IL-12 Fc fusion protein and uses thereof

Abstract

The present invention is directed to novel non-naturally occurring IL-12 variants, homodimeric IL-12 Fc fusion proteins, and heterodimeric IL-12 Fc fusion protein compositions, and methods of making and using such compositions.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 476,687, filed December 22, 2022, and U.S. Provisional Application No. 63 / 368,740, filed July 18, 2022, each of which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy, created on July 17, 2023, has the filename "54100_4001.xml" and is 336,708 bytes in size.

[0003] This application relates generally to compositions and methods for modulating interleukin-12 (IL-12)-mediated signal transduction. In particular, the disclosure provides novel non-naturally occurring IL-12 variant polypeptides and fusion proteins in which the IL-12 p35 subunit contains novel amino acid substitutions that reduce binding affinity to interleukin-12 receptor β2 (IL-12Rβ2), as well as methods of making and using the non-naturally occurring IL-12 variant polypeptides and fusion proteins.

[0004] IL-12 is a potent proinflammatory cytokine produced by antigen-presenting cells, such as 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 it contains a heterodimeric cytokine. IL-12 is composed of an alpha (α)-subunit (encoded by the IL12A gene; also referred to herein as the "IL-12 p35 subunit"; precursor sequence: SEQ ID NO: 1 (shown in FIG. 1); mature sequence: SEQ ID NO: 2 (shown in FIG. 1)) and a beta (β)-subunit (encoded by the IL12B gene; also referred to herein as the "IL-12 p40 subunit"; precursor sequence: SEQ ID NO: 3 (shown in FIG. 1); mature sequence: SEQ ID NO: 4 (shown in FIG. 1)), which assemble to form a 70,000 dalton (70 kDa) disulfide-linked heterodimer. The assembly of IL-12 p35 and p40 subunits forms the biologically active IL-12 heterodimer. IL-12 receptor, IL-12R, is a type I cytokine receptor containing a β-1 subunit (IL-12Rβ1: SEQ ID NO:5 (shown in FIG. 2); extracellular domain: SEQ ID NO:6 (shown in FIG. 2)) and a β-2 subunit (IL-12Rβ2: SEQ ID NO:7 (shown in FIG. 2); extracellular domain: SEQ ID NO:8 (shown in FIG. 2)). The IL-12 p40 subunit has binding affinity for IL-12Rβ1, and the IL-12 p35 subunit has binding affinity for IL-12Rβ2.

[0005] Binding of IL-12 to IL-12R results in the phosphorylation of intracellular signal transducer and activator of transcription 4 (STAT4), triggering signaling pathways that (i) induce TH1 cell differentiation, (ii) enhance the activation and cytotoxicity of T cells and natural killer (NK) cells, (iii) inhibit or reprogram immunosuppressive cells, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), and (iv) induce the production of large amounts of interferon-γ (IFNγ), which is cytostatic / cytotoxic, antiangiogenic, and can upregulate major histocompatibility complex (MHC) class 1 and MHC class 2 molecules on tumor cells, enabling immune recognition.

[0006] As a result, IL-12 has demonstrated potent antitumor activity against various malignancies in preclinical studies. However, systemic administration of wild-type IL-12 to humans can result in significant toxicity, including death in studies, due to overactivation of circulating immune cells. Furthermore, activated immune cells undergo cell proliferation, which contributes to the short serum half-life of administered IL-12 due to target-mediated pharmacokinetics. The biologically active form of human IL-12 (i.e., a heterodimeric complex containing the IL-12 p35 and IL-12 p40 subunits) has been reported to have an in vivo half-life of as short as 5 hours when administered as a therapeutic compound. In recent years, cytokine engineering has emerged as a promising strategy for tailoring cytokines with desired activity and reduced toxicity. Therefore, there is a need for further approaches to improve the properties of IL-12 for use as a therapeutic agent. In particular, there is an unmet need for novel IL-12 variant polypeptides and / or fusion proteins that (i) bind to IL-12R with altered binding efficiency or affinity such that the therapeutic properties of IL-12 (e.g., recognition and elimination of target cells, such as cancer cells) are maintained and the undesirable side effects of IL-12 are reduced or eliminated, and (ii) have increased serum half-life. Summary of the Invention

[0007] In one aspect, the disclosure provides a non-naturally occurring IL-12 variant comprising: a) a variant IL-12 p35 subunit, wherein the variant IL-12 p35 subunit comprises one or more amino acid substitutions selected from the group comprising: Y40A, T43A, D126A, P127A, R129A, K168A, and K170A; and b) an IL-12 p40 subunit.

[0008] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises two or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0009] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises three or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0010] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises four or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0011] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises five or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0012] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises six or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0013] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises seven or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0014] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and D126A.

[0015] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and P127A.

[0016] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and T43A.

[0017] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, D126A and P127A.

[0018] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, T43A, D126A and P127A.

[0019] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and R129A.

[0020] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and K168A.

[0021] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and K170A.

[0022] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, P127A and R129A.

[0023] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, P127A and K168A.

[0024] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, P127A and K170A.

[0025] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue Y40. In some further embodiments, the substitution mutation at amino acid residue Y40 is selected from the group comprising: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

[0026] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 177-187.

[0027] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue D126. In some further embodiments, the substitution mutation at amino acid residue D126 is selected from the group comprising: D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

[0028] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue P127. In some further embodiments, the substitution mutation at amino acid residue P127 is selected from the group comprising P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

[0029] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue R129. In some further embodiments, the substitution mutation at amino acid residue R129 is selected from the group comprising: R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

[0030] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue K168. In some further embodiments, the substitution mutation at amino acid residue K168 is selected from the group comprising K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

[0031] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue K170. In some further embodiments, the substitution mutation at amino acid residue K170 is selected from the group comprising K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, and K170W.

[0032] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit comprises a first substitution mutation selected from the group comprising Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

[0033] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit further comprises a second substitution mutation.

[0034] In a further embodiment, in accordance with the above, the second substitution mutation is D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D126W, P127A, P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I , R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R1 29Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, K168Y, K170A, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, and K170W.

[0035] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 199-247 or SEQ ID NOs: 279-290.

[0036] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit may further comprise a C74S substitution mutation.

[0037] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit comprises a variant IL-12 p40 subunit.

[0038] In a further embodiment, and in accordance with the above, the variant IL-12 p40 subunit comprises one or more amino acid substitutions selected from the group comprising C177S, C252S, and C177S / C252S.

[0039] In a further embodiment, in accordance with any of the above, the variant IL-12 p35 subunit comprises an amino acid sequence selected from the group comprising SEQ ID NOs: 24-86 and 103-166, and the IL-12 p40 subunit comprises an amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.

[0040] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.

[0041] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 96 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.

[0042] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 97 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 97 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.

[0043] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 98 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.

[0044] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 99 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 99 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.

[0045] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 24-86 and 103-166, and the IL-12 p40 subunit consists of an amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.

[0046] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit comprises SEQ ID NO: 87 and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group comprising: Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0047] In a further embodiment, in accordance with any of the above, the one or more amino acid substitutions in the variant IL-12 p35 subunit improve the half-life compared to the half-life of a reference IL-12, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0048] In a further embodiment, and in accordance with any of the above, the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

[0049] In a further embodiment, according to any of the above, in a further embodiment, in any of the above, the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the IL-12 p40 subunit.

[0050] In a further embodiment, in accordance with the above, the non-naturally occurring IL-12 variant further comprises a linker comprising an amino acid sequence selected from the group comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the IL-12 p40 subunit.

[0051] In a further embodiment, and in accordance with any of the above, the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

[0052] In a further embodiment, in accordance with the above, the non-naturally occurring IL-12 variant further comprises a linker domain comprising an amino acid sequence selected from the group comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

[0053] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit comprises an additional amino acid substitution.

[0054] In a further embodiment there is provided a non-naturally occurring IL-12 variant for use in treating cancer in a subject in accordance with any of the above.

[0055] In another aspect, the present disclosure provides one or more nucleic acids encoding a non-naturally occurring IL-12 variant according to any of the above aspects and embodiments.

[0056] In another aspect, the present disclosure provides a host cell comprising one or more nucleic acids encoding a non-naturally occurring IL-12 variant according to any of the above aspects and embodiments.

[0057] In another aspect, the disclosure provides a method for producing a non-naturally occurring IL-12 variant, comprising culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the non-naturally occurring IL-12 variant is produced, wherein the one or more nucleic acids or vectors comprise one or more of the nucleic acids described in the above aspects and embodiments.

[0058] In a further embodiment, in accordance with the above, the method further comprises isolating and / or purifying the non-naturally occurring IL-12 variant produced.

[0059] In a further embodiment, and in accordance with any of the above, the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

[0060] In a further embodiment, the non-naturally occurring IL-12 variant produced in accordance with any of the above has an altered binding affinity to interleukin-12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0061] In further embodiments, the non-naturally occurring IL-12 variant produced in accordance with the above has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by an assay.

[0062] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0063] In a further embodiment, the non-naturally occurring IL-12 variant produced in accordance with any of the above has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of the reference IL-12 is detectable.

[0064] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0065] In further embodiments, the non-naturally occurring IL-12 variant produced in accordance with any of the above is about 0.5 to about 50.0 fold less potent than a reference IL-12 as measured by an assay, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, about 30.0 to about 35.0 fold less potent, about 35.0 fold less potent, or about 35.0 fold less potent. 1 / 100 to about 1 / 400, about 1 / 400 to about 1 / 450, about 1 / 450 to about 1 / 500, about 1 / 500 to about 1 / 1000, about 1 / 1000 to about 1 / 2000, about 1 / 2000 to about 1 / 3000, about 1 / 3000 to about 1 / 4000, about 1 / 4000 to about 1 / 5000, about 1 / 5000 to about 1 / 6000, about 1 / 6000 to about 1 / 7000, about 1 / 7000 to about 1 / 8000, about 1 / 8000 1 in to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 1 in 8000.0, about 1 in 8000.0 to about 1 in 9000.0, about 9000 and having a potency reduced by 1 / 0.0 to about 1 / 10,000.0, about 1 / 10,000.0 to about 1 / 50,000.0, about 1 / 50,000.0 to about 1 / 100,000.0, about 1 / 100,000.0 to about 1 / 200,000.0, about 1 / 200,000.0 to about 1 / 300,000.0, about 1 / 300,000.0, or less, wherein the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0066] In a further embodiment, the assay comprises an IL-12 HEK reporter assay, in accordance with the above.

[0067] In further embodiments, the non-naturally occurring IL-12 variant produced in accordance with any of the above has an ability to stimulate IFNγ production that is at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, about 30.0 to about 35.0 fold less than that of a reference IL-12, as measured by an assay. 1 in, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 800. 1 / 0 to about 1 / 900.0, about 1 / 900.0 to about 1 / 1000.0, about 1 / 1000.0 to about 1 / 2000.0, about 1 / 2000.0 to about 1 / 3000.0, about 1 / 3000.0 to about 1 / 4000.0, about 1 / 4000.0 to about 1 / 5000.0, about 1 / 5000.0 to about 1 / 6000.0, about 1 / 6000.0 to about 1 / 7000.0, about 1 / 7000.0 to about 1 / 8000.0, about 1 / 8000.0 to about 1 / 9000.0, about 1 / 9000.0 and / or a reference IL-12 having an ability to stimulate IFNγ production that is reduced to about 1 in 10,000.0, about 1 in 10,000.0 to about 1 in 50,000.0, about 1 in 50,000.0 to about 1 in 100,000.0, about 1 in 100,000.0 to about 1 in 200,000.0, about 1 in 200,000.0 to about 1 in 300,000.0, about 1 in 300,000.0, or less, wherein the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0068] In further embodiments, in accordance with the above, the assay comprises one or more of: (i) an intracellular cytokine staining assay; (ii) a Luminex bead-based cytokine release assay; (iii) an ELISA; or (iv) an ELISpot assay.

[0069] In another aspect, the disclosure provides a heterodimeric Fc-fusion protein comprising: a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first Fc domain; and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the second Fc domain, wherein optionally the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first and second Fc domains and / or (ii) reduce or inhibit effector function.

[0070] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises one or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0071] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises two or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0072] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises three or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0073] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises four or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0074] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises five or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0075] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises six or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0076] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises seven or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0077] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and D126A.

[0078] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and P127A.

[0079] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and T43A.

[0080] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, D126A and P127A.

[0081] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, T43A, D126A and P127A.

[0082] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and R129A.

[0083] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K168A.

[0084] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K170A.

[0085] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A and R129A.

[0086] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A and K168A.

[0087] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A and K170A.

[0088] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue Y40. In some further embodiments, the substitution mutation at amino acid residue Y40 is selected from the group comprising: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

[0089] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 177-187.

[0090] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue D126. In some further embodiments, the substitution mutation at amino acid residue D126 is selected from the group comprising: D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

[0091] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue P127. In some further embodiments, the substitution mutation at amino acid residue P127 is selected from the group comprising P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

[0092] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue R129. In some further embodiments, the substitution mutation at amino acid residue R129 is selected from the group comprising: R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

[0093] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue K168. In some further embodiments, the substitution mutation at amino acid residue K168 is selected from the group comprising K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

[0094] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue K170. In some further embodiments, the substitution mutation at amino acid residue K170 is selected from the group comprising K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, and K170W.

[0095] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a first substitution mutation selected from the group comprising Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

[0096] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain further comprises a second substitution mutation.

[0097] In a further embodiment, in accordance with the above, the second substitution mutation is D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D126W, P127A, P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I , R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R1 29Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, K168Y, K170A, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, and K170W.

[0098] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 199-247 or SEQ ID NOs: 279-290.

[0099] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain may further comprise a C74S substitution mutation.

[0100] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

[0101] In a further embodiment, and in accordance with the above, the variant IL-12 p40 subunit domain comprises one or more amino acid substitutions selected from the group comprising C177S, C252S, and C177S / C252S.

[0102] In a further embodiment, in accordance with any of the above, the first Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and the second Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0103] In a further embodiment, in accordance with any of the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from the group comprising SEQ ID NOs:24-86 and 103-166, the first Fc domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, the IL-12 p40 subunit domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, and the second Fc domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0104] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0105] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 96 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0106] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 97 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 97 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 97 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 97 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0107] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 98 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0108] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 99 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 99 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 99 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0109] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 24-86 and 103-166, the first Fc domain consists of an amino acid sequence selected from the group comprising SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, the IL-12 p40 subunit domain consists of an amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, and the second Fc domain consists of an amino acid sequence selected from the group comprising SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0110] In a further embodiment, in accordance with any of the above, the variant IL-12 p35 subunit domain comprises SEQ ID NO:87 and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A; the first Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0111] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises additional amino acid substitutions.

[0112] In another aspect, the disclosure provides a heterodimeric Fc-fusion protein comprising: a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the N-terminus of the variant IL-12 p35 subunit domain is covalently linked to the C-terminus of the first Fc domain; and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the N-terminus of the IL-12 p40 subunit domain is covalently linked to the C-terminus of the second Fc domain, wherein optionally the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first and second Fc domains and / or (ii) reduce or inhibit effector function.

[0113] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises one or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0114] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises two or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0115] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises three or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0116] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises four or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0117] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises five or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0118] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises six or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0119] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises seven or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0120] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and D126A.

[0121] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and P127A.

[0122] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and T43A.

[0123] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, D126A and P127A.

[0124] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, T43A, D126A and P127A.

[0125] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and R129A.

[0126] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K168A.

[0127] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K170A.

[0128] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A and R129A.

[0129] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A and K168A.

[0130] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A and K170A.

[0131] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue Y40. In some further embodiments, the substitution mutation at amino acid residue Y40 is selected from the group comprising: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

[0132] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 177-187.

[0133] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue D126. In some further embodiments, the substitution mutation at amino acid residue D126 is selected from the group comprising: D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

[0134] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue P127. In some further embodiments, the substitution mutation at amino acid residue P127 is selected from the group comprising P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

[0135] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue R129. In some further embodiments, the substitution mutation at amino acid residue R129 is selected from the group comprising: R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

[0136] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue K168. In some further embodiments, the substitution mutation at amino acid residue K168 is selected from the group comprising K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

[0137] In further embodiments, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue K170. In some further embodiments, the substitution mutation at amino acid residue K170 is selected from the group comprising K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, and K170W.

[0138] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises a first substitution mutation selected from the group comprising Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

[0139] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain further comprises a second substitution mutation.

[0140] In a further embodiment, in accordance with the above, the second substitution mutation is D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D126W, P12 7A, P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P1 27R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R1 29L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R129Y, K168A, K 168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K 168Q, K168S, K168T, K168W, K168Y, K170A, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

[0141] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 199-247 or SEQ ID NOs: 279-290.

[0142] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain may further comprise a C74S substitution mutation.

[0143] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

[0144] In a further embodiment, and in accordance with the above, the variant IL-12 p40 subunit domain comprises one or more amino acid substitutions selected from the group comprising C177S, C252S, and C177S / C252S.

[0145] In a further embodiment, in accordance with any of the above, the first Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and the second Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0146] In a further embodiment, in accordance with any of the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from the group comprising SEQ ID NOs:24-86 and 103-166, the first Fc domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, the IL-12 p40 subunit domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, and the second Fc domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0147] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0148] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 96 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0149] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 97 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 97 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 97 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 97 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0150] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 98 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0151] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 99 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:24-87, or SEQ ID NO:103-166; the first Fc domain comprises an amino acid sequence having at least 99 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99 percent sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 99 percent sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0152] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit domain consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 24-86 and 103-166, the first Fc domain consists of an amino acid sequence selected from the group comprising SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, the IL-12 p40 subunit domain consists of an amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, and the second Fc domain consists of an amino acid sequence selected from the group comprising SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0153] In a further embodiment, in accordance with any of the above, the variant IL-12 p35 subunit domain comprises SEQ ID NO:87 and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A; the first Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; and the second Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0154] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises additional amino acid substitutions.

[0155] In a further embodiment there is provided a heterodimeric Fc-fusion protein for use in treating cancer in a subject in accordance with any of the above.

[0156] In another aspect, the present disclosure provides one or more nucleic acids encoding a heterodimeric Fc-fusion protein according to any of the above aspects and embodiments.

[0157] In another aspect, the present disclosure provides a host cell comprising one or more nucleic acids encoding a heterodimeric Fc-fusion protein according to any of the above aspects and embodiments.

[0158] In another aspect, the present disclosure provides a host cell comprising one or more nucleic acids encoding a heterodimeric Fc-fusion protein according to any of the above aspects and embodiments.

[0159] In another aspect, the disclosure provides a method of producing a heterodimeric Fc-fusion protein, comprising culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the heterodimeric Fc-fusion protein is produced, wherein the one or more nucleic acids or vectors comprise one or more of the nucleic acids described in the above aspects and embodiments, and further wherein the heterodimeric Fc-fusion protein produced has an increased half-life compared to the half-life of a reference IL-12, wherein the IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

[0160] In a further embodiment, in accordance with the above, the method further comprises isolating and / or purifying the produced heterodimeric Fc-fusion protein.

[0161] In a further embodiment, the heterodimeric Fc-fusion protein produced in accordance with any of the above has an altered binding affinity for interleukin-12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0162] In further embodiments, the heterodimeric Fc-fusion proteins produced as described above have a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by an assay.

[0163] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0164] In a further embodiment, the heterodimeric Fc-fusion protein produced in accordance with any of the above has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

[0165] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0166] In further embodiments, the heterodimeric Fc-fusion protein produced in accordance with any of the above is about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, about 30.0 to about 35.0 fold less potent, about 35.0 to about 40.0 fold less potent, or about 40.0 fold less potent. 1 in 0.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 800.0 1 / 100 to about 1 / 900, about 1 / 900 to about 1 / 1000, about 1 / 1000 to about 1 / 2000, about 1 / 2000 to about 1 / 3000, about 1 / 3000 to about 1 / 4000, about 1 / 4000 to about 1 / 5000, about 1 / 5000 to about 1 / 6000, about 1 / 6000 to about 1 / 7000, about 1 / 7000 to about 1 / 8000, about 1 / 8000 to about 1 / 9000, about 1 / 9000 and having a potency reduced by 1 / 0.0 to about 1 / 10,000.0, about 1 / 10,000.0 to about 1 / 50,000.0, about 1 / 50,000.0 to about 1 / 100,000.0, about 1 / 100,000.0 to about 1 / 200,000.0, about 1 / 200,000.0 to about 1 / 300,000.0, about 1 / 300,000.0, or less, wherein the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0167] In a further embodiment, the assay comprises an IL-12 HEK reporter assay, in accordance with the above.

[0168] In further embodiments, the heterodimeric Fc-fusion protein produced in accordance with any of the above has an ability to stimulate IFNγ production of a reference IL-12 as measured by an assay that is at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, or about 30.0 to about 35 fold less than that of a reference IL-12. 1 in 0.0, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 800 1 / 0.0 to about 1 / 900.0, about 1 / 900.0 to about 1 / 1000.0, about 1 / 1000.0 to about 1 / 2000.0, about 1 / 2000.0 to about 1 / 3000.0, about 1 / 3000.0 to about 1 / 4000.0, about 1 / 4000.0 to about 1 / 5000.0, about 1 / 5000.0 to about 1 / 6000.0, about 1 / 6000.0 to about 1 / 7000.0, about 1 / 7000.0 to about 1 / 8000.0, about 1 / 8000.0 to about 1 / 9000.0, about 1 / 9000.0 and having an ability to stimulate IFNγ production that is reduced by 1 to about 10,000.0 fold, about 1 to about 10,000.0 fold, about 1 to about 50,000.0 fold, about 1 to about 100,000.0 fold, about 1 to about 100,000.0 fold, about 1 to about 200,000.0 fold, about 1 to about 200,000.0 fold, about 1 to about 300,000.0 fold, about 300,000.0 fold, or more, wherein the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, or IL-12 Fc fusion proteins.

[0169] In further embodiments, in accordance with the above, the assay comprises one or more of: (i) an intracellular cytokine staining assay; (ii) a Luminex bead-based cytokine release assay; (iii) an ELISA; or (iv) an ELISpot assay.

[0170] In another aspect, the disclosure provides a first fusion construct comprising: a) (i) a variant IL-12 p35 subunit domain selected from the group consisting of SEQ ID NOs: 24, 30-34, 49, 52, 53, 65, 103, 104, 112, 177-247; (ii) a first Fc domain selected from the group consisting of SEQ ID NOs: 12 and 13; and (iii) a linker comprising SEQ ID NO: 15, wherein the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the linker and the C-terminus of the linker is covalently linked to the N-terminus of the first Fc domain; and b) (i) a (variant) IL-12 p40 subunit domain selected from the group consisting of SEQ ID NOs: 4, 89, and 90. (ii) a second Fc domain selected from the group consisting of SEQ ID NOs: 13 and 12; and (iii) a linker comprising SEQ ID NO: 15, wherein the C-terminus of the (variant) IL-12 p40 subunit domain is covalently linked to the N-terminus of the linker and the C-terminus of the linker is covalently linked to the N-terminus of the second Fc domain.

[0171] In a further embodiment, in accordance with the above, the variant IL-12 p35 subunit may further comprise a C74S substitution mutation and the (variant) IL-12 p40 subunit may further comprise a C177S substitution mutation such that the interchain disulfide bond between the variant IL-12 p35 subunit domain and the (variant) IL-12 p40 subunit domain is eliminated.

[0172] In one aspect, the disclosure provides a) a variant IL-12 p35 subunit, wherein the variant IL-12 p35 subunit comprises a first amino acid substitution mutation, wherein the first amino acid substitution is selected from the group comprising: Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, K170T, and b) a non-naturally occurring IL-12 variant comprising an IL-12 p40 subunit.

[0173] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L, and K170T.

[0174] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40E; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168I, K168T, and R129A.

[0175] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40G; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168I, K168T, and R129A.

[0176] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168D, K168I, and K168T.

[0177] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40S; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168I, K168T, K170A, K170L, K170T, and R129A.

[0178] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0179] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0180] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170T; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0181] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 24, 34, 103, 104, 109, 179, 180, 183, 185, 186, 194, 196, 233, 234, 238, 240, 243, 245, and 248-278.

[0182] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0183] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit comprises a variant IL-12 p40 subunit, wherein the variant IL-12 p40 subunit comprises one or more amino acid substitutions selected from the group comprising C177S, C252S, and C177S / C252S.

[0184] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit comprises any of SEQ ID NOs: 4, 88, 89, and 90.

[0185] In a further embodiment, and in accordance with any of the above, the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

[0186] In a further embodiment, and in accordance with any of the above, the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the IL-12 p40 subunit.

[0187] In a further embodiment, in accordance with the above, the non-naturally occurring IL-12 variant further comprises a linker domain comprising an amino acid sequence selected from the group comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently linked to the N-terminus of the IL-12 p40 subunit.

[0188] In a further embodiment, and in accordance with any of the above, the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

[0189] In a further embodiment, in accordance with the above, the non-naturally occurring IL-12 variant further comprises a linker domain comprising an amino acid sequence selected from the group comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

[0190] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit comprises one or more additional amino acid substitutions.

[0191] In a further embodiment there is provided a non-naturally occurring IL-12 variant for use in treating cancer in a subject in accordance with any of the above.

[0192] In another aspect, the present disclosure provides one or more nucleic acids encoding a non-naturally occurring IL-12 variant according to any of the above aspects and embodiments.

[0193] In another aspect, the present disclosure provides a host cell comprising one or more nucleic acids encoding a non-naturally occurring IL-12 variant according to any of the above aspects and embodiments.

