IL-12 Fc fusion proteins and uses thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 476,687, filed on December 22, 2022, and U.S. Provisional Application No. 63 / 368,740, filed on July 18, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on July 17, 2023, is named 54100_4001.xml, and is 336,708 bytes in size. Background Art

[0005] The present application generally relates to compositions and methods for modulating interleukin 12 (IL-12)-mediated signal transduction. Specifically, the present disclosure provides novel, non-naturally occurring IL-12 variant polypeptides and fusion proteins, wherein the IL-12 p35 subunit comprises novel amino acid substitutions that reduce binding affinity to interleukin 12 receptor β2 (IL-12Rβ2), and methods of making and using non-naturally occurring IL-12 variant polypeptides and fusion proteins.

[0006] IL-12 is a potent pro-inflammatory cytokine that is produced by antigen presenting cells, such as, for example, dendritic cells, macrophages, and neutrophils. IL-12 belongs to the IL-12 cytokine family. The IL-12 cytokine family is unique in that they contain heterodimeric cytokines. IL-12 consists of an alpha-(α-) subunit (encoded by the IL12A gene; also referred to herein as "IL-12 p35 subunit"; precursor sequence - SEQ ID NO: 1 (e.g. Figure 1 Mature sequence-SEQ ID NO:2 (as shown in Figure 1 )) and beta-(β-) subunit (encoded by IL12B gene; also referred to herein as "IL-12 p40 subunit"); precursor sequence - SEQ ID NO: 3 (as shown in Figure 1 Mature sequence-SEQ ID NO:4 (as shown); Figure 1 ) which assemble to form a 70,000 Dalton (70 kDa) disulfide-linked heterodimer. After the IL-12 p35 and p40 subunits assemble, a biologically active IL-12 heterodimer is formed. The IL-12 receptor IL-12R is a type I cytokine receptor that contains a β-1 subunit (IL-12Rβ1-SEQ ID NO: 5 (as shown) Figure 2Extracellular domain-SEQ ID NO:6 (as shown); Figure 2 As shown)) and β-2 subunit (IL-12Rβ2-SEQ ID NO:7 (as Figure 2 Extracellular domain-SEQ ID NO:8 (as shown); Figure 2 The IL-12 p40 subunit has a binding affinity for IL-12Rβ1, while the IL-12 p35 subunit has a binding affinity for IL-12Rβ2.

[0007] Binding of IL-12 to IL-12R leads to phosphorylation of intracellular signal transducer and activator of transcription 4 (STAT4) and triggers a signaling pathway that: (i) induces TH1 cell differentiation, (ii) increases the activation and cytotoxic capacity of T cells and natural killer (NK) cells, (iii) inhibits or reprograms immunosuppressive cells, such as, for example, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), and (iv) induces the production of large amounts of interferon gamma (IFNγ), which has cytostatic / cytotoxic, anti-angiogenic properties, and can upregulate major histocompatibility complex (MHC) class 1 and MHC class 2 molecules on tumor cells for immune recognition.

[0008] Therefore, IL-12 has been shown to have potent antitumor activity against a range of malignant tumors in preclinical studies. However, due to the overactivation of circulating immune cells, systemic administration of wild-type IL-12 in humans may lead to severe toxicity, including death in experiments. In addition, due to target-mediated drug disposal, activated immune cells undergo cell proliferation, resulting in a shorter serum half-life of the administered IL-12. It is reported that the biologically active form of human IL-12 (i.e., a heterodimer complex comprising IL-12 p35 subunit and IL-12 p40 subunit) has a half-life as low as 5 hours in vivo when administered as a therapeutic compound. In recent years, cytokine engineering has become a promising strategy that can customize cytokines with desired activity and reduced toxicity. Therefore, additional methods are needed to improve the properties of IL-12 for use in therapeutic agents. In particular, there is an unmet need for novel IL-12 variant polypeptides and / or fusion proteins that (i) bind to IL-12R with improved binding efficiency or affinity such that the therapeutic properties of IL-12 (e.g., recognition and elimination of target cells, such as, for example, cancer cells) are maintained and the negative side effects of IL-12 are reduced or eliminated and (ii) have increased serum half-life. Summary of the invention

[0009] In one aspect, the present 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 consisting of: Y40A, T43A, D126A, P127A, R129A, K168A and K170A; and b) an IL-12 p40 subunit.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and D126A.

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

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

[0019] 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.

[0020] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A, T43A, D126A and P127A.

[0021] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and R129A.

[0022] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and K168A.

[0023] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises the amino acid substitutions Y40A and K170A.

[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 R129A.

[0025] 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.

[0026] 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.

[0027] In further embodiments and according to 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 consisting of: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S and Y40T.

[0028] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises any one of SEQ ID NOs: 177-187.

[0029] In further embodiments and according to 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 consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V and D126W.

[0030] In further embodiments and according to any of the above, the variant IL-12 p35 subunit comprises a substitution mutation at amino acid residue P 127. In some further embodiments, 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.

[0031] In further embodiments and according to 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.

[0032] In further embodiments and according to 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 consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W and K168Y.

[0033] In further embodiments and according to 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.

[0034] In a further embodiment and according to 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.

[0035] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit further comprises a second substitution mutation.

[0036] In a further embodiment and in accordance with the above, the second substitution mutation is selected from the group comprising 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, R129N, 29H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129 V, R129W, R129Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K K170I, K170M, K170P, K170S, K170T, K170V, and K170W.

[0037] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit comprises any one of SEQ ID NOs: 199-247 or SEQ ID NOs: 279-290.

[0038] 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.

[0039] In a further embodiment and according to any of the above, the IL-12 p40 subunit comprises a variant IL-12 p40 subunit.

[0040] 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.

[0041] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and SEQ ID NOs: 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.

[0042] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87 or SEQ ID NO: 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.

[0043] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87 or SEQ ID NO: 103-166, and the IL-12 p40 subunit 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.

[0044] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87 or SEQ ID NO: 103-166, and the IL-12 p40 subunit 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.

[0045] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87 or SEQ ID NO: 103-166, and the IL-12 p40 subunit 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.

[0046] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87 or SEQ ID NO: 103-166, and the IL-12 p40 subunit 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.

[0047] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and SEQ ID NOs: 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.

[0048] In a further embodiment and according to 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.

[0049] In a further embodiment and according to any of the above, the one or more amino acid substitutions of 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 the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0050] In a further embodiment and according to 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, wherein the Fc domain comprises one or more amino acid sequences 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, (ii) albumin, (iii) one or more unstructured biodegradable polypeptides ("XTEN"), or (iv) polyethylene glycol (PEG).

[0051] In a further embodiment and according to any of the above, wherein the C-terminus of the variant IL-12 p35 subunit is covalently attached to the N-terminus of the IL-12 p40 subunit.

[0052] In a further embodiment and 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, and wherein the C-terminus of the variant IL-12 p35 subunit is covalently attached to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently attached to the N-terminus of the IL-12 p40 subunit.

[0053] In a further embodiment, according to any of the above, the C-terminus of the IL-12 p40 subunit is covalently attached to the N-terminus of the variant IL-12 p35 subunit.

[0054] In a further embodiment and 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, and wherein the C-terminus of the IL-12 p40 subunit is covalently attached to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently attached to the N-terminus of the variant IL-12 p35 subunit.

[0055] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit comprises additional amino acid substitutions.

[0056] In a further embodiment and in accordance with any of the above, a non-naturally occurring IL-12 variant is provided for use in treating cancer in a subject.

[0057] 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.

[0058] 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.

[0059] In another aspect, the present disclosure provides a method of producing a non-naturally occurring IL-12 variant, the method comprising: culturing a host cell with one or more nucleic acids or vectors under conditions that produce the non-naturally occurring IL-12 variant, wherein the one or more nucleic acids or vectors comprise one or more nucleic acids described in the above aspects and embodiments.

[0060] In a further embodiment and in accordance with the above, the method further comprises isolating and / or purifying the produced non-naturally occurring IL-12 variant.

[0061] In a further embodiment and according to 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, wherein the Fc domain comprises one or more amino acid sequences 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, (ii) albumin, (iii) one or more unstructured biodegradable polypeptides ("XTEN"), or (iv) polyethylene glycol (PEG).

[0062] In a further embodiment and according to any of the above, the non-naturally occurring IL-12 variant is produced with an altered binding affinity for 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 the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0063] In further embodiments and in accordance with the above, the non-naturally occurring IL-12 variants produced 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 the reference IL-12 as determined by an assay.

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

[0065] In a further embodiment and according to any of the above, the non-naturally occurring IL-12 variant produced has a binding affinity for IL-12Rβ2 that is below the minimum detectable level of the assay, and the binding affinity of the reference IL-12 is detectable as determined by the assay.

[0066] In a further embodiment and in accordance with the above, the assay comprises an SPR assay.

[0067] In further embodiments and according to any of the above, the potency of the non-naturally occurring IL-12 variant produced is reduced by 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 45.0-fold to about 46.0-fold, about 47.0-fold to about 48.0-fold, about 49.0-fold to about 50.0-fold, about 50.0-fold to about 51.0-fold, about 51.0-fold to about 52.0-fold, about 52.0-fold to about 53.0-fold, about 53.0-fold to about 54.0-fold 0.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 900. 0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by an assay.

[0068] In a further embodiment and in accordance with the above, the assay comprises an IL-12 HEK reporter gene assay.

[0069] In further embodiments and in accordance with any of the above, the ability of the non-naturally occurring IL-12 variant produced to stimulate IFNγ production is reduced by 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, or about 35.0-fold to about 36.0-fold compared to the ability of the reference IL-12 to stimulate IFNγ production. fold, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times .0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times .0-fold to about 10,000.0-fold, about 10,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by the assay.

[0070] In further embodiments and 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.

[0071] In another aspect, 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 C-terminus of the variant IL-12 p35 subunit domain is covalently attached 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 attached to the N-terminus of the second Fc domain; optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[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 D126A.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[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 R129A.

[0085] 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.

[0086] 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.

[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 R129A.

[0088] 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.

[0089] 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.

[0090] In further embodiments and according to 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 consisting of: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S and Y40T.

[0091] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 177-187.

[0092] In further embodiments and according to 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 consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V and D126W.

[0093] In further embodiments and according to 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 consisting of P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R and P127S.

[0094] In further embodiments and according to 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.

[0095] In further embodiments and according to 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 consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W and K168Y.

[0096] In further embodiments and according to 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 consisting of K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V and K170W.

[0097] In a further embodiment and according to 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.

[0098] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a second substitution mutation.

[0099] In a further embodiment and in accordance with the above, the second substitution mutation is selected from the group comprising 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, R129N, 29H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129 V, R129W, R129Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K K170I, K170M, K170P, K170S, K170T, K170V, and K170W.

[0100] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 199-247 or SEQ ID NOs: 279-290.

[0101] 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.

[0102] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

[0103] 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.

[0104] In a further embodiment and according to any of the above, the first Fc domain comprises an amino acid 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 an amino acid 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.

[0105] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and SEQ ID NOs: 103-166; 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; 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; and 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.

[0106] In a further embodiment and 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 one 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.

[0107] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 96% sequence identity to any one 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% 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% 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% 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 and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 97% sequence identity to any one 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% 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% 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% 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 and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 98% sequence identity to any one 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% 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% 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% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

[0110] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 99% sequence identity to any one 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% 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 the above, 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 SEQ ID NOs: 103-166; 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; 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; and 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.

[0112] In a further embodiment and according to 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: NO:13 has an amino acid sequence with at least 99% sequence identity.

[0113] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises additional amino acid substitutions.

[0114] In another aspect, 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 N-terminus of the variant IL-12 p35 subunit domain is covalently attached 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 attached to the C-terminus of the second Fc domain; optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[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 D126A.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[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 R129A.

[0128] 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.

[0129] 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.

[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 R129A.

[0131] 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.

[0132] 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.

[0133] In further embodiments and according to 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 consisting of: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S and Y40T.

[0134] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 177-187.

[0135] In further embodiments and according to 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 consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V and D126W.

[0136] In further embodiments and according to 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 consisting of P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R and P127S.

[0137] In further embodiments and according to 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.

[0138] In further embodiments and according to 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 consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W and K168Y.

[0139] In further embodiments and according to 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 consisting of K170C, K170D, K170E, K170G, K170I, K170M, K170P, K170S, K170T, K170V and K170W.

[0140] In a further embodiment and according to 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.

[0141] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a second substitution mutation.

[0142] In a further embodiment and in accordance with the above, the second substitution mutation is selected from the group comprising 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, R12 9Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K1 70S, K170T, K170V, K170F, K170L, K170N, and K170W.

[0143] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 199-247 or SEQ ID NOs: 279-290.

[0144] 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.

[0145] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

[0146] 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.

[0147] In a further embodiment and according to any of the above, the first Fc domain comprises an amino acid 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 an amino acid 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.

[0148] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and SEQ ID NOs: 103-166; 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; 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; and 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.

[0149] In a further embodiment and 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 one 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.

[0150] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 96% sequence identity to any one 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% 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% 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% 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 and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 97% sequence identity to any one 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% 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% 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% 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 and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 98% sequence identity to any one 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% 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% 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% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

[0153] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain comprises an amino acid sequence having at least 99% sequence identity to any one 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% 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 the above, 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 SEQ ID NOs: 103-166; 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; 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; and 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.

[0155] In a further embodiment and according to 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: NO:13 has an amino acid sequence with at least 99% sequence identity.

[0156] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises additional amino acid substitutions.

[0157] In a further embodiment and in accordance with any of the above, a heterodimeric Fc fusion protein is provided for use in treating cancer in a subject.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] In another aspect, the present disclosure provides a method for producing a heterodimeric Fc fusion protein, the method comprising: culturing a host cell with one or more nucleic acids or vectors under conditions where the heterodimeric Fc fusion protein is produced, wherein the one or more nucleic acids or vectors comprise one or more nucleic acids as 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 the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0162] In a further embodiment and in accordance with the above, the method further comprises isolating and / or purifying the produced heterodimeric Fc fusion protein.

[0163] In a further embodiment and according to any of the above, the resulting heterodimeric Fc fusion protein has an altered binding affinity for interleukin 12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0164] In further embodiments and in accordance with the above, the binding affinity of the resulting heterodimeric Fc fusion protein to IL-12Rβ2 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 determined by an assay.

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

[0166] In a further embodiment and according to any of the above, the binding affinity of the generated heterodimeric Fc fusion protein to IL-12Rβ2 is below the minimum detectable level of the assay, and the binding affinity of the reference IL-12 is detectable as determined by the assay.

[0167] In a further embodiment and in accordance with the above, the assay comprises an SPR assay.

[0168] In further embodiments and according to any of the above, the potency of the resulting heterodimeric Fc fusion protein is reduced by 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 45.0-fold to about 50.0-fold, about 40.0-fold to about 55.0-fold, about 45.0-fold to about 50.0-fold, about 45 ... 0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times , about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0-fold, about 10,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by the assay.

[0169] In a further embodiment and in accordance with the above, the assay comprises an IL-12 HEK reporter gene assay.

[0170] In further embodiments and according to any of the above, the ability of the resulting heterodimeric Fc fusion protein to stimulate IFNγ production is reduced by 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 as compared to the ability of a reference IL-12 to stimulate IFNγ production. times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 0-fold to about 10,000.0-fold, about 10,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by an assay.

[0171] In further embodiments and 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.

[0172] In another aspect, the present disclosure provides a heterodimeric Fc fusion protein comprising: a) a first fusion construct comprising: (i) a variant IL-12 p35 subunit domain, wherein the variant IL-12 p35 subunit domain is 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, wherein the first Fc domain is selected from the group consisting of SEQ ID NOs: 12 and 13, and (iii) a linker, wherein the linker comprises SEQ ID NO: 15, the C-terminus of the variant IL-12 p35 subunit domain is covalently attached to the N-terminus of the linker, and the C-terminus of the linker is covalently attached to the N-terminus of the first Fc domain; and b) a second fusion construct comprising: (i) a (variant) IL-12 p40 subunit domain, wherein the (variant) IL-12 p40 subunit is selected from the group consisting of SEQ ID NOs: NO:4, 89 and 90, (ii) a second Fc domain, wherein the second Fc domain is selected from the group consisting of SEQ ID NO:13 and 12 and (iii) a linker, wherein the linker comprises SEQ ID NO:15, the C-terminus of the (variant) IL-12 p40 subunit domain is covalently attached to the N-terminus of the linker, and the C-terminus of the linker is covalently attached to the N-terminus of the second Fc domain.

[0173] In a further embodiment and 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 removed.

[0174] In one aspect, the present disclosure provides a non-naturally occurring IL-12 variant comprising: a) a variant IL-12p35 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 consisting of: Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, K170T; and b) an IL-12p40 subunit.

[0175] In a further embodiment and 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.

[0176] In a further embodiment and 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.

[0177] In a further embodiment and 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.

[0178] In a further embodiment and 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.

[0179] In a further embodiment and 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.

[0180] In a further embodiment and 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.

[0181] In a further embodiment and 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.

[0182] In a further embodiment and 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.

[0183] In further embodiments and in accordance with the above, the variant IL-12 p35 subunit comprises any one 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.

[0184] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0185] In a further embodiment and according to 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.

[0186] In a further embodiment and according to any of the above, the IL-12 p40 subunit comprises any one of SEQ ID NO: 4, 88, 89 and 90.

[0187] In a further embodiment and according to 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, wherein the Fc domain comprises one or more amino acid sequences 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, (ii) albumin, (iii) one or more unstructured biodegradable polypeptides ("XTEN"), or (iv) polyethylene glycol (PEG).

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

[0189] In a further embodiment and 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, and wherein the C-terminus of the variant IL-12 p35 subunit is covalently attached to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently attached to the N-terminus of the IL-12 p40 subunit.

[0190] In a further embodiment and according to any of the above, the C-terminus of the IL-12 p40 subunit is covalently attached to the N-terminus of the variant IL-12 p35 subunit.

[0191] In a further embodiment and 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, and wherein the C-terminus of the IL-12 p40 subunit is covalently attached to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently attached to the N-terminus of the variant IL-12 p35 subunit.

[0192] In a further embodiment and according to any of the above, the variant 11-12 p35 subunit comprises one or more additional amino acid substitutions.

[0193] In a further embodiment and in accordance with any of the above, a non-naturally occurring IL-12 variant is provided for use in treating cancer in a subject.

[0194] 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.

[0195] 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.

[0196] In another aspect, the disclosure provides a method of producing a non-naturally occurring IL-12 variant, the method comprising: culturing a host cell with one or more nucleic acids or vectors under conditions that produce the non-naturally occurring variant, wherein: i) the one or more nucleic acids or vectors comprise one or more nucleic acids as described in the aspects and embodiments above, and ii) at least one substitution mutation of the variant IL-12 p35 subunit improves the half-life compared to the half-life of a reference IL-12.

[0197] In a further embodiment and in accordance with the above, the method further comprises isolating and / or purifying the produced non-naturally occurring IL-12 variant.

[0198] In a further embodiment and according to 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, wherein the Fc domain comprises one or more amino acid sequences 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, (ii) albumin, (iii) one or more unstructured biodegradable polypeptides ("XTEN"), or (iv) polyethylene glycol (PEG).

[0199] In a further embodiment and according to any of the above, the non-naturally occurring IL-12 variant is produced with an altered binding affinity for interleukin 12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0200] In further embodiments and in accordance with the above, the non-naturally occurring IL-12 variants produced 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 the reference IL-12 as determined by an assay.

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

[0202] In a further embodiment and according to any of the above, the non-naturally occurring IL-12 variant produced has a binding affinity for IL-12Rβ2 that is below the minimum detectable level of the assay, and the binding affinity of the reference IL-12 is detectable as determined by the assay.

[0203] In a further embodiment and in accordance with the above, the assay comprises an SPR assay.

[0204] In further embodiments and according to any of the above, the potency of the non-naturally occurring IL-12 variant produced is reduced by 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, compared to the potency of the reference IL-12. 0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8 , about 10,000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more as determined by the assay.

[0205] In a further embodiment and in accordance with the above, the assay comprises an IL-12 HEK reporter gene assay.

[0206] In further embodiments and according to any of the above, the ability of the non-naturally occurring IL-12 variant produced to stimulate IFNγ production is reduced by 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 as compared to the ability of the reference IL-12 to stimulate IFNγ production. times, about 30.0 times to about 35.0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times. 0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 1000.0 times. 0-fold to about 8000.0-fold, about 8000.0-fold to about 9000.0-fold, about 9000.0-fold to about 10,000.0-fold, about 10,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more as determined by an assay.

[0207] In further embodiments and 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.

[0208] In a further embodiment and according to any of the above, 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.

[0209] In one aspect, 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: i) the C-terminus of the variant IL-12 p35 subunit domain is covalently attached 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 consisting of: 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: i) the C-terminus of the variant IL-12 p35 subunit domain is covalently attached 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 consisting of: Y40A, Y40E, Y40G, Y40P, Y40R, Y40S, K170A, K170P, K170T; The C-terminus of the p40 subunit domain is covalently attached to the N-terminus of the second Fc domain, optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

[0210] In a further embodiment and 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.

[0211] In a further embodiment and 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.

[0212] In a further embodiment and 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.

[0213] In a further embodiment and 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.

[0214] In a further embodiment and 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.

[0215] In a further embodiment and 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.

[0216] In a further embodiment and 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.

[0217] In a further embodiment and 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.

[0218] In further embodiments and according to any of the above, the variant IL-12 p35 subunit domain comprises any one 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.

[0219] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0220] In a further embodiment and according to 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.

[0221] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 4, 88, 89 and 90.

[0222] In a further embodiment and according to any of the above, i) the first Fc domain comprises an amino acid 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 an amino acid 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.

[0223] In a further embodiment and according to 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 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; and iii) the C-terminus of the variant IL-12 p35 subunit domain is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the first Fc domain.

[0224] In a further embodiment and according to 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 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; and iii) the C-terminus of the IL-12 p40 subunit domain is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the second Fc domain.

[0225] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises one or more additional amino acid substitutions.

[0226] In a further embodiment and in accordance with any of the above, a heterodimeric Fc fusion protein is provided for use in treating cancer in a subject.

[0227] 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.

[0228] 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.

[0229] In another aspect, the present disclosure provides a method for producing a heterodimeric Fc fusion protein, the method comprising: culturing a host cell with one or more nucleic acids or vectors under conditions that produce a heterodimeric Fc fusion protein, wherein: i) the one or more nucleic acids or vectors comprise one or more nucleic acids as described in the aspects and embodiments above, and ii) at least one substitution mutation in the variant IL-12p35 subunit domain improves the half-life compared to the half-life of a reference IL-12.

[0230] In a further embodiment and in accordance with the above, the method further comprises isolating and / or purifying the produced heterodimeric Fc fusion protein.

[0231] In a further embodiment and according to any of the above, the resulting heterodimeric Fc fusion protein has an altered binding affinity for interleukin 12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0232] In further embodiments and in accordance with the above, the binding affinity of the resulting heterodimeric Fc fusion protein to IL-12Rβ2 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 determined by an assay.

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

[0234] In a further embodiment and according to any of the above, the binding affinity of the generated heterodimeric Fc fusion protein to IL-12Rβ2 is below the minimum detectable level of the assay, and the binding affinity of the reference IL-12 is detectable as determined by the assay.

[0235] In a further embodiment and in accordance with the above, the assay comprises an SPR assay.

[0236] In further embodiments and according to any of the above, the potency of the resulting heterodimeric Fc fusion protein is reduced by 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 30.0-fold to about 35.0-fold, about 35.0-fold to about 36.0-fold, about 37.0-fold to about 38.0-fold, about 39.0-fold to about 40.0-fold, about 41.0-fold to about 42.0-fold, about 43.0-fold to about 44.0-fold, about 45.0-fold to about 46.0-fold, about 47.0-fold to about 48.0-fold, about 49.0-fold to about 50.0-fold, about 50.0-fold to about 51.0-fold, about 51.0-fold to about 52.0-fold, about 52.0-fold to about 53.0-fold, about 53.0-fold to about 54.0-fold about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times , about 100,000.0 times, about 150,000.0 times, about 200,000.0 times, about 300,000.0 times, about 400,000.0 times, or more, as determined by an assay.

[0237] In a further embodiment and in accordance with the above, the assay comprises an IL-12 HEK reporter gene assay.

[0238] In a further embodiment and according to any of the above, the ability of the resulting heterodimeric Fc fusion protein to stimulate IFNγ production is reduced by 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, or about 35.0-fold to about 40.0-fold, compared to the ability of a reference IL-12 to stimulate IFNγ production. about 30.0 times to about 35.0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times , about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about fold to about 8000.0 fold, about 8000.0 fold to about 9000.0 fold, about 9000.0 fold to about 10,000.0 fold, about 10,000.0 fold to about 50,000.0 fold, about 50,000.0 fold to about 100,000.0 fold, about 100,000.0 fold to about 200,000.0 fold, about 200,000.0 fold to about 300,000.0 fold, about 300,000.0 fold or more as determined by an assay.

[0239] In further embodiments and 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.

[0240] In a further embodiment and according to any of the above, 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.

[0241] In one aspect, 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: i) the C-terminus of the first Fc domain is covalently attached 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 consisting of: 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 attached to the N-terminus of the variant IL-12 p35 subunit domain, The N-terminus of the p40 subunit domain, optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

[0242] In a further embodiment and 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.

[0243] In a further embodiment and 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.

[0244] In a further embodiment and 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.

[0245] In a further embodiment and 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.

[0246] In a further embodiment and 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.

[0247] In a further embodiment and 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.

[0248] In a further embodiment and 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.

[0249] In a further embodiment and 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.

[0250] In further embodiments and according to any of the above, the variant IL-12 p35 subunit domain comprises any one 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.

[0251] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0252] In a further embodiment and according to 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.

[0253] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain comprises any one of SEQ ID NOs: 4, 88, 89 and 90.

[0254] In a further embodiment and according to any of the above, i) the first Fc domain comprises an amino acid 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 an amino acid 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.

[0255] In a further embodiment and according to 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 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; and iii) the C-terminus of the first Fc domain is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the variant IL-12 p35 subunit domain.

[0256] In a further embodiment and according to 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 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; and iii) the C-terminus of the second Fc domain is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the IL-12 p40 subunit domain.

[0257] In a further embodiment and according to any of the above, the variant IL-12 p35 subunit domain comprises one or more additional amino acid substitutions.

[0258] In a further embodiment and in accordance with any of the above, a heterodimeric Fc fusion protein is provided for use in treating cancer in a subject.

[0259] 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.

[0260] 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.

[0261] In another aspect, the present disclosure provides a method for producing a heterodimeric Fc fusion protein, the method comprising: culturing a host cell with one or more nucleic acids or vectors under conditions that produce a heterodimeric Fc fusion protein, wherein: i) the one or more nucleic acids or vectors comprise one or more nucleic acids as described in the aspects and embodiments above, and ii) at least one substitution mutation in the variant IL-12p35 subunit domain improves the half-life compared to the half-life of a reference IL-12.

[0262] In a further embodiment and in accordance with the above, the method further comprises isolating and / or purifying the produced heterodimeric Fc fusion protein.

[0263] In a further embodiment and according to any of the above, the resulting heterodimeric Fc fusion protein has an altered binding affinity for interleukin 12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

[0264] In further embodiments and in accordance with the above, the binding affinity of the resulting heterodimeric Fc fusion protein to IL-12Rβ2 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 determined by an assay.

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

[0266] In a further embodiment and according to any of the above, the binding affinity of the generated heterodimeric Fc fusion protein to IL-12Rβ2 is below the minimum detectable level of the assay, and the binding affinity of the reference IL-12 is detectable as determined by the assay.

[0267] In a further embodiment and in accordance with the above, the assay comprises an SPR assay.

[0268] In further embodiments and according to any of the above, the potency of the resulting heterodimeric Fc fusion protein is reduced by 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 30.0-fold to about 35.0-fold, about 35.0-fold to about 36.0-fold, about 37.0-fold to about 38.0-fold, about 39.0-fold to about 40.0-fold, about 41.0-fold to about 42.0-fold, about 43.0-fold to about 44.0-fold, about 45.0-fold to about 46.0-fold, about 47.0-fold to about 48.0-fold, about 49.0-fold to about 50.0-fold, about 50.0-fold to about 51.0-fold, about 51.0-fold to about 52.0-fold, about 52.0-fold to about 53.0-fold, about 53.0-fold to about 54.0-fold about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times , about 100,000.0 times, about 150,000.0 times, about 200,000.0 times, about 300,000.0 times, about 400,000.0 times, or more, as determined by an assay.

[0269] In a further embodiment and in accordance with the above, the assay comprises an IL-12 HEK reporter gene assay.

[0270] In a further embodiment and according to any of the above, the ability of the resulting heterodimeric Fc fusion protein to stimulate IFNγ production is reduced by 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, or about 35.0-fold to about 40.0-fold, compared to the ability of a reference IL-12 to stimulate IFNγ production. about 30.0 times to about 35.0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times , about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about fold to about 8000.0 fold, about 8000.0 fold to about 9000.0 fold, about 9000.0 fold to about 10,000.0 fold, about 10,000.0 fold to about 50,000.0 fold, about 50,000.0 fold to about 100,000.0 fold, about 100,000.0 fold to about 200,000.0 fold, about 200,000.0 fold to about 300,000.0 fold, about 300,000.0 fold or more as determined by an assay.

[0271] In further embodiments and 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.

[0272] In a further embodiment and according to any of the above, 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.

[0273] In another aspect, the present disclosure provides a heterodimeric Fc fusion protein comprising: a) a first fusion construct comprising: 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 attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached 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 comprises one or more amino acid substitutions 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 / K 170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / 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, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K17 0A / K168I, K170A / K168T, K170A / R129E, K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E;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 IL-12 p40 subunit domain is covalently attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached 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, and iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89, optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.;

[0274] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0275] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

[0276] In another aspect, the present disclosure provides a heterodimeric Fc fusion protein comprising: a) a first fusion construct comprising: 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 attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached 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 comprises one or more amino acid substitutions 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 / K 170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / 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, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K17 0A / K168I, K170A / K168T, K170A / R129E, K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E;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 IL-12 p40 subunit domain is covalently attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached 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, and iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89, optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.;

[0277] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0278] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

[0279] In another aspect, the present disclosure provides a heterodimeric Fc fusion protein comprising: a) a first fusion construct comprising: 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 attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached 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 comprises one or more amino acid substitutions 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 / K 170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / 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, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K17 0A / K168I, K170A / K168T, K170A / R129E, K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E;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 attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached 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, and iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89, optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.;

[0280] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0281] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation.

[0282] In another aspect, the present disclosure provides a heterodimeric Fc fusion protein comprising: a) a first fusion construct comprising: 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 attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached 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 comprises one or more amino acid substitutions 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 / K 170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129A, Y40G, Y40G / K170A, Y40G / K168A, Y40G / 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, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K17 0A / K168I, K170A / K168T, K170A / R129E, K170P, K170P / K168A, K170P / K168I, K170P / K168T, K170P / R129E, K170T, K170T / K168A, K170T / K168I, K170T / K168T, and K170T / R129E;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 attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached 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, and iv) the IL-12 p40 subunit domain comprises SEQ ID NO: 89, optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.;

[0283] In a further embodiment and in accordance with the above, the variant IL-12 p35 subunit domain further comprises a C74S substitution mutation.

[0284] In a further embodiment and according to any of the above, the IL-12 p40 subunit domain further comprises a C177S substitution mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0286] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the invention are utilized, and in which the accompanying drawings (also referred to herein as "Fig.", "FIG.", "Figure," "Figures," "Figs." and "FIGs."):

[0287] Figure 1 Shown is a sequence listing of human wild-type IL-12 subunits α and β (precursor and mature form sequences).

[0288] Figure 2 Sequence listings of human wild-type IL-12Rβ1 (+ extracellular domain sequence) and IL-12Rβ2 (+ extracellular domain sequence) are shown.

[0289] Figure 3 Exemplary sequences of human IgGl Fc domains (Glm allotype, and two sets of knob / hole mutants) are shown.

[0290] Figure 4 A list of exemplary domain linker sequences is shown.

[0291] Figure 5 Shown is a list of variant IL-12 p35 subunit sequences containing one mutation.

[0292] Fig. 6A and 6B Shown is a list of variant IL-12 p35 subunit sequences containing two mutations.

[0293] Fig. 7A and 7B Shown is a list of variant IL-12 p35 subunit sequences containing three mutations.

[0294] Fig. 8A and 8B Shown is a list of variant IL-12 p35 subunit sequences containing four mutations.

[0295] Fig. 9 Shown is a list of variant IL-12 p35 subunit sequences comprising: (i) five mutations, (ii) six mutations, and (iii) the C74S mutation.

[0296] Fig.10 A list of variant IL-12 p40 subunit sequences is shown.

[0297] Figures 11A-11E A list of exemplary heterodimeric Fc fusion proteins and related sequences is shown.

[0298] Fig.12 A schematic diagram of an exemplary IL-12 Fc fusion protein in a monovalent format is shown.

[0299] Fig.13 Size exclusion (upper graph) and cation exchange (lower graph) chromatographic profiles of wild-type IL-12 heterodimeric Fc fusion protein (also referred to herein as "wild-type IL-12 Fc," "IL-12 Fcwt," or "IL-12 Fcw.t." or "WT") are shown. Fractions pooled for further purification and / or analysis are indicated by rectangles.

[0300] Fig.14 Shown are reducing (+DTT; left panel) and non-reducing (-DTT; right panel) SDS-PAGE analysis of purified wild-type IL-12 Fc.

[0301] Fig.15 The interaction model between IL-12 and IL-12Rβ2 is shown. The IL-12 p35 subunit, IL-12 p40 subunit and IL-12Rβ2 are shown in cartoon representations, and the IL-12 p35 subunit residues selected for mutational analysis are shown in sticks.

[0302] Fig.16A and 16BShown are reducing (+DTT; 16A) and non-reducing (-DTT; 16B) SDS-PAGE analyses of purified heterodimeric IL-12 Fc fusion proteins, wherein the IL-12 p35 subunit domain comprises one mutation and the Fc domain comprises knob and hole mutations that promote heterodimerization of the Fc domains.

[0303] Fig.17 Shown are SPR sensorgrams (i.e., a plot of the SPR response (in response units (RU)) versus time (in seconds) generated from an SPR instrument) representing binding of 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000, and 2000 nM IL-12Rβ2 to immobilized wild-type IL-12Fc ("wt"; top row) or mutants (middle and bottom rows), as indicated.

[0304] Fig.18 Summary of SPR data for IL-12Rβ2 binding to wild-type IL-12 Fc ("wt") or a heterodimeric IL-12 Fc fusion protein containing a mutation in the IL-12 p35 subunit domain is shown. The amount of IL-12 Fc immobilized on the chip (captured) and the dissociation constant (K) are provided. D As used herein, when referring to SPR assay results, the term "weak" refers to K D The value is below the lowest detectable level of the assay used (i.e., K cannot be accurately determined with respect to the lower detection limit of the assay). D value).

[0305] Fig.19 The activity of commercially available IL-12 (Miltenyi; top row) or heterodimeric IL-12 Fc fusion proteins containing a mutation in the IL-12 p35 subunit domain (middle and bottom rows) compared to wild-type IL-12 Fc ("wt") in a HEK-Blue IL-12 reporter gene assay is shown. Symbols and error bars indicate the mean and SD, respectively. For symbols, solid circles show the mean of wild-type IL-12 Fc, while open squares show the mean of commercially available IL-12 or heterodimeric IL-12 Fc fusion proteins.

[0306] Fig. 20 EC values ​​for commercial IL-12 (Miltenyi), wild-type IL-12 Fc ("wt"), and heterodimeric IL-12 Fc fusion protein using the HEK-Blue IL-12 assay are shown. 50 Summary of values ​​and fold changes (relative to wt) for IL-12 p35 subunit domains containing a mutation.

[0307] Fig.21 The activity of commercial IL-12 (Miltenyi; top row) and heterodimeric IL-12 Fc fusion proteins in primary T cell IFNγ release assays compared to wild-type IL-12 Fc ("wt"), wherein the IL-12 p35 subunit domain contains a mutation (middle and bottom rows). Symbols and error bars indicate the mean and SD, respectively. For symbols, solid circles show the mean of wild-type IL-12 Fc, while open squares show the mean of commercial IL-12 or heterodimeric IL-12 Fc fusion proteins.

[0308] Fig. 22 EC values ​​for commercial IL-12 (Miltenyi), wild-type IL-12 Fc ("wt"), and heterodimeric IL-12 Fc fusion protein using a primary T cell IFNγ release assay are shown. 50 Summary of values ​​and fold changes (relative to wt) for IL-12 p35 subunit domains containing a mutation.

[0309] Fig.23 Shown are reducing (+DTT; left panel) and non-reducing (-DTT; right panel) SDS-PAGE analyses of purified heterodimeric IL-12 Fc fusion proteins, wherein the IL-12 p35 subunit domain comprises two, three, or four mutations and the Fc domain comprises knob and hole mutations that promote heterodimerization of the Fc domains.

[0310] Fig.24A and 24B The activity of commercially available IL-12 (Miltenyi) or heterodimeric IL-12 Fc fusion proteins in which the IL-12p35 subunit domain comprises one, two, three or four mutations compared to wild-type IL-12 Fc ("wt") in a HEK-Blue IL-12 reporter gene assay is shown. Symbols and error bars indicate the mean and SD, respectively. For symbols, solid circles show the mean of wild-type IL-12 Fc, while open squares show the mean of commercially available IL-12 or heterodimeric IL-12 Fc fusion proteins.

[0311] Fig.25 EC values ​​for commercial IL-12 (Miltenyi), wild-type IL-12 Fc ("wt"), and heterodimeric IL-12 Fc fusion protein using the HEK-Blue IL-12 assay are shown. 50 Summary of values ​​and fold changes (relative to wt) in IL-12 p35 subunit domains containing one, two, three, or four mutations.

[0312] Fig.26The activity of heterodimeric IL-12 Fc fusion proteins in primary T cell IFNγ release assays is shown, wherein the IL-12 p35 subunit domain contains two, three or four mutations (top and bottom rows). Symbols and error bars indicate mean values ​​and SD, respectively. For symbols, solid circles show the mean values ​​of wild-type IL-12 Fc, while hollow squares show the mean values ​​of heterodimeric IL-12 Fc fusion proteins.

[0313] Fig. 27 EC values ​​of wild-type IL-12 Fc ("wt") and heterodimeric IL-12 Fc fusion protein obtained using a primary T cell IFNγ release assay are shown. 50 Summary of values ​​and fold changes (relative to wt) in IL-12 p35 subunits containing two, three, or four mutations.

[0314] Fig.28 Shown are SPR sensorgrams (i.e., a plot of the SPR response generated from an SPR instrument in response units (RU) versus time in seconds) representing binding of 0.7, 2.1, 6.2, 18.5, 55.6, 167, 500 nM IL-12Rβ2 to immobilized wild-type IL-12 Fc ("wt"; left) or IL-12 Fc K170A mutant ("K170A"; right), as indicated.

[0315] Fig.29A and 29B Shown is the activity of 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 ("WT") in a HEK-Blue IL-12 reporter assay. Symbols and error bars indicate the mean and SD, respectively. For symbols, solid circles show the mean of wild-type IL-12 Fc, while open squares show the mean of heterodimeric IL-12 Fc mutant fusion proteins.

[0316] Fig.30 EC values ​​for commercial IL-12 (Miltenyi), wild-type IL-12 Fc ("wt"), and heterodimeric IL-12 Fc fusion protein using the HEK-Blue IL-12 assay are shown. 50 Summary of values ​​and fold changes (relative to wt) in IL-12 p35 subunit domains containing one, two, or three mutations.

[0317] Fig.31The activity of heterodimeric IL-12 Fc fusion proteins in primary T cell IFNγ release assays is shown, wherein the IL-12 p35 subunit domain contains one, two or three mutations. Symbols and error bars indicate the mean and SD, respectively. For symbols, solid circles show the mean of wild-type IL-12 Fc, while open squares show the mean of heterodimeric IL-12 Fc mutant fusion proteins.

[0318] Fig.32 The EC of heterodimeric IL-12 Fc fusion protein obtained using primary T cell IFNγ release assay is shown. 50 Summary of values ​​and fold changes (relative to wt) in IL-12p35 subunit domains containing one, two, or three mutations.

[0319] Fig.33 A schematic diagram of an exemplary IL-12 Fc fusion protein in a monovalent format is shown.

[0320] Fig.34 Shown are cation exchange (upper panel) and analytical size exclusion (lower panel) chromatographic profiles of wild-type IL-12 heterodimeric Fc fusion protein (also referred to herein as "wild-type IL-12 Fc," "IL-12 Fc wt," or "IL-12 Fc wt," or "WT"). Fractions pooled for functional assays or analyses are indicated by rectangles.

[0321] Fig.35A and 35B Shown are reducing (+DTT; 35A) and non-reducing (-DTT; 35B) SDS-PAGE analyses of heterodimeric IL-12 Fc fusion proteins containing one, two, or three mutations in the IL-12 p35 subunit domain.

[0322] Fig.36 Shown are the activities of commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc fusion proteins in which the IL-12 p35 subunit domains comprise one, two, or three mutations compared to wild-type IL-12 Fc ("WT") in a HEK-Blue IL-12 reporter assay. Symbols and error bars indicate the mean and SD, respectively. For symbols, solid circles show the mean of wild-type IL-12 Fc, while open squares show the mean of commercially available IL-12 or heterodimeric IL-12 Fc mutant fusion proteins.

[0323] Fig.37 EC values ​​for commercial IL-12 (Miltenyi), wild-type IL-12 Fc ("wt"), and heterodimeric IL-12 Fc fusion protein using the HEK-Blue IL-12 assay are shown.50 Summary of values ​​and fold changes (relative to wt) in IL-12 p35 subunit domains containing one, two, or three mutations.

[0324] Fig.38 The activity of commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc fusion proteins in primary T cell IFNγ release assays is shown, wherein the IL-12 p35 subunit domain contains one, two or three mutations. Symbols and error bars indicate the mean and SD, respectively. For symbols, solid circles show the mean of wild-type IL-12 Fc, while open squares show the mean of commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc mutant fusion proteins.

[0325] Fig.39 The EC values ​​of commercially available IL-12 (Miltenyi) and heterodimeric IL-12 Fc fusion protein obtained using a primary T cell IFNγ release assay are shown. 50 Summary of values ​​and fold changes (relative to wt) in IL-12 p35 subunit domains containing one, two, or three mutations.

[0326] Fig.40A and 40B The expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39 and granzyme B on CD8+ T cells derived from a donor ("Donor 245") after culture in the presence or absence of 1, 5 or 10 ng / ml wild-type IL-12 Fc or 10 ng / ml heterodimeric IL-12 Fc fusion protein containing mutations within the IL-12 p35 subunit domain is shown, as indicated. The horizontal line represents the mean of two replicate measurements (symbols).

[0327] Fig.41A and 41B The expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39 and granzyme B on CD8+ T cells derived from a donor ("Donor 247") after culture in the presence or absence of 1, 5 or 10 ng / ml wild-type IL-12 Fc or 10 ng / ml heterodimeric IL-12 Fc fusion protein containing mutations within the IL-12 p35 subunit domain is shown, as indicated. The horizontal line represents the mean of two replicate measurements (symbols).

[0328] Fig.42A and 42BThe expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39 and granzyme B on CD4+ T cells derived from a donor ("Donor 245") after culture in the presence or absence of 1, 5 or 10 ng / ml wild-type IL-12 Fc or 10 ng / ml heterodimeric IL-12 Fc fusion protein containing mutations within the IL-12 p35 subunit domain is shown, as indicated. The horizontal line represents the mean of two replicate measurements (symbols).

[0329] Fig.43A and 43B The expression of Lag-3, PD-1, CD27, CD45RA / CD45RO, TCF1, CD62L, CD39 and granzyme B on CD4+ T cells derived from a donor ("Donor 247") after culture in the presence or absence of 1, 5 or 10 ng / ml wild-type IL-12 Fc or 10 ng / ml heterodimeric IL-12 Fc fusion protein containing mutations within the IL-12 p35 subunit domain is shown, as indicated. The horizontal line represents the mean of two replicate measurements (symbols).

[0330] Fig.44 The principal component analysis (PCA) of CD8+T cells based on the complete phenotypic marker panel described in the text is shown. The donor batch effect is removed using the limma "removeBatchEffect" function, and the corrected MFI values ​​of each functional marker are analyzed using the limma "plotMDS" function to obtain the PCA graph. The PC1 component most relevant to the efficacy of the variant is shown as a box plot, where the horizontal line represents the median from three donor samples, and each sample is repeated (symbols).

[0331] Fig.45 The principal component analysis (PCA) of CD4+T cells based on the complete phenotypic marker panel described in the text is shown. The donor batch effect is removed using the limma "removeBatchEffect" function, and the corrected MFI values ​​of each functional marker are analyzed using the limma "plotMDS" function to obtain the PCA graph. The PC2 component most relevant to the variant efficacy is shown as a box plot, where the horizontal line represents the median from three donor samples, and each sample is repeated (symbols).

[0332] Fig.46 Shown in the injection of 10x10 6Twelve days after the incubation of freshly isolated human PBMCs with wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12p35 subunit domain, the number of CD4+ cells (upper graph), the number of CD8+ cells (middle graph), and the CD4:CD8 ratio (defined as the number of CD4+ cells divided by the number of CD8+ cells; lower graph) present in 1 μl of mouse plasma, as indicated. The horizontal lines represent the average (symbols) of measurements made from three mice.

[0333] Fig.47 Shown in the injection of 10x10 6 Expression of PD-1, Lag-3, Tim-3, CD95, Ki67, granzyme B, and CD45RA / CD45RO on CD4+ T cells derived from mouse plasma twelve days after administration of freshly isolated human PBMCs and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12p35 subunit domain. Horizontal lines represent the mean (symbols) of measurements performed from three mice.

[0334] Fig.48 Shown in the injection of 10x10 6 Expression of PD-1, Lag-3, Tim-3, CD95, Ki67, granzyme B, and CD45RA / CD45RO on CD8+ T cells derived from mouse plasma twelve days after freshly isolated human PBMCs and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12p35 subunit domain. Horizontal lines represent the mean (symbols) of measurements performed from three mice.

[0335] Fig.49 Shown is the injection of 10x10 6 IFNγ concentrations present in mouse plasma of freshly isolated human PBMCs and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12 p35 subunit domain, as indicated. IL-12 Fc was injected at 0.01 mg / kg (upper graph) or 0.1 mg / kg (lower graph) at time 0 hours and time 168 hours, as indicated by downward arrows. As indicated in the graph, data points were collected at multiple time points.

[0336] Fig.50 Shown is the injection of 10x10 6IL-12 Fc concentrations present in mouse plasma of freshly isolated human PBMCs and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12 p35 subunit domain, as indicated. IL-12 Fc was injected at time 0 hours and time 168 hours at 0.01 mg / kg (upper graph) or 0.1 mg / kg (lower graph), as indicated by downward arrows. As indicated in the graph, data points were collected at multiple time points.

[0337] Fig.51 The results show that the injection of 2.5x10 6 Seven days after the induction of T cell receptor alpha constant (TRAC) knockout, 1G4 transduced primary T cells and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12 p35 subunit domain, the number of CD4+ cells and CD8+ cells present in 1 μl of mouse blood and the expression of granzyme B, Lag-3, PD-1, T-bet, CD45RA+ / CD45RO- and Ki-67 on CD8+ T cells, as indicated. The horizontal line represents the median (symbol) of measurements performed on five to six mice per group.

[0338] Fig.52 The results show that the injection of 2.5x10 6 Fourteen days after the induction of TRAC knockout, 1G4 transduced primary T cells and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12 p35 subunit domain, the number of CD4+ cells and CD8+ cells present in 1 μl of mouse blood and the expression of granzyme B, Lag-3, PD-1, T-bet, CD45RA+ / CD45RO- and Ki67 on CD8+ T cells, as indicated. The horizontal line represents the median (symbol) of measurements made on five to six mice per group.

[0339] Fig.53 The results show that on days 1, 7, 14, and 21, 2.5 x 10 6 IL-12 Fc concentrations present in plasma of mice with TRAC knockout, 1G4-transduced primary T cells and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12 p35 subunit domain, as indicated. Symbols and error bars represent the mean and standard deviation, respectively, of measurements made on plasma from five to six mice via Luminex assay.

[0340] Fig.54 The results show that on days 1, 7, 14, and 21, 2.5 x 10 6IFNγ concentrations present in plasma of mice with TRAC knockout, 1G4-transduced primary T cells and wild-type IL-12 Fc or heterodimeric IL-12Fc fusion proteins containing mutations within the IL-12 p35 subunit domain, as indicated. Symbols and error bars represent the mean and standard deviation, respectively, of measurements made on plasma from five to six mice via Luminex assay.

[0341] Fig.55 Tumor growth curves are shown on days 0, 7, and 14 for NSG mice (n=5-6 per treatment group) injected subcutaneously with 2×10 5 HCT116 human colorectal cancer cells expressing HLA-A2 / NY-ESO-1A4 variants were then treated with 2.5x10 6 TRAC knockout, 1G4-transduced primary T cells were treated with wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12 p35 subunit domain, as indicated. Symbols and error bars represent the mean and standard error of the mean, respectively.

[0342] Fig.56 Shown are tumor measurements on day 18 of NSG mice (n=5-6 per treatment group) injected subcutaneously with 2x10 5 HCT116 human colorectal cancer cells expressing HLA-A2 / NY-ESO-1A4 variants were then treated with 2.5x10 6 TRAC knockout, 1G4-transduced primary T cells and wild-type IL-12 Fc or heterodimeric IL-12 Fc fusion proteins containing mutations within the IL-12p35 subunit domain were treated as indicated. Symbols represent tumor volumes of individual mice, while black lines represent medians for each treatment group. Statistics were calculated using one-way ANOVA. ns indicates not significant, ** indicates P < 0.01.

[0343] Fig.57A and 57B Shown are non-reducing SDS-PAGE analyses of protein A purified heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain, as indicated.

[0344] Fig.58A and 58B Shown are reducing SDS-PAGE analyses of protein A purified heterodimeric IL-12 Fc fusion proteins containing one or two mutations in the IL-12 p35 subunit domain, as indicated.

[0345] Fig.59 AlphaLISA measurements of IFNγ in culture medium of activated T cells that were unstimulated or incubated with protein A purified expi-CHO medium ("no DNA") or 20 pM wild-type IL-12 Fc ("WT") or 20 pM heterodimeric IL-12 Fc fusion protein containing a mutation in the IL-12 p35 subunit domain at amino acid position 40, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced following treatment with IL-12 Fc Y40Y.

[0346] Fig.60 AlphaLISA measurements of IFNγ in culture medium of activated T cells are shown, which were not stimulated or incubated with protein A purified expi-CHO medium ("no DNA") or 80 pM wild-type IL-12 Fc ("WT") or 80 pM heterodimeric IL-12 Fc fusion protein, wherein the IL-12 p35 subunit domain contains a Y40A mutation and additional mutations at amino acid position 126, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12Fc Y40A / D126D.

[0347] Fig.61 AlphaLISA measurements of IFNγ in culture medium of activated T cells are shown, which were not stimulated or incubated with protein A purified expi-CHO medium ("no DNA") or 80 pM wild-type IL-12 Fc ("WT") or 80 pM heterodimeric IL-12 Fc fusion protein containing a Y40A mutation and additional mutations at amino acid position 127 in the IL-12 p35 subunit domain, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12Fc Y40A / P127P.

[0348] Fig.62 AlphaLISA measurements of IFNγ in culture medium of activated T cells are shown, which were not stimulated or incubated with protein A purified expi-CHO medium ("no DNA") or 80 pM wild-type IL-12 Fc ("WT") or 80 pM heterodimeric IL-12 Fc fusion protein, wherein the IL-12 p35 subunit domain contains a Y40A mutation and additional mutations at amino acid position 129, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12Fc Y40A / R129R.

[0349] Fig.63AlphaLISA measurements of IFNγ in culture medium of activated T cells are shown, which were not stimulated or incubated with protein A purified expi-CHO medium ("no DNA") or 80 pM wild-type IL-12 Fc ("WT") or 80 pM heterodimeric IL-12 Fc fusion protein, wherein the IL-12 p35 subunit domain contains a Y40A mutation and additional mutations at amino acid position 168, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced after treatment with IL-12Fc Y40A / K168K.

[0350] Fig.64 AlphaLISA measurements of IFNγ in culture medium of activated T cells that were unstimulated or incubated with protein A purified expi-CHO medium ("no DNA") or 20 pM wild-type IL-12 Fc ("WT") or 20 pM heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit contains a mutation at amino acid position 170, as indicated on the x-axis. The horizontal line indicates the amount of IFNγ produced following treatment with IL-12 Fc K170K.

[0351] Fig.65 Shown are the activities of wild-type IL-12 Fc and heterodimeric IL-12 Fc fusion proteins in primary T cell IFNγ release assays, wherein the IL-12 p35 subunit domain comprises a Y40X single mutant (upper panel) or a Y40A / K168X double mutant (lower panel). Symbols and error bars indicate mean and SD, respectively.

[0352] Fig.66 The EC of heterodimeric IL-12 Fc fusion protein obtained using primary T cell IFNγ release assay is shown. 50 Summary of values ​​and fold changes (relative to wt) in IL-12p35 subunit domains containing one or two mutations.

[0353] Fig.67 Thermal unfolding curves of WT IL-12 Fc and heterodimeric IL-12 Fc fusion proteins are shown, wherein the IL-12 p35 subunit domain comprises Y40A, D126A, R129A, P127A, K168A or K170A as indicated. The solid line represents the average of three independent replicates.

[0354] Fig.68The percentage activity of heterodimeric IL-12 Fc fusion proteins in primary T cell IFNγ release assays is shown, wherein the IL-12 p35 subunit domain contains one or two mutations as indicated. The percentage activity refers to the activity of samples after incubation at a given temperature (indicated in the title of each group) relative to the activity of untreated or 48°C treated samples (as indicated on the y-axis of each group).

[0355] Fig.69 Shown is a summary of the individual melting temperatures (TM1 and TM2) of WT 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 differential scanning fluorimetry (DSF).

[0356] Fig.70 Shown are the activities of three different batches of WT IL-12 Fc (WT1, WT2 and WT3) and heterodimeric IL-12 Fc fusion proteins in primary T cell IFNγ release assays, wherein the IL-12 p35 subunit domain contains one or two mutations. Symbols and error bars indicate mean and SD, respectively.

[0357] Fig.71 Shown are the EC values ​​of three different batches of WT IL-12 Fc (WT1, WT2, and WT3) and heterodimeric IL-12 Fc fusion protein obtained using the primary T cell IFNγ release assay in four separate donors. 50 Summary of values ​​and fold changes (relative to wt) in IL-12 p35 subunit domains containing one or two mutations.

[0358] Fig.72 Killing curves generated by incubating TRAC knockout, 1G4-transduced primary T cells (effectors) with HLA-A2 / NY-ESO A4 variants / GFP-transduced HCT116 cells (targets) at an effector to target cell ratio of 2.5:1 are shown. Effectors were incubated overnight with 0-10 ng / ml WT IL-12 Fc prior to assay setup, as indicated by the symbols. The number of remaining target cells (depicted as GFP confluence normalized to 0 h) is plotted over time.

[0359] Fig.73Shown is a summary of the results of killing assays, wherein TRAC knockout, 1G4 transduced primary T cells (effectors) and HLA-A2 / NY-ESO A4 variants / GFP transduced HCT116 cells (targets) were incubated with an effector to target cell ratio of 2.5:1. Before the assay was set, the effector was incubated overnight with 0-10 ng / ml WT IL-12 Fc (transparent bars) or a single determined concentration of heterodimer IL-12 Fc fusion protein, wherein the IL-12 p35 subunit domain comprises one or two mutations (shaded bars), as indicated. Bar graphs and error bars represent the mean and standard deviation of the number of target cells remaining at 8 hours (depicted as GFP confluence). Horizontal dotted lines represent the number of target cells remaining after incubation with 0 and 10 ng / ml WT IL-12 Fc (upper and lower horizontal dotted lines, respectively).

[0360] Fig.74 Shown is a summary of the interpolated fold changes in activity of heterodimeric IL-12 Fc fusion proteins relative to WT IL-12 Fc as determined by T cell killing assays, wherein the IL-12 p35 subunit domain contains one or two mutations.

[0361] Fig.75 Shown is a summary of the individual melting temperatures (TM1 and TM2) of WT 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.

[0362] Fig.76 The EC values ​​of WT IL-12 Fc and heterodimeric IL-12 Fc fusion protein obtained using the primary T cell IFNγ release assay are shown. 50 Summary of values ​​and fold changes (relative to wt) in IL-12 p35 subunit domains containing one or two mutations.

[0363] Fig.77 A schematic diagram of an exemplary IL-12 Fc fusion protein in a monovalent format is shown.

[0364] Figures 78A-78G A list of additional variant IL-12 p35 subunit sequences is shown.

[0365] Figures 79A-79D A list of additional exemplary heterodimeric Fc fusion proteins and related sequences is shown.

[0366] Figures 80A-80MA list of additional variant IL-12 p35 subunit sequences (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) is shown.

[0367] Fig.81 The sequences of "Single-chain trimer of HLA-A2, β2-microglobulin and NY-ESO-1A4 variant peptide" and "1G4 T cell receptor β chain-P2A-1G4 T cell receptor α chain" are shown.

[0368] Fig.82 Shown are the activities of two different configurations of WT IL-12 Fc (p40-Fc (knob) / p35-Fc (hole) and p40-Fc (hole) / p35-Fc (knob)) and a heterodimeric IL-12 Fc fusion protein in which the IL-12 p35 subunit domain contains two mutations in a primary T cell IFNγ release assay. Symbols and error bars indicate mean and SD, respectively.

[0369] Fig.83 The EC values ​​of two different configurations of WT IL-12 Fc (p40-Fc (knob) / p35-Fc (hole) and p40-Fc (hole) / p35-Fc (knob)) and heterodimeric IL-12 Fc fusion protein obtained using primary T cell IFNγ release assay are shown. 50 Summary of values ​​and fold changes (relative to wt) for IL-12 p35 subunit domains containing two mutations. DETAILED DESCRIPTION

[0370] The description is presented to enable one of ordinary skill in the art to prepare and use the invention, and is provided in the context of a patent application and its requirements. The subsection headings used herein are only for organizational purposes and are not interpreted as limiting the subject matter. Although various embodiments of the invention disclosed herein have been shown and described herein, such embodiments are provided only by way of example, which will be apparent to those skilled in the art. Many modifications, variations and substitutions may now be made by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used to practice any one of the present invention set forth herein.

[0371] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety to the extent relevant.

[0372] I. Definitions

[0373] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For the purposes of the present disclosure, the following terms are defined below. The definitions provided are intended to apply to a given term as well as other derivative linguistic rephrasings and grammatical equivalents of the term.

[0374] As used herein, the term "protein" refers to at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more covalently attached amino acids, including proteins, polypeptides, oligopeptides and peptides. When a "biologically functional molecule" comprises two or more proteins, such as, for example, IL-12 (which comprises an IL-12 p35 polypeptide and an IL-12 p40 polypeptide), the individual proteins comprising the two or more proteins may each be referred to as a "subunit", "monomer" or "domain", and the biologically functional molecule may be referred to as a "complex". In some embodiments, two or more proteins of a functional complex are non-covalently attached. In some embodiments, two or more proteins of a functional complex are covalently attached, such as, for example, through a disulfide bond.

[0375] As used herein, the term "cytokine" refers to a large class of proteins, such as, for example, chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, etc., which are secreted by a first cell and affect one or more cells by binding of the secreted cytokine to receptors on one or more cells. Cytokines can participate in autocrine, paracrine, juxtacrine and / or endocrine signaling.

[0376] 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 the amino acid sequence) that has not been intentionally modified.

[0377] As used herein, the term "IL-12 p35 subunit" refers to human wild-type IL-12 p35 polypeptide, whether native 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. Figure 1 The amino acid sequence of the human wild-type IL-12 p35 subunit is depicted (precursor: SEQ ID NO: 1; mature: SEQ ID NO: 2). In the context of the fusion protein, IL-12

[0378] The p35 subunit may also be referred to as an "IL-12 p35 subunit domain," wherein the IL-12 p35 subunit domain comprises at least a portion of the amino acid sequence encoding the IL-12 p35 subunit.

[0379] As used herein, the term "IL-12 p40 subunit" refers to human wild-type IL-12 p40 polypeptide, whether native or recombinant. Thus, 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. Figure 1 The amino acid sequence of the human wild-type IL-12 p40 subunit is depicted (precursor: SEQ ID NO: 3; mature: SEQ ID NO: 4). In the context of a fusion protein, the IL-12 p40 subunit may also be referred to as an "IL-12 p40 subunit domain", wherein the IL-12 p40 subunit domain comprises at least 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 wherein the IL-12 p40 subunit comprises a wild-type IL-12 p40 polypeptide or a variant IL-12 p40 subunit.

[0380] As used herein, the term "single chain" refers to a molecule comprising two or more protein domains linearly connected by peptide bonds. In some embodiments, the biologically functional IL-12 is a single-chain IL-12 complex (sc-IL-12) (i.e., IL-12 p35 subunit and 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 connected to the N-terminus of the IL-12 p40 subunit (sc-IL-12 (p35 / p40)). In yet further embodiments, sc-IL-12 (p35 / p40) further comprises a linker, wherein the C-terminus of the IL-12 p35 subunit is connected to the N-terminus of the linker, and the C-terminus of the linker is connected to the N-terminus of the IL-12 p40 subunit. In other embodiments, the C-terminus of the IL-12 p40 subunit is connected to the N-terminus of the IL-12 p35 subunit (sc-IL-12 (p40 / p35)). In yet further embodiments, 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.

[0381] As used herein, the term "residue" refers to a position in a protein and its associated amino acid identity. For example, cysteine ​​252 (also known as Cys252 or C252) is the residue at position 252.

[0382] As used herein, the term "parent protein" refers to a "reference" protein, an amino acid sequence encoding a reference protein, or a DNA sequence encoding an amino acid sequence encoding a reference protein. In some embodiments, the reference protein includes a wild-type protein, an amino acid sequence encoding a wild-type protein, and / or a nucleic acid sequence encoding an amino acid sequence encoding a wild-type protein. In some embodiments, the reference protein includes a human wild-type protein, an amino acid sequence encoding a human wild-type protein, and / or a nucleic acid sequence encoding an amino acid sequence encoding a human wild-type protein. In some embodiments, the reference protein includes a (human) wild-type protein conjugated to an Fc domain.

[0383] As used herein, the term "variant protein", "protein variant" or "variant" refers to a protein that differs from a parent protein in properties due to at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more modifications. The term may refer to the protein itself, a composition comprising the protein, an amino acid sequence encoding the protein, or a DNA sequence encoding the amino acid sequence. 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 having one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid substitutions, deletions and / or insertions compared to the amino acid sequence of a reference IL-12 p35 subunit, such as, for example, 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 having one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid substitutions, deletions and / or insertions compared to the amino acid sequence of a reference IL-12 p40 subunit, such as, for example, a (human) wild-type IL-12 p40 subunit.

[0384] As used herein, the term "modification" refers to amino acid substitutions, insertions and / or deletions in a polypeptide sequence, or changes in the part chemically linked to the protein. As used herein, the term "amino acid modification" refers to amino acid substitutions, insertions and / or deletions in a polypeptide sequence. There are no particular limitations on the position of the amino acid modification and the number of amino acids that can be modified in the amino acid sequence.

[0385] It will be understood that modifications to the IL-12 p35 subunit (domain) described throughout refer to modifications made to the mature form of the sequence (SEQ ID NO: 2) and / or variants thereof, and not to the precursor sequence (SEQ ID NO: 1). The precursor sequence of the IL-12 p35 subunit comprises an additional 22 amino acid residues at the N-terminus, which comprises the following sequence: MCPARSSLLLVATLVLLDHLSLA. However, the listed modifications to the mature form of the IL-12 p35 subunit may be made to the precursor form of the IL-12 p35 subunit after correcting the position of the modification to take into account the additional leading amino acid residues in the precursor sequence. For example, the substitution modification Y40A is disclosed herein as a potential modification to the mature sequence of the IL-12 p35 subunit, but may also refer to a substitution modification of the IL-12 p35 subunit precursor sequence comprising Y62A.

[0386] Similarly, modifications to the IL-12 p40 subunit (domain) described throughout are intended to refer to modifications made to the mature form of the sequence (SEQ ID NO: 4) and / or variants thereof, rather than to the precursor sequence (SEQ ID NO: 3). The precursor sequence of the IL-12 p40 subunit comprises an additional 22 amino acid residues at the N-terminus, which comprises the following sequence: MCHQQLVISWFSLVFLASPLVA. However, it will also be understood that the listed modifications to the mature form of the IL-12 p40 subunit can be made to the precursor form of the IL-12 p40 subunit after correcting the position of the modification to take into account the additional leading amino acid residues in the precursor sequence. For example, the substitution modification C177S is disclosed herein as a potential modification to the mature sequence of the IL-12 p40 subunit, but may also refer to a substitution modification of the IL-12 p40 subunit precursor sequence comprising C199S.

[0387] As used herein, the term "amino acid substitution" or "substitution" refers to replacing an amino acid at a specific position in a parent polypeptide sequence with a different amino acid. For example, Y40A means that the tyrosine at position 40 is replaced by alanine at the same position. In some embodiments, the replacement is an amino acid that is non-naturally present at a specific position, and the amino acid is non-naturally present in an organism or in any organism. For clarity, a protein that is engineered to change a nucleic acid coding sequence but does not change the resulting amino acid (e.g., CCU (encoding proline) is exchanged for CCC (still encoding proline)) is not an "amino acid substitution". In other words, although a new gene encoding the same protein is produced, if the protein has the same amino acid as its starting position at a specific position, this is not an amino acid substitution.

[0388] As used herein, the term "amino acid insertion" or "insertion" refers to the addition of an amino acid residue or sequence at a specific position of a parent polypeptide sequence. For example, -40A indicates the insertion of alanine after position 40 and before position 41. As a separate example, D8EPKSS or -8EPKSS indicates the insertion of the sequence Glu-Pro-Lys-Ser-Ser after position 8 and before position 9.

[0389] As used herein, the term "amino acid deletion" or "deletion" refers to the removal of an amino acid or sequence at a specific position of a parent polypeptide sequence. For example, Y40-, Y40#, Y40(), or Y40del indicates a deletion of tyrosine at position 40. As a separate example, EPKSS8-, EPKSS8#, EPKSS8del indicates a deletion of the sequence Glu-Pro-Lys-Ser-Ser starting from position 8.

[0390] As used herein, the term "non-naturally occurring protein" or "non-naturally occurring protein variant" refers to a variant protein that differs from a parent protein by at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more non-isotypic modifications. For example, since the IL-12 p35 subunit of IL-12 does not contain alanine at position 40, the substitution Y40A is considered a non-naturally occurring IL-12 p35 variant (or more generally, a non-naturally occurring IL-12 variant). Non-isotypic modifications to a protein may be referred to as "non-naturally occurring modifications".

[0391] 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 the amino acid residues (or nucleic acids encoding amino acid residues) in a specific sequence (such as, for example, the amino acid sequence of a parent protein) after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for determining percent sequence identity can be achieved by various means within the skill of the art, such as, for example, using publicly available computer software (e.g., BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires achieving maximum alignment over the full length of the compared sequences. 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.

[0392] As used herein, the term "half-life" of an agent may refer to such activity when applied to the serum of an organism or a subject, or the time in a tissue, or relative to any other defined time point, the time required for an agent to lose half of its pharmacological, physiological or other activity."half-life" may also refer to such amount or concentration when applied to the serum of an organism or a subject, or relative to any other defined time point, the time required for the amount or concentration of an agent to reduce the amount of half of the initial amount applied to the serum of an organism or a subject or the tissue. Half-life can be measured in serum and / or any one or more selected tissues.

[0393] As used herein, the terms "host cell" and "recombinant cell" refer to individual cells or cell cultures that can be or have been recipients of any recombinant vector or isolated polynucleotide. Host cells can be transfected, transformed, transduced or infected cells of any origin, including but not limited to prokaryotic cells, eukaryotic cells, mammalian cells, avian cells, insect cells, plant cells or bacterial cells, or can be any source that can be used to propagate nucleic acids described herein. Host cells include cells transfected or infected in vivo or in vitro with the recombinant vectors or polynucleotides of the present invention. Host cells comprising the recombinant vectors of the present invention may be referred to as "recombinant host cells".

[0394] Host cells may include, but are not limited to, cells of mammals, plants, insects, fungi, and bacteria. Bacterial cells include, but are not limited to, cells of gram-positive bacteria (such as, for example, species of Bacillus, Streptomyce, and Staphylococcus), and gram-negative bacterial cells, such as, for example, cells of Escherichia and Pseudomonas. Fungal cells may include, but are not limited to, yeast cells, such as, for example, Saccharomyces, Pichia pastoris, and Hansenula polymorpha. Insect cells may include, but are not limited to, cells of Drosophila and Sf9 cells. Plant cells include, but are not limited to, cells from crops, medicinal or ornamental plants, or bulbs. Mammalian cells suitable for use in the present invention include, but are not limited to, epithelial cell lines (e.g., porcine epithelial cells), osteosarcoma cell lines, neuroblastoma cell lines, epithelial cancers, glial cells, hepatic cell lines, Chinese hamster ovary (CHO) cells, COS cells, BHK cells, HeLa cells, D3 cells of mouse embryonic stem cells (mESC) lines, 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.

[0395] As used herein, the terms "cell", "cell culture", "cell line" and "host cell" refer not only to a specific cell, cell culture, cell line or host cell, but also to the progeny or potential progeny of such a cell, cell culture, cell line or host cell, regardless of the number of transfers or passages in culture. It should be understood that not all progeny are completely identical to the parent cell. This is because certain modifications may occur in the progeny due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so that the progeny may not actually be identical to the parent cell, but still be included in the scope of the terms as used herein, as long as the progeny retains the same function or substantially the same function as the original cell, cell culture, cell line or host cell.

[0396] As used herein, the term "medium" or "culture medium" includes any culture medium, solution, solid, semisolid or rigid support that can support or hold any host cell, including but not limited to bacterial host cells, yeast or fungal host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli host cells, Pseudomonas host cells, etc. and cell contents. Thus, the term can encompass culture media in which host cells are grown (e.g., culture media from which polypeptides have been secreted, including culture media before, during or after a proliferation step). The term can also encompass buffers or other reagents containing host cell lysates, such as, for example, when polynucleotides are produced intracellularly and host cells are lysed or destroyed to release polypeptides.

[0397] As used herein, the term "fusion protein" refers to the covalent bonding of at least two proteins or protein domains. Fusion proteins may include artificial sequences, such as, for example, domain joints, variant Fc domains, variant IL-12 p35 subunit domains, (variants) 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, which is generally connected (optionally through a domain joint, as described herein) to one or more different protein domains. In some embodiments, the C-terminus of the Fc domain is connected to the N-terminus of one or more different protein domains (optionally through a domain joint, wherein the C-terminus of the Fc domain is connected to the N-terminus of the domain joint, and the C-terminus of the domain joint is connected to the N-terminus of one or more different protein domains). In some embodiments, the N-terminus of the Fc domain is connected to the C-terminus of one or more different protein domains (optionally through a domain joint, wherein the C-terminus of one or more protein domains is connected to the N-terminus of the domain joint, and the C-terminus of the domain joint is connected to the N-terminus of the Fc domain). Thus, an "IL-12 Fc fusion" comprises an Fc region (optionally via a domain linker) linked 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 "heterodimer Fc fusion protein" or a "homodimer Fc fusion protein".

[0398] As used herein, the term "heterodimer 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 wherein 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 Fc domain and the second Fc domain. In some embodiments, the first Fc domain and / or the second Fc domain comprise one or more modifications that change Fc binding. In some embodiments, the first Fc domain and / or the second Fc domain comprise one or more modifications that change Fc half-life. In some embodiments, the first Fc domain and / or the second Fc domain comprise one or more modifications that change the first Fc domain and / or the second Fc domain and neonatal Fc receptor (FcRn) binding. In certain further embodiments, the first Fc domain and / or the second Fc domain comprise one or more modifications that increase half-life and / or binding to FcRn. In some embodiments, the first IL-12 subunit domain includes a variant IL-12 p35 subunit domain, and the second IL-12 subunit domain includes an IL-12 p40 subunit domain or a variant IL-12 p40 subunit domain. In some embodiments, the first IL-12 subunit domain includes an IL-12 p40 subunit domain or a variant IL-12 p40 subunit domain, and the second IL-12 subunit domain includes a variant IL-12 p35 subunit domain. In some embodiments, the C-terminus of the first IL-12 subunit domain is connected to the N-terminus of the first Fc domain (optionally through a domain linker), and the C-terminus of the second IL-12 subunit domain is connected to the N-terminus of the second Fc domain (optionally through a domain linker). In some embodiments, the C-terminus of the first Fc domain is linked to the N-terminus of the first IL-12 subunit domain (optionally through a domain linker), and the C-terminus of the second Fc domain is linked to the N-terminus of the second IL-12 subunit domain (optionally through a domain linker).

[0399] As used herein, the term "homodimer fusion protein" refers to a complex comprising two identical instances of a fusion construct, wherein an individual instance of the fusion construct comprises an Fc domain and one or more protein domains (optionally connected by a domain joint). In some embodiments, for two instances of a fusion construct, the C-terminus of the Fc domain is connected to the N-terminus of one or more protein domains (optionally through a domain joint). In some embodiments, for two instances of a fusion construct, the C-terminus of one or more protein domains is connected to the N-terminus of the Fc domain (optionally through a domain joint). In some embodiments, the same instance of the Fc domain comprises one or more modifications that change Fc binding. In some embodiments, the same instance of the Fc domain comprises one or more modifications that change the half-life of the Fc domain. In some embodiments, the same instance of the Fc domain comprises one or more modifications that change the same instance of the Fc domain and the neonatal Fc receptor (FcRn) binding. In certain further embodiments, the same instance of the domain comprises one or more modifications that increase half-life and / or binding to FcRn.

[0400] As used herein, the term "isolated" when used to describe the various polypeptides disclosed herein refers to a polypeptide that has been identified and separated and / or recovered from a cell (e.g., a host cell and / or cell line) or cell culture in which it is expressed. Typically, an isolated polypeptide will be 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 (such as, for example, host cell proteins).

[0401] As used herein, the term "Fc", "Fc region" or "Fc domain" refers to a polypeptide comprising the CH2-CH3 domain of an immunoglobulin G (IgG) molecule, and in some cases includes all or part of a hinge and variants thereof. In the EU numbering of human IgG1, the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Therefore, the definition of "Fc domain" includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3) or fragments thereof. In this context, an "Fc fragment" may contain fewer amino acids from the N-terminus or the C-terminus or both, but still retains the ability to form a dimer with another Fc domain or Fc fragment, which can be detected using standard methods, typically based on size (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). Herein, unless otherwise specified, "Fc domain" generally refers to the CH2-CH3 domain of human IgG1 (and optionally all or part of a hinge). Various human IgG1 Fc domains are described below, at least some of which contain one or more modifications. For clarity, although modifications are discussed herein primarily in the context of human IgG1 Fc domains for brevity, it is clearly contemplated that mutations in other immunoglobulins (such as, for example, IgG2, IgG3, IgG4, IgA, IgM, and IgE) may occur at residue positions corresponding to mutations in human IgG1 described herein. Those skilled in the relevant art will be able to readily determine residue positions in other immunoglobulins corresponding to the human IgG1 modifications described herein.

[0402] As used herein, the term "vector" refers to a nucleic acid molecule or sequence that can transfer or transport another nucleic acid molecule. The nucleic acid molecule transferred is usually connected to (for example, inserted into) a carrier nucleic acid molecule. Generally, when associated with an appropriate control element, the vector can replicate. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integration vectors. "Expression vector" is a vector that contains a regulatory region so that a DNA sequence and its fragments can be expressed in vitro and / or in vivo. The vector may include a sequence that directs autonomous replication in the cell or may include a sequence that is sufficient to allow integration into the host cell DNA. Useful vectors include, but are not limited to, plasmids (for example, DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, but are not limited to, replication-defective retroviruses and slow viruses. In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle to transfer genes into cells.

[0403] As used herein, the term "recombinant" in relation to nucleic acid molecules refers to polynucleotides of genomic, cDNA, viral, semisynthetic and / or synthetic origin that, by virtue of their origin or manipulation, are not related to all or part of the polynucleotides with which they are associated in nature. In relation to proteins or polypeptides, the term "recombinant" as used refers to a polypeptide produced by expression of a recombinant polynucleotide. In relation to host cells, the term "recombinant" as used refers to a host cell into which a recombinant polynucleotide or a vector comprising a recombinant polynucleotide has been introduced.

[0404] As used herein, the term "operably connected" refers to a physical or functional linkage between two or more elements (e.g., a polypeptide sequence or a polynucleotide sequence) that allows them to operate in their intended manner. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (such as, for example, a promoter) is a functional connection that enables the polynucleotide of interest to be expressed. In this sense, the term "operably connected" refers to the positioning of a regulatory region and a coding sequence to be transcribed, so that the regulatory region effectively regulates the transcription or translation of the coding sequence of interest. Therefore, if a promoter can mediate the transcription of a nucleic acid sequence, it is operably linked to the nucleic acid sequence. It should be understood that the elements that are operably connected can be continuous or non-continuous. In the context of a polypeptide, "operably connected" refers to a physical linkage (e.g., directly or indirectly connected) between an amino acid sequence (e.g., different segments, modules or domains) to provide the activity of a polypeptide.

[0405] As used herein, the term "binding affinity" refers to the "strength" of binding of a given molecule (such as, for example, 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 (such as, for example, IL-12Rβ2) and / or the rate at which the molecule associates and / or dissociates with its ligand. Binding affinity is often expressed in terms of the 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 can be determined by any suitable method known in the art, such as, for example, surface plasmon resonance (SPR) assay, enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Biacore assay, KinExA assay, etc.

[0406] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a non-naturally occurring IL-12 variant, a homodimeric IL-12 Fc fusion protein, and / or a heterodimeric IL-12 Fc fusion protein) to elicit a response at a certain dose or concentration in a given biological system or experimental environment. The potency of the non-naturally occurring IL-12 variant, homodimeric IL-12 Fc fusion protein, and / or heterodimeric IL-12 Fc fusion protein disclosed herein can be determined by any suitable method known in the art, such as, for example, an IL-12 HEK reporter gene assay (e.g., InvivoGen's IL-12 HEK reporter gene assay (Catalog No. hkb-il12)), a ligand binding assay (e.g., ELISA or flow cytometry), and / or a functional assay. Typically, a change in potency can be graphically displayed as a shift in the response curve to the left or right compared to a control. A shift in the response curve to the right generally indicates a decrease in potency, while a shift in the response curve to the left generally indicates an increase in potency.

[0407] As used herein, the term "activity" refers to a specific response elicited by a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a non-naturally occurring IL-12 variant, a homodimeric IL-12 Fc fusion protein and / or a heterodimeric IL-12 Fc fusion protein) in a specific biological system or experimental environment at a given dose or concentration. 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 determined by any suitable method known in the art, such as, for example, a ligand binding assay and / or a functional assay. Typically, a change in activity can be graphically displayed as an upward or downward shift in a response curve compared to a control. An upward shift in a response curve generally indicates an increase in activity, while a downward shift in a response curve generally indicates a decrease in activity. As described in further detail below, a change in activity may or may not be associated with a change in potency and / or caused by a change in potency.

[0408] As used herein, the term "manufacturability" refers to any property that may affect the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, etc., at a scale and quantity sufficient for administration to an individual. Examples of properties that affect manufacturability include, but are not limited to, stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a non-naturally occurring IL-12 variant, a homodimeric IL-12 Fc fusion protein, and / or a heterodimeric IL-12 Fc fusion protein).

[0409] As used herein, the term "stability" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, 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 that it had when manufactured within specific limits and / or storage and / or usage parameters. The stability 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 determined by any suitable method known in the art, such as, for example, ELISA, Western blot, Biacore assay, SDS-PAGE, size exclusion chromatography, dynamic light scattering, differential scanning calorimetry, and differential scanning fluorimetry.

[0410] As used herein, the term "expression yield" refers to the amount or quantity of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, non-naturally occurring IL-12 variants, homodimeric IL-12 Fc fusion proteins and / or heterodimeric IL-12 Fc fusion proteins) produced using a prokaryotic or eukaryotic host system. Recombinant expression of proteins, cytokines, fusion proteins, antibodies, etc. is well known in the relevant art. Any suitable method can be used to quantify or determine expression yield, such as, for example, UV absorption measurement, colorimetric assays (e.g., Bradford assay, BCA assay, and Lowry assay), and fluorescence assays.

[0411] As used herein, the term "subject" or "individual" for therapeutic purposes refers to any animal classified as a mammal (including but not limited to humans, primates and / or non-human primates), domestic animals, farm animals, zoo animals, research animals, sports animals and / or pet animals, such as dogs, horses, cats, cows, etc.

[0412] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an antigen" includes mixtures of antigens; reference to "a pharmaceutically acceptable carrier" includes mixtures of two or more such carriers, etc. Thus, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein.

[0413] In addition, when used herein, "and / or" should be regarded as a specific disclosure of each of two specific features or components with or without the other. Therefore, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A (alone)" and "B (alone)".

[0414] As used herein, the term "about" value (or parameter) refers to ±10% of a specified value. When referring to a range of values ​​(or parameters), the term "about" refers to +10% of the upper limit and -10% of the lower limit of the specified range of values. When providing a range of values, it is understood that each intermediate value between the upper and lower limits of the range and any other intermediate values ​​specified or in the specified range are included in the scope of the present disclosure. In the case where the specified range includes an upper limit and / or a lower limit, a range that does not include one of those included limits is also included in the present disclosure.

[0415] It should be understood that certain features of the present disclosure described in the context of separate embodiments for the sake of clarity may also be provided in combination with a single embodiment. Conversely, various features of the present disclosure described in the context of a single embodiment for the sake of simplicity may also be provided separately or in any suitable sub-combination. All combinations of embodiments belonging to the present disclosure are specifically encompassed in the present disclosure and disclosed herein just as each and every combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed in the present disclosure and disclosed herein just as each and every such sub-combination is individually and explicitly disclosed herein.

[0416] II. Overview

[0417] The present invention relates to novel, non-naturally occurring IL-12 variants and fusion proteins, wherein the IL-12 p35 subunit comprises novel amino acid substitutions that reduce binding affinity to IL-12Rβ2, and methods of making and using the non-naturally occurring IL-12 variants and fusion proteins.

[0418] As described above, IL-12 is composed of a covalently linked α chain (p35 subunit; IL-12 p35 subunit) and a β chain (p40 subunit; IL-12 p40 subunit) to form a biologically functional IL-12 complex. IL-12 exerts its cell signaling function by binding to a dimeric IL-12 receptor complex composed of IL-12Rβ1 and IL-12Rβ2, thereby leading to STAT4 phosphorylation and initiating multiple downstream signaling pathways, including but not limited to inducing IFNγ secretion. Systemic administration of wild-type IL-12 may lead to severe toxicity, including death, due to excessive activation of circulating immune cells. In addition, due to target-mediated drug disposal, cell proliferation of activated immune cells results in a shorter serum half-life of the administered IL-12.

[0419] In some embodiments, the compositions and methods described herein reduce toxicity associated with IL-12 therapy by providing novel IL-12 variants with reduced binding affinity for IL-12Rβ2. In further embodiments, the compositions and methods described herein utilize IL-12 Fc fusion proteins, which in still further embodiments comprise such novel IL-12 variants.

[0420] 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, the half-life can be further improved by fusing the novel IL-12 variants to one or more Fc domains (such as, for example, one or more Fc domains encoded by the amino acid sequences SEQ ID NOs: 9-13, Fc domains comprising 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).

[0421] 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 for IL-12Rβ2.

[0422] III. Composition

[0423] One skilled in the art will appreciate that 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 manufacture described below, or also in the methods of use described below.

[0424] A. Interleukin 12 (IL-12) variants

[0425] 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. The non-naturally occurring IL-12 variants and various configurations of the variant IL-12 p35 subunit and IL-12 p40 subunit are described in detail below.

[0426] 1. p35 subunit :

[0427] 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, wherein the 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) 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.

[0428] 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, two or more amino acid substitutions include T43A / K168A. In some embodiments, two or more amino acid substitutions include D126A / P127A. In some embodiments, two or more amino acid substitutions include D126A / R129A. In some embodiments, two or more amino acid substitutions include D126A / K168A. In some embodiments, two or more amino acid substitutions include P127A / R129A. In some embodiments, two or more amino acid substitutions include P127A / K168A. In some embodiments, two or more amino acid substitutions include R129A / K168A. In some embodiments, two or more amino acid substitutions include Y40A / K170A. In some embodiments, two or more amino acid substitutions include T43A / K170A. In some embodiments, two or more amino acid substitutions include D126A / K170A. In some embodiments, two or more amino acid substitutions include P127A / K170A. In some embodiments, the two or more amino acid substitutions include R129A / K170A. In some embodiments, the two or more amino acid substitutions include K168A / K170A.

[0429] 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, three or more amino acid substitutions include Y40A / T43A / D126A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / P127A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / R129A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / P127A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / R129A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / P127A / R129A. In some embodiments, three or more amino acid substitutions include Y40A / P127A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / R129A / K168A. In some embodiments, three or more amino acid substitutions include T43A / D126A / P127A. In some embodiments, three or more amino acid substitutions include T43A / D126A / R129A. In some embodiments, three or more amino acid substitutions include T43A / D126A / K168A. In some embodiments, three or more amino acid substitutions include T43A / P127A / R129A. In some embodiments, three or more amino acid substitutions include T43A / P127A / K168A. In some embodiments, three or more amino acid substitutions include T43A / R129A / K168A. In some embodiments, three or more amino acid substitutions include D126A / P127A / R129A. In some embodiments, three or more amino acid substitutions include D126A / P127A / K168A. In some embodiments, three or more amino acid substitutions include D126A / R129A / K168A. In some embodiments, three or more amino acid substitutions include P127A / R129A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / K170A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / K170A. In some embodiments, three or more amino acid substitutions include Y40A / P127A / K170A.In some embodiments, three or more amino acid substitutions include Y40A / R129A / K170A. In some embodiments, three or more amino acid substitutions include Y40A / K168A / K170A. In some embodiments, three or more amino acid substitutions include T43A / D126A / K170A. In some embodiments, