surface antigen is CCR7, and the target cell comprises: (i) naïve CD4+ T cell(s), (ii) naïve CD8+ T cell(s), or (iii) naïve regulatory T cell(s). In other further embodiments, the cell surface antigen is CCR8, and the target cell comprises: (i) activated and / or exhausted CD4+ T cell(s), or (ii) activated and / or exhausted regulatory T cell(s).III. Compositions

[398] As will be appreciated by one skilled in the art, any of the aspects and embodiments of the compositions described herein can be used in any of the aspects and / or embodiments of the methods of making described below or in the methods of use also described below.A. Low PotencyInterleukin 12 (IL-12) FcFusion Proteins

[399] The present disclosure provides several aspects and embodiments comprising low potency IL-12 Fc fusion proteins (sometimes referred to herein as an “IL-12 Fc fusion protein” for the sake of brevity). The IL-12 Fc fusion proteins described herein comprise: (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 reduces binding affinity of the resultant IL-12 Fc fusion protein for IL-12Rβ2 as compared to a reference IL-12, wherein the set of amino acid substitutions is selected from a group including: (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 a target antigen selected from a group including: (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 comprises 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 comprises a mouse target antigen. In some embodiments, the ABD comprises a humanized ABD.

[400] As will be appreciated by those skilled in the art, a subject IL-12 Fc fusion protein can take on one of several different configurations. In some instances, the IL-12 Fc fusion protein is configured such that the ABD is in a Fab format (i.e., the IL-12 Fc fusion protein comprises a variable heavy (VH) domain, a variable light (VL) domain, a constant heavy (CH1) domain, and a constant light (CL) domain). In some other instances, the IL-12 Fc fusion protein is configured such that the ABD is in an scFv format (i.e., the IL-12 Fc fusion protein comprises, from N-terminus to C-terminus, a VH domain, a domain linker, and a VL domain; or the reverse where the IL-12 Fc fusion protein comprises, from N-terminus to C-terminus, a VL domain, a domain linker, and a VH domain). In still other instances, the IL-12 Fc fusion protein is configured such that the ABD is in a nanobody format (i.e., the IL-12 Fc fusion protein comprises a VHH domain, wherein the VHH domain comprises a complementarity determining region (CDR) 1, a CDR2, and a CDR3, wherein the CDR1, CDR2, and CDR3 bind to a target antigen).

[401] Due to the modular nature of the IL-12 Fc fusion protein’s configuration, a discussion of exemplary components comprising a subject IL-12 Fc fusion protein is first provided below (such as, for example, variant IL-12 p35 subunit domains, (variant) IL-12 p40 subunit domains, ABDs, VH domains, VL domains, Fc domains, domain linkers, and the like), followed by a discussion of exemplary IL-12 Fc fusion protein configurations finding utility in some of the embodiments described herein.1. Interleukin 12:

[402] The present disclosure provides IL-12 Fc fusion proteins 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 reduces binding affinity of the resultant IL-12 Fc fusion protein for IL-12Rβ2 as compared to a reference IL-12, wherein the set of amino acid substitutions is selected from a group including: (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 a target antigen selected from a group including: (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.

[403] 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 on separate monomers of the IL-12 Fc fusion protein (e.g., a first monomer of the IL-12 Fc fusion protein comprises the first IL-12 subunit domain and the second monomer comprises the second IL-12 subunit domain, or the reverse where the first monomer comprises the second IL-12 subunit domain and the second monomer comprises 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 on the same monomer of the IL-12 Fc fusion protein, often in a single chain format linked by a domain linker (e.g., the first monomer comprises the first and second IL-12 subunit domains and the second monomer does not comprise an IL-12 subunit domain; the second monomer comprises the first and second IL-12 subunit domains and the first monomer does not comprise an IL-12 subunit domain).