[0194] In another aspect, the disclosure provides a method of producing a non-naturally occurring IL-12 variant, comprising culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the non-naturally occurring variant is produced, wherein i) the one or more nucleic acids or vectors comprise one or more nucleic acids described in the above aspects and embodiments, and ii) at least one substitution mutation in the variant IL-12 p35 subunit improves the half-life compared to the half-life of a reference IL-12.

[0195] In a further embodiment, in accordance with the above, the method further comprises isolating and / or purifying the non-naturally occurring IL-12 variant produced.

[0196] In a further embodiment, and in accordance with any of the above, the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

[0197] In a further embodiment, the non-naturally occurring IL-12 variant produced in accordance with any of the above has an altered binding affinity for interleukin-12 receptor beta 2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0198] In further embodiments, the non-naturally occurring IL-12 variant produced in accordance with the above has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by an assay.

[0199] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0200] In a further embodiment, the non-naturally occurring IL-12 variant produced in accordance with any of the above has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of the reference IL-12 is detectable.

[0201] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0202] In further embodiments, the non-naturally occurring IL-12 variant produced in accordance with any of the above is about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, about 30.0 to about 40.0 fold less potent, or about 40.0 to about 50.0 fold less potent, as measured by an assay relative to a reference IL-12. about 1 in 35.0, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, About 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 7000.0 The potency is reduced to 1 in to about 1 in 8000.0, about 1 in 8000.0 to about 1 in 9000.0, about 1 in 9000.0 to about 1 in 10,000.0, about 1 in 10,000.0 to about 1 in 50,000.0, about 1 in 50,000.0 to about 100,000.0, about 1 in 100,000.0 to about 1 in 200,000.0, about 1 in 200,000.0 to about 1 in 300,000.0, about 1 in 300,000.0, or less.

[0203] In a further embodiment, the assay comprises an IL-12 HEK reporter assay, in accordance with the above.

[0204] In further embodiments, the non-naturally occurring IL-12 variant produced in accordance with any of the above has an ability to stimulate IFNγ production that is at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold less than that of a reference IL-12, as measured by an assay. About 1 in 30.0 to about 1 in 35.0, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 700.0 1, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 80 The present invention has the ability to stimulate IFNγ production reduced to about 1 / 00.0, about 1 / 8000.0 to about 1 / 9000.0, about 1 / 9000.0 to about 1 / 10,000.0, about 1 / 10,000.0 to about 1 / 50,000.0, about 1 / 50,000.0 to about 1 / 100,000.0, about 1 / 100,000.0 to about 1 / 200,000.0, about 1 / 200,000.0 to about 1 / 300,000.0, about 1 / 300,000.0, or less.

[0205] In further embodiments, in accordance with the above, the assay comprises one or more of: (i) an intracellular cytokine staining assay; (ii) a Luminex bead-based cytokine release assay; (iii) an ELISA; or (iv) an ELISpot assay.

[0206] In a further embodiment, in accordance with any of the above, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0207] In one aspect, the disclosure provides a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein i) the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first Fc domain, ii) the variant IL-12 p35 subunit domain comprises a first amino acid substitution mutation, and iii) the first amino acid substitution mutation is selected from the group comprising: Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, K170T, and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the IL-12 The present invention provides a heterodimeric Fc fusion protein comprising the second fusion construct, wherein the C-terminus of the p40 subunit domain is covalently linked to the N-terminus of the second Fc domain, and optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains and / or (ii) reduces or inhibits effector function.

[0208] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L, and K170T.

[0209] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40E; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168I, K168T, and R129A.

[0210] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40G; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168I, K168T, and R129A.

[0211] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168D, K168I, and K168T.

[0212] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40S; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168I, K168T, K170A, K170L, K170T, and R129A.

[0213] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0214] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0215] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170T; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0216] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 24, 34, 103, 104, 109, 179, 180, 183, 185, 186, 194, 196, 233, 234, 238, 240, 243, 245, and 248-278.

[0217] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0218] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, wherein the variant IL-12 p40 subunit domain comprises one or more amino acid substitutions selected from the group comprising C177S, C252S, and C177S / C252S.

[0219] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain comprises any of SEQ ID NOs: 4, 88, 89, and 90.

[0220] In a further embodiment, in accordance with any of the above, i) the first Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and ii) the second Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0221] In a further embodiment, in accordance with any of the above, i) the first fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; and iii) the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the first Fc domain.

[0222] In a further embodiment, in accordance with any of the above, i) the second fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; and iii) the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the second Fc domain.

[0223] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises one or more additional amino acid substitutions.

[0224] In a further embodiment there is provided a heterodimeric Fc-fusion protein for use in treating cancer in a subject in accordance with any of the above.

[0225] In another aspect, the present disclosure provides one or more nucleic acids encoding a heterodimeric Fc-fusion protein according to any of the above aspects and embodiments.

[0226] In another aspect, the present disclosure provides a host cell comprising one or more nucleic acids encoding a heterodimeric Fc-fusion protein according to any of the above aspects and embodiments.

[0227] In another aspect, the disclosure provides a method of producing a heterodimeric Fc-fusion protein, comprising culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the heterodimeric Fc-fusion protein is produced, wherein i) the one or more nucleic acids or vectors comprise one or more nucleic acids described in the above aspects and embodiments, and ii) at least one substitution mutation in the variant IL-12 p35 subunit domain improves the half-life compared to the half-life of a reference IL-12.

[0228] In a further embodiment, in accordance with the above, the method further comprises isolating and / or purifying the produced heterodimeric Fc-fusion protein.

[0229] In a further embodiment, the heterodimeric Fc-fusion protein produced in accordance with any of the above has an altered binding affinity for interleukin-12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0230] In further embodiments, the heterodimeric Fc-fusion proteins produced as described above have a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by an assay.

[0231] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0232] In a further embodiment, the heterodimeric Fc-fusion protein produced in accordance with any of the above has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

[0233] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0234] In further embodiments, the heterodimeric Fc-fusion protein produced in accordance with any of the above is about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, or about 30.0 fold less potent, relative to a reference IL-12 as measured by an assay. to about 1 in 35.0, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0 , about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 7000.0 The potency is reduced to 1 / 8000, about 1 / 8000 to about 1 / 9000, about 1 / 9000 to about 1 / 10,000, about 1 / 10,000 to about 1 / 50,000, about 1 / 50,000 to about 1 / 100,000, about 1 / 100,000 to about 1 / 200,000, about 1 / 200,000 to about 1 / 300,000, about 1 / 300,000, or less.

[0235] In a further embodiment, the assay comprises an IL-12 HEK reporter assay, in accordance with the above.

[0236] In further embodiments, the heterodimeric Fc-fusion protein produced in accordance with any of the above has an ability to stimulate IFNγ production of a reference IL-12 as measured by an assay that is at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, or about 25.0 to about 30.0 fold less than that of a reference IL-12. , about 1 in 30.0 to about 1 in 35.0, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 700.0 1 in, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 8 The present invention has the ability to stimulate IFNγ production reduced to about 1 / 10,000, about 1 / 8,000 to about 1 / 9,000, about 1 / 9,000 to about 1 / 10,000, about 1 / 10,000 to about 1 / 50,000, about 1 / 50,000 to about 1 / 100,000, about 1 / 100,000 to about 1 / 200,000, about 1 / 200,000 to about 1 / 300,000, about 1 / 300,000, or less.

[0237] In further embodiments, in accordance with the above, the assay comprises one or more of: (i) an intracellular cytokine staining assay; (ii) a Luminex bead-based cytokine release assay; (iii) an ELISA; or (iv) an ELISpot assay.

[0238] In a further embodiment, in accordance with any of the above, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0239] In one aspect, the disclosure provides a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein i) the C-terminus of the first Fc domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain, ii) the variant IL-12 p35 subunit domain comprises a first amino acid substitution mutation, and iii) the first amino acid substitution mutation is selected from the group comprising: Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, K170T; and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the C-terminus of the second Fc domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain. Provided is a heterodimeric Fc fusion protein comprising the second fusion construct covalently linked to the N-terminus of a p40 subunit domain, wherein optionally the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains and / or (ii) reduces or inhibits effector function.

[0240] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L, and K170T.

[0241] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40E; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168I, K168T, and R129A.

[0242] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40G; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168I, K168T, and R129A.

[0243] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K170A, K168A, K168D, K168I, and K168T.

[0244] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is Y40S; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168I, K168T, K170A, K170L, K170T, and R129A.

[0245] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0246] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0247] In a further embodiment, in accordance with the above, i) the first amino acid substitution mutation is K170T; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group comprising K168A, K168I, K168T, and R129E.

[0248] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 24, 34, 103, 104, 109, 179, 180, 183, 185, 186, 194, 196, 233, 234, 238, 240, 243, 245, and 248-278.

[0249] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0250] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, wherein the variant IL-12 p40 subunit domain comprises one or more amino acid substitutions selected from the group comprising C177S, C252S, and C177S / C252S.

[0251] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain comprises any of SEQ ID NOs: 4, 88, 89, and 90.

[0252] In a further embodiment, in accordance with any of the above, i) the first Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and ii) the second Fc domain comprises amino acids selected from the group comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0253] In a further embodiment, in accordance with any of the above, i) the first fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; and iii) the C-terminus of the first Fc domain is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain.

[0254] In a further embodiment, in accordance with any of the above, i) the second fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; and iii) the C-terminus of the second Fc domain is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the IL-12 p40 subunit domain.

[0255] In a further embodiment, and in accordance with any of the above, the variant IL-12 p35 subunit domain comprises one or more additional amino acid substitutions.

[0256] In a further embodiment there is provided a heterodimeric Fc-fusion protein for use in treating cancer in a subject in accordance with any of the above.

[0257] In another aspect, the present disclosure provides one or more nucleic acids encoding a heterodimeric Fc-fusion protein according to any of the above aspects and embodiments.

[0258] In another aspect, the present disclosure provides a host cell comprising one or more nucleic acids encoding a heterodimeric Fc-fusion protein according to any of the above aspects and embodiments.

[0259] In another aspect, the disclosure provides a method of producing a heterodimeric Fc-fusion protein, comprising culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the heterodimeric Fc-fusion protein is produced, wherein i) the one or more nucleic acids or vectors comprise one or more nucleic acids described in the above aspects and embodiments, and ii) at least one substitution mutation in the variant IL-12 p35 subunit domain improves the half-life compared to the half-life of a reference IL-12.

[0260] In a further embodiment, in accordance with the above, the method further comprises isolating and / or purifying the produced heterodimeric Fc-fusion protein.

[0261] In a further embodiment, the heterodimeric Fc-fusion protein produced in accordance with any of the above has an altered binding affinity for interleukin-12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0262] In further embodiments, the heterodimeric Fc-fusion proteins produced as described above have a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by an assay.

[0263] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0264] In a further embodiment, the heterodimeric Fc-fusion protein produced in accordance with any of the above has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

[0265] In a further embodiment, in accordance with the above, the assay comprises an SPR assay.

[0266] In further embodiments, the heterodimeric Fc-fusion protein produced in accordance with any of the above is about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, or about 30.0 fold less potent, relative to a reference IL-12 as measured by an assay. to about 1 in 35.0, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0 , about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 7000.0 The potency is reduced to 1 / 8000, about 1 / 8000 to about 1 / 9000, about 1 / 9000 to about 1 / 10,000, about 1 / 10,000 to about 1 / 50,000, about 1 / 50,000 to about 1 / 100,000, about 1 / 100,000 to about 1 / 200,000, about 1 / 200,000 to about 1 / 300,000, about 1 / 300,000, or less.

[0267] In a further embodiment, the assay comprises an IL-12 HEK reporter assay, in accordance with the above.

[0268] In further embodiments, the heterodimeric Fc-fusion protein produced in accordance with any of the above has an ability to stimulate IFNγ production of a reference IL-12 as measured by an assay that is at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, or about 25.0 to about 30.0 fold less than that of a reference IL-12. , about 1 in 30.0 to about 1 in 35.0, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 700.0 1 in, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 8 The present invention has the ability to stimulate IFNγ production reduced to about 1 / 10,000, about 1 / 8,000 to about 1 / 9,000, about 1 / 9,000 to about 1 / 10,000, about 1 / 10,000 to about 1 / 50,000, about 1 / 50,000 to about 1 / 100,000, about 1 / 100,000 to about 1 / 200,000, about 1 / 200,000 to about 1 / 300,000, about 1 / 300,000, or less.

[0269] In further embodiments, in accordance with the above, the assay comprises one or more of: (i) an intracellular cytokine staining assay; (ii) a Luminex bead-based cytokine release assay; (iii) an ELISA; or (iv) an ELISpot assay.

[0270] In a further embodiment, in accordance with any of the above, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0271] In another aspect, the disclosure provides a first fusion construct comprising: a) a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain, wherein i) the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first linker domain and the C-terminus of the first linker domain is covalently linked to the N-terminus of the first Fc domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 12; and iv) the variant IL-12 The p35 subunit domain contains the following amino acids: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40 E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / K 168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I , Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K17 0T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, K170P, K and b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain, wherein i) the IL-12and (iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89; and optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits effector function.

[0272] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0273] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

[0274] In another aspect, the disclosure provides a first fusion construct comprising: a) a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain, wherein i) the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first linker domain and the C-terminus of the first linker domain is covalently linked to the N-terminus of the first Fc domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 13; and iv) the variant IL-12 The p35 subunit domain contains the following amino acids: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40 E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / K 168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I , Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K17 0T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, K170P, K and b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain, wherein i) the IL-12and (iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89; and optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits effector function.

[0275] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0276] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

[0277] In another aspect, the disclosure provides a first fusion construct comprising: a) a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain, wherein i) the C-terminus of the first Fc domain is covalently linked to the N-terminus of the first linker domain and the C-terminus of the first linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 12; and iv) the variant IL-12 The p35 subunit domain contains the following amino acids: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40 E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / K 168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I , Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K17 0T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, K170P, K and b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain, wherein i) the C-terminus of the second Fc domain is covalently linked to the N-terminus of the second linker domain, andand ii) the second linker domain comprises SEQ ID NO: 15; iii) the second Fc domain comprises SEQ ID NO: 13; and iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89. Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains and / or (ii) reduces or inhibits effector function.

[0278] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0279] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

[0280] In another aspect, the disclosure provides a first fusion construct comprising: a) a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain, wherein i) the C-terminus of the first Fc domain is covalently linked to the N-terminus of the first linker domain and the C-terminus of the first linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 13; and iv) the variant IL-12 The p35 subunit domain contains the following amino acids: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40 E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / K 168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I , Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K17 0T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, K170P, K and b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain, wherein i) the C-terminus of the second Fc domain is covalently linked to the N-terminus of the second linker domain, andand iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89. The present invention provides a heterodimeric Fc fusion protein comprising the second fusion construct, wherein the first Fc domain and the second Fc domain comprise a first linker domain and a second Fc domain covalently linked to the N-terminus of the first Fc domain and the second Fc domain; ii) the second linker domain comprises SEQ ID NO: 15; iii) the second Fc domain comprises SEQ ID NO: 12; and iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89, wherein optionally the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains and / or (ii) reduces or inhibits effector function.

[0281] In a further embodiment, and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0282] In a further embodiment, and in accordance with any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

[0283] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood 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 referred to herein as "Fig.", "FIG.", "Figure," "Figures," "Figs.", and "FIGs."). [Brief explanation of the drawings]

[0284] [Figure 1] 1 shows a list of the sequences of human wild-type IL-12 subunits α and β (precursor and mature forms). [Figure 2] The sequences of human wild-type IL-12Rβ1 (+extracellular domain sequence) and IL-12Rβ2 (+extracellular domain sequence) are listed. [Figure 3]Exemplary sequences of the human IgG1 Fc domain (G1m allotype and two sets of knob / hole variants) are shown. [Figure 4] A list of exemplary domain linker sequences is provided. [Figure 5] A list of variant IL-12 p35 subunit sequences containing one mutation is shown. [Figure 6A] A list of variant IL-12 p35 subunit sequences containing two mutations is shown. [Figure 6B] A list of variant IL-12 p35 subunit sequences containing two mutations is shown. [Figure 7A] A list of variant IL-12 p35 subunit sequences containing three mutations is shown. [Figure 7B] A list of variant IL-12 p35 subunit sequences containing three mutations is shown. [Figure 8A] A list of variant IL-12 p35 subunit sequences containing four mutations is shown. [Figure 8B] A list of variant IL-12 p35 subunit sequences containing four mutations is shown. [Figure 9] Shown are lists of (i) variant IL-12 p35 subunit sequences containing five mutations, (ii) variant IL-12 p35 subunit sequences containing six mutations, and (iii) variant IL-12 p35 subunit sequences containing the C74S mutation. [Figure 10] A list of variant IL-12 p40 subunit sequences is provided. [Figure 11A] A list of exemplary heterodimeric Fc fusion protein sequences and related sequences is provided. [Figure 11B] A list of exemplary heterodimeric Fc fusion protein sequences and related sequences is provided. [Figure 11C] A list of exemplary heterodimeric Fc fusion protein sequences and related sequences is provided. [Figure 11D] A list of exemplary heterodimeric Fc fusion protein sequences and related sequences is provided. [Figure 11E] A list of exemplary heterodimeric Fc fusion protein sequences and related sequences is provided. [Figure 12] FIG. 1 shows a schematic diagram of an exemplary IL-12 Fc fusion protein in a monovalent format. [Figure 13] 1 shows size-exclusion (upper panel) and cation-exchange (lower panel) profiles of wild-type IL-12 heterodimeric Fc fusion protein (also referred to herein as "wild-type IL-12 Fc," "IL-12 Fc wild-type," or "IL-12 Fc wt," or "WT"). Fractions pooled for further purification and / or analysis are indicated by rectangles. [Figure 14] Shown are reducing (+DTT; left panel) and non-reducing (-DTT; right panel) SDS-PAGE analyses of purified wild-type IL-12 Fc. [Figure 15] 1 shows a model of the interaction between IL-12 and IL-12Rβ2. The IL-12 p35 subunit, IL-12 p40 subunit, and IL-12Rβ2 are shown in schematic representation, with residues of the IL-12 p35 subunit selected for mutational analysis shown as sticks. [Figure 16A] 1 shows a reducing SDS-PAGE analysis (+DTT) of purified heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit domain contains one mutation and the Fc domain contains knob and hole mutations that promote heterodimerization of the Fc domain. [Figure 16B] 1 shows non-reducing SDS-PAGE analysis (-DTT) of purified heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit domain contains one mutation and the Fc domain contains knob and hole mutations that promote heterodimerization of the Fc domain. [Figure 17]1 shows SPR sensorgrams (i.e., plots of SPR response in response units (RU) generated by the SPR instrument versus time in seconds) illustrating binding of 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1,000, and 2,000 nM IL-12Rβ2 to immobilized wild-type IL-12 Fc ("wild-type"; top row) or the indicated mutants (middle and bottom rows). [Figure 18]

[0023] Figure 1 shows a summary of SPR data for binding of IL-12Rβ2 to wild-type IL-12 Fc ("wild-type") or a heterodimeric IL-12 Fc fusion protein containing one mutation in the IL-12 p35 subunit domain. The amount of IL-12 Fc immobilized on the chip (capture) and the dissociation constant (KD) are shown. As used herein, when referring to the results of an SPR assay, the term "weak" refers to a KD value below the lower limit of detection of the assay used (i.e., the KD value cannot be accurately determined due to the lower limit of detection of the assay). [Figure 19] Figure 1 shows the activity of commercially available IL-12 (Miltenyi; top row) or heterodimeric IL-12 Fc fusion proteins containing a single mutation in the IL-12 p35 subunit domain (middle and bottom rows) compared to wild-type IL-12 Fc ("wild-type") in a HEK-Blue IL-12 reporter assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for commercially available IL-12 or heterodimeric IL-12 Fc fusion proteins. [Figure 20] A summary of the EC50 values and fold change (compared to wild type) for commercially available IL-12 (Miltenyi), wild-type IL-12 Fc ("wild type"), and a heterodimeric IL-12 Fc fusion protein containing one mutation in the IL-12 p35 subunit domain using the HEK-Blue IL-12 assay is shown. [Figure 21]Figure 1 shows the activity of commercially available IL-12 (Miltenyi; top row) and heterodimeric IL-12 Fc fusion proteins containing a single mutation in the IL-12 p35 subunit domain (middle and bottom rows) compared to wild-type IL-12 Fc ("wild-type") in a primary T cell IFNγ release assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for commercially available IL-12 or heterodimeric IL-12 Fc fusion proteins. [Figure 22] A summary of the EC50 values and fold change (compared to wild type) for commercially available IL-12 (Miltenyi), wild-type IL-12 Fc ("wild type"), and a heterodimeric IL-12 Fc fusion protein containing one mutation in the IL-12 p35 subunit domain using a primary T cell IFNγ release assay is shown. [Figure 23] Shown are reducing (+DTT; left panel) and non-reducing (-DTT; right panel) SDS-PAGE analyses of purified heterodimeric IL-12 Fc fusion proteins in which the IL-12 p35 subunit domain contains two, three, or four mutations and the Fc domain contains knob and hole mutations that promote heterodimerization of the Fc domain. [Figure 24A] Figure 1 shows the activity of commercially available IL-12 (Miltenyi) or heterodimeric IL-12 Fc fusion proteins containing one, two, three, or four mutations in the IL-12 p35 subunit domain compared to wild-type IL-12 Fc ("wild-type") in a HEK-Blue IL-12 reporter assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for heterodimeric IL-12 Fc mutant fusion proteins. [Figure 24B]Figure 1 shows the activity of commercially available IL-12 (Miltenyi) or heterodimeric IL-12 Fc fusion proteins containing one, two, three, or four mutations in the IL-12 p35 subunit domain compared to wild-type IL-12 Fc ("wild-type") in a HEK-Blue IL-12 reporter assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for commercially available IL-12 or heterodimeric IL-12 Fc fusion proteins. [Figure 25] Figure 1 shows a summary of the EC50 values and fold changes (compared to wild-type) for commercially available IL-12 (Miltenyi), wild-type IL-12 Fc ("wild-type"), and heterodimeric IL-12 Fc fusion proteins containing one, two, three, or four mutations in the IL-12 p35 subunit domain using the HEK-Blue IL-12 assay. [Figure 26]

[0039] Figure 1 shows the activity of heterodimeric IL-12 Fc fusion proteins (top and bottom rows) containing two, three, or four mutations in the IL-12 p35 subunit domain in a primary T cell IFNγ release assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for heterodimeric IL-12 Fc fusion proteins. [Figure 27] A summary of the EC50 values and fold changes (compared to wild-type) for wild-type IL-12 Fc ("wild-type") and heterodimeric IL-12 Fc fusion proteins containing two, three, or four mutations in the IL-12 p35 subunit using a primary T cell IFNγ release assay is shown. [Figure 28]Shown are SPR sensorgrams (i.e., plots of SPR response in response units (RU) generated by the SPR instrument versus time in seconds) showing binding of 0.7, 2.1, 6.2, 18.5, 55.6, 167, and 500 nM IL-12Rβ2 to immobilized wild-type IL-12 Fc (“wild-type”; left) or the indicated IL-12 Fc K170A mutant (“K170A”; right). [Figure 29A] Figure 1 shows the activity of heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain compared to wild-type IL-12 Fc ("WT") in a HEK-Blue IL-12 reporter assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for heterodimeric IL-12 Fc mutant fusion proteins. [Figure 29B] Figure 1 shows the activity of heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain compared to wild-type IL-12 Fc ("WT") in a HEK-Blue IL-12 reporter assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for heterodimeric IL-12 Fc mutant fusion proteins. [Figure 30] A summary of the EC50 values and fold changes (compared to wild type) for commercially available IL-12 (Miltenyi), wild-type IL-12 Fc ("wild type"), and heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain using the HEK-Blue IL-12 assay is shown. [Figure 31]

[0033] Figure 1 shows the activity of heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain in a primary T cell IFNγ release assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for heterodimeric IL-12 Fc mutant fusion proteins. [Figure 32] A summary of the EC50 values and fold changes (compared to wild type) of heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain using a primary T cell IFNγ release assay is shown. [Figure 33] FIG. 1 shows a schematic diagram of an exemplary IL-12 Fc fusion protein in a monovalent format. [Figure 34] Cation exchange chromatography (upper panel) and analytical size-exclusion chromatography (lower panel) profiles of wild-type IL-12 heterodimeric Fc fusion protein (also referred to herein as "wild-type IL-12 Fc," "IL-12 Fc wild-type," or "IL-12 Fc wt," or "WT"). Fractions pooled for further functional assays or analysis are indicated by rectangles. [Figure 35A] 1 shows a reducing SDS-PAGE analysis (+DTT) of heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain. [Figure 35B] 1 shows non-reducing SDS-PAGE analysis (-DTT) of heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain. [Figure 36]Figure 1 shows the activity of commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain compared to wild-type IL-12 Fc ("WT") in a HEK-Blue IL-12 reporter assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for commercially available IL-12 or heterodimeric IL-12 Fc mutant fusion proteins. [Figure 37] A summary of the EC50 values and fold changes (compared to wild type) for commercially available IL-12 (Miltenyi), wild-type IL-12 Fc ("wild type"), and heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain using the HEK-Blue IL-12 assay is shown. [Figure 38] Figure 1 shows the activity of commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain in a primary T cell IFNγ release assay. Symbols and error bars represent the mean and SD, respectively. For symbols, filled circles represent the mean value for wild-type IL-12 Fc, and open squares represent the mean value for commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc mutant fusion proteins. [Figure 39] A summary of the EC50 values and fold changes (compared to wild type) of commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain using a primary T cell IFNγ release assay is shown. [Figure 40A]Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD8+ T cells from a donor ("Donor 245") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 40B] Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD8+ T cells from a donor ("Donor 245") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 41A] Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD8+ T cells from a donor ("Donor 247") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 41B] Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD8+ T cells from a donor ("Donor 247") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 42A] Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD4+ T cells from a donor ("Donor 245") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 42B] Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD4+ T cells from a donor ("Donor 245") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 43A] Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD4+ T cells from a donor ("Donor 247") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 43B]Figure 1 shows the expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39, and granzyme B on CD4+ T cells from a donor ("Donor 247") after culture in the presence or absence of 1, 5, or 10 ng / ml of wild-type IL-12 Fc or 10 ng / ml of the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. Horizontal lines represent the mean of two duplicate measurements (symbols). [Figure 44] Principal component analysis (PCA) of CD8+ T cells based on the full panel of phenotypic markers described in the text is shown. Donor batch effects were removed using the "removeBatchEffect" function in limma, and the corrected MFI values of each functional marker were analyzed using the "plotMDS" function in limma to obtain a PCA plot. The PC1 components that correlated best with variant potency are shown as box-and-whisker plots. Here, the horizontal line represents the median derived from three donor samples (symbols), each analyzed in duplicate. [Figure 45] Principal component analysis (PCA) of CD4+ T cells based on the full panel of phenotypic markers described in the text is shown. Donor batch effects were removed using the "removeBatchEffect" function in limma, and the corrected MFI values for each functional marker were analyzed using the "plotMDS" function in limma to generate PCA plots. The PC2 components most correlated with variant potency are shown as box-and-whisker plots. Here, the horizontal line represents the median derived from three donor samples (symbols), each analyzed in duplicate. [Figure 46]The numbers of CD4+ cells (upper panel), CD8+ cells (middle panel), and CD4:CD8 ratios (defined as the number of CD4+ cells divided by the number of CD8+ cells; lower panel) present in 1 μl of mouse plasma 12 days after injection of 10 × 10 freshly isolated human PBMCs and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations within the IL-12 p35 subunit domain are shown. Horizontal lines represent the mean values of measurements (symbols) performed in three mice. [Figure 47] Figure 1 shows the expression of PD-1, Lag-3, Tim-3, CD95, Ki67, granzyme B, and CD45RA / CD45RO on CD4 T cells derived from 10 x 10 freshly isolated human PBMCs and plasma of mice 12 days after injection with either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations within the IL-12 p35 subunit domain. Horizontal lines represent the mean values of measurements (symbols) performed in three mice. [Figure 48] Figure 1 shows the expression of PD-1, Lag-3, Tim-3, CD95, Ki67, granzyme B, and CD45RA / CD45RO on CD8+ T cells from 10 x 10 freshly isolated human PBMCs and plasma of mice 12 days after injection with either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations within the IL-12 p35 subunit domain. Horizontal lines represent the mean values of measurements (symbols) performed in three mice. [Figure 49] Figure 1 shows the concentration of IFNγ present in the plasma of mice injected with 10 x 10 freshly isolated human PBMCs at time 0 and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. IL-12 Fc was injected at either 0.01 mg / kg (upper panel) or 0.1 mg / kg (lower panel) at time 0 and 168 hours, as indicated by the downward arrows. Data points were collected across multiple time points as indicated on the graph. [Figure 50] Figure 1 shows the concentration of IL-12 Fc present in the plasma of mice injected with 10 x 10 freshly isolated human PBMCs at time 0 and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain. IL-12 Fc was injected at either 0.01 mg / kg (upper panel) or 0.1 mg / kg (lower panel) at time 0 and 168 hours, as indicated by the downward arrows. Data points were collected across multiple time points as indicated on the graph. [Figure 51] Figure 1 shows the number of CD4+ and CD8+ cells present in 1 μl of mouse blood 7 days after injection of 2.5 × 106 T cell receptor alpha chain constant region (TRAC) knockout, 1G4-transduced primary T cells, and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations within the IL-12 p35 subunit domain, as well as the expression of granzyme B, Lag-3, PD-1, T-bet, CD45RA+ / CD45RO-, and Ki-67 on CD8+ T cells. Horizontal lines represent the median values of measurements (symbols) performed in 5–6 mice per group. [Figure 52] The numbers of CD4+ and CD8+ cells present in 1 μl of mouse blood 14 days after injection of 2.5 × 106 TRAC knockout, 1G4-transduced primary T cells, and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain, as well as the expression of granzyme B, Lag-3, PD-1, T-bet, CD45RA+ / CD45RO-, and Ki67 on CD8+ T cells are shown. Horizontal lines represent the median values of measurements (symbols) performed in 5–6 mice per group. [Figure 53]Concentrations of IL-12 Fc present in the plasma of mice injected with 2.5 x 10 TRAC knockout, 1G4-transduced primary T cells and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations within the IL-12 p35 subunit domain on days 1, 7, 14, and 21. Symbols and error bars represent the mean and standard deviation, respectively, of measurements by Luminex assay performed on plasma from 5–6 mice. [Figure 54] Figure 1 shows the concentration of IFNγ present in the plasma of mice injected with 2.5 x 10 TRAC knockout, 1G4-transduced primary T cells and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations in the IL-12 p35 subunit domain on days 1, 7, 14, and 21. Symbols and error bars represent the mean and standard deviation, respectively, of measurements by Luminex assay performed on plasma from 5–6 mice. [Figure 55] Tumor growth curves are shown for NSG mice (n=5-6 per treatment group) that were subcutaneously injected with 2 x 10 HCT116 human colorectal carcinoma cells expressing the HLA-A2 / NY-ESO-1 A4 variant on day -14, followed by 2.5 x 10 TRAC knockout, 1G4-transduced primary T cells on day 0, and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations within the IL-12 p35 subunit domain on days 0, 7, and 14. Symbols and error bars represent the mean and standard error of the mean, respectively. [Figure 56]Tumor measurements at day 18 are shown for NSG mice (n=5-6 per treatment group) that were subcutaneously injected with 2x10 HCT116 human colorectal carcinoma cells expressing the HLA-A2 / NY-ESO-1 A4 variant on day -14, followed by 2.5x10 TRAC knockout, 1G4-transduced primary T cells on day 0 and either wild-type IL-12 Fc or the indicated heterodimeric IL-12 Fc fusion proteins with mutations within the IL-12 p35 subunit domain. Symbols represent tumor volumes for individual mice, and black lines represent the median within each treatment group. Statistics were calculated using one-way ANOVA. ns indicates not significant, and ** indicates P<0.01. [Figure 57A] 1 shows non-reducing SDS-PAGE analysis of protein A purified indicated heterodimeric IL-12 Fc fusion proteins with one or two mutations in the IL-12 p35 subunit domain. [Figure 57B] 1 shows non-reducing SDS-PAGE analysis of protein A purified indicated heterodimeric IL-12 Fc fusion proteins with one or two mutations in the IL-12 p35 subunit domain. [Figure 58A] 1 shows reducing SDS-PAGE analysis of protein A purified indicated heterodimeric IL-12 Fc fusion proteins with one or two mutations in the IL-12 p35 subunit domain. [Figure 58B] 1 shows reducing SDS-PAGE analysis of protein A purified indicated heterodimeric IL-12 Fc fusion proteins with one or two mutations in the IL-12 p35 subunit domain. [Figure 59]AlphaLISA measurements of IFNγ in the culture medium of activated T cells that were unstimulated or incubated with protein A-purified Expi-CHO medium ("No DNA") or either 20 pM wild-type IL-12 Fc ("WT") or 20 pM heterodimeric IL-12 Fc fusion protein with the IL-12 p35 subunit domain bearing the mutation at amino acid position 40 indicated on the x-axis are shown. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12 Fc Y40Y. [Figure 60] AlphaLISA measurements of IFNγ in the culture medium of activated T cells that were either unstimulated or incubated with Protein A-purified Expi-CHO medium ("No DNA") or with 80 pM wild-type IL-12 Fc ("WT") or 80 pM heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit domain has a Y40A mutation and an additional mutation at amino acid position 126, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12 Fc Y40A / D126D. [Figure 61] AlphaLISA measurements of IFNγ in the culture medium of activated T cells that were either unstimulated or incubated with Protein A-purified Expi-CHO medium ("No DNA") or with 80 pM wild-type IL-12 Fc ("WT") or 80 pM heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit domain has a Y40A mutation and an additional mutation at amino acid position 127, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12 Fc Y40A / P127P. [Figure 62]AlphaLISA measurements of IFNγ in the culture medium of activated T cells that were either unstimulated or incubated with Protein A-purified Expi-CHO medium ("No DNA") or with 80 pM wild-type IL-12 Fc ("WT") or 80 pM heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit domain has a Y40A mutation and an additional mutation at amino acid position 129, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12 Fc Y40A / R129R. [Figure 63] AlphaLISA measurements of IFNγ in the culture medium of activated T cells that were either unstimulated or incubated with Protein A-purified Expi-CHO medium ("No DNA") or with 80 pM of either wild-type IL-12 Fc ("WT") or 80 pM of a heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit domain has a Y40A mutation and an additional mutation at amino acid position 168, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12 Fc Y40A / K168K. [Figure 64] AlphaLISA measurements of IFNγ in the culture medium of activated T cells that were unstimulated or incubated with protein A-purified Expi-CHO medium ("No DNA") or either 20 pM wild-type IL-12 Fc ("WT") or 20 pM of a heterodimeric IL-12 Fc fusion protein bearing the mutation at amino acid position 170 of the IL-12 p35 subunit indicated on the x-axis are shown. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12 Fc K170K. [Figure 65] Figure 1 shows the activity of wild-type IL-12 Fc and heterodimeric IL-12 Fc fusion proteins containing the Y40X single mutation (upper panel) or the Y40A / K168X double mutation (lower panel) in the IL-12 p35 subunit domain in a primary T cell IFNγ release assay. Symbols and error bars represent the mean and SD, respectively. [Figure 66]A summary of the EC50 values and fold changes (compared to wild type) of heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain using a primary T cell IFNγ release assay is shown. [Figure 67] 1 shows the thermal denaturation profiles of wild-type IL-12 Fc and the indicated heterodimeric IL-12 Fc fusion proteins in which the IL-12 p35 subunit domain contains either Y40A, D126A, R129A, P127A, K168A, or K170A. The solid line represents the average of three independent replicates. [Figure 68] Figure 1 shows the percentage activity of the indicated heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain in a primary T cell IFNγ release assay. Percent activity refers to the activity of the sample after incubation at a given temperature (indicated in the title of each panel) relative to the activity of an untreated sample or a sample treated at 48°C (indicated on the y-axis of each panel). [Figure 69] A summary of the individual melting temperatures (TM1 and TM2) of wild-type IL-12 Fc and heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain, as measured by differential scanning fluorimetry (DSF), is shown. [Figure 70] Figure 1 shows the activity of three different batches of wild-type IL-12 Fc (WT1, WT2, and WT3) and heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain in a primary T cell IFNγ release assay. Symbols and error bars represent the mean and SD, respectively. [Figure 71] A summary of the EC50 values and fold changes (compared to wild type) for three different batches of wild-type IL-12 Fc (WT1, WT2, and WT3) and heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain using a primary T cell IFNγ release assay in four separate donors is shown. [Figure 72]Killing curves generated by incubation of TRAC knockout, 1G4-transduced primary T cells (effectors) with HLA-A2 / NY-ESO A4 variant / GFP-transduced HCT116 cells (targets) at an effector-to-target cell ratio of 2.5:1 are shown. Prior to assay setup, effectors were incubated overnight with 0–10 ng / ml wild-type IL-12 Fc, as indicated by the symbols. The number of remaining target cells (shown as GFP confluence normalized to 0 h) is plotted as a function of time. [Figure 73] This figure shows a summary of the results of a killing assay in which TRAC knockout, 1G4-transduced primary T cells (effectors) were incubated with HLA-A2 / NY-ESO A4 variant / GFP-transduced HCT116 cells (targets) at an effector-to-target cell ratio of 2.5:1. Prior to assay setup, effectors were incubated overnight with either 0–10 ng / ml wild-type IL-12 Fc (blank bars) or a single, predefined concentration of the indicated heterodimeric IL-12 Fc fusion protein containing one or two mutations in the IL-12 p35 subunit domain (shaded bars). The bars and error bars represent the mean and standard deviation of the number of target cells (shown as GFP confluence) remaining at 8 h. The horizontal dashed lines represent the number of target cells remaining after incubation with 0 and 10 ng / ml wild-type IL-12 Fc (top and bottom horizontal dashed lines, respectively). [Figure 74] A summary of the fold change in the interpolated activity of heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain compared to wild-type IL-12 Fc is shown, as determined by a T cell killing assay. [Figure 75] A summary of the individual melting temperatures (TM1 and TM2) of wild-type IL-12 Fc and heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain as determined by DSF is shown. [Figure 76]A summary of the EC50 values and fold changes (compared to wild type) for wild-type IL-12 Fc and heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain using a primary T cell IFNγ release assay is shown. [Figure 77] FIG. 1 shows a schematic diagram of an exemplary IL-12 Fc fusion protein in a monovalent format. [Figure 78A] A list of additional variant IL-12 p35 subunit sequences is provided. [Figure 78B] A list of additional variant IL-12 p35 subunit sequences is provided. [Figure 78C] A list of additional variant IL-12 p35 subunit sequences is provided. [Figure 78D] A list of additional variant IL-12 p35 subunit sequences is provided. [Figure 78E] A list of additional variant IL-12 p35 subunit sequences is provided. [Figure 78F] A list of additional variant IL-12 p35 subunit sequences is provided. [Figure 78G] A list of additional variant IL-12 p35 subunit sequences is provided. [Figure 79A] A list of additional exemplary heterodimeric Fc-fusion protein sequences and related sequences is provided. [Figure 79B] A list of additional exemplary heterodimeric Fc-fusion protein sequences and related sequences is provided. [Figure 79C] A list of additional exemplary heterodimeric Fc-fusion protein sequences and related sequences is provided. [Figure 79D] A list of additional exemplary heterodimeric Fc-fusion protein sequences and related sequences is provided. [Figure 80A]A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80B] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80C] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80D] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80E] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80F] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80G] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80H] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80I] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80J] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80K] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80L] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 80M] A list of additional variant IL-12 p35 subunit sequences is shown (alanine mutations, selected non-alanine mutations, combinations of selected alanine mutations with selected non-alanine mutations, and combinations of selected non-alanine mutations with additional non-alanine mutations). [Figure 81] The sequences of "HLA-A2, β2-microglobulin, and NY-ESO-1 A4 variant peptide single-chain trimer" and "1G4 T cell receptor β chain-P2A-1G4 T cell receptor α chain" are shown. [Figure 82]Figure 1 shows the activity of two different configurations of wild-type IL-12 Fc (p40-Fc(knob) / p35-Fc(hole) and p40-Fc(hole) / p35-Fc(knob)) and a heterodimeric IL-12 Fc fusion protein containing two mutations in the IL-12 p35 subunit domain in a primary T cell IFNγ release assay. Symbols and error bars represent the mean and SD, respectively. [Figure 83] A summary of the EC50 values and fold changes (compared to wild type) for two different configurations of wild-type IL-12 Fc (p40-Fc(knob) / p35-Fc(hole) and p40-Fc(hole) / p35-Fc(knob)) and a heterodimeric IL-12 Fc fusion protein containing two mutations in the IL-12 p35 subunit domain using a primary T cell IFNγ release assay is shown. DETAILED DESCRIPTION OF THE INVENTION

[0285] The description is provided 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 headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. While various embodiments of the disclosed invention(s) have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will 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 can be used in practicing any of the invention(s) described herein.

[0286] With respect to related art, all patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated by reference in their entirety.

[0287] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In keeping with the scope of this disclosure, the following terms are defined below. The definitions provided are intended to apply to a given term as well as other derivative linguistic and grammatical equivalents of that term.

[0288] As used herein, the term "protein" refers to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. When a "biologically functional molecule" includes two or more proteins, such as, for example, IL-12 (including IL-12 p35 and IL-12 p40 polypeptides), the individual proteins included in the two or more proteins may be referred to as "subunits," "monomers," or "domains," respectively, and the biologically functional molecule may be referred to as a "complex." In some embodiments, the two or more proteins of a functional complex may be non-covalently linked. In some embodiments, the two or more proteins of a functional complex are covalently linked, for example, by disulfide bonds.

[0289] As used herein, the term "cytokine" refers to a wide variety of proteins, e.g., chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, etc., that are secreted by a first cell and cause an effect on one or more cells upon binding of the secreted cytokine to a receptor on the cell or cells. Cytokines can be involved in autocrine, paracrine, juxtocrine, and / or endocrine signaling.

[0290] As used herein, the term "wild-type" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A wild-type protein has an amino acid sequence (or a nucleotide sequence encoding that amino acid sequence) that has not been intentionally modified.

[0291] As used herein, the term "IL-12 p35 subunit" refers to human wild-type IL-12 p35 polypeptide, whether natural or recombinant. Thus, IL-12 p35 subunit refers to recombinantly produced IL-12 p35 polypeptide, synthetically produced IL-12 p35 polypeptide, and IL-12 p35 extracted from cells or tissues. The amino acid sequence of human wild-type IL-12 p35 subunit is shown in Figure 1 (precursor: SEQ ID NO:1; mature: SEQ ID NO:2). In the context of a fusion protein, the IL-12 p35 subunit may also be referred to as an "IL-12 p35 subunit domain," where the IL-12 p35 subunit domain comprises at least a portion of the amino acid sequence encoding the IL-12 p35 subunit.

[0292] As used herein, the term "IL-12 p40 subunit" refers to human wild-type IL-12 p40 polypeptide, whether natural or recombinant. Accordingly, IL-12 p40 subunit refers to recombinantly produced IL-12 p40 polypeptide, synthetically produced IL-12 p40 polypeptide, and IL-12 p40 extracted from cells or tissues. The amino acid sequence of human wild-type IL-12 p40 subunit (precursor: SEQ ID NO:3; mature: SEQ ID NO:4) is shown in FIG. 1. In the context of a fusion protein, the IL-12 p40 subunit may also be referred to as an "IL-12 p40 subunit domain," where the IL-12 p40 subunit domain comprises at least a portion of the amino acid sequence encoding the IL-12 p40 subunit. As used herein, the phrase "(variant) IL-12 p40 subunit" is used to disclose embodiments in which the IL-12 p40 subunit comprises a wild-type IL-12 p40 polypeptide or a variant IL-12 p40 subunit.

[0293] 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 and the IL-12 p40 subunit are fused to form a single peptide chain). In further embodiments, the C-terminus of the IL-12 p35 subunit is linked to the N-terminus of the IL-12 p40 subunit (sc-IL-12(p35 / p40)). In yet other 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 linked to the N-terminus of the IL-12 p35 subunit (sc-IL-12(p40 / p35)). In yet other embodiments, 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.

[0294] 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 the residue at position 252.

[0295] As used herein, the term "parent protein" refers to a "reference" protein, an amino acid sequence encoding the reference protein, or a DNA sequence encoding an amino acid sequence encoding the reference protein. In some embodiments, the reference protein includes a wild-type protein, an amino acid sequence encoding the wild-type protein, and / or a nucleic acid sequence encoding an amino acid sequence encoding the wild-type protein. In some embodiments, the reference protein includes a human wild-type protein, an amino acid sequence encoding the human wild-type protein, and / or a nucleic acid sequence encoding an amino acid sequence encoding the human wild-type protein. In some embodiments, the reference protein includes a (human) wild-type protein conjugated to an Fc domain.

[0296] As used herein, the terms "variant protein," "protein variant," or "variant" refer to a protein that differs from that of a parent protein by at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-five, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, or more modifications. The term can refer to the protein itself, a composition comprising the protein, the amino acid sequence encoding it, or the DNA sequence encoding 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 a "variant" of an IL-12 p35 subunit domain) refers to a polypeptide in which there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, deletions, and / or insertions compared to the amino acid sequence of a reference IL-12 p35 subunit, e.g., a (human) wild-type IL-12 p35 subunit. Furthermore, a "variant" of an IL-12 p40 subunit (or a "variant" of an IL-12 p40 subunit domain) refers to a polypeptide in which there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, deletions, and / or insertions compared to the amino acid sequence of a reference IL-12 p40 subunit, e.g., a (human) wild-type IL-12 p40 subunit.

[0297] As used herein, the term "modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or a change in 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) at which the amino acid(s) are modified and the number of amino acid(s) that can be modified in the amino acid sequence are not particularly limited.

[0298] As will be understood, the modifications of 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, rather than the precursor sequence (SEQ ID NO:1). The precursor sequence of the IL-12 p35 subunit contains an additional 22 amino acid residues at the N-terminus, constituting the following sequence: MCPARSLLLVATLVLLDHLSLA. However, the listed modifications of the mature form of the IL-12 p35 subunit may also be made to the precursor form of the IL-12 p35 subunit after correcting the position of the specified modification to take into account the additional preceding amino acid residues in the precursor sequence. For example, the substitution modification Y40A is disclosed herein as a candidate modification of the IL-12 p35 subunit mature sequence, but can also refer to substitution modifications of the IL-12 p35 subunit precursor sequence, including Y62A.

[0299] Similarly, modifications of 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, rather than the precursor sequence (SEQ ID NO:3). The precursor sequence of the IL-12 p40 subunit contains an additional 22 amino acid residues at the N-terminus that constitute the following sequence: MCHQQLVISWFSLVFLASPLVA. However, it will be understood that the listed modifications of the mature form of the IL-12 p40 subunit may also be made to the precursor form of the IL-12 p40 subunit after correcting the position of the specified modification to take into account the additional preceding amino acid residues in the precursor sequence. For example, the substitution modification C177S is disclosed herein as a candidate modification of the IL-12 p40 subunit mature sequence, but can also refer to substitution modifications of the IL-12 p40 subunit precursor sequence, including C199S.

[0300] As used herein, the term "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. For example, Y40A indicates the substitution of a tyrosine at position 40 with an alanine at the same position. In some embodiments, the substitution is with an amino acid that does not naturally occur at the particular position and does not naturally occur in the organism or any organism. For clarity, a protein that has been engineered to change the nucleic acid coding sequence but not the resulting amino acid (e.g., replacing CCU (which encodes proline) with CCC (which still encodes proline)) is not an "amino acid substitution." In other words, if a new gene encoding the same protein is created, but the protein has the same amino acid at the particular position where it begins, it is not an amino acid substitution.

[0301] As used herein, the term "amino acid insertion" or "insertion" refers to the addition of an amino acid residue or amino acid sequence at a particular position within a parent polypeptide sequence. For example, -40A refers to the insertion of an alanine after position 40 and before position 41. As another example, D8EPKSS or -8EPKSS indicates the insertion of the sequence Glu-Pro-Lys-Ser-Ser before position 9 and the insertion of the sequence Glu-Pro-Lys-Ser-Ser after position 8.

[0302] As used herein, the term "amino acid deletion" or "deletion" refers to the removal of an amino acid or sequence at a particular position within a parent polypeptide sequence. For example, Y40-, Y40#, Y40(), or Y40del refers to the deletion of a tyrosine at position 40. As another example, EPKSS8-, EPKSS8#, EPKSS8del indicate the deletion of the sequence Glu-Pro-Lys-Ser-Ser starting at position 8.

[0303] As used herein, the term "non-naturally occurring protein" or "non-naturally occurring protein variant" refers to a variant protein that differs from that of a parent protein by at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-five, thirty, forty, fifty, sixty-one, seventy-eight, ninety-one, one hundred, or more modifications that are not isotypic. For example, because the IL-12 p35 subunit of IL-12 does not contain an alanine at position 40, a Y40A substitution is considered a non-naturally occurring IL-12 p35 variant (or more generally, a non-naturally occurring IL-12 variant). Modifications of proteins that are not isotypic may be referred to as "non-naturally occurring modifications."

[0304] 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 the percentage of amino acid residues (or nucleic acids encoding amino acid residues) in a candidate sequence that are identical to those in a particular sequence, e.g., the amino acid sequence of a parent protein, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; any conservative substitutions are not considered part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways within the skill of the art, 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 assessing alignment, including any algorithms necessary to achieve maximum alignment over the entire 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.

[0305] As used herein, the term "half-life" of an agent can refer to the time required for the agent to lose half of its pharmacological, physiological, or other activity when administered to the serum or tissues of an organism or subject, or compared to any other predetermined time point. "Half-life" can also refer to the time required for the amount or concentration of an agent to decrease to half of the starting amount administered to the serum or tissues of an organism or subject, or compared to such amount or concentration when administered to the serum or tissues of an organism or subject, or compared to any other predetermined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0306] As used herein, the terms "host cell" and "recombinant cell" refer to an individual cell or cell culture that can be or has been host to any recombinant vector(s) or isolated polynucleotide(s). Host cells can be transfected, transformed, transduced, or infected cells of any origin, including but not limited to prokaryotic, eukaryotic, mammalian, avian, insect, plant, or bacterial cells, or can be of any origin that can be used to propagate a nucleic acid described herein. Host cells include cells that have been transfected or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. Host cells containing a recombinant vector of the invention may be referred to as "recombinant host cells."

[0307] Host cells may include, but are not limited to, mammalian, plant, insect, fungal, and bacterial cells. Bacterial cells include, but are not limited to, gram-positive bacterial cells, such as Bacillus, Streptomyces, and Staphylococcus species, and gram-negative bacterial cells, such as Escherichia and Pseudomonas cells. Fungal cells may include, but are not limited to, yeast cells, such as Saccharomyces, Pichia pastoris, and Hansenula polymorpha. Insect cells may include, but are not limited to, Drosophila cells and Sf9 cells. Plant cells include, but are not limited to, cells derived from crop, medicinal, or ornamental plants or bulbs. Mammalian cells suitable 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, hepatic cell lines, Chinese hamster ovary (CHO) cells, COS cells, BHK cells, HeLa cells, mouse embryonic stem cell (mESC) line D3 cells, human embryonic stem cells (e.g., HS293 cells and BG01V cells), NIH 3T3 cells, human embryonic kidney (HEK) 293T cells, human mesenchymal stem cells (hMSCs), and the like.

[0308] As used herein, the terms "cell," "cell culture," "cell line," and "host cell" refer not only to a particular cell, cell culture, cell line, or host cell, regardless of the number of transfers or passages in culture, but also to the progeny or future progeny of such a cell, cell culture, cell line, or host cell. It should be understood that not all progeny are necessarily identical to the parent cell. This is because certain modifications may occur in subsequent generations due to either mutation (e.g., deliberate or accidental mutation) or environmental influences (e.g., methylation or other epigenetic modifications), such that the progeny may not, in fact, be identical to the parent cell; however, so long as the progeny retain the same or substantially the same functionality as that of the original cell, cell culture, cell line, or host cell, they are still encompassed within the scope of the term as used herein.

[0309] As used herein, the term "culture medium" or "culture medium" encompasses any culture medium, solution, solid, semi-solid, or rigid support capable of supporting or containing any host cell and cellular contents, 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. Thus, the term can encompass the medium in which a host cell is grown (e.g., the medium into which a polypeptide is secreted, including the medium before, during, or after the growth step). The term can also encompass buffers or other reagents containing host cell lysates, such as when a polynucleotide is produced intracellularly and the host cells are lysed or disrupted to release the polypeptide.

[0310] As used herein, the term "fusion protein" refers to the covalent linkage of at least two proteins or protein domains. Fusion proteins may include artificial sequences, such as domain linkers, variant Fc domains, variant IL-12 p35 subunit domains, (variant) IL-12 p40 subunit domains, etc., as described herein. As used herein, the term "Fc fusion protein" refers to a protein comprising an Fc domain normally linked to one or more different protein domains (optionally via a domain linker as described herein). In some embodiments, the C-terminus of the Fc domain is linked to the N-terminus of one or more different protein domains (optionally linked via a domain linker, where 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 via a domain linker, where 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). Thus, an "IL-12 Fc fusion" comprises an Fc region linked (optionally via a domain linker) to a variant IL-12 p35 subunit domain, an IL-12 p40 subunit domain, a variant IL-12 p40 subunit domain, sc-IL-12, sc-IL-12(p35 / p40), and / or sc-IL-12(p40 / p35). An "Fc fusion protein" may refer to a "heterodimeric Fc fusion protein" or a "homodimeric Fc fusion protein."

[0311] As used herein, the term "heterodimeric Fc-fusion protein" refers to a complex comprising a first fusion construct and a second fusion construct, wherein the first fusion construct comprises a first Fc domain and a first IL-12 subunit domain, and the second fusion construct comprises a second Fc domain and a second IL-12 subunit domain. In some embodiments, the first Fc domain and 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 comprise one or more modifications that alter Fc binding. In some embodiments, the first Fc domain and / or the second Fc domain comprise one or more modifications that alter Fc half-life. In some embodiments, the first Fc domain and / or the second Fc domain comprise one or more modifications that alter 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 enhance binding to FcRn. 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 (optionally via a domain linker) to the N-terminus of the first Fc domain, and the C-terminus of the second IL-12 subunit domain is linked (optionally via a domain linker) to the N-terminus of the second Fc domain.In some embodiments, the C-terminus of the first Fc domain is linked (optionally via a domain linker) to the N-terminus of the first IL-12 subunit domain, and the C-terminus of the second Fc domain is linked (optionally via a domain linker) to the N-terminus of the second IL-12 subunit domain.

[0312] As used herein, the term "homodimeric fusion protein" refers to a complex comprising two identical fusion constructs, where each fusion construct comprises an Fc domain and one or more protein domains (optionally linked via a domain linker). In some embodiments, for both fusion constructs, the C-terminus of the Fc domain is linked to the N-terminus of one or more protein domains (optionally via a domain linker). In some embodiments, for both fusion constructs, the C-terminus of one or more protein domains is linked to the N-terminus of the Fc domain (optionally via a domain linker). In some embodiments, the identical Fc domains comprise one or more modifications that alter Fc binding. In some embodiments, the identical Fc domains comprise one or more modifications that alter Fc half-life. In some embodiments, the identical Fc domains comprise one or more modifications that alter binding of the identical Fc domain to the neonatal Fc receptor (FcRn). In certain further embodiments, the identical domains comprise one or more modifications that increase half-life and / or enhance binding to FcRn.

[0313] As used herein, the term "isolated," when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified, separated, and / or recovered from the cell (e.g., host cell and / or cell line) or cell culture in which it is expressed. Typically, an isolated polypeptide is prepared by at least one purification step. As used herein, the term "isolated protein" refers to a protein that is substantially free of other proteins from cell culture, e.g., host cell proteins.

[0314] As used herein, the terms "Fc," "Fc region," or "Fc domain" refer to a polypeptide comprising the CH2-CH3 domain of an immunoglobulin G (IgG) molecule, and optionally including all or part of the hinge, and variants thereof. In the EU numbering system for human IgG1, the CH2-CH3 domain comprises amino acids 231-447, and the hinge is amino acids 216-230. Thus, the definition of an "Fc domain" encompasses both amino acids 231-447 (CH2-CH3) or amino acids 216-447 (hinge-CH2-CH3), or fragments thereof. An "Fc fragment" in this context may contain fewer amino acids from either or both of the N- and C-termini, but generally still retains the ability to form a dimer with another Fc domain or Fc fragment, as can be detected using standard size-based methods (e.g., non-denaturing chromatography, size-exclusion chromatography, etc.). Unless specifically defined, "Fc domain" herein generally refers to the CH2-CH3 domain of human IgG1 (and optionally all or part of the hinge). Various human IgG1 Fc domains are described below, at least some of which include one or more modifications. For clarity, while for brevity, modifications are discussed herein primarily in the context of the human IgG1 Fc domain, it is expressly intended that mutations in other immunoglobulins, e.g., IgG2, IgG3, IgG4, IgA, IgM, and IgE, can be made at residue positions corresponding to the mutations in human IgG1 described herein. One of skill in the relevant art will readily be able to determine residue positions in other immunoglobulins that correspond to the human IgG1 modifications described herein.

[0315] As used herein, the term "vector" refers to a nucleic acid molecule or sequence capable of introducing or transporting another nucleic acid molecule. The introduced nucleic acid molecule is usually linked (e.g., inserted) into the vector nucleic acid molecule. Typically, a vector is capable of replication when linked to appropriate regulatory elements. The term "vector" encompasses cloning and expression vectors, as well as viral and integrating vectors. An "expression vector" is a vector that contains regulatory regions, thereby enabling the expression of DNA sequences and fragments thereof in vitro and / or in vivo. A vector may contain a sequence that directs autonomous replication in a cell or may contain a sequence sufficient to enable integration into host cell DNA. Useful vectors include, but are not limited to, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, but are not limited to, replication-deficient retroviruses and lentiviruses. In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle to introduce genes into cells.

[0316] 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 that is not associated, by its own source or manipulation, with all or part of a polynucleotide with which it is associated in nature. The term "recombinant," when used with respect to a protein or polypeptide, refers to a polypeptide produced by expression of a recombinant polynucleotide. The term "recombinant," when used with respect to a host cell, refers to a host cell into which a recombinant polynucleotide or a vector containing a recombinant polynucleotide has been introduced.

[0317] As used herein, the term "operably linked" refers to a physical or functional linkage between two or more components (e.g., polypeptide or polynucleotide sequences) that enables them to function in their intended manner. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that allows expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to the arrangement of a regulatory region and a coding sequence to be transcribed such that the regulatory region is effective to regulate the transcription or translation of the coding sequence of interest. Thus, a promoter is operably linked to a nucleic acid sequence if it is capable of effecting transcription of the nucleic acid sequence. It should be understood that operably linked components may or may not be contiguous. In the context of a polypeptide, "operably linked" refers to a physical linkage (e.g., a direct or indirect linkage) between amino acid sequences (e.g., different segments, modules, or domains) to effect a described activity of the polypeptide.

[0318] As used herein, the term "binding affinity" refers to the "strength" of binding of a given molecule (e.g., a non-naturally occurring IL-12 variant, a homodimeric IL-12 Fc fusion protein, and / or a heterodimeric IL-12 Fc fusion protein) to its ligand (e.g., IL-12Rβ2) and / or the rate at which a molecule associates with and / or dissociates from its ligand. Binding affinity is often measured using a dissociation constant (K D The binding activity of the non-naturally occurring IL-12 variants, homodimeric IL-12 Fc fusion proteins, and / or heterodimeric IL-12 Fc fusion proteins of the present disclosure may be analyzed 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, a Biacore assay, a KinExA assay, etc.

[0319] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant, homodimeric IL-12 Fc fusion protein, and / or heterodimeric IL-12 Fc fusion protein) to elicit a response at a particular dose or concentration in a given biological system or experimental setting. The potency of the non-naturally occurring IL-12 variants, homodimeric IL-12 Fc fusion proteins, and / or heterodimeric IL-12 Fc fusion proteins of the present disclosure can be analyzed by any suitable method known in the art, such as an IL-12 HEK reporter assay (e.g., InvivoGen's IL-12 HEK reporter assay (catalog number: hkb-il12)), a ligand binding assay (e.g., ELISA or flow cytometry), and / or a functional assay. Typically, a change in potency can be demonstrated visually as a leftward or rightward shift in a response curve compared to a control. A rightward shift in the response curve generally indicates a decrease in efficacy, while a leftward shift in the response curve generally indicates an increase in efficacy.

[0320] As used herein, the term "activity" refers to the specific response of a given protein, cytokine, fusion protein, antibody, etc. (e.g., non-naturally occurring IL-12 variant, homodimeric IL-12 Fc fusion protein, and / or heterodimeric IL-12 Fc fusion protein) elicited at a given dose or concentration in a particular biological system or experimental setting. The activity of the non-naturally occurring IL-12 variant, homodimeric IL-12 Fc fusion protein, and / or heterodimeric IL-12 Fc fusion protein of the present disclosure can be analyzed by any suitable method known in the art, such as ligand binding assays and / or functional assays. Generally, changes in activity can be visually demonstrated as an upward or downward shift in a response curve compared to a control. An upward shift in the response curve generally indicates increased activity, while a downward shift in the response curve generally indicates decreased activity. As detailed below, a change in activity may be related to and / or caused by a change in potency, or may be unrelated to and / or uncaused by a change in potency.

[0321] As used herein, the term "manufacturability" refers to any characteristic that may affect the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, etc. on a scale and in quantities sufficient for administration to an individual. Examples of characteristics that affect manufacturability include, but are not limited to, the stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant, a homodimeric IL-12 Fc fusion protein, and / or a heterodimeric IL-12 Fc fusion protein).

[0322] As used herein, the term "stability" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant, a homodimeric IL-12 Fc fusion protein, and / or a heterodimeric IL-12 Fc fusion protein) to retain the same properties and characteristics as it possessed at the time of manufacture, within certain limits and / or storage and / or use parameters. The stability of the non-naturally occurring IL-12 variants, homodimeric IL-12 Fc fusion proteins, and / or heterodimeric IL-12 Fc fusion proteins of the present disclosure can be analyzed by any suitable method known in the art, such as ELISA, Western blot, Biacore assay, SDS-PAGE, size exclusion chromatography, dynamic light scattering, differential scanning calorimetry, and differential scanning fluorimetry.

[0323] As used herein, the term "expression yield" refers to the amount or content of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant, a homodimeric IL-12 Fc fusion protein, and / or a heterodimeric IL-12 Fc fusion protein) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, etc. is well known in the relevant technical field. Any suitable method for quantifying or determining expression yield may be used, such as UV absorbance measurement, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorimetric analysis.

[0324] As used herein, the term "subject" or "individual" for purposes of treatment refers to any animal classified as a mammal (including, but not limited to, humans, primates, and / or non-human primates), domestic animal, livestock, zoo animal, research animal, sport animal, and / or companion animal, e.g., dog, horse, cat, cow, etc.

[0325] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "antigens" includes mixtures of antigens, reference to "a pharmaceutically acceptable carrier" includes mixtures of two or more such carriers, and the like. Thus, the terms "a" (a "an"), "one or more," and "at least one" can be used interchangeably herein.

[0326] Furthermore, "and / or," when used herein, should be considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or," as used herein in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0327] As used herein, the term "about" a numerical value (or parameter) refers to ±10% of the specified numerical value. When referring to a range of numerical values (or parameters), the term "about" refers to +10% of the upper limit and -10% of the lower limit of the specified numerical range. When a range of numerical values is stated, it is understood that each intervening value between the upper and lower limits of that range, as well as any other stated or intervening value within that stated range, is encompassed within the scope of the present disclosure. When a stated range includes the upper and / or lower limits, ranges excluding any of those included limits are also encompassed within the present disclosure.

[0328] It will be understood that certain features of the present disclosure, which are described in separate embodiments for clarity, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are described for brevity in a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present disclosure are specifically embraced by this application and disclosed herein, just as if every combination were individually and explicitly disclosed herein. In addition, all subcombinations of the various embodiments and components thereof are also specifically embraced by this disclosure and disclosed herein, just as if every such subcombination were individually and explicitly disclosed herein.

[0329] II. Overview The present invention is directed to novel non-naturally occurring IL-12 variants and fusion proteins in which the IL-12 p35 subunit contains novel amino acid substitutions that reduce binding affinity to IL-12Rβ2, as well as methods of making and using the non-naturally occurring IL-12 variants and fusion proteins.

[0330] As described above, IL-12 is composed of an α chain (p35 subunit; IL-12 p35 subunit) and a β chain (p40 subunit; IL-12 p40 subunit), which are covalently linked to form a biologically functional IL-12 complex. IL-12 exerts its cell signaling function by binding to the dimeric IL-12 receptor complex composed of IL-12Rβ1 and IL-12Rβ2, resulting in the phosphorylation of STAT4 and the initiation of multiple downstream signaling pathways, including, but not limited to, the induction of IFNγ secretion. Systemic administration of wild-type IL-12 can result in significant toxicity, including death, due to the overactivation of circulating immune cells. Furthermore, activated immune cells undergo cell proliferation, which contributes to the short serum half-life of administered IL-12 due to target-mediated pharmacokinetics.

[0331] In some embodiments, the compositions and methods described herein reduce toxicity associated with IL-12 administration by providing novel IL-12 variants with reduced binding affinity to IL-12Rβ2. In further embodiments, the compositions and methods described herein utilize IL-12 Fc fusion proteins, which in yet other embodiments comprise such novel IL-12 variants.

[0332] In some embodiments, the compositions and methods described herein address the short half-life of IL-12 by providing novel IL-12 variants with reduced binding affinity to IL-12Rβ2. In further embodiments, half-life is further improved by fusion of one or more Fc domains of the novel IL-12 variants (e.g., one or more of the Fc domains encoded by amino acid sequences SEQ ID NOs: 9-13, an Fc domain containing one or more modifications that result in altered binding to the neonatal Fc receptor (FcRn), etc.), one or more albumins, one or more unstructured biodegradable polypeptides ("XTEN"), or one or more polyethylene glycols (PEGs).

[0333] In some embodiments, the compositions and methods described herein address the short half-life of IL-12 by providing an IL-12 Fc fusion protein with reduced binding affinity to IL-12Rβ2.

[0334] III. Composition As will be appreciated by one of skill in the art, any of the aspects and embodiments of the compositions described herein may be used in any of the aspects and / or embodiments of the methods of making described below or the methods of using also described below.

[0335] A. Interleukin-12 (IL-12) variants In one aspect, the present disclosure provides a non-naturally occurring IL-12 variant comprising a) a variant IL-12 p35 subunit, and b) an IL-12 p40 subunit. Various configurations of non-naturally occurring IL-12 variants and variant IL-12 p35 and IL-12 p40 subunits are detailed below.

[0336] 1. p35 subunit: According to any of the aspects and embodiments described herein, the disclosure provides a) a variant IL-12 p35 subunit, the variant IL-12 p35 subunit comprising one or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A, and b) a non-naturally occurring IL-12 variant comprising an IL-12 p40 subunit. In some embodiments, the one or more amino acid substitutions comprise Y40A. In some embodiments, the one or more amino acid substitutions comprise T43A. In some embodiments, the one or more amino acid substitutions comprise D126A. In some embodiments, the one or more amino acid substitutions comprise P127A. In some embodiments, the one or more amino acid substitutions comprise R129A. In some embodiments, the one or more amino acid substitutions comprise K168A. In some embodiments, the one or more amino acid substitutions comprise K170A.

[0337] In some embodiments, the variant IL-12 p35 subunit comprises two or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the two or more amino acid substitutions comprise Y40A / T43A. In some embodiments, the two or more amino acid substitutions comprise Y40A / D126A. In some embodiments, the two or more amino acid substitutions comprise Y40A / P127A. In some embodiments, the two or more amino acid substitutions comprise Y40A / R129A. In some embodiments, the two or more amino acid substitutions comprise Y40A / K168A. In some embodiments, the two or more amino acid substitutions comprise T43A / D126A. In some embodiments, the two or more amino acid substitutions comprise T43A / P127A. In some embodiments, the two or more amino acid substitutions comprise T43A / R129A. In some embodiments, the two or more amino acid substitutions comprise T43A / K168A. In some embodiments, the two or more amino acid substitutions comprise D126A / P127A. In some embodiments, the two or more amino acid substitutions comprise D126A / R129A. In some embodiments, the two or more amino acid substitutions comprise D126A / K168A. In some embodiments, the two or more amino acid substitutions comprise P127A / R129A. In some embodiments, the two or more amino acid substitutions comprise P127A / K168A. In some embodiments, the two or more amino acid substitutions comprise R129A / K168A. In some embodiments, the two or more amino acid substitutions comprise Y40A / K170A. In some embodiments, the two or more amino acid substitutions comprise T43A / K170A. In some embodiments, the two or more amino acid substitutions comprise D126A / K170A. In some embodiments, the two or more amino acid substitutions comprise P127A / K170A. In some embodiments, the two or more amino acid substitutions comprise R129A / K170A. In some embodiments, the two or more amino acid substitutions comprise K168A / K170A.

[0338] In some embodiments, the variant IL-12 p35 subunit comprises three or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / D126A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / P127A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / R129A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / K168A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / P127A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / R129A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / K168A. In some embodiments, the three or more amino acid substitutions comprise Y40A / P127A / R129A. In some embodiments, the three or more amino acid substitutions comprise Y40A / P127A / K168A. In some embodiments, the three or more amino acid substitutions comprise Y40A / R129A / K168A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / P127A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / R129A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / K168A. In some embodiments, the three or more amino acid substitutions comprise T43A / P127A / R129A. In some embodiments, the three or more amino acid substitutions comprise T43A / P127A / K168A. In some embodiments, the three or more amino acid substitutions comprise T43A / R129A / K168A. In some embodiments, the three or more amino acid substitutions comprise D126A / P127A / R129A. In some embodiments, the three or more amino acid substitutions comprise D126A / P127A / K168A. In some embodiments, the three or more amino acid substitutions comprise D126A / R129A / K168A. In some embodiments, the three or more amino acid substitutions comprise P127A / R129A / K168A.In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / P127A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / P127A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise D126A / P127A / K170A. In some embodiments, the three or more amino acid substitutions comprise D126A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise D126A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise P127A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise P127A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise R129A / K168A / K170A.

[0339] In some embodiments, the variant IL-12 p35 subunit comprises four or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / P127A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / R129A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / P127A / R129A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / P127A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / P127A / R129A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / P127A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / P127A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / P127A / R129A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / P127A / K168A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise T43A / P127A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise D126A / P127A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / P127A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / R129A / K170A.In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / P127A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / P127A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / P127A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / P127A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / P127A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / P127A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise D126A / P127A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise D126A / P127A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise D126A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise P127A / R129A / K168A / K170A.

[0340] In some embodiments, the variant IL-12 p35 subunit comprises five or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / P127A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / P127A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / P127A / R129A / K168A / K170A.In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / P127A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / P127A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise T43A / P127A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise D126A / P127A / R129A / K168A / K170A.

[0341] In some embodiments, the variant IL-12 p35 subunit comprises six or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K168A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / R129A / K168A / K170A. In some embodiments, the six or more amino acid substitutions comprise T43A / D126A / P127A / R129A / K168A / K170A.

[0342] In some embodiments, the variant IL-12 p35 subunit comprises seven or more amino acid substitutions selected from the group including Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0343] In some embodiments, the variant IL-12 p35 subunit is selected from the group consisting of SEQ ID NO:24 (Y40A), SEQ ID NO:25 (T43A), SEQ ID NO:26 (D126A), SEQ ID NO:27 (P127A), SEQ ID NO:28 (R129A), SEQ ID NO:29 (K168A), SEQ ID NO:30 (Y40A / T43A), SEQ ID NO:31 (Y40A / D126A), SEQ ID NO:32 (Y40A / P127A), SEQ ID NO:33 (Y40A / R129A), SEQ ID NO:34 (Y40A / K168A), SEQ ID NO:35 (T43A / D126A), SEQ ID NO:36 (T43A / P127A), SEQ ID NO:37 (R129A), SEQ ID NO:38 (K168A), SEQ ID NO:39 (K168A), SEQ ID NO:40 (Y40A / T43A), SEQ ID NO:41 (Y40A / D126A), SEQ ID NO:42 (Y40A / P127A), SEQ ID NO:43 (Y40A / R129A), SEQ ID NO:44 (Y40A / K168A), SEQ ID NO:45 (T43A / D126A), SEQ ID NO:46 (T43A / P127A), SEQ ID NO:47 (R129A), SEQ ID NO:48 (K168A), SEQ ID NO:49 (K168A), SEQ ID NO:50 (Y40A / T43A), SEQ ID NO:51 (Y40A / D126A), SEQ ID NO:52 (Y40A / P127A), SEQ 27A), SEQ ID NO: 37 (T43A / R129A), SEQ ID NO: 38 (T43A / K168A), SEQ ID NO: 39 (D126A / P127A), SEQ ID NO: 40 (D126A / R129A), SEQ ID NO: 41 (D126A / K168A), SEQ ID NO: 42 (P127A / R129A), SEQ ID NO: 43 (P127A / K168A), SEQ ID NO: 44 (R129A / K168A), SEQ ID NO: 45 (Y40A / T43A / D126A), SEQ ID NO: 46 (Y40A / T43A / P127A), SEQ ID NO: 47 (Y40A / T43A / R129A), SEQ ID NO: 48 (Y40A / T43A / K168A), SEQ ID NO: 49 (Y40A / D126A / P127A), SEQ ID NO: 50 (Y40A / D126A / R129A), SEQ ID NO: 51 (Y40A / D126A / K168A), SEQ ID NO: 52 (Y40A / P127A / R129A), SEQ ID NO: 53 (Y40A / P127A / K168A), SEQ ID NO: 54 (Y40A / R129A / K168A), SEQ ID NO: 55 (T43A / D126A / P127A), SEQ ID NO: 56 (T43A / D126A / R129A), SEQ ID NO: 57 (T43A / D126A / K168A ), SEQ ID NO: 58 (T43A / P127A / R129A), SEQ ID NO: 59 (T43A / P127A / K168A), SEQ ID NO: 60 (T43A / R129A / K168A), SEQ ID NO: 61 (D126A / P127A / R129A), SEQ ID NO: 62 (D126A / P127A / K168A), SEQ ID NO: 63 (D126A / R129A / K168A), SEQ ID NO: 64 (P127A / R129A / K168A), SEQ ID NO: 65 (Y40A / T43A / D126A / P127A), SEQ ID NO: 66 (Y40A / T43A / D126A / R129A),SEQ ID NO: 67 (Y40A / T43A / D126A / K168A), SEQ ID NO: 68 (Y40A / T43A / P127A / R129A), SEQ ID NO: 69 (Y40A / T43A / P127A / K168A), SEQ ID NO: 70 (Y40A / T43A / R129A / K168A), SEQ ID NO: 71 (Y40A / D126A / P127A / R129A), SEQ ID NO: 72 (Y40A / D126A / P127A / K168A), SEQ ID NO: 73 (Y40A / D126A / R129A / K168A), SEQ ID NO: 74 (Y40A / P127A / R129A / K168A), SEQ ID NO: No. 75 (T43A / D126A / P127A / R129A), SEQ ID NO: 76 (T43A / D126A / P127A / K168A), SEQ ID NO: 77 (T43A / D126A / R129A / K168A), SEQ ID NO: 78 (T43A / P127A / R129A / K168A), SEQ ID NO: 79 (D126A / P127A / R129A / K168A), SEQ ID NO: 80 (Y40A / T43A / D126A / P127A / R129A), SEQ ID NO: 81 (Y40A / T43A / D126A / P127A / K168A), SEQ ID NO: 82 (Y40A / D126A / P127A / R129A / K168A), SEQ ID NO: 83 (Y40A / T43A / P127A / R129A / K168A), SEQ ID NO: 84 (Y40A / T43A / D126A / R129A / K168A), SEQ ID NO: 85 (T43A / D126A / P127A / R129A / K168A), SEQ ID NO: 103 (K170A), SEQ ID NO: 104 (Y40A / K170A), SEQ ID NO: 105 (T43A / K170A), SEQ ID NO: 106 (D126A / K170A), SEQ ID NO: 107 (P127A / K170A), SEQ ID NO: 108 (R129A / K170A), SEQ ID NO: 10 9 (K168A / K170A), SEQ ID NO: 110 (Y40A / T43A / K170A), SEQ ID NO: 111 (Y40A / D126A / K170A), SEQ ID NO: 112 (Y40A / P127A / K170A), SEQ ID NO: 113 (Y40A / R129A / K170A), SEQ ID NO: 114 (Y40A / K168A / K170A), SEQ ID NO: 115 (T43A / D126A / K170A), SEQ ID NO: 116 (T43A / P127A / K170A), SEQ ID NO: 117 (T43A / R129A / K170A), SEQ ID NO: 118 (T43A / K168A / K170A),SEQ ID NO: 119 (D126A / P127A / K170A), SEQ ID NO: 120 (D126A / R129A / K170A), SEQ ID NO: 121 (D126A / K168A / K170A), SEQ ID NO: 122 (P127A / R129A / K170A), SEQ ID NO: 123 (P127A / K168A / K170A), SEQ ID NO: 124 (R129A / K168A / K170A), SEQ ID NO: 125 (Y40A / T43A / D126A / K170A), SEQ ID NO: 126 (Y40A / T43A / P127A / K170A), SEQ ID NO: 127 (Y40A / T43A / R129 A / K170A), SEQ ID NO: 128 (Y40A / T43A / K168A / K170A), SEQ ID NO: 129 (Y40A / D126A / P127A / K170A), SEQ ID NO: 130 (Y40A / D126A / R129A / K170A), SEQ ID NO: 131 (Y40A / D126A / K168A / K170A), SEQ ID NO: 132 (Y40A / P127A / R129A / K170A), SEQ ID NO: 133 (Y40A / P127A / K168A / K170A), SEQ ID NO: 134 (Y40A / R129A / K168A / K170A), SEQ ID NO: 135 (T43A / D1 26A / P127A / K170A), SEQ ID NO: 136 (T43A / D126A / R129A / K170A), SEQ ID NO: 137 (T43A / D126A / K168A / K170A), SEQ ID NO: 138 (T43A / P127A / R129A / K170A), SEQ ID NO: 139 (T43A / P127A / K168A / K170A), SEQ ID NO: 140 (T43A / R129A / K168A / K170A), SEQ ID NO: 141 (D126A / P127A / R129A / K170A), SEQ ID NO: 142 (D126A / P127A / K168A / K170A), SEQ ID NO: 143 (D126A / R129A / K168A / K170A), SEQ ID NO: 144 (P127A / R129A / K168A / K170A), SEQ ID NO: 145 (Y40A / T43A / D126A / P127A / K170A), SEQ ID NO: 146 (Y40A / T43A / D126A / R129A / K170A), SEQ ID NO: 147 (Y40A / T43A / D126A / K168A / K170A), SEQ ID NO: 148 (Y40A / T43A / P127A / R129A / K170A), SEQ ID NO: 149 (Y40A / T43A / P127A / K168A / K170A),SEQ ID NO: 150 (Y40A / T43A / R129A / K168A / K170A), SEQ ID NO: 151 (Y40A / D126A / P127A / R129A / K170A), SEQ ID NO: 152 (Y40A / D126A / P127A / K168A / K170A), SEQ ID NO: 153 (Y40A / D126A / R129A / K168A / K170A), SEQ ID NO: 154 (Y40A / P127A / R129A / K168A / K170A) , SEQ ID NO: 155 (T43A / D126A / P127A / R129A / K170A), SEQ ID NO: 156 (T43A / D126A / P127A / K168A / K170A), SEQ ID NO: 157 (T43A / D126A / R129A / K168A / K170A), SEQ ID NO: 158 (T43A / P127A / R129A / K168A / K170A), SEQ ID NO: 159 (D126A / P127A / R129A / K168A / K17 0A), SEQ ID NO: 160 (Y40A / T43A / D126A / P127A / R129A / K170A), SEQ ID NO: 161 (Y40A / T43A / D126A / P127A / K168A / K170A), SEQ ID NO: 162 (Y40A / D126A / P127A / R129A / K168A / K170A), SEQ ID NO: 163 (Y40A / T43A / P127A / R129A / K168A / K170A), SEQ ID NO: 164 (Y40 165 (T43A / D126A / P127A / R129A / K168A / K170A), SEQ ID NO: 166 (Y40A / T43A / D126A / P127A / R129A / K168A / K170A), and SEQ ID NO: 86 (Y40A / T43A / D126A / P127A / R129A / K168A).

[0344] In some embodiments, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 (wild-type precursor), SEQ ID NO:2 (wild-type mature), SEQ ID NOs:24-86, SEQ ID NOs:103-166, and SEQ ID NO:87 (C74S) (as shown in Figures 1, 5-9, 78, and 80).

[0345] In some embodiments, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue Y40. In some further embodiments, the substitution mutation at amino acid residue Y40 is selected from the group comprising: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 177-187.

[0346] In some embodiments, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue D126. In some further embodiments, the substitution mutation at amino acid residue D126 is selected from the group including D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

[0347] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 199-214.

[0348] In some embodiments, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue P127. In some further embodiments, the substitution mutation at amino acid residue P127 is selected from the group comprising P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

[0349] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 279-290.

[0350] In some embodiments, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue R129. In some further embodiments, the substitution mutation at amino acid residue R129 is selected from the group including R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

[0351] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 215-231.

[0352] In some embodiments, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue K168. In some further embodiments, the substitution mutation at amino acid residue K168 is selected from the group including K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

[0353] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 232-247.

[0354] In some embodiments, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue K170. In some further embodiments, the substitution mutation at amino acid residue K170 is selected from the group comprising: K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 188-198 or 306-308.

[0355] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising K170L and K170T, hi some embodiments, the variant IL-12 p35 subunit comprises either SEQ ID NO: 248 or 249.

[0356] In some embodiments, the variant IL-12 p35 subunit comprises (i) a first substitution mutation selected from the group consisting of Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T, and (ii) D126A, D126C, D126E, D126F, D126G, D 126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D 126W, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R1 29M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R129Y, K168A, K168C, K1 68D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K16 and a second substitution mutation selected from the group including K170A, K170B, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

[0357] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40E and further comprises a second substitution mutation selected from the group comprising K170A, K168A, K168I, K168T, and R129A. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 250-253 or 311.

[0358] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40G and further comprises a second substitution mutation selected from the group comprising K170A, K168A, K168I, K168T, and R129A. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 254-257 or 309.

[0359] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40P and further comprises a second substitution mutation selected from the group comprising K170A, K168A, K168D, K168I, and K168T. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 258-261 or 310.

[0360] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising Y40S and further comprises a second substitution mutation selected from the group comprising K168I, K168T, K170A, K170L, K170T, and R129A, hi some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 262-267.

[0361] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising K170A and further comprises a second substitution mutation selected from the group comprising K168I, K168T, and R129E, hi some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 268-270.

[0362] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising K170P and further comprises a second substitution mutation selected from the group comprising K168A, K168I, K168T, and R129E, hi some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 271-274.

[0363] In some embodiments, the variant IL-12 p35 subunit comprises a first substitution mutation comprising K170T and further comprises a second substitution mutation selected from the group comprising K168A, K168I, K168T, and R129E. In some embodiments, the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 275-278.

[0364] In some embodiments, the variant IL-12 p35 subunit may further comprise a C74S substitution mutation.

[0365] In addition to the novel, non-naturally occurring IL-12 p35 variants described above, additional modifications may be included in the IL-12 p35 variants disclosed herein. Non-limiting examples of residues that may be modified include Q20, N21, Q35, E38, F39, P41, S44, E45, E46, E50, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, T69, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, M98, A99, L12 4, M125, K128, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, F166, Y167, I171, R181, I182, R183, V185, T186, D188, R189, V190, M191, S192, Y193, N195, A196, and S197. In some embodiments, the variant IL-12 p35 subunit described above, below, or in any of Figures 1, 5-9, 78, and 80 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 described above, below, or in any of Figures 1, 5-9, 78, and 80 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. In some embodiments, the variant IL-12 p35 subunit comprises SEQ ID NO: 87 (as shown in FIG. 9 ) and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group including: Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0366] Mutations in the variant IL-12 p35 subunit may result in changes in one or more of the following parameters: (i) binding affinity, (ii) potency, (iii) activity, (iv) manufacturability, or (v) stability, although changes in one or more of the above parameters (e.g., binding affinity) may not necessarily correlate with changes in one or more of the other parameters (e.g., potency or activity). In some embodiments, one or more amino acid substitutions in the variant IL-12 p35 subunit result in changes in binding affinity for IL-12Rβ2 compared to the binding affinity of a reference IL-12. In some embodiments, the one or more amino acid substitutions decrease the binding affinity of the variant IL-12 p35 subunit for IL-12Rβ2 compared to the reference IL-12. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein. In certain exemplary embodiments, one or more amino acid substitutions in the variant IL-12 p35 subunit do not affect binding affinity, but may result in changes in one or more of the following parameters: (i) potency, (ii) activity, (iii) manufacturability, (iv) stability, or (v) any combination thereof.

[0367] A variety of assay formats can be used to select non-naturally occurring IL-12 variants that bind to a ligand of interest (e.g., IL-12Rβ2 and / or IL-12Rβ1). Non-limiting examples include solid-phase ELISA immunoassays, immunoprecipitation, Biacore assays, KinExA assays, fluorescence-activated cell sorting (FACS), Octet assays, Western blot analysis, etc. The binding activity of non-naturally occurring IL-12 variants of the present disclosure can be analyzed by any suitable method known in the art, such as surface plasmon resonance (SPR) assays, enzyme-linked immunosorbent assays (ELISAs), ELISpot assays, Biacore assays, KinExA assays, etc.

[0368] Those skilled in the art will appreciate that binding affinity can also be used as a measure of the "strength" of the non-covalent interaction between two binding partners (e.g., a variant IL-12 p35 subunit and IL-12Rβ2). The binding affinity between two molecules is determined by the dissociation constant (K D ) can be quantified by determining K D can be determined by measuring the kinetics of complex formation and dissociation using a suitable assay known in the art, for example, an SPR assay. The rate constants corresponding to the association and dissociation of a monovalent complex are the association rate constant k, respectively. a (or k on ) and dissociation rate constant k d (or k off ) is called K D is the formula K D =k d / k a By k a and k d The value of the dissociation constant can be determined directly by well-known methods.

[0369] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant) to elicit a response at a particular dose or concentration in a given biological system or experimental setting. The potency of the non-naturally occurring IL-12 variants of the present disclosure can be analyzed by any suitable method known in the art, such as an IL-12 HEK reporter assay (e.g., InvivoGen's IL-12 HEK reporter assay (catalog number: hkb-il12)), a ligand binding assay (e.g., ELISA or flow cytometry), and / or a functional assay. Typically, a change in potency can be visually demonstrated as a leftward or rightward shift in a response curve compared to a control. A rightward shift in the response curve generally indicates a decrease in potency, while a leftward shift in the response curve generally indicates an increase in potency. As used herein, the term "activity" refers to the specific response of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant) elicited at a given dose or concentration in a particular biological system or experimental setting. The activity of the non-naturally occurring IL-12 variants of the present disclosure can be analyzed by any suitable method known in the art, for example, ligand binding assays and / or functional assays. Generally, changes in activity can be visually demonstrated as an upward or downward shift in a response curve compared to a control. An upward shift in the response curve generally indicates increased activity, while a downward shift in the response curve generally indicates decreased activity.

[0370] As used herein, the term "manufacturability" refers to any characteristic that may affect the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, etc., on a scale and in quantities sufficient for administration to an individual. Examples of characteristics that affect manufacturability include, but are not limited to, the stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant). As used herein, the term "stability" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant) to retain the same properties and characteristics as it possessed at the time of manufacture, within specified limits and / or storage and / or use parameters. The stability of the non-naturally occurring IL-12 variants of the present disclosure can be analyzed by any suitable method known in the art, such as 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 "expression yield" refers to the amount or content of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a non-naturally occurring IL-12 variant) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, etc. is well known in the relevant technical field. Any suitable method for quantifying or determining expression yield may be used, such as UV absorbance measurement, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorimetric analysis.

[0371] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit has a binding affinity for IL-12Rβ2 that is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or more compared to the binding affinity of a reference IL-12, as measured by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0372] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by an assay. In some embodiments, the assay includes an SPR assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0373] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit has a binding affinity for IL-12Rβ2 that is below the lower limit of detection of the assay, and the binding affinity of the reference IL-12 for IL-12Rβ2 is between or equal to the lower or upper limit of detection of the assay (i.e., the binding affinity of the reference IL-12 can be equal to the lower limit of detection, the upper limit of detection, or a value between the lower and upper limits of detection; in other words, the binding affinity is "detectable"). In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0374] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit is at least about 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold, 11.0-fold, 12.0-fold, 13.0-fold, 14.0-fold, 15.0-fold, 16.0-fold, 17.0-fold, 18.0-fold, 19.0-fold, 20.0-fold, 21.0-fold, 22.0-fold, 23.0-fold, 24.0-fold, 25.0-fold, 26.0-fold, 27.0-fold, 28.0-fold, 29.0-fold, 30.0-fold, 31.0-fold, 32.0-fold, 33.0-fold, 34.0-fold, 35.0-fold, 36.0-fold, 37.0-fold, 38.0-fold, 39.0-fold, 40.0-fold, 41.0 1 in 3.1, 1 in 3.2, 1 in 3.3, 1 in 3.4, 1 in 3.5, 1 in 3.6, 1 in 3.7, 1 in 3.8, 1 in 3.9, 1 in 4.0, 1 in 4.1, 1 in 4.2, 1 in 4.3, 1 in 4.4, 1 in 4.5, 1 in 4.6, 1 in 4.7, 1 in 4.8, 1 in 4.9, 1 in 5.0, 1 in 5.1, 1 in 5.2, 1 in 5.3, 1 in 5.4, 1 in 5.5, 1 in 6.0, 1 in 7.0, 1 in 8.0, 1 in 9.0, 1 in 10.0, 1 in 11.0 , 1 in 12.0, 1 in 13.0, 1 in 14.0, 1 in 15.0, 1 in 16.0, 1 in 17.0, 1 in 18.0, 1 in 19.0, 1 in 20.0, 1 in 21.0, 1 in 22.0, 1 in 23.0, 1 in 24.0, 1 in 25.0, 1 in 30.0, 1 in 35.0, 1 in 40.0, 1 in 45.0, 1 in 50.0, 1 in 100.0, 1 in 150.0, 1 in 200.0, 1 in 250.0, 1 in 300.0, 1 in 350.0, 1 in 400.0, 45 1 in 0.0, 1 in 500.0, 1 in 550.0, 1 in 600.0, 1 in 650.0, 1 in 700.0, 1 in 750.0, 1 in 800.0, 1 in 850.0, 1 in 900.0, 1 in 950.0, 1 in 1000.0, 1 in 2000.0, 1 in 3000.0, 1 in 4000.0, 1 in 5000.0, 1 in 6000.0, 1 in 7000.0, 1 in 8000.0, 1 in 9000.0, 1 in 10,000.0, or less. In some embodiments, the assay comprises an IL-12 HEK reporter assay.In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0375] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit is at least about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, about 30.0 to about 35.0 fold less potent, about 35.0 to about 4 fold less potent, as measured by an assay. 1 in 0.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 80 1 in 0.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 800 The IL-12 has a potency reduced to about 1 / 0.0, about 1 / 8000.0 to about 1 / 9000.0, about 1 / 9000.0 to about 1 / 10,000.0, about 1 / 10,000.0 to about 1 / 50,000.0, about 1 / 50,000.0 to about 1 / 100,000.0, about 1 / 100,000.0 to about 1 / 200,000.0, about 1 / 200,000.0 to about 1 / 300,000.0, about 1 / 300,000.0, or less. In some embodiments, the assay includes an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0376] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit has a reduced ability to stimulate STAT4 signaling compared to a reference IL-12, as measured by an assay. The reduced ability to stimulate STAT4 signaling may be manifested by a reduced maximal response observed and / or an EC 50 "A" may refer to a change in the value of a non-naturally occurring IL-12 variant. In some embodiments, the ability of the non-naturally occurring IL-12 variant to stimulate STAT4 signaling is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or more compared to a reference IL-12, as measured by an assay. In some embodiments, the assay includes an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0377] In some embodiments, the ability of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit 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 measured by an assay. In some embodiments, the assay includes an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0378] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit has a reduced ability to stimulate IFNγ production compared to a reference IL-12, as measured by an assay. The reduced ability to stimulate IFNγ production may be manifested by a reduced maximal response observed and / or a reduced EC 50In some embodiments, the ability of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit to stimulate IFNγ production may be at least about 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, or greater than that of a reference IL-12 as measured by an assay. 1 in, 3.0 in, 3.1 in, 3.2 in, 3.3 in, 3.4 in, 3.5 in, 3.6 in, 3.7 in, 3.8 in, 3.9 in, 4.0 in, 4.1 in, 4.2 in, 4.3 in, 4.4 in, 4.5 in, 4.6 in, 4.7 in, 4.8 in, 4.9 in, 5.0 in, 5.1 in, 5.2 in, 5.3 in, 5.4 in, 5.5 in, 6.0 in, 7.0 in, 8.0 in, 9.0 in, 10.0 in , 1 in 11.0, 1 in 12.0, 1 in 13.0, 1 in 14.0, 1 in 15.0, 1 in 16.0, 1 in 17.0, 1 in 18.0, 1 in 19.0, 1 in 20.0, 1 in 21.0, 1 in 22.0, 1 in 23.0, 1 in 24.0, 1 in 25.0, 1 in 30.0, 1 in 35.0, 1 in 40.0, 1 in 45.0, 1 in 50.0, 1 in 100.0, 1 in 150.0, 1 in 200.0, 1 in 250.0, 1 in 300.0, 1 in 350.0, 400. 1 in 0, 1 in 450.0, 1 in 500.0, 1 in 550.0, 1 in 600.0, 1 in 650.0, 1 in 700.0, 1 in 750.0, 1 in 800.0, 1 in 850.0, 1 in 900.0, 1 in 950.0, 1 in 1000.0, 1 in 2000.0, 1 in 3000.0, 1 in 4000.0, 1 in 5000.0, 1 in 6000.0, 1 in 7000.0, 1 in 8000.0, 1 in 9000.0, 1 in 10,000.0, or less.In some embodiments, the assay includes an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA, or an ELISpot assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0379] In some embodiments, the ability of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit to stimulate IFNγ production is at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, about 30.0 to about 35.0 fold, about 1 in 35.0 to approximately 1 in 40.0, approximately 1 in 40.0 to approximately 1 in 45.0, approximately 1 in 45.0 to approximately 1 in 50.0, approximately 1 in 50.0 to approximately 1 in 100.0, approximately 1 in 100.0 to approximately 1 in 200.0, approximately 1 in 200.0 to approximately 1 in 300.0, approximately 1 in 300.0 to approximately 1 in 400.0, approximately 1 in 400.0 to approximately 1 in 500.0, approximately 1 in 500.0 to approximately 1 in 600.0, approximately 1 in 600.0 to approximately 1 in 700.0, approximately 700. 1 / 0 to about 1 / 800.0, about 1 / 800.0 to about 1 / 900.0, about 1 / 900.0 to about 1 / 1000.0, about 1 / 1000.0 to about 1 / 2000.0, about 1 / 2000.0 to about 1 / 3000.0, about 1 / 3000.0 to about 1 / 4000.0, about 1 / 4000.0 to about 1 / 5000.0, about 1 / 5000.0 to about 1 / 6000.0, about 1 / 6000.0 to about 1 / 7000.0, about 7000.0 In some embodiments, the assay is reduced to about 1 part in 8000.0, about 1 part in 8000.0 to about 1 part in 9000.0, about 1 part in 9000.0 to about 10,000.0, about 1 part in 10,000.0 to about 1 part in 50,000.0, about 1 part in 50,000.0 to about 100,000.0, about 1 part in 100,000.0 to about 1 part in 200,000.0, about 1 part in 200,000.0 to about 1 part in 300,000.0, about 1 part in 300,000.0, or less. In some embodiments, the assay includes an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA, or an ELISpot assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0380] 2. p40 subunit: According to any of the aspects and embodiments described herein, the disclosure provides non-naturally occurring IL-12 variants, including: a) a variant IL-12 p35 subunit, wherein the variant IL-12 p35 subunit comprises one or more amino acid substitutions selected from the group comprising: Y40A, T43A, D126A, P127A, R129A, K168A, and K170A; and b) an IL-12 p40 subunit.

[0381] In some embodiments, the IL-12 p40 subunit comprises a variant IL-12 p40 subunit.

[0382] In some embodiments, the IL-12 p40 subunit comprises a variant IL-12 p40 subunit, and in further embodiments, the variant IL-12 p40 subunit comprises one or more amino acid substitutions selected from the group including C177S, C252S, and C177S / C252S.

[0383] In some embodiments, the (variant) IL-12 p40 subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 (wild-type precursor), SEQ ID NO:4 (wild-type mature), SEQ ID NO:88 (C177S), SEQ ID NO:89 (C252S), and SEQ ID NO:90 (C177S / C252S) (as shown in Figures 1 and 10).

[0384] In some embodiments, the (variant) IL-12 p40 subunit comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90 (as shown in Figures 1 and 10).

[0385] In addition to the novel, non-naturally occurring IL-12 p35 variants described above, various modifications of the IL-12 p40 subunit are known in the art. Non-limiting examples of residues that may be modified include E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, S49, T54, Q56, I55, Q56, K58, E59, F60, G61, D62, Y 66, E73, H77, K84, E86, D87, G88, I89, W90, D93, K99, E100, K102, N103, K104, T105, F106, R108, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, E164, Y165, E166 , S168, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, M189, H1 94, K195, L196, K197, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E 235, T242, P243, S245, Y246, F247, S248, C252, Q256, K258, S259, K260, R261, E262, K264, D265, V267, D270, N281, S283, S285, R287, Q289, D290, R291, Y292, Y293, and E299. In some embodiments, the (variant) IL-12 p40 subunit of any of the above, below, or Figures 1 and 10 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more additional amino acid substitutions.In some embodiments, the (variant) IL-12 p40 subunit of any of the above, below, or Figures 1 and 10 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 41 to about 45, about 46 to about 50, about 51 to about 55, about 56 to about 60, about 1 to about 10, about 11 to about 20, about 21 to about 30, about 31 to about 40, about 41 to about 50, about 51 to about 60, about 1 to about 20, or about 21 to about 40, or about 41 to about 60 additional amino acid substitutions.

[0386] 3. Single Chain IL-12 Complex and Domain Linker: According to any of the aspects and embodiments described herein, the present disclosure provides a non-naturally occurring IL-12 variant comprising: a) a variant IL-12 p35 subunit; and b) an IL-12 p40 subunit.

[0387] In some embodiments, the non-naturally occurring IL-12 variant comprises a single-chain IL-12 complex (sc-IL-12) in which a variant IL-12 p35 subunit and a (variant) IL-12 p40 subunit are linked. In further embodiments, the subunits are linked to each other using a domain linker (also referred to herein as a "linker" or "linker domain"). In still other embodiments, the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 (as shown in FIG. 4).

[0388] In some embodiments, the non-naturally occurring IL-12 variant comprises a single-chain IL-12 complex (sc-IL-12) in which a variant IL-12 p35 subunit and a (variant) IL-12 p40 subunit are linked. In further embodiments, the C-terminus of the IL-12 p35 subunit is linked to the N-terminus of the (variant) IL-12 p40 subunit (sc-IL-12(p35 / p40)). In yet other embodiments, sc-IL-12(p35 / p40) further comprises a linker domain, wherein the C-terminus of the IL-12 p35 subunit is linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is linked to the N-terminus of the (variant) IL-12 p40 subunit. In still further embodiments, the linker domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 (as shown in Figure 4).

[0389] In some embodiments, the non-naturally occurring IL-12 variant comprises a single-chain IL-12 complex (sc-IL-12) in which a variant IL-12 p35 subunit and a (variant) IL-12 p40 subunit are linked. In some embodiments, the C-terminus of the (variant) IL-12 p40 subunit is linked to the N-terminus of the IL-12 p35 subunit (sc-IL-12(p40 / p35)). In yet other embodiments, sc-IL-12(p40 / p35) further comprises a linker domain, wherein the C-terminus of the (variant) IL-12 p40 subunit is linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is linked to the N-terminus of the IL-12 p35 subunit. In still further embodiments, the linker domain comprises an amino acid selected from the group comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 (as shown in Figure 4).

[0390] In some embodiments, the subunits are not linked via a linker domain.

[0391] Any of the sc-IL-12 complexes described herein can be utilized to generate heterodimeric and / or homodimeric IL-12 Fc fusion proteins, which are described in further detail below.

[0392] 4. Compositions with improved half-life: According to any of the aspects and embodiments described herein, the present disclosure provides a non-naturally occurring IL-12 variant comprising: a) a variant IL-12 p35 subunit; and b) an IL-12 p40 subunit.

[0393] In some embodiments, non-naturally occurring IL-12 variants are provided, wherein one or more amino acid substitutions in the variant IL-12 p35 subunit improve the half-life compared to the half-life of a reference IL-12. In some embodiments, the half-life of the non-naturally occurring IL-12 variant is 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, or 2.9-fold improved compared to the half-life of the reference IL-12. , 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5.0-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold or more. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein, or any combination thereof.

[0394] Various methods for measuring the half-life of a drug, such as IL-12, are known in the art. A person skilled in the relevant art would be able to easily determine and use any number of such methods for measuring changes in half-life. In some embodiments, at least one sample selected from the group including: (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, is used to measure half-life. One, two, three, four, five, six, seven, eight, nine, ten, or more samples (as described above) may be used to measure half-life.

[0395] In some embodiments, the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or the (variant) IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13 (as shown in FIG. 3 ); (ii) albumin; (iii) one or more unstructured biodegradable polypeptides (“XTEN”); or (iv) polyethylene glycol (PEG).

[0396] 5. Nucleic acids and vectors: According to any of the aspects and embodiments described herein, the present disclosure provides nucleic acid compositions encoding (i) a non-naturally occurring IL-12 variant, (ii) a variant IL-12 p35 subunit, (iii) a (variant) IL-12 p40 subunit, (iv) an sc-IL-12 complex, and (v) a domain linker, as described above.

[0397] In some embodiments, one or more of the nucleic acids encoding the components of the present invention are incorporated into an expression cassette or expression vector. It will be understood by those skilled in the art that an expression cassette generally comprises a construct of genetic material containing a coding sequence and sufficient regulatory information to direct proper transcription and / or translation of the coding sequence in recipient cells in vivo and / or ex vivo. Typically, the expression cassette can be inserted into a vector for targeting to a desired host cell and / or within an individual. Thus, in some embodiments, an expression cassette of the present disclosure comprises a coding sequence for a polypeptide as disclosed herein, operably linked to one or a combination of expression control elements, such as a promoter, and optionally other nucleic acid sequences that affect the transcription or translation of the coding sequence (e.g., an origin of replication, a selectable marker, a ribosome binding site, an inducer, etc.).

[0398] In some embodiments, the nucleotide sequence is incorporated into an expression vector. The term "vector" will be understood by those skilled in the art to generally refer to a recombinant polynucleotide construct designed for introduction between host cells and capable of being used for transformation (e.g., introduction of heterologous DNA into a host cell). Thus, in some embodiments, a vector may be a replicon, such as a plasmid, phage, or cosmid, into which another DNA fragment may be inserted to cause replication of the inserted fragment. In some embodiments, an expression vector may be an integrating vector.

[0399] In some embodiments, the expression vector may be a viral vector. As understood by those skilled in the art, the term "viral vector" is broadly used to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that typically contains viral-derived nucleic acid elements that facilitate the transfer or integration of the nucleic acid molecule into a cell's genome, or a viral particle that mediates the transfer of the nucleic acid, and may further include oncolytic viruses (both naturally occurring and those recombinantly produced or modified in a laboratory or clinical setting). Viral particles typically contain, in addition to the nucleic acid of interest, various viral components and, optionally, host cell components. The term viral vector may refer to either a virus or viral particle capable of transferring a nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. In some embodiments, the viral vector is a baculoviral vector, retroviral vector, or lentiviral vector. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, including long terminal repeats (LTRs), primarily derived from lentiviruses, a genus of retroviruses.

[0400] Thus, also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acids encoding any of the non-naturally occurring IL-12 variants, variant IL-12 p35 subunits, (variant) IL-12 p40 subunits, sc-IL-12 complexes, and / or domain linkers disclosed herein. The nucleic acids can be contained within a vector capable of directing their expression, for example, in cells transformed / transduced with the vector. Suitable vectors for use in prokaryotic and eukaryotic cells are known in the art and are commercially available or readily prepared by one of ordinary skill in the art.

[0401] As will be understood by those skilled in the art, the composition of the nucleic acids will depend on the configuration of the non-naturally occurring IL-12 variant. Thus, for example, if the configuration requires three amino acid sequences (e.g., a non-naturally occurring IL-12 variant in which variant IL-12 p35 and (variant) IL-12 p40 subunits are linked using a domain linker), the three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, for other configurations, if only two nucleic acids are required, they can be incorporated into one or two expression vectors.

[0402] DNA vectors can be introduced into host cells, e.g., eukaryotic or prokaryotic cells, by conventional transformation or transfection techniques, including, but not limited to, one or more of the following: transfection, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic transfer, nucleoporation, hydrodynamic shock, infection, and the like.

[0403] Viral vectors that may be used in the present disclosure include, but are not limited to, baculovirus vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, herpes viruses, simian virus 40 (SV40), bovine papillomavirus vectors, and the like.

[0404] 6. Recombinant Cells and Cell Cultures: In another aspect, provided herein is a cell culture comprising at least one recombinant cell as disclosed herein (also referred to herein as a "host cell") and a culture medium. Typically, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned cells and species are known in the art. Accordingly, a cell culture comprising at least one recombinant cell as disclosed herein is also within the scope of the present application. Suitable methods and systems for producing and maintaining cell cultures are known in the art.

[0405] Host cells may be used preparatively to propagate one or more nucleic acids encoding (i) a non-naturally occurring IL-12 variant, (ii) a variant IL-12 p35 subunit, (iii) a (variant) IL-12 p40 subunit, (iv) an sc-IL-12 complex, and (v) a domain linker, and combinations and / or components thereof. Host cells may include prokaryotic or eukaryotic cells specifically intended for the production of a non-naturally occurring IL-12 variant. Non-limiting examples of host cells include bacterial cells (e.g., Gram-positive bacterial cells (e.g., Bacillus, Streptomyces, and Staphylococcus species) or Gram-negative bacterial cells (e.g., Escherichia and Pseudomonas cells)), fungal or yeast cells (e.g., Saccharomyces, Pichia pastoris, and Hansenula polymorpha), insect cells (e.g., Drosophila cells and Sf9 cells), plant cells (e.g., cells from crop, medicinal, or ornamental plants or bulbs), mammalian cells (e.g., epithelial cell lines, osteosarcoma cell lines, neuroblastoma cell lines, epithelial cancer, glial cells, hepatic cell lines, Chinese hamster ovary (CHO) cells, COS cells, BHK cells, HeLa cells, mouse embryonic stem cell (mESC) line D3 cells, human embryonic stem cells (e.g., HS293 cells and BG01V cells), NIH 3T3 cells, human embryonic kidney (HEK) 293T cells, human mesenchymal stem cells (hMSCS), etc.

[0406] B. Heterodimeric IL-12 Fc fusion protein In one aspect, the disclosure provides a heterodimeric Fc-fusion protein comprising: a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first Fc domain; and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the second Fc domain.

[0407] In another aspect, the disclosure provides a heterodimeric Fc-fusion protein comprising: a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the N-terminus of the variant IL-12 p35 subunit domain is covalently linked to the C-terminus of the first Fc domain; and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the N-terminus of the IL-12 p40 subunit domain is covalently linked to the C-terminus of the second Fc domain.

[0408] Heterodimeric Fc fusion proteins, as well as various configurations of variant IL-12 p35 subunit domains, IL-12 p40 subunit domains, and Fc domains, are detailed below.

[0409] 1. p35 subunit domain: According to any of the aspects and embodiments described herein, the present disclosure provides a heterodimeric Fc-fusion protein comprising: a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the variant IL-12 p35 subunit domain is covalently linked to the N-terminus or C-terminus of the first Fc domain; and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the IL-12 p40 subunit domain is covalently linked to the N-terminus or C-terminus of the second Fc domain.

[0410] In some embodiments, the variant IL-12 p35 subunit domain comprises one or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the one or more amino acid substitutions comprise Y40A. In some embodiments, the one or more amino acid substitutions comprise T43A. In some embodiments, the one or more amino acid substitutions comprise D126A. In some embodiments, the one or more amino acid substitutions comprise P127A. In some embodiments, the one or more amino acid substitutions comprise R129A. In some embodiments, the one or more amino acid substitutions comprise K168A. In some embodiments, the one or more amino acid substitutions comprise K170A.

[0411] In some embodiments, the variant IL-12 p35 subunit domain comprises two or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the two or more amino acid substitutions comprise Y40A / T43A. In some embodiments, the two or more amino acid substitutions comprise Y40A / D126A. In some embodiments, the two or more amino acid substitutions comprise Y40A / P127A. In some embodiments, the two or more amino acid substitutions comprise Y40A / R129A. In some embodiments, the two or more amino acid substitutions comprise Y40A / K168A. In some embodiments, the two or more amino acid substitutions comprise T43A / D126A. In some embodiments, the two or more amino acid substitutions comprise T43A / P127A. In some embodiments, the two or more amino acid substitutions comprise T43A / R129A. In some embodiments, the two or more amino acid substitutions comprise T43A / K168A. In some embodiments, the two or more amino acid substitutions comprise D126A / P127A. In some embodiments, the two or more amino acid substitutions comprise D126A / R129A. In some embodiments, the two or more amino acid substitutions comprise D126A / K168A. In some embodiments, the two or more amino acid substitutions comprise P127A / R129A. In some embodiments, the two or more amino acid substitutions comprise P127A / K168A. In some embodiments, the two or more amino acid substitutions comprise R129A / K168A. In some embodiments, the two or more amino acid substitutions comprise Y40A / K170A. In some embodiments, the two or more amino acid substitutions comprise T43A / K170A. In some embodiments, the two or more amino acid substitutions comprise D126A / K170A. In some embodiments, the two or more amino acid substitutions comprise P127A / K170A. In some embodiments, the two or more amino acid substitutions comprise R129A / K170A. In some embodiments, the two or more amino acid substitutions comprise K168A / K170A.

[0412] In some embodiments, the variant IL-12 p35 subunit domain comprises three or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / D126A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / P127A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / R129A. In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / K168A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / P127A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / R129A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / K168A. In some embodiments, the three or more amino acid substitutions comprise Y40A / P127A / R129A. In some embodiments, the three or more amino acid substitutions comprise Y40A / P127A / K168A. In some embodiments, the three or more amino acid substitutions comprise Y40A / R129A / K168A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / P127A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / R129A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / K168A. In some embodiments, the three or more amino acid substitutions comprise T43A / P127A / R129A. In some embodiments, the three or more amino acid substitutions comprise T43A / P127A / K168A. In some embodiments, the three or more amino acid substitutions comprise T43A / R129A / K168A. In some embodiments, the three or more amino acid substitutions comprise D126A / P127A / R129A. In some embodiments, the three or more amino acid substitutions comprise D126A / P127A / K168A. In some embodiments, the three or more amino acid substitutions comprise D126A / R129A / K168A. In some embodiments, the three or more amino acid substitutions comprise P127A / R129A / K168A.In some embodiments, the three or more amino acid substitutions comprise Y40A / T43A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / D126A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / P127A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise Y40A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / D126A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / P127A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise T43A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise D126A / P127A / K170A. In some embodiments, the three or more amino acid substitutions comprise D126A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise D126A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise P127A / R129A / K170A. In some embodiments, the three or more amino acid substitutions comprise P127A / K168A / K170A. In some embodiments, the three or more amino acid substitutions comprise R129A / K168A / K170A.

[0413] In some embodiments, the variant IL-12 p35 subunit domain comprises four or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / P127A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / R129A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / P127A / R129A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / P127A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / P127A / R129A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / P127A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / P127A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / P127A / R129A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / P127A / K168A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise T43A / P127A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise D126A / P127A / R129A / K168A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / D126A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / P127A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / R129A / K170A.In some embodiments, the four or more amino acid substitutions comprise Y40A / T43A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / P127A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / D126A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / P127A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / P127A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise Y40A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / P127A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / D126A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / P127A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / P127A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise T43A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise D126A / P127A / R129A / K170A. In some embodiments, the four or more amino acid substitutions comprise D126A / P127A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise D126A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions comprise P127A / R129A / K168A / K170A.

[0414] In some embodiments, the variant IL-12 p35 subunit domain comprises five or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / P127A / R129A / K168A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / D126A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / P127A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / T43A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / P127A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / D126A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / P127A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise Y40A / P127A / R129A / K168A / K170A.In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / P127A / R129A / K170A. In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / P127A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise T43A / D126A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise T43A / P127A / R129A / K168A / K170A. In some embodiments, the five or more amino acid substitutions comprise D126A / P127A / R129A / K168A / K170A.

[0415] In some embodiments, the variant IL-12 p35 subunit domain comprises six or more amino acid substitutions selected from the group comprising Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K168A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A / K170A. In some embodiments, the six or more amino acid substitutions comprise Y40A / T43A / D126A / R129A / K168A / K170A. In some embodiments, the six or more amino acid substitutions comprise T43A / D126A / P127A / R129A / K168A / K170A.

[0416] In some embodiments, the variant IL-12 p35 subunit domain comprises seven or more amino acid substitutions selected from the group including Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0417] In some embodiments, the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and 103-166 (as shown in Figures 5-9, 78, and 80).

[0418] In some embodiments, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, and SEQ ID NOs:103-166 (as shown in Figures 1, 5-9, 78, and 80).

[0419] In some embodiments, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue Y40. In some further embodiments, the substitution mutation at amino acid residue Y40 is selected from the group comprising: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 177-187.

[0420] In some embodiments, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue D126. In some further embodiments, the substitution mutation at amino acid residue D126 is selected from the group including D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

[0421] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 199-214.

[0422] In some embodiments, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue P127. In some further embodiments, the substitution mutation at amino acid residue P127 is selected from the group including P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

[0423] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 279-290.

[0424] In some embodiments, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue R129. In some further embodiments, the substitution mutation at amino acid residue R129 is selected from the group including R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

[0425] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 215-231.

[0426] In some embodiments, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue K168. In some further embodiments, the substitution mutation at amino acid residue K168 is selected from the group including K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

[0427] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 232-247.

[0428] In some embodiments, the variant IL-12 p35 subunit domain comprises a substitution mutation at amino acid residue K170. In some further embodiments, the substitution mutation at amino acid residue K170 is selected from the group comprising: K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 188-198 or 306-308.

[0429] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising Y40A and further comprises a second substitution mutation selected from the group comprising K170L and K170T, hi some embodiments, the variant IL-12 p35 subunit domain comprises either SEQ ID NO: 248 or 249.

[0430] In some embodiments, the variant IL-12 p35 subunit domain comprises (i) a first substitution mutation selected from the group consisting of Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T, and (ii) D126A, D126C, D126E, D126F, D126G, D126H, D126I, D126J, D126K, D126N, D126P, D126Q, D126R, D126S, D126T ... G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V , D126W, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R129Y, K168A, K168C, K 168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K1 and a second substitution mutation selected from the group including K168S, K168T, K168W, K168Y, K170A, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

[0431] 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 the group comprising K170A, K168A, K168I, K168T, and R129A. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 250-253 or 311.

[0432] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising Y40G and further comprises a second substitution mutation selected from the group comprising K170A, K168A, K168I, K168T, and R129A. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 254-257 or 309.

[0433] 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 the group comprising K170A, K168A, K168D, K168I, and K168T. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 258-261 or 310.

[0434] 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 the group comprising K168I, K168T, K170A, K170L, K170T, and R129A, hi some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 262-267.

[0435] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising K170A and further comprises a second substitution mutation selected from the group comprising K168I, K168T, and R129E, hi some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 268-270.

[0436] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising K170P and further comprises a second substitution mutation selected from the group comprising K168A, K168I, K168T, and R129E, hi some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 271-274.

[0437] In some embodiments, the variant IL-12 p35 subunit domain comprises a first substitution mutation comprising K170T and further comprises a second substitution mutation selected from the group comprising K168A, K168I, K168T, and R129E. In some embodiments, the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 275-278.

[0438] In some embodiments, the variant IL-12 p35 subunit domain may further comprise a C74S substitution mutation.

[0439] In addition to the novel heterodimeric Fc fusion proteins described above, additional modifications of various residues are known in the relevant technical field. Non-limiting examples of residues that can be modified include Q20, N21, Q35, E38, F39, P41, S44, E45, E46, E50, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, T69, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, M98, A99, and L12. 4, M125, K128, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, F166, Y167, I171, 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 of any of the above, below, or Figures 1, 5-9, 78, and 80 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 above, below, or in any of Figures 1, 5-9, 78, and 80 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. In some embodiments, the variant IL-12 p35 subunit domain comprises SEQ ID NO: 87 (as shown in FIG. 9 ) and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group including: Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0440] Mutations in the variant IL-12 p35 subunit domain may result in changes in one or more of the following parameters: (i) binding affinity, (ii) potency, (iii) activity, (iv) manufacturability, or (v) stability, although changes in one or more of the above parameters (e.g., binding affinity) may not necessarily correlate with changes in one or more of the other parameters (e.g., potency or activity). In some embodiments, one or more amino acid substitutions in the variant IL-12 p35 subunit domain result in changes in binding affinity for IL-12Rβ2 compared to the binding affinity of a reference IL-12. In some embodiments, the one or more amino acid substitutions decrease the binding affinity of the variant IL-12 p35 subunit domain for IL-12Rβ2 compared to the reference IL-12. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein, or any combination thereof. In other embodiments, one or more amino acid substitutions in the variant IL-12 p35 subunit do not affect binding affinity, but may result in changes in one or more of the following parameters: (i) potency, (ii) activity, (iii) manufacturability, (iv) stability, or (v) any combination thereof.

[0441] Various assay formats can be used to select heterodimeric 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 immunoassays, immunoprecipitation, Biacore assays, KinExA assays, fluorescence-activated cell sorting (FACS), Octet assays, Western blot analysis, etc. The binding activity of the heterodimeric Fc-fusion proteins of the present disclosure can be analyzed by any suitable method known in the art, such as surface plasmon resonance (SPR) assays, enzyme-linked immunosorbent assays (ELISAs), ELISpot assays, Biacore assays, KinExA assays, etc.

[0442] Those skilled in the art will appreciate that binding affinity can also be used as a measure of the "strength" of the non-covalent interaction between two binding partners (e.g., a variant IL-12 p35 subunit domain and IL-12Rβ2). The binding affinity between two molecules is determined by the dissociation constant (K D ) can be quantified by determining K D can be determined by measuring the kinetics of complex formation and dissociation using a suitable assay known in the art, for example, an SPR assay. The rate constants corresponding to the association and dissociation of a monovalent complex are the association rate constant k, respectively. a (or k on ) and dissociation rate constant k d (or k off ) is called K D is the formula K D =k d / k a By k a and k d The value of the dissociation constant can be determined directly by well-known methods.

[0443] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a heterodimeric Fc-fusion protein) to elicit a response at a particular dose or concentration in a given biological system or experimental setting. The potency of the heterodimeric Fc-fusion proteins of the present disclosure can be analyzed by any suitable method known in the art, such as an IL-12 HEK reporter assay (e.g., InvivoGen's IL-12 HEK reporter assay (catalog number: hkb-il12)), a ligand binding assay (e.g., ELISA or flow cytometry), and / or a functional assay. Typically, changes in potency can be visually demonstrated as a leftward or rightward shift in a response curve compared to a control. A rightward shift in the response curve generally indicates a decrease in potency, while a leftward shift in the response curve generally indicates an increase in potency. As used herein, the term "activity" refers to the specific response of a given protein, cytokine, fusion protein, antibody, etc. (e.g., heterodimeric Fc-fusion protein) elicited at a given dose or concentration in a particular biological system or experimental setting. The activity of the heterodimeric Fc-fusion proteins of the present disclosure can be analyzed by any suitable method known in the art, such as ligand binding assays and / or functional assays. Generally, changes in activity can be visually demonstrated as an upward or downward shift in a response curve compared to a control. An upward shift in the response curve generally indicates increased activity, while a downward shift in the response curve generally indicates decreased activity.

[0444] As used herein, the term "manufacturability" refers to any characteristic that may affect the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, etc., on a scale and in quantities sufficient for administration to an individual. Examples of characteristics that affect manufacturability include, but are not limited to, the stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a heterodimeric Fc-fusion protein). As used herein, the term "stability" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (e.g., a heterodimeric Fc-fusion protein) to retain the same properties and characteristics as it possessed at the time of manufacture within specified limits and / or storage and / or use parameters. The stability of the heterodimeric Fc-fusion proteins of the present disclosure can be analyzed by any suitable method known in the art, such as 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 "expression yield" refers to the amount or content of a given protein, cytokine, fusion protein, antibody, etc. (e.g., heterodimeric Fc-fusion protein) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, etc. is well known in the relevant technical field. Any suitable method for quantifying or determining expression yield can be used, such as UV absorption measurement, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorimetric analysis.

[0445] In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain has a binding affinity for IL-12Rβ2 that is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more compared to the binding affinity of a reference IL-12 as measured by an SPR assay. In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by SPR assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

[0446] In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain has a binding affinity for IL-12Rβ2 that is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or more compared to the binding affinity of a reference IL-12, as measured by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

[0447] In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12, as measured by an assay. In some embodiments, the assay includes an SPR assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

[0448] In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain has a binding affinity for IL-12Rβ2 that is below the lower limit of detection of the assay, and the binding affinity of the reference IL-12 for IL-12Rβ2 is between or equal to the lower or upper limit of detection of the assay (i.e., the binding affinity of the reference IL-12 can be equal to the lower limit of detection, the upper limit of detection, or a value between the lower and upper limits of detection; in other words, the binding affinity is "detectable"). In some embodiments, the assay includes an SPR assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

[0449] In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain exhibits at least about 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold reduction in potency compared to a reference IL-12 as measured by an assay. 1 in, 3.1 in, 3.2 in, 3.3 in, 3.4 in, 3.5 in, 3.6 in, 3.7 in, 3.8 in, 3.9 in, 4.0 in, 4.1 in, 4.2 in, 4.3 in, 4.4 in, 4.5 in, 4.6 in, 4.7 in, 4.8 in, 4.9 in, 5.0 in, 5.1 in, 5.2 in, 5.3 in, 5.4 in, 5.5 in, 6.0 in, 7.0 in, 8.0 in, 9.0 in, 10.0 in, 11.0 in 1 in 12.0, 1 in 13.0, 1 in 14.0, 1 in 15.0, 1 in 16.0, 1 in 17.0, 1 in 18.0, 1 in 19.0, 1 in 20.0, 1 in 21.0, 1 in 22.0, 1 in 23.0, 1 in 24.0, 1 in 25.0, 1 in 30.0, 1 in 35.0, 1 in 40.0, 1 in 45.0, 1 in 50.0, 1 in 100.0, 1 in 150.0, 1 in 200.0, 1 in 250.0, 1 in 300.0, 1 in 350.0, 1 in 400.0, 4 1 in 50.0, 1 in 500.0, 1 in 550.0, 1 in 600.0, 1 in 650.0, 1 in 700.0, 1 in 750.0, 1 in 800.0, 1 in 850.0, 1 in 900.0, 1 in 950.0, 1 in 1000.0, 1 in 2000.0, 1 in 3000.0, 1 in 4000.0, 1 in 5000.0, 1 in 6000.0, 1 in 7000.0, 1 in 8000.0, 1 in 9000.0, 1 in 10,000.0, or less. In some embodiments, the assay comprises an IL-12 HEK reporter assay.In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0450] In some embodiments, the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain is at least about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, about 30.0 to about 35.0 fold less potent, about 35.0 fold less potent, compared to a reference IL-12 as measured by an assay. to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 80 The IL-12 has a reduced potency of about 1 / 00.0, about 1 / 8000.0 to about 1 / 9000.0, about 1 / 9000.0 to about 1 / 10,000.0, about 1 / 10,000.0 to about 1 / 50,000.0, about 1 / 50,000.0 to about 1 / 100,000.0, about 1 / 100,000.0 to about 1 / 200,000.0, about 1 / 200,000.0 to about 1 / 300,000.0, about 1 / 300,000.0, or less. In some embodiments, the assay includes an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0451] In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain has a reduced ability to stimulate STAT4 signaling compared to a reference IL-12, as measured by an assay. The reduced ability to stimulate STAT4 signaling is manifested by a reduced observed maximal response and / or EC 50 The term "ability" may refer to a change in the value of the heterodimeric Fc-fusion protein. In some embodiments, the ability of the heterodimeric Fc-fusion protein to stimulate STAT4 signaling is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or more compared to a reference IL-12, as measured by an assay. In some embodiments, the assay includes an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

[0452] In some embodiments, the ability of the heterodimeric Fc-fusion protein and / or 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 measured by an assay. In some embodiments, the assay includes an IL-12 HEK reporter assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

[0453] In some embodiments, the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain has a reduced ability to stimulate IFNγ production compared to a reference IL-12, as measured by an assay. The reduced ability to stimulate IFNγ production can be measured by a reduced maximal response observed and / or a reduced EC 50In some embodiments, the ability of the heterodimeric Fc-fusion protein and / or variant IL-12 p35 subunit domain to stimulate IFNγ production as measured by an assay may be at least about 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.10-fold, 3.11-fold, 3.12-fold, 3.13-fold, 3.14-fold, 3.15-fold, 3.16-fold, 3.17-fold, 3.18-fold, 3.19-fold, 3.20-fold, 3.21-fold, 3.22-fold, 3.23-fold, 3.24-fold, 3.25-fold, 3.26-fold, 3.27-fold, 3.28-fold, 3.29-fold, 3.30-fold, 3.31-fold, 3.32-fold, 3.33-fold, 3.34-fold, 3.35-fold, 3.36-fold, 3.37-fold, 3.38-fold, 3.39-fold, 3.40-fold, 3.41-fold, 3.42-fold, 3.43-fold, 3.44-fold, 1 in 0.9, 1 in 3.0, 1 in 3.1, 1 in 3.2, 1 in 3.3, 1 in 3.4, 1 in 3.5, 1 in 3.6, 1 in 3.7, 1 in 3.8, 1 in 3.9, 1 in 4.0, 1 in 4.1, 1 in 4.2, 1 in 4.3, 1 in 4.4, 1 in 4.5, 1 in 4.6, 1 in 4.7, 1 in 4.8, 1 in 4.9, 1 in 5.0, 1 in 5.1, 1 in 5.2, 1 in 5.3, 1 in 5.4, 1 in 5.5, 1 in 6.0, 1 in 7.0, 1 in 8.0, 9.0, 10.0 1 in 11.0, 1 in 12.0, 1 in 13.0, 1 in 14.0, 1 in 15.0, 1 in 16.0, 1 in 17.0, 1 in 18.0, 1 in 19.0, 1 in 20.0, 1 in 21.0, 1 in 22.0, 1 in 23.0, 1 in 24.0, 1 in 25.0, 1 in 30.0, 1 in 35.0, 1 in 40.0, 1 in 45.0, 1 in 50.0, 1 in 100.0, 1 in 150.0, 1 in 200.0, 1 in 250.0, 1 in 300.0, 1 in 350.0, 400 1 in 0.0, 1 in 450.0, 1 in 500.0, 1 in 550.0, 1 in 600.0, 1 in 650.0, 1 in 700.0, 1 in 750.0, 1 in 800.0, 1 in 850.0, 1 in 900.0, 1 in 950.0, 1 in 1000.0, 1 in 2000.0, 1 in 3000.0, 1 in 4000.0, 1 in 5000.0, 1 in 6000.0, 1 in 7000.0, 1 in 8000.0, 1 in 9000.0, 1 in 10,000.0, or less.In some embodiments, the assay includes an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA, or an ELISpot assay. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0454] In some embodiments, the ability of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain to stimulate IFNγ production is at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, or about 30.0 to about 35.0 fold less than a reference IL-12 as measured by an assay. 1, about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 70 1 in 0.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 7000.0 In some embodiments, the cytokine release is reduced to about 1 in 1 to about 1 in 8000.0, about 1 in 8000.0 to about 1 in 9000.0, about 1 in 9000.0 to about 10,000.0, about 1 in 10,000.0 to about 1 in 50,000.0, about 1 in 50,000.0 to about 100,000.0, about 1 in 100,000.0 to about 1 in 200,000.0, about 1 in 200,000.0 to about 1 in 300,000.0, about 1 in 300,000.0, or less. In some embodiments, the assay includes an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA, or an ELISpot assay.In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0455] In some embodiments, heterodimeric Fc fusion proteins and / or variant IL-12 p35 subunit domains are provided, wherein one or more amino acid substitutions in the variant IL-12 p35 subunit domain improve half-life compared to the half-life of a reference IL-12. In some embodiments, fusion of a variant IL-12 p35 subunit domain and / or a (variant) IL-12 p40 subunit domain improves half-life compared to the reference half-life. In some embodiments, heterodimeric Fc fusion proteins and / or variant IL-12 The half-lives of the p35 subunit domain were 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, and 3.1-fold, respectively, compared to the half-life of the reference IL-12. In some embodiments, the reference IL-12 is shortened or lengthened by 1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4.0 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold, 5.0 fold, 5.1 fold, 5.2 fold, 5.3 fold, 5.4 fold, 5.5 fold, 6.0 fold, 7.0 fold, 8.0 fold, 9.0 fold, 10.0 fold or more. In some embodiments, the reference IL-12 includes one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, an IL-12 Fc fusion protein, or any combination thereof.

[0456] Various methods for measuring the half-life of a drug, such as IL-12, are known in the art. A person skilled in the relevant art would be able to easily determine and use any number of such methods for measuring changes in half-life. In some embodiments, at least one sample selected from the group including: (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, is used to measure half-life. One, two, three, four, five, six, seven, eight, nine, ten, or more samples (as described above) may be used to measure half-life.

[0457] 2. p40 subunit domain: According to any of the aspects and embodiments described herein, the present disclosure provides a heterodimeric Fc-fusion protein comprising: a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the variant IL-12 p35 subunit domain is covalently linked to the N-terminus or C-terminus of the first Fc domain; and b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the IL-12 p40 subunit domain is covalently linked to the N-terminus or C-terminus of the second Fc domain.

[0458] In some embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

[0459] In some embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, and in further embodiments, the variant IL-12 p40 subunit domain comprises one or more amino acid substitutions selected from the group including C177S, C252S, and C177S / C252S.

[0460] In some embodiments, the (variant) IL-12 p40 subunit domain comprises an amino acid sequence selected from the group comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90 (as shown in Figures 1 and 10).

[0461] In some embodiments, the (variant) IL-12 p40 subunit domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90 (as shown in Figures 1 and 10).

[0462] In addition to the novel heterodimeric Fc-fusion proteins described above, various modifications of the IL-12 p40 subunit domain are known in the art and may be further included in the heterodimeric Fc-fusion proteins described herein. Non-limiting examples of residues that may be modified include E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, S49, T54, Q56, I55, Q56, K58, E59, F60, G61, D62, Y 66, E73, H77, K84, E86, D87, G88, I89, W90, D93, K99, E100, K102, N103, K104, T105, F106, R108, E110, N113, Y114, D129,...

Claims

1. a) a first fusion construct comprising a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain; i) the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently linked to the N-terminus of the first Fc domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 13; iv) the variant IL-12 p35 subunit domain is selected from the group consisting of Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y4 0G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y4 0S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, the first fusion construct comprising one or more amino acid substitutions selected from the group consisting of K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E; b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain; i) the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently linked to the N-terminus of the second Fc domain; ii) the second linker domain comprises SEQ ID NO: 15; iii) the second Fc domain comprises SEQ ID NO: 12; iv) the second fusion construct, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

89. A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

2. a) a first fusion construct comprising a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain; i) the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently linked to the N-terminus of the first Fc domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 12; iv) the variant IL-12 p35 subunit domain is selected from the group consisting of Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y4 0G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y4 0S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, the first fusion construct comprising one or more amino acid substitutions selected from the group consisting of K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E; b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain; i) the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently linked to the N-terminus of the second Fc domain; ii) the second linker domain comprises SEQ ID NO: 15; iii) the second Fc domain comprises SEQ ID NO: 13; iv) the second fusion construct, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

89. A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

3. a) a first fusion construct comprising a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain; i) the C-terminus of the first Fc domain is covalently linked to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 12; iv) the variant IL-12 p35 subunit domain is selected from the group consisting of Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y4 0G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y4 0S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, the first fusion construct comprising one or more amino acid substitutions selected from the group consisting of K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E; b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain; i) the C-terminus of the second Fc domain is covalently linked to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently linked to the N-terminus of the IL-12 p40 subunit domain; ii) the second linker domain comprises SEQ ID NO: 15; iii) the second Fc domain comprises SEQ ID NO: 13; iv) the second fusion construct, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

89. A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

4. a) a first fusion construct comprising a variant IL-12 p35 subunit domain, a first linker domain, and a first Fc domain; i) the C-terminus of the first Fc domain is covalently linked to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain; ii) the first linker domain comprises SEQ ID NO: 15; iii) the first Fc domain comprises SEQ ID NO: 13; iv) the variant IL-12 p35 subunit domain is selected from the group consisting of Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y4 0G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y40S / K168T, Y40S / K170A, Y40S / K170L, Y4 0S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, the first fusion construct comprising one or more amino acid substitutions selected from the group consisting of K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E; b) a second fusion construct comprising an IL-12 p40 subunit domain, a second linker domain, and a second Fc domain; i) the C-terminus of the second Fc domain is covalently linked to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently linked to the N-terminus of the IL-12 p40 subunit domain; ii) the second linker domain comprises SEQ ID NO: 15; iii) the second Fc domain comprises SEQ ID NO: 12; iv) the second fusion construct, wherein the IL-12 p40 subunit domain comprises SEQ ID NO:

89. A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

5. 5. The heterodimeric Fc-fusion protein of claim 1, wherein said variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

6. The heterodimeric Fc-fusion protein of any one of claims 1 to 5, wherein the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

7. a) a variant IL-12 p35 subunit, said variant IL-12 p35 subunit comprising a first amino acid substitution mutation, said first amino acid substitution being selected from the group consisting of Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, and K170T; b) the IL-12 p40 subunit; and A non-naturally occurring IL-12 variant comprising:

8. i) the first amino acid substitution mutation is Y40A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L, and K170T.

9. i) the first amino acid substitution mutation is Y40E; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K170A, K168A, K168I, K168T, and R129A.

10. i) the first amino acid substitution mutation is Y40G; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K170A, K168A, K168I, K168T, and R129A.

11. i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K170A, K168A, K168D, K168I, and K168T.

12. i) the first amino acid substitution mutation is Y40S; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K168I, K168T, K170A, K170L, K170T, and R129A.

13. i) the first amino acid substitution mutation is K170A; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

14. i) the first amino acid substitution mutation is K170P; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

15. i) the first amino acid substitution mutation is K170T; ii) the variant IL-12 p35 subunit further comprises a second substitution mutation; 8. The non-naturally occurring IL-12 variant of claim 7, wherein iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

16. 8. The non-naturally occurring IL-12 variant of claim 7, wherein the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 24, 34, 103, 104, 109, 179, 180, 183, 185, 186, 194, 196, 233, 234, 238, 240, 243, 245, and 248-278.

17. The non-naturally occurring IL-12 variant of any one of claims 7 to 16, wherein the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

18. 18. The non-naturally occurring IL-12 variant of any one of claims 7 to 17, wherein the IL-12 p40 subunit comprises a variant IL-12 p40 subunit, wherein the variant IL-12 p40 subunit comprises one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

19. The non-naturally occurring IL-12 variant of any one of claims 7 to 18, wherein the IL-12 p40 subunit comprises any of SEQ ID NOs: 4, 88, 89, and 90.

20. 20. The non-naturally occurring IL-12 variant of any one of claims 7-19, wherein the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or the IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

21. 21. The non-naturally occurring IL-12 variant of any one of claims 7 to 20, wherein the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the IL-12 p40 subunit.

22. 22. The non-naturally occurring IL-12 variant of claim 21, further comprising a linker domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently linked to the N-terminus of the IL-12 p40 subunit.

23. 21. The non-naturally occurring IL-12 variant of any one of claims 7 to 20, wherein the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

24. 24. The non-naturally occurring IL-12 variant of claim 23, further comprising a linker domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

25. The non-naturally occurring IL-12 variant of any one of claims 7 to 24, wherein the variant IL-12 p35 subunit comprises one or more additional amino acid substitutions.

26. A composition comprising the non-naturally occurring IL-12 variant of any one of claims 7 to 25 for use in treating cancer in a subject.

27. One or more nucleic acids encoding the non-naturally occurring IL-12 variant of any one of claims 7 to 25.

28. 28. A host cell comprising one or more nucleic acids of claim 27.

29. 1. A method for producing a non-naturally occurring IL-12 variant, comprising: culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the non-naturally occurring IL-12 variant is produced; i) the one or more nucleic acids or vectors comprise one or more nucleic acids according to claim 27; ii) the method, wherein at least one substitution mutation in said variant IL-12 p35 subunit improves the half-life compared to the half-life of a reference IL-12.

30. 30. The method of claim 29, further comprising isolating and / or purifying the non-naturally occurring IL-12 variant.

31. 31. The method of claim 29 or 30, wherein the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or the IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

32. 32. The method of any one of claims 29 to 31, wherein the produced non-naturally occurring IL-12 variant has an altered binding affinity for interleukin-12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

33. 33. The method of claim 32, wherein the produced non-naturally occurring IL-12 variant has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12 as measured by an assay.

34. 34. The method of claim 33, wherein the assay comprises an SPR assay.

35. 33. The method of claim 32, wherein the produced non-naturally occurring IL-12 variant has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

36. 36. The method of claim 35, wherein the assay comprises an SPR assay.

37. The produced non-naturally occurring IL-12 variant is about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, about 30.0 to about 35.0 fold, about 35.0 to about 40 fold less potent than a reference IL-12 as measured by an assay. 1 in 0.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 1 in 8000.0, about 8000.0 32. The method of any one of claims 29-31, wherein the potency is reduced by from about 1 part in to about 1 part in 9000.0, from about 1 part in 9000.0 to about 10,000.0, from about 10,000.0 to about 1 part in 50,000.0, from about 1 part in 50,000.0 to about 100,000.0, from about 100,000.0 to about 1 part in 200,000.0, from about 1 part in 200,000.0 to about 1 part in 300,000.0, about 1 part in 300,000.0, or less.

38. 38. The method of claim 37, wherein the assay comprises an IL-12 HEK reporter assay.

39. and / or wherein the produced non-naturally occurring IL-12 variant has an ability to stimulate IFNγ production of at least about 0.5-fold to about 50.0-fold, about 0.5-fold to about 5.0-fold, about 5.0-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 25.0-fold, about 25.0-fold to about 30.0-fold, about 30.0-fold to about 35.0-fold, about 35.0-fold to about 40.0-fold, about 40.0-fold to about 5 ... 1 / 0 to about 1 / 40.0, about 1 / 40.0 to about 1 / 45.0, about 1 / 45.0 to about 1 / 50.0, about 1 / 50.0 to about 1 / 100.0, about 1 / 100.0 to about 1 / 200.0, about 1 / 200.0 to about 1 / 300.0, about 1 / 300.0 to about 1 / 400.0, about 1 / 400.0 to about 1 / 500.0, about 1 / 500.0 to about 1 / 600.0, about 1 / 600.0 to about 1 / 700.0, about 1 / 700.0 to about 1 / 800.0 , about 1 in 800.0 to about 1 in 900.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 1 in 8000.0, about 1 in 8000.0 to about 9 32. The method of any one of claims 29 to 31, wherein the antibody has an ability to stimulate IFNγ production that is reduced to about 1 in 000.0, about 1 in 9000.0 to about 10,000.0, about 1 in 10,000.0 to about 1 50,000.0, about 1 in 50,000.0 to about 100,000.0, about 1 in 100,000.0 to about 1 200,000.0, about 1 in 200,000.0 to about 1 300,000.0, about 1 300,000.0, or less.

40. 40. The method of claim 39, wherein the assay comprises one or more of: (i) an intracellular cytokine staining assay, (ii) a Luminex bead-based cytokine release assay, (iii) an ELISA, or (iv) an ELISpot assay.

41. 41. The method of any one of claims 29-40, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

42. a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, i) the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first Fc domain; ii) the variant IL-12 p35 subunit domain comprises a first amino acid substitution mutation; iii) the first fusion construct, wherein the first amino acid substitution mutation is selected from the group consisting of Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, K170T; b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the second Fc domain; and A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

43. i) the first amino acid substitution mutation is Y40A; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L, and K170T.

44. i) the first amino acid substitution mutation is Y40E; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K170A, K168A, K168I, K168T, and R129A.

45. i) the first amino acid substitution mutation is Y40G; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K170A, K168A, K168I, K168T, and R129A.

46. i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K170A, K168A, K168D, K168I, and K168T.

47. i) the first amino acid substitution mutation is Y40S; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168I, K168T, K170A, K170L, K170T, and R129A.

48. i) the first amino acid substitution mutation is K170A; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

49. i) the first amino acid substitution mutation is K170P; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

50. i) the first amino acid substitution mutation is K170T; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

51. 43. The heterodimeric Fc-fusion protein of claim 42, wherein the variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 24, 34, 103, 104, 109, 179, 180, 183, 185, 186, 194, 196, 233, 234, 238, 240, 243, 245, and 248-278.

52. 52. The heterodimeric Fc-fusion protein of any one of claims 42 to 51, wherein said variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

53. 52. The heterodimeric Fc-fusion protein of any one of claims 42 to 51, wherein said IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, said variant IL-12 p40 subunit domain comprising one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

54. 54. The heterodimeric Fc-fusion protein of any one of claims 42 to 53, wherein the IL-12 p40 subunit domain comprises any of SEQ ID NOs: 4, 88, 89, and 90.

55. i) the first Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; ii) the second Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

56. i) the first fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; iii) the C-terminus of said variant IL-12 p35 subunit domain is covalently linked to the N-terminus of said linker domain, and the C-terminus of said linker domain is covalently linked to the N-terminus of said first Fc domain.

57. i) the second fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; iii) the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently linked to the N-terminus of the second Fc domain.

58. 58. The heterodimeric Fc-fusion protein of any one of claims 42 to 57, wherein said variant IL-12 p35 subunit domain comprises one or more additional amino acid substitutions.

59. 59. A composition comprising the heterodimeric Fc-fusion protein of any one of claims 42 to 58 for use in treating cancer in a subject.

60. One or more nucleic acids encoding the heterodimeric Fc-fusion protein of any one of claims 42 to 58.

61. 61. A host cell comprising one or more nucleic acids of claim 60.

62. 1. A method for producing a heterodimeric Fc-fusion protein, comprising: culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the heterodimeric Fc-fusion protein is produced; i) the one or more nucleic acids or vectors comprise one or more nucleic acids of claim 60; ii) the method, wherein at least one substitution mutation in the variant IL-12 p35 subunit domain improves the half-life compared to the half-life of a reference IL-12.

63. 63. The method of claim 62, further comprising isolating and / or purifying the heterodimeric Fc-fusion protein.

64. 64. The method of claim 62 or 63, wherein the produced heterodimeric Fc-fusion protein has an altered binding affinity for interleukin-12 receptor beta 2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

65. 65. The method of claim 64, wherein the produced heterodimeric Fc-fusion protein has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12 as measured by an assay.

66. 66. The method of claim 65, wherein the assay comprises an SPR assay.

67. 65. The method of claim 64, wherein the produced heterodimeric Fc-fusion protein has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

68. 68. The method of claim 67, wherein the assay comprises an SPR assay.

69. and / or wherein the produced heterodimeric Fc-fusion protein is about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, about 30.0 to about 35.0 fold less potent, about 35.0 to about 40.0 fold less potent, or about 40.0 to about 50.0 fold less potent, as measured by an assay relative to a reference IL-12. about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 800.0 1, from about 1 in 800.0 to about 1 in 900.0, from about 1 in 900.0 to about 1 in 1000.0, from about 1 in 1000.0 to about 1 in 2000.0, from about 1 in 2000.0 to about 1 in 3000.0, from about 1 in 3000.0 to about 1 in 4000.0, from about 1 in 4000.0 to about 1 in 5000.0, from about 1 in 5000.0 to about 1 in 6000.0, from about 1 in 6000.0 to about 1 in 7000.0, from about 1 in 7000.0 to about 1 in 8000.0, 64. The method of claim 62 or 63, wherein the compound has a reduced potency of from 0.0 to about 1 in 9000.0, from about 1 in 9000.0 to about 10,000.0, from about 10,000.0 to about 1 in 50,000.0, from about 1 in 50,000.0 to about 100,000.0, from about 100,000.0 to about 1 in 200,000.0, from about 1 in 200,000.0 to about 1300,000.0, about 1 in 300,000.0, or less.

70. 70. The method of claim 69, wherein the assay comprises an IL-12 HEK reporter assay.

71. the produced heterodimeric Fc-fusion protein has an ability to stimulate IFNγ production as measured by an assay at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, about 30.0 to about 35.0 fold lower than that of a reference IL-12; about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 800.0 1 in, from about 1 in 800.0 to about 1 in 900.0, from about 1 in 900.0 to about 1 in 1000.0, from about 1 in 1000.0 to about 1 in 2000.0, from about 1 in 2000.0 to about 1 in 3000.0, from about 1 in 3000.0 to about 1 in 4000.0, from about 1 in 4000.0 to about 1 in 5000.0, from about 1 in 5000.0 to about 1 in 6000.0, from about 1 in 6000.0 to about 1 in 7000.0, from about 1 in 7000.0 to about 1 in 8000.0, and about 8000.0 64. The method of claim 62 or 63, wherein the ability to stimulate IFNγ production is reduced by a factor of 1 to about 9000.0, about 9000.0 to about 10,000.0, about 10,000.0 to about 50,000.0, about 50,000.0 to about 100,000.0, about 100,000.0 to about 200,000.0, about 200,000.0 to about 300,000.0, about 300,000.0, or more.

72. 72. The method of claim 71, wherein the assay comprises one or more of: (i) an intracellular cytokine staining assay, (ii) a Luminex bead-based cytokine release assay, (iii) an ELISA, or (iv) an ELISpot assay.

73. 73. The method of any one of claims 62-72, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

74. a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, i) the C-terminus of the first Fc domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit domain; ii) the variant IL-12 p35 subunit domain comprises a first amino acid substitution mutation; iii) the first fusion construct, wherein the first amino acid substitution mutation is selected from the group consisting of Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, K170T; b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the C-terminus of the second Fc domain is covalently linked to the N-terminus of the IL-12 p40 subunit domain; and A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

75. i) the first amino acid substitution mutation is Y40A; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L, and K170T.

76. i) the first amino acid substitution mutation is Y40E; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; 75. The heterodimeric Fc-fusion protein of claim 74, wherein iii) said second substitution mutation is selected from the group consisting of K170A, K168A, K168I, K168T, and R129A.

77. i) the first amino acid substitution mutation is Y40G; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; 75. The heterodimeric Fc-fusion protein of claim 74, wherein iii) said second substitution mutation is selected from the group consisting of K170A, K168A, K168I, K168T, and R129A.

78. i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K170A, K168A, K168D, K168I, and K168T.

79. i) the first amino acid substitution mutation is Y40S; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; 75. The heterodimeric Fc-fusion protein of claim 74, wherein iii) said second substitution mutation is selected from the group consisting of K168I, K168T, K170A, K170L, K170T, and R129A.

80. i) the first amino acid substitution mutation is K170A; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

81. i) the first amino acid substitution mutation is K170P; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

82. i) the first amino acid substitution mutation is K170T; ii) the variant IL-12 p35 subunit domain further comprises a second substitution mutation; iii) the second substitution mutation is selected from the group consisting of K168A, K168I, K168T, and R129E.

83. 75. The heterodimeric Fc-fusion protein of claim 74, wherein said variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 24, 34, 103, 104, 109, 179, 180, 183, 185, 186, 194, 196, 233, 234, 238, 240, 243, 245, and 248-278.

84. 84. The heterodimeric Fc-fusion protein of any one of claims 74 to 83, wherein said variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

85. 85. The heterodimeric Fc-fusion protein of any one of claims 74 to 84, wherein said IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, said variant IL-12 p40 subunit domain comprising one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

86. 86. The heterodimeric Fc-fusion protein of any one of claims 74 to 85, wherein said IL-12 p40 subunit domain comprises any of SEQ ID NOs: 4, 88, 89, and 90.

87. i) the first Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; ii) the second Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

88. i) the first fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; iii) the C-terminus of said first Fc domain is covalently linked to the N-terminus of said linker domain, and the C-terminus of said linker domain is covalently linked to the N-terminus of said variant IL-12 p35 subunit domain.

89. i) the second fusion construct further comprises a linker domain; ii) the linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; iii) the C-terminus of said second Fc domain is covalently linked to the N-terminus of said linker domain, and the C-terminus of said linker domain is covalently linked to the N-terminus of said IL-12 p40 subunit domain.

90. 90. The heterodimeric Fc-fusion protein of any one of claims 74 to 89, wherein said variant IL-12 p35 subunit domain comprises one or more additional amino acid substitutions.

91. 91. A composition comprising the heterodimeric Fc-fusion protein of any one of claims 74 to 90 for use in treating cancer in a subject.

92. One or more nucleic acids encoding the heterodimeric Fc-fusion protein of any one of claims 74 to 90.

93. 93. A host cell comprising one or more nucleic acids of claim 92.

94. 1. A method for producing a heterodimeric Fc-fusion protein, comprising: culturing a host cell harboring one or more nucleic acids or vectors under conditions such that the heterodimeric Fc-fusion protein is produced; i) the one or more nucleic acids or vectors comprise one or more nucleic acids of claim 92; ii) the method, wherein at least one substitution mutation in the variant IL-12 p35 subunit domain improves the half-life compared to the half-life of a reference IL-12.

95. 95. The method of claim 94, further comprising isolating and / or purifying said heterodimeric Fc-fusion protein.

96. 96. The method of claim 94 or 95, wherein the produced heterodimeric Fc-fusion protein has an altered binding affinity for interleukin-12 receptor beta 2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

97. 97. The method of claim 96, wherein the produced heterodimeric Fc-fusion protein has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12 as measured by an assay.

98. 98. The method of claim 97, wherein the assay comprises an SPR assay.

99. 97. The method of claim 96, wherein the produced heterodimeric Fc-fusion protein has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

100. 100. The method of claim 99, wherein the assay comprises an SPR assay.

101. and / or wherein the produced heterodimeric Fc-fusion protein is about 0.5 to about 50.0 fold less potent, about 0.5 to about 5.0 fold less potent, about 5.0 to about 10.0 fold less potent, about 10.0 to about 15.0 fold less potent, about 15.0 to about 20.0 fold less potent, about 20.0 to about 25.0 fold less potent, about 25.0 to about 30.0 fold less potent, about 30.0 to about 35.0 fold less potent, about 35.0 to about 40.0 fold less potent, or about 40.0 to about 50.0 fold less potent, as measured by an assay relative to a reference IL-12. about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 800.0 1, from about 1 in 800.0 to about 1 in 900.0, from about 1 in 900.0 to about 1 in 1000.0, from about 1 in 1000.0 to about 1 in 2000.0, from about 1 in 2000.0 to about 1 in 3000.0, from about 1 in 3000.0 to about 1 in 4000.0, from about 1 in 4000.0 to about 1 in 5000.0, from about 1 in 5000.0 to about 1 in 6000.0, from about 1 in 6000.0 to about 1 in 7000.0, from about 1 in 7000.0 to about 1 in 8000.0, 96. The method of claim 94 or 95, wherein the potency is reduced by from 0.0 to about 1 in 9000.0, from about 1 in 9000.0 to about 10,000.0, from about 10,000.0 to about 1 in 50,000.0, from about 1 in 50,000.0 to about 100,000.0, from about 100,000.0 to about 1 in 200,000.0, from about 1 in 200,000.0 to about 1300,000.0, about 1 in 300,000.0, or less.

102. 102. The method of claim 101, wherein the assay comprises an IL-12 HEK reporter assay.

103. the produced heterodimeric Fc-fusion protein has an ability to stimulate IFNγ production as measured by an assay at least about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, about 30.0 to about 35.0 fold lower than that of a reference IL-12; about 1 in 35.0 to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 800.0 1 in, from about 1 in 800.0 to about 1 in 900.0, from about 1 in 900.0 to about 1 in 1000.0, from about 1 in 1000.0 to about 1 in 2000.0, from about 1 in 2000.0 to about 1 in 3000.0, from about 1 in 3000.0 to about 1 in 4000.0, from about 1 in 4000.0 to about 1 in 5000.0, from about 1 in 5000.0 to about 1 in 6000.0, from about 1 in 6000.0 to about 1 in 7000.0, from about 1 in 7000.0 to about 1 in 8000.0, and about 8000.0 96. The method of claim 94 or 95, wherein the ability to stimulate IFNγ production is reduced by a factor of 1 to about 9000.0, about 9000.0 to about 10,000.0, about 10,000.0 to about 50,000.0, about 50,000.0 to about 100,000.0, about 100,000.0 to about 200,000.0, about 200,000.0 to about 300,000.0, about 300,000.0, or more.

104. 104. The method of claim 103, wherein the assay comprises one or more of: (i) an intracellular cytokine staining assay, (ii) a Luminex bead-based cytokine release assay, (iii) an ELISA, or (iv) an ELISpot assay.

105. 105. The method of any one of claims 94-104, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

106. a) a variant IL-12 p35 subunit, wherein said variant IL-12 p35 subunit comprises one or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A; and b) the IL-12 p40 subunit; and A non-naturally occurring IL-12 variant comprising:

107. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises two or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

108. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises three or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

109. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises four or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

110. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises five or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

111. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises six or more amino acid substitutions consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

112. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and D126A.

113. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and P127A.

114. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and T43A.

115. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, D126A, and P127A.

116. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, T43A, D126A, and P127A.

117. 117. The non-naturally occurring IL-12 variant of any one of claims 106 to 116, wherein the IL-12 p40 subunit comprises a variant IL-12 p40 subunit.

118. 118. The non-naturally occurring IL-12 variant of claim 117, wherein the variant IL-12 p40 subunit comprises one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

119. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:24-86 and 103-166, and the IL-12 p40 subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:

90.

120. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, and SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:

90.

121. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises an amino acid sequence having at least 96% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 96% sequence identity to any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:

90.

122. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises an amino acid sequence having at least 97 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 97 percent sequence identity to any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:

90.

123. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises an amino acid sequence having at least 98 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 98 percent sequence identity to any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:

90.

124. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises an amino acid sequence having at least 99 percent sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166, and the IL-12 p40 subunit comprises an amino acid sequence having at least 99 percent sequence identity to any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:

90.

125. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:24-86 and 103-166, and the IL-12 p40 subunit consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:

90.

126. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises SEQ ID NO:87 and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

127. 127. The non-naturally occurring IL-12 variant of any one of claims 106-126, wherein one or more amino acid substitutions in the variant IL-12 p35 subunit improve the half-life compared to the half-life of a reference IL-12, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

128. 128. The non-naturally occurring IL-12 variant of any one of claims 106-127, wherein the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or the IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

129. 129. The non-naturally occurring IL-12 variant of any one of claims 106 to 128, wherein the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the IL-12 p40 subunit.

130. 130. The non-naturally occurring IL-12 variant of claim 129, further comprising a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the variant IL-12 p35 subunit is covalently linked to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently linked to the N-terminus of the IL-12 p40 subunit.

131. 129. The non-naturally occurring IL-12 variant of any one of claims 106 to 128, wherein the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

132. 132. The non-naturally occurring IL-12 variant of claim 131, further comprising a linker domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, wherein the C-terminus of the IL-12 p40 subunit is covalently linked to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently linked to the N-terminus of the variant IL-12 p35 subunit.

133. 133. The non-naturally occurring IL-12 variant of any one of claims 106 to 132, wherein the variant IL-12 p35 subunit comprises an additional amino acid substitution.

134. 134. A composition comprising the non-naturally occurring IL-12 variant of any one of claims 106 to 133 for use in treating cancer in a subject.

135. 134. One or more nucleic acids encoding the non-naturally occurring IL-12 variant of any one of claims 106 to 133.

136. 136. A host cell comprising one or more nucleic acids of claim 135.

137. 1. A method for producing a non-naturally occurring IL-12 variant, comprising:

136. The method of claim 135, comprising culturing a host cell harboring one or more nucleic acids or vectors under conditions in which the non-naturally occurring IL-12 variant is produced, wherein the one or more nucleic acids or vectors comprise one or more nucleic acids of claim 135.

138. 138. The method of claim 137, further comprising isolating and / or purifying the non-naturally occurring IL-12 variant produced.

139. 139. The method of any one of claims 137 or 138, wherein the non-naturally occurring IL-12 variant further comprises one or more of the following fused to the variant IL-12 p35 subunit and / or the IL-12 p40 subunit: (i) an Fc domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (ii) albumin; (iii) one or more unstructured biodegradable polypeptides ("XTEN"); or (iv) polyethylene glycol (PEG).

140. 140. The method of any one of claims 137-139, wherein the produced non-naturally occurring IL-12 variant has an altered binding affinity to interleukin-12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

141. 141. The method of claim 140, wherein the produced non-naturally occurring IL-12 variant has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12 as measured by an assay.

142. 142. The method of claim 141, wherein the assay comprises an SPR assay.

143. 141. The method of claim 140, wherein the produced non-naturally occurring IL-12 variant has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

144. 144. The method of claim 143, wherein the assay comprises an SPR assay.

145. As measured by the assay, the non-naturally occurring IL-12 variants described above have a potency of about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, about 30.0 to about 35.0 fold, about 35.0 to about 40.0 fold, about 4 fold, 1 in 0.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 900.0 1 in, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 1 in 8000.0, about 1 in 8000.0 to about 1 in 9000.0, about 9000.0 140. The method of any one of claims 137-139, wherein the reference IL-12 has a potency reduced by a factor of 1 to about 10,000.0, about 10,000.0 to about 50,000.0, about 50,000.0 to about 100,000.0, about 100,000.0 to about 200,000.0, about 200,000.0 to about 300,000.0, about 300,000.0, or less, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

146. 146. The method of claim 145, wherein the assay comprises an IL-12 HEK reporter assay.

147. As measured by an assay, the non-naturally occurring IL-12 variants described above have an ability to stimulate IFNγ production that is at least about 0.5-fold to about 50.0-fold, about 0.5-fold to about 5.0-fold, about 5.0-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 25.0-fold, about 25.0-fold to about 30.0-fold, about 30.0-fold to about 35.0-fold, about 35.0-fold to about 4. 1 in 0.0, from about 1 in 40.0 to about 1 in 45.0, from about 1 in 45.0 to about 1 in 50.0, from about 1 in 50.0 to about 1 in 100.0, from about 1 in 100.0 to about 1 in 200.0, from about 1 in 200.0 to about 1 in 300.0, from about 1 in 300.0 to about 1 in 400.0, from about 1 in 400.0 to about 1 in 500.0, from about 1 in 500.0 to about 1 in 600.0, from about 1 in 600.0 to about 1 in 700.0, from about 1 in 700.0 to about 1 in 800.0, from about 1 in 800.0 to about 900 1 in 0.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 1 in 8000.0, about 1 in 8000.0 to about 1 in 9000.0, about 1 in 9000.0 to about 1 140. The method of any one of claims 137-139, wherein the reference IL-12 has an ability to stimulate IFNγ production that is reduced to about 1 in 0,000.0, about 1 in 10,000.0 to about 1 in 50,000.0, about 1 in 50,000.0 to about 100,000.0, about 1 in 100,000.0 to about 1 in 200,000.0, about 1 in 200,000.0 to about 1 in 300,000.0, about 1 in 300,000.0, or less, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

148. 148. The method of claim 147, wherein the assay comprises one or more of: (i) an intracellular cytokine staining assay, (ii) a Luminex bead-based cytokine release assay, (iii) an ELISA, or (iv) an ELISpot assay.

149. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises seven or more amino acid substitutions consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

150. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and R129A.

151. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and K168A.

152. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and K170A.

153. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, P127A, and R129A.

154. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, P127A, and K168A.

155. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, P127A, and K170A.

156. 107. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12 p35 subunit comprises a non-alanine substitution mutation at any of amino acid residues Y40, D126, P127, R129, K168, or K170.

157. 157. The non-naturally occurring IL-12 variant of claim 156, wherein the substitution mutation at amino acid residue Y40 is selected from the group consisting of Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

158. 157. The non-naturally occurring IL-12 variant of claim 156, wherein the substitution mutation at amino acid residue D126 is selected from the group consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

159. 157. The non-naturally occurring IL-12 variant of claim 156, wherein the substitution mutation at amino acid residue P127 is selected from the group consisting of P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

160. 157. The non-naturally occurring IL-12 variant of claim 156, wherein the substitution mutation at amino acid residue R129 is selected from the group consisting of R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

161. 157. The non-naturally occurring IL-12 variant of claim 156, wherein the substitution mutation at amino acid residue K168 is selected from the group consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

162. 157. The non-naturally occurring IL-12 variant of claim 156, wherein the substitution mutation at amino acid residue K170 is selected from the group consisting of K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

163. 157. The non-naturally occurring IL-12 variant of claim 156, wherein the variant IL-12 p35 subunit comprises any of SEQ ID NOs: 177-198.

164. a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first Fc domain; b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the second Fc domain; and A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

165. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises one or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

166. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises two or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

167. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises three or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

168. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises four or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

169. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises five or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

170. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises six or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

171. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and D126A.

172. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and P127A.

173. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and T43A.

174. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, D126A, and P127A.

175. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, T43A, D126A, and P127A.

176. 176. The heterodimeric Fc-fusion protein of any one of claims 164 to 175, wherein said IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

177. 177. The heterodimeric Fc-fusion protein of claim 176, wherein said variant IL-12 p40 subunit domain comprises one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

178. 178. The heterodimeric Fc-fusion protein of any one of claims 164 to 177, wherein said first Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and said second Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

179. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and 103-166; ii) the first Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:

13. The heterodimeric Fc-fusion protein of claim 164.

180. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

181. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 96% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

182. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 97% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

183. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 98% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

184. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 99% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

185. i) the variant IL-12 p35 subunit domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and 103-166; ii) the first Fc domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; iii) the IL-12 p40 subunit domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90; iv) the second Fc domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:

13. The heterodimeric Fc-fusion protein of claim 164.

186. i) the variant IL-12 p35 subunit domain comprises SEQ ID NO:87 and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A; ii) the first Fc domain comprises amino acids having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

187. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises seven or more amino acid substitutions consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

188. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and R129A.

189. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K168A.

190. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K170A.

191. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A, and R129A.

192. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A, and K168A.

193. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A, and K170A.

194. 165. The heterodimeric Fc-fusion protein of claim 164, wherein said variant IL-12 p35 subunit domain comprises a non-alanine substitution mutation at any of amino acid residues Y40, D126, P127, R129, K168, or K170.

195. 195. The heterodimeric Fc-fusion protein of claim 194, wherein the substitution mutation at amino acid residue Y40 is selected from the group consisting of Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

196. 195. The heterodimeric Fc-fusion protein of claim 194, wherein the substitution mutation at amino acid residue D126 is selected from the group consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

197. 195. The heterodimeric Fc-fusion protein of claim 194, wherein the substitution mutation at amino acid residue P127 is selected from the group consisting of P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

198. 195. The heterodimeric Fc-fusion protein of claim 194, wherein the substitution mutation at amino acid residue R129 is selected from the group consisting of R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

199. 195. The heterodimeric Fc-fusion protein of claim 194, wherein the substitution mutation at amino acid residue K168 is selected from the group consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

200. The heterodimeric Fc fusion protein of claim 194, wherein the substitution mutation at amino acid residue K170 is selected from the group consisting of K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

201. 195. The heterodimeric Fc-fusion protein of claim 194, wherein said variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 177-198.

202. 202. The heterodimeric Fc-fusion protein of any one of claims 164 to 201, wherein said variant IL-12 p35 subunit domain comprises an additional amino acid substitution.

203. a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the N-terminus of the variant IL-12 p35 subunit domain is covalently linked to the C-terminus of the first Fc domain; b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the N-terminus of the IL-12 p40 subunit domain is covalently linked to the C-terminus of the second Fc domain; and A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

204. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises one or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

205. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises two or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

206. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises three or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

207. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises four or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

208. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises five or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

209. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises six or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

210. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and D126A.

211. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and P127A.

212. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and T43A.

213. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, D126A, and P127A.

214. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, T43A, D126A, and P127A.

215. 215. The heterodimeric Fc-fusion protein of any one of claims 203 to 214, wherein said IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

216. 216. The heterodimeric Fc-fusion protein of claim 215, wherein said variant IL-12 p40 subunit domain comprises one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

217. 217. The heterodimeric Fc-fusion protein of any one of claims 203 to 216, wherein said first Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and said second Fc domain comprises amino acids selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

218. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and 103-166; ii) the first Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90; iv) The heterodimeric Fc-fusion protein of claim 203, wherein the second Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:

13.

219. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

220. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 96% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

221. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 97% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

222. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 98% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

223. i) the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 99% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOs:24-87, or SEQ ID NOs:103-166; ii) the first Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

224. i) the variant IL-12 p35 subunit domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and 103-166; ii) the first Fc domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; iii) the IL-12 p40 subunit domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90; iv) The heterodimeric Fc-fusion protein of claim 203, wherein the second Fc domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:

13.

225. i) the variant IL-12 p35 subunit domain comprises SEQ ID NO:87 and further comprises one, two, three, four, five, six, or all seven amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A; ii) the first Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; iii) the IL-12 p40 subunit domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90; iv) the second Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

13.

226. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises seven or more amino acid substitutions consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

227. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and R129A.

228. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K168A.

229. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A and K170A.

230. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A, and R129A.

231. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A, and K168A.

232. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises the amino acid substitutions Y40A, P127A, and K170A.

233. 204. The heterodimeric Fc-fusion protein of claim 203, wherein said variant IL-12 p35 subunit domain comprises a non-alanine substitution mutation at any of amino acid residues Y40, D126, P127, R129, K168, or K170.

234. 234. The heterodimeric Fc-fusion protein of claim 233, wherein the substitution mutation at amino acid residue Y40 is selected from the group consisting of Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T.

235. 234. The heterodimeric Fc-fusion protein of claim 233, wherein the substitution mutation at amino acid residue D126 is selected from the group consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

236. 234. The heterodimeric Fc-fusion protein of claim 233, wherein the substitution mutation at amino acid residue P127 is selected from the group consisting of P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

237. 234. The heterodimeric Fc-fusion protein of claim 233, wherein the substitution mutation at amino acid residue R129 is selected from the group consisting of R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, and R129Y.

238. 234. The heterodimeric Fc-fusion protein of claim 233, wherein the substitution mutation at amino acid residue K168 is selected from the group consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

239. 234. The heterodimeric Fc-fusion protein of claim 233, wherein the substitution mutation at amino acid residue K170 is selected from the group consisting of K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

240. 234. The heterodimeric Fc-fusion protein of claim 233, wherein said variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 177-198.

241. 241. The heterodimeric Fc-fusion protein of any one of claims 203-240, wherein said variant IL-12 p35 subunit domain comprises an additional amino acid substitution.

242. 242. A composition comprising the heterodimeric Fc-fusion protein of any one of claims 164 to 241 for use in treating cancer in a subject.

243. 242. One or more nucleic acids encoding the heterodimeric Fc-fusion protein of any one of claims 164-241.

244. 244. A host cell comprising one or more nucleic acids of claim 243.

245. 1. A method for producing a heterodimeric Fc-fusion protein, comprising: culturing a host cell harboring one or more nucleic acids or vectors under conditions such that said heterodimeric Fc-fusion protein is produced, wherein said nucleic acid or vector comprises one or more nucleic acids of claim 243; The method, wherein the produced heterodimeric Fc-fusion protein has an increased half-life compared to the half-life of a reference IL-12, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

246. 246. The method of claim 245, further comprising isolating and / or purifying the produced heterodimeric Fc-fusion protein.

247. 247. The method of any one of claims 245 or 246, wherein the produced heterodimeric Fc-fusion protein has an altered binding affinity for interleukin-12 receptor beta 2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

248. 248. The method of claim 247, wherein the produced heterodimeric Fc-fusion protein has a binding affinity for IL-12Rβ2 that 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 the binding affinity of a reference IL-12 as measured by an assay.

249. 249. The method of claim 248, wherein the assay comprises an SPR assay.

250. 248. The method of claim 247, wherein the produced heterodimeric Fc-fusion protein has a binding affinity for IL-12Rβ2, as measured by the assay, that is below the lower limit of detection of the assay, and the binding affinity of a reference IL-12 is detectable.

251. 251. The method of claim 250, wherein the assay comprises an SPR assay.

252. As measured by the assay, the produced heterodimeric Fc-fusion protein has a potency of about 0.5 to about 50.0 fold, about 0.5 to about 5.0 fold, about 5.0 to about 10.0 fold, about 10.0 to about 15.0 fold, about 15.0 to about 20.0 fold, about 20.0 to about 25.0 fold, about 25.0 to about 30.0 fold, about 30.0 to about 35.0 fold, or about 35.0 to about 40.0 fold lower than that of a reference IL-12. , about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 900 1 in 0.0, about 1 in 900.0 to about 1 in 1000.0, about 1 in 1000.0 to about 1 in 2000.0, about 1 in 2000.0 to about 1 in 3000.0, about 1 in 3000.0 to about 1 in 4000.0, about 1 in 4000.0 to about 1 in 5000.0, about 1 in 5000.0 to about 1 in 6000.0, about 1 in 6000.0 to about 1 in 7000.0, about 1 in 7000.0 to about 1 in 8000.0, about 1 in 8000.0 to about 1 in 9000.0, about 9000.0 247. The method of any one of claims 245 or 246, wherein the reference IL-12 has a potency reduced by a factor of 1 to about 10,000.0, about 10,000.0 to about 50,000.0, about 50,000.0 to about 100,000.0, about 100,000.0 to about 200,000.0, about 200,000.0 to about 300,000.0, about 300,000.0, or less, wherein the reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

253. 253. The method of claim 252, wherein the assay comprises an IL-12 HEK reporter assay.

254. As measured by an assay, the produced heterodimeric Fc-fusion protein has an ability to stimulate IFNγ production that is at least about 0.5 to about 50.0 fold lower, about 0.5 to about 5.0 fold lower, about 5.0 to about 10.0 fold lower, about 10.0 to about 15.0 fold lower, about 15.0 to about 20.0 fold lower, about 20.0 to about 25.0 fold lower, about 25.0 to about 30.0 fold lower, about 30.0 to about 35.0 fold lower, or about 35.0 fold lower than that of a reference IL-12. to about 1 in 40.0, about 1 in 40.0 to about 1 in 45.0, about 1 in 45.0 to about 1 in 50.0, about 1 in 50.0 to about 1 in 100.0, about 1 in 100.0 to about 1 in 200.0, about 1 in 200.0 to about 1 in 300.0, about 1 in 300.0 to about 1 in 400.0, about 1 in 400.0 to about 1 in 500.0, about 1 in 500.0 to about 1 in 600.0, about 1 in 600.0 to about 1 in 700.0, about 1 in 700.0 to about 1 in 800.0, about 1 in 800.0 to about 9 1 in 00.0, from about 1 in 900.0 to about 1 in 1000.0, from about 1 in 1000.0 to about 1 in 2000.0, from about 1 in 2000.0 to about 1 in 3000.0, from about 1 in 3000.0 to about 1 in 4000.0, from about 1 in 4000.0 to about 1 in 5000.0, from about 1 in 5000.0 to about 1 in 6000.0, from about 1 in 6000.0 to about 1 in 7000.0, from about 1 in 7000.0 to about 1 in 8000.0, from about 1 in 8000.0 to about 1 in 9000.0, from about 1 in 9000.0 to about 247. The method of any one of claims 245 or 246, having an ability to stimulate IFNγ production reduced by a factor of 10,000.0, about 10,000.0 to about 150,000.0, about 150,000.0 to about 100,000.0, about 100,000.0 to about 1200,000.0, about 1200,000.0 to about 1300,000.0, about 1300,000.0, or more, wherein said reference IL-12 comprises one or more of wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc-fusion protein.

255. 255. The method of claim 254, wherein the assay comprises one or more of: (i) an intracellular cytokine staining assay, (ii) a Luminex bead-based cytokine release assay, (iii) an ELISA, or (iv) an ELISpot assay.

256. a) a variant IL-12 p35 subunit, said variant IL-12 p35 subunit comprising a first amino acid substitution mutation, said first amino acid substitution mutation being selected from the group consisting of Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40S, and Y40T; b) the IL-12 p40 subunit; and A non-naturally occurring IL-12 variant comprising:

257. The variant IL-12 p35 subunit further comprises a second substitution mutation, wherein the second substitution mutation is selected from the group consisting of D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D126W, P1 27A, P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129 M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R129Y, K168A, K168C, K168D, K1 68E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, 257. The non-naturally occurring IL-12 variant of claim 256, selected from the group consisting of K168W, K168Y, K170A, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

258. 257. The non-naturally occurring IL-12 variant of claim 256, wherein said variant IL-12 p35 subunit comprises any of SEQ ID NOs: 199-247 or 279-290.

259. a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the C-terminus of the variant IL-12 p35 subunit domain is covalently linked to the N-terminus of the first Fc domain, and wherein the variant IL-12 p35 subunit domain comprises a first amino acid substitution mutation, wherein the first amino acid substitution is selected from the group consisting of Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40S, and Y40T; b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the C-terminus of the IL-12 p40 subunit domain is covalently linked to the N-terminus of the second Fc domain; and A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

260. the variant IL-12 p35 subunit domain further comprises a second substitution mutation, the second substitution mutation being D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D126W, P127A, P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127 R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R12 9M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R129Y, K168A, K168C, K168D, K 168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, 260. The heterodimeric Fc-fusion protein of claim 259, selected from the group consisting of K168W, K168Y, K170A, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

261. 260. The heterodimeric Fc-fusion protein of claim 259, wherein said variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 199-247 or 279-290.

262. a) a first fusion construct comprising a variant IL-12 p35 subunit domain and a first Fc domain, wherein the N-terminus of the variant IL-12 p35 subunit domain is covalently linked to the C-terminus of the first Fc domain, and wherein the variant IL-12 p35 subunit domain comprises a first amino acid substitution mutation, wherein the first amino acid substitution mutation is selected from the group consisting of Y40A, Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40S, and Y40T; b) a second fusion construct comprising an IL-12 p40 subunit domain and a second Fc domain, wherein the N-terminus of the IL-12 p40 subunit domain is covalently linked to the C-terminus of the second Fc domain; and A heterodimeric Fc fusion protein comprising: Optionally, the first Fc domain and the second Fc domain comprise a modification that (i) promotes heterodimerization of the first and second Fc domains, and / or (ii) reduces or inhibits an effector function.

263. the variant IL-12 p35 subunit domain further comprises a second substitution mutation, the second substitution mutation being D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D126W, P127A, P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127 R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R12 9M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R129Y, K168A, K168C, K168D, K 168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, 263. The heterodimeric Fc-fusion protein of claim 262, selected from the group consisting of K168W, K168Y, K170A, K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

264. 263. The heterodimeric Fc-fusion protein of claim 262, wherein said variant IL-12 p35 subunit domain comprises any of SEQ ID NOs: 199-247 or 279-290.

265. 259. The non-naturally occurring IL-12 variant of any one of claims 106-119, 149-163, or 256-258, wherein the variant IL-12 p35 subunit further comprises a C74S substitution mutation.

266. 266. The non-naturally occurring IL-12 variant of claim 265, wherein said IL-12 p40 subunit comprises a variant IL-12 p40 subunit, said variant IL-12 p40 subunit comprising one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

267. 265. The heterodimeric Fc-fusion protein of any one of claims 164-241 or 259-264, wherein said variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

268. 268. The heterodimeric Fc-fusion protein of claim 267, wherein said IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain, said variant IL-12 p40 subunit domain comprising one or more amino acid substitutions selected from the group consisting of C177S, C252S, and C177S / C252S.

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