[404] In some instances where the first and second IL-12 subunit domains are on separate monomers of the IL-12 Fc fusion protein, the first and second IL-12 subunit domains may be configured such that they are on the N-terminus of their respective monomers, or they may be configured such that they are on the C-terminus of their respective monomers. In instances where the first and second IL-12 subunit domain are on one monomer of the IL-12 Fc fusion protein, a single chain IL-12 comprising a first IL-12 subunit domain, a domain linker, and a second IL-12 subunit domain may be configured such that the single chain IL-12 is on the N-terminus of a monomer or configured such that the single chain IL-12 is on the C-terminus of the monomer. In either configuration, the variant p35 subunit domain may be on the N-terminus of the single chain IL-12 and the (variant) p40 subunit domain is on the C-terminus of the single chain IL-12, or the (variant) p40 subunit domain may be on the N-terminus of the single chain IL-12 and the variant p35 subunit domain is on the C-terminus of the single chain IL-12.

[405] While exemplary IL-12 Fc fusion proteins are generally set forth such that either: (1) the first IL-12 subunit domain and the second IL-12 subunit domain are on the same monomer of a subject IL-12 Fc fusion protein (covalently linked using a domain linker in several embodiments (e.g., from N-terminus to 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 on separate monomers of a subject IL-12 Fc fusion protein (e.g., a first monomer and a second monomer, respectively, or the reverse), it is also expressly contemplated that a subject IL-12 Fc fusion protein can be configured such that the first IL-12 subunit domain is covalently attached to a monomer of the subject IL-12 Fc fusion protein and the second IL-12 subunit domain is expressed as a soluble protein, wherein the soluble IL-12 subunit interacts with / binds the IL-12 subunit covalently attached to a monomer of the subject IL-12 Fc fusion protein (or the reverse, where the first IL-12 subunit domain is expressed as a soluble protein and the second IL-12 subunit domain is covalently attached to a monomer of a subject IL-12 Fc fusion protein). Exemplary configurations wherein one IL-12 subunit is covalently attached to a subject IL-12 Fc fusion protein and one IL-12 subunit is expressed as a soluble fragment is described in, for example, PCT Publication No. WO 2023 / 242769 A1, which is incorporated by reference in its entirety.

[406] Binding affinities to IL-12Rβ2 can be assessed using any number of assays known to those skilled in the relevant art. Non-limiting examples of suitable assays for evaluating binding affinity include: a surface plasmon resonance (SPR) assay, an enzyme-linked immunosorbent assay (ELISA), an ELISpot assay, Biacore assays, KinExA assays, flow cytometry, and the like.a. p35 Subunit Domain:

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

[408] In some embodiments, the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from a group including any one of: (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 in Fig. 5.

[409] In some embodiments, the variant IL-12 p35 subunit domain comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an amino acid sequence selected from a group including any one of SEQ ID NOs: 17-21 (as shown in Fig. 5).

[410] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation, comprising: Y40P, and further comprises a second substitution mutation selected from a group including: K168A, and K170A. In some embodiments, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 17 and 18, as shown in Fig. 5.

[411] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation, comprising: Y40S, and further comprises a second substitution mutation selected from a group including: K170T, and K170A. In some embodiments, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 19 and 20, as shown in Fig. 5.

[412] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation, comprising: Y40E, and further comprises a second substitution mutation selected from a group including: K170A. In some embodiments, the variant IL-12 p35 subunit domain comprises SEQ ID NO: 21, as shown in Fig. 5.

[413] In some embodiments, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation as compared to SEQ ID NO: 2 (finding particular utility in embodiments where the subject IL-12 Fc fusion protein comprises a variant IL-12 p40 subunit domain that comprises C177S or C177S / C252S substitution mutation(s)).

[414] In addition to the IL-12 Fc fusion proteins described above, additional modifications to various residues are known in relevant arts. Non-limiting examples of residues that may be modified 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, M125, 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, the (variant) IL-12 p35 subunit domain described herein or in the figures (see, e.g., Figs. 1, 3, and 5) comprises 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 (variant) IL-12 p35 subunit domain described herein or in the figures (see, e.g., Figs. 1, 3, and 5) comprises 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.

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

[416] A variety of assay formats may be used to select IL-12 Fc fusion proteins that bind to a ligand of interest (e.g., IL-12Rβ2 and / or IL-12Rβ1). Non-limiting examples include: solid-phase ELISA immunoassay, immunoprecipitation, a Biacore assay, a KinExA assay, fluorescence-activated cell sorting (FACS), an Octet assay, Western blot analysis, and the like. The binding activity of the IL-12 Fc fusion proteins of the disclosure can be assayed by any suitable method known in the art, such as, for example, a surface plasmon resonance (SPR) assay, an enzyme-linked immunosorbent assay (ELISA), an ELISpot assay, Biacore assays, KinExA assays, and the like.

[417] One of ordinary skill in the art will appreciate that binding affinity can also be used as a measure of the “strength” of a non-covalent interaction between two binding partners (e.g., a variant IL-12 p35 subunit domain and IL-12Rβ2). Binding affinity between two molecules may be quantified by determination of the dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using suitable assays known in the art, such as, for example, an SPR assay. The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or kon) and dissociation rate constant kd (or koff), respectively. KD is related to ka and kd through the equation: KD = kd / ka. The value of the dissociation constant can be determined directly by well-known methods.

[418] As used herein, the term “potency” refers to the ability of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) to elicit a response at a certain dose or concentration in a given biological system or experimental setting. The potency of the IL-12 Fc fusion proteins of the disclosure can be assayed by any suitable method known in the art, such as, for example, an IL-12 HEK reporter assay (e.g., InvivoGen’s IL-12 HEK reporter assay (Catalog No. hkb-il12)), a ligand-binding assay (e.g., ELISA or flow cytometry), and / or a functional assay (e.g., AlphaLISA). Generally, changes in potency may be demonstrated graphically as a leftward or rightward shift in a response curve compared to a control. A rightward shift of the response curve is generally indicative of a reduction in potency, whereas as a leftward shift of the response curve is generally indicative of an increase in potency. As used herein, the term “activity” refers to the specific response of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) elicited in a particular biological system or experimental setting at a given dose or concentration. The activity of the IL-12 Fc fusion proteins of the disclosure can be assayed by any suitable method known in the art, such as, for example, a ligand-binding assay and / or a functional assay. Generally, changes in activity may be demonstrated graphically as an upward or downward shift in a response curve compared to a control. An upward shift of the response curve is generally indicative of an increase in activity, whereas a downward shift of the response curve is generally indicative of a decrease in activity.

[419] As used herein, the term “manufacturability” refers to any property that may impact the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, and the like at a scale and quantity sufficient for administration to an individual. Examples of properties that impact manufacturability include, but are not limited to, the stability, purity, aggregation levels, and / or yield of expression of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein). As used herein, the term “stability” refers to the ability of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) to retain the same properties and characteristics that it possessed at the time of its manufacture within specified limits and / or storage and / or use parameters. The stability of the IL-12 Fc fusion proteins of the disclosure can be assayed 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 fluorimetry. As used herein, the term “yield of expression” refers to the amount or quantity of a given protein, cytokine, fusion protein, antibody, and the like (such as, for example, an IL-12 Fc fusion protein) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, and the like are well-known in relevant arts. Any suitable method for quantitating or determining a yield of expression may be employed, such as, for example, UV absorption measurements, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorometric assays.

[420] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has binding affinity for IL-12Rβ2 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 compared to binding affinity of a reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[421] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has binding affinity for IL-12Rβ2 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 binding affinity of a reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[422] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has binding affinity for IL-12Rβ2 reduced by at least about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2.0-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3.0-fold, about 3.1-fold, about 3.2-fold, about 3.3-fold, about 3.4-fold, about 3.5-fold, about 3.6-fold, about 3.7-fold, about 3.8-fold, about 3.9-fold, about 4.0-fold, about 4.1-fold, about 4.2-fold, about 4.3-fold, about 4.4-fold, about 4.5-fold, about 4.6-fold, about 4.7-fold, about 4.8-fold, about 4.9-fold, about 5.0-fold, about 5.1-fold, about 5.2-fold, about 5.3-fold, about 5.4-fold, about 5.5-fold, about 6.0-fold, about 7.0-fold, about 8.0-fold, about 9.0-fold, about 10.0-fold, about 20.0-fold, about 30.0-fold, about 40.0-fold, 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, about 1,000.0-fold, about 2,000.0-fold, about 3,000.0-fold, about 4,000.0-fold, about 5,000.0-fold, about 6,000.0-fold, about 7,000.0-fold, about 8,000.0-fold, about 9,000.0-fold, about 10,000.0-fold, about 20,000.0-fold, about 30,000.0-fold, about 40,000.0-fold, about 50,000.0-fold, about 100,000.0-fold, about 150,000.0-fold, about 200,000.0-fold, about 250,000.0-fold, about 300,000.0-fold, about 350,000.0-fold, about 400,000.0-fold, about 450,000.0-fold, about 500,000.0-fold or more compared to binding affinity of a reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[423] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has binding affinity for IL-12Rβ2 reduced 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 10.0-fold, about 10.0-fold to about 20.0-fold, about 20.0-fold to about 30.0-fold, about 30.0-fold to about 40.0-fold, about 40.0-fold to about 50.0-fold, about 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 to about 600.0-fold, about 600.0-fold to about 650.0-fold, about 650.0-fold to about 700.0-fold, about 700.0-fold to about 750.0-fold, about 750.0-fold to about 800.0-fold, about 800.0-fold to about 850.0-fold, about 850.0-fold to about 900.0-fold, about 900.0-fold to about 950.0-fold, about 950.0-fold to about 1,000.0-fold, about 1,000.0-fold to about 2,000.0-fold, about 2,000.0-fold to about 3,000.0-fold, about 3,000.0-fold to about 4,000.0-fold, about 4,000.0-fold to about 5,000.0-fold, about 5,000.0-fold to about 6,000.0-fold, about 6,000.0-fold to about 7,000.0-fold, about 7,000.0-fold to about 8,000.0-fold, about 8,000.0-fold to about 9,000.0-fold, about 9,000.0-fold to about 10,000.0-fold, about 10,000.0-fold to about 20,000.0-fold, about 20,000.0-fold to about 30,000.0-fold, about 30,000.0-fold to about 40,000.0-fold, about 40,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 150,000.0-fold, about 150,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 250,000.0-fold, about 250,000.0-fold to about 300,000.0-fold, about 300,000.0-fold to about 350,000.0-fold, about 350,000.0-fold to about 400,000.0-fold, about 400,000.0-fold to about 450,000.0-fold, about 450,000.0-fold to about 500,000.0-fold or higher compared to binding affinity of a reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[424] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has binding affinity for IL-12Rβ2 below the lower level of detectability of an assay, and binding affinity of a reference IL-12 for IL-12Rβ2 is between or equivalent to the lower or upper level of detectability of an assay (i.e., the binding affinity of the reference IL-12 may be equal to the lower level of detectability, the upper level of detectability, or a value between the lower and upper levels of detectability; in other words, the binding affinity is “detectable” for the reference IL-12 and the binding affinity is “undetectable” for the IL-12 Fc fusion protein). In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[425] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has potency 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, about 1,000.0-fold, about 2,000.0-fold, about 3,000.0-fold, about 4,000.0-fold, about 5,000.0-fold, about 6,000.0-fold, about 7,000.0-fold, about 8,000.0-fold, about 9,000.0-fold, about 10,000.0-fold, about 20,000.0-fold, about 30,000.0-fold, about 40,000.0-fold, about 50,000.0-fold, about 100,000.0-fold, about 200,000-fold, or about 300,000-fold or more compared to potency of a reference IL-12, as determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[426] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has potency 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 to about 600.0-fold, about 600.0-fold to about 650.0-fold, about 650.0-fold to about 700.0-fold, about 700.0-fold to about 750.0-fold, about 750.0-fold to about 800.0-fold, about 800.0-fold to about 850.0-fold, about 850.0-fold to about 900.0-fold, about 900.0-fold to about 950.0-fold, about 950.0-fold to about 1,000.0-fold, about 1,000.0-fold to about 2,000.0-fold, about 2,000.0-fold to about 3,000.0-fold, about 3,000.0-fold to about 4,000.0-fold, about 4,000.0-fold to about 5,000.0-fold, about 5,000.0-fold to about 6,000.0-fold, about 6,000.0-fold to about 7,000.0-fold, about 7,000.0-fold to about 8,000.0-fold, about 8,000.0-fold to about 9,000.0-fold, about 9,000.0-fold to about 10,000.0-fold, about 10,000.0-fold to about 20,000.0-fold, about 20,000.0-fold to about 30,000.0-fold, about 30,000.0-fold to about 40,000.0-fold, about 40,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 60,000.0-fold, about 60,000.0-fold to about 70,000.0-fold, about 70,000.0-fold to about 80,000.0-fold, about 80,000.0-fold to about 90,000.0-fold, about 90,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, or about 200,000.0-fold to about 300,000.0-fold or higher compared to potency of a reference IL-12 as determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[427] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain exhibits a potency that is too weak to accurately be determined, and potency of a reference IL-12 is between or equivalent to the lower or upper level of detectability of an assay (i.e., the potency of the reference IL-12 may be equal to the lower level of detectability, the upper level of detectability, or a value between the lower and upper levels of detectability; in other words, the potency is “detectable” for the reference IL-12 and the potency is “undetectable” for the IL-12 Fc fusion protein). In some embodiments, the assay comprises an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[428] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has a reduced capability to stimulate STAT4 signaling compared to a reference IL-12, as determined by an assay. A reduction in the capability to stimulate STAT4 signaling may refer to a reduction in the maximal response observed and / or shifting in the EC50 value. In some embodiments, the capability 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 an assay. In some embodiments, the assay comprises an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

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

[430] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain has a reduced capability to stimulate IFNγ production compared to a reference IL-12, as determined by an assay. A reduction in the capability to stimulate IFNγ production may refer to a reduction in the maximal response observed and / or shifting in the EC50 value. In some embodiments, the capability of the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain to stimulate IFNγ production is reduced by 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, about 1,000.0-fold, about 2,000.0-fold, about 3,000.0-fold, about 4,000.0-fold, about 5,000.0-fold, about 6,000.0-fold, about 7,000.0-fold, about 8,000.0-fold, about 9,000.0-fold, about 10,000.0-fold, about 20,000.0-fold, about 30,000.0-fold, about 40,000.0-fold, about 50,000.0-fold, about 100,000.0-fold, about 200,000-fold, or about 300,000-fold or more compared to a reference IL-12, as determined by an assay. In some embodiments, the assay comprises an AlphaLISA assay, an intracellular cytokine stain 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: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[431] In some embodiments, the capability 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 to about 600.0-fold, about 600.0-fold to about 650.0-fold, about 650.0-fold to about 700.0-fold, about 700.0-fold to about 750.0-fold, about 750.0-fold to about 800.0-fold, about 800.0-fold to about 850.0-fold, about 850.0-fold to about 900.0-fold, about 900.0-fold to about 950.0-fold, about 950.0-fold to about 1,000.0-fold, about 1,000.0-fold to about 2,000.0-fold, about 2,000.0-fold to about 3,000.0-fold, about 3,000.0-fold to about 4,000.0-fold, about 4,000.0-fold to about 5,000.0-fold, about 5,000.0-fold to about 6,000.0-fold, about 6,000.0-fold to about 7,000.0-fold, about 7,000.0-fold to about 8,000.0-fold, about 8,000.0-fold to about 9,000.0-fold, about 9,000.0-fold to about 10,000.0-fold, about 10,000.0-fold to about 20,000.0-fold, about 20,000.0-fold to about 30,000.0-fold, about 30,000.0-fold to about 40,000.0-fold, about 40,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 60,000.0-fold, about 60,000.0-fold to about 70,000.0-fold, about 70,000.0-fold to about 80,000.0-fold, about 80,000.0-fold to about 90,000.0-fold, about 90,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, or about 200,000.0-fold to about 300,000.0-fold or higher compared to a reference IL-12, as determined by an assay. In some embodiments, the assay comprises an AlphaLISA assay, an intracellular cytokine stain 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: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[432] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain exhibits a capability to stimulate IFNγ production that is too weak to accurately be determined, and capability to stimulate IFNγ production of a reference IL-12 is between or equivalent to the lower or upper level of detectability of an assay (i.e., the potency of the reference IL-12 may be equal to the lower level of detectability, the upper level of detectability, or a value between the lower and upper levels of detectability; in other words, the capability to stimulate IFNγ production is “detectable” for the reference IL-12 and the capability to stimulate IFNγ production is “undetectable” for the IL-12 Fc fusion protein). In some embodiments, the assay comprises an AlphaLISA assay, an intracellular cytokine stain 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: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[433] In some embodiments, IL-12 Fc fusion protein and / or variant IL-12 p35 subunit domains are provided, wherein one or more amino acid substitutions of the variant IL-12 p35 subunit domain improves half-life, as compared to half-life of a reference IL-12. In some embodiments, the fusion of the variant IL-12 p35 subunit domain and / or the (variant) IL-12 p40 subunit domain improves half-life, as compared to half-life of a reference. In some embodiments, the half-life of the IL-12 Fc fusion protein and / or variant IL-12 p35 subunit domain is decreased or increased 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, as compared to the half-life of a reference IL-12. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[434] In some embodiments, IL-12 Fc fusion protein and / or variant IL-12 p35 subunit domains are provided, wherein one or more amino acid substitutions of the variant IL-12 p35 subunit domain improves half-life, as compared to half-life of a reference IL-12. In some embodiments, the fusion of the variant IL-12 p35 subunit domain and / or the (variant) IL-12 p40 subunit domain improves half-life, as compared to half-life of a reference. In some embodiments, the half-life of the IL-12 Fc fusion protein and / or variant IL-12 p35 subunit domain is decreased or increased 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-fold to about 8.0-fold, about 8.0-fold to about 9.0-fold, about 9.0-fold to about 10.0-fold, about 10.0-fold to about 15.0-fold, about 15.0-fold to about 20.0-fold, about 20.0-fold to about 30.0-fold, about 30.0-fold to about 40.0-fold, about 40.0-fold to about 50.0-fold, or about 50.0-fold to about 100.0-fold or more, as compared to the half-life of a reference IL-12. In some embodiments, the reference IL-12 comprises one or more of the following: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[435] Various methods for determining the half-life of an agent, such as, for example, IL-12, are known in the art. Those skilled in the relevant art will readily be able to determine and employ any number of such methods for determining changes in half-life. In some embodiments, at least one sample selected from a group comprising: (i) one or more blood samples, (ii) one or more plasma samples, (iii) one or more serum samples, (iv) one or more tissue samples, and (v) any combination thereof, are utilized to measure half-life. To measure the half-life, one, two, three, four, five, six, seven, eight, nine, 10 or more samples (as described above) may be utilized to measure half-life.

[436] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain exhibits a resistance to tachyphylaxis of 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 an assay. In some embodiments, the assay comprises: a measurement of plasma IFNγ using an AlphaLISA 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: a wild-type IL-12, a human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein that does not comprise one or more amino acid substitutions selected from a group including: (a) Y40S / K170A, (b) Y40S / K170T, (c) Y40P / K168A, (d) Y40P / K170A, and (e) Y40E / K170A, or any combination thereof.

[437] In some embodiments, the IL-12 Fc fusion protein and / or the variant IL-12 p35 subunit domain exhibits a resistance to tachyphylaxis of about 10% to about 100%, about 10% to about 50%, about 20% to about 70%, about 30% to about 80%, about 40% to about 90%, about 50% to about 100%, about 20% to about 50%, about 40% to about 70%, about 30% to about 60%, about 40% to about 100%, about 20% to about 80%, or about 10% to about 90% compared to a reference IL-12, as determined by an assay. In some embodiments, the assay comprises: a measurement of plasma IFNγ using an AlphaLISA 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: a wi...

Claims

1. An IL-12 Fc fusion protein, comprising:(a) a first monomer comprising, from N-terminus to 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-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 a set 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 N-terminus to C-terminus: (i) a variable heavy (VH) domain, (ii) a constant heavy (CH1) domain, and (iii) a second Fc domain; and(c) a third monomer comprising, from N-terminus to C-terminus: (i) a variable light (VL) domain, and (ii) a constant light (CL) domain, wherein the VH domain and VL domain form an antigen binding domain (ABD) that binds a 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 NOs: 17-21.

3. The IL-12 Fc fusion protein according to claim 1 or 2, wherein: (i) the IL-12 p40 subunit domain comprises SEQ ID NO: 4, or(ii) the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, wherein the variant IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 23-25.

4. The IL-12 Fc fusion protein according to claim 3, wherein:(A) the variant IL-12 p40 subunit domain comprises SEQ ID NO: 23 or SEQ ID NO: 25; and (B) the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

5. The IL-12 Fc fusion protein according to any one of claims 1-4, wherein: (i) the first Fc domain comprises any one of SEQ ID NOs: 26-30; and (ii) the second Fc domain comprises any one of SEQ ID NOs: 26-30.

6. The IL-12 Fc fusion protein according to claim 5, wherein:(1) the first Fc domain further comprises a set of substitution mutations comprising T366W / S354C, and the second Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C; or(2) the first Fc domain further comprises a set of substitution mutations comprising T366S / L368A / Y407V / Y349C, and the second Fc domain further comprises a set of substitution mutations comprising T366W / S354C.

7. The IL-12 Fc fusion protein according to claim 6, wherein:(A) the ABD binds a target antigen comprising human OX40; and(B) the ABD comprises a VH / VL domain pair selected from the group consisting of: (i) SEQ ID NOs: 152 and 143, respectively, (ii) SEQ ID NOs: 132 and 133, respectively, (iii) SEQ ID NOs: 137 and 138, respectively, (iv) SEQ ID NOs: 142 and 143, respectively, (v) SEQ ID NOs: 147 and 148, respectively, (vi) SEQ ID NOs: 130 and 131, respectively, (vii) SEQ ID NOs: 154 and 143, respectively, (viii) SEQ ID NOs: 156 and 143, respectively, (ix) SEQ ID NOs: 158 and 148, respectively, (x) SEQ ID NOs: 160 and 148, respectively, (xi) SEQ ID NOs: 162 and 148, respectively, (xii) SEQ ID NOs: 164 and 148, respectively, (xiii) SEQ ID NOs: 166 and 148, respectively, (xiv) SEQ ID NOs: 168 and 148, respectively, and (xv) SEQ ID NOs: 170 and 171, respectively.

8. The IL-12 Fc fusion protein according to any one of claims 1-7, wherein the ABD binds an extracellular domain (ECD) of the target antigen.

9. The IL-12 Fc fusion protein according to any one of claims 1-8, wherein the first domain linker and / or the second domain linker comprises any one of SEQ ID NOs: 31-40.

10. The IL-12 Fc fusion protein according to any one of claims 1-9, wherein:(i) the first monomer comprises the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence with at least about 95% or more identity to SEQ ID NO: 49;(ii) the second monomer comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence with at least about 95% or more identity to SEQ ID NO: 153; and(iii) the third monomer comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence with at least about 95% or more identity to SEQ ID NO: 145.

11. A nucleic acid composition comprising one or more nucleic acids encoding an IL-12 Fc fusion protein according to any one of claims 1-10.

12. An expression vector comprising the one or more nucleic acids of claim 11.

13. A host cell transformed with the expression vector of claim 12.

14. A method of making an IL-12 Fc fusion protein, comprising:(a) culturing the host cell of claim 13 under conditions wherein the IL-12 Fc fusion protein is expressed; and(b) isolating and / or purifying the IL-12 Fc fusion protein.

15. A method of treating a cancer in a human subject comprising administering the IL-12 Fc fusion protein according to any one of claims 1-10 to a human subject in need thereof, wherein:(A) the IL-12 Fc fusion protein binds to one or more target cell(s) that expresses: (1) IL-12Rβ2, and (2) human OX40; and(B) the one or more target cell(s) comprise: (i) activated and / or exhausted CD8+ T cell(s), (ii) activated and / or exhausted CD4+ T cell(s), (iii) activated and / or exhausted NK cell(s), or (iv) activated and / or exhausted regulatory T cell(s). 

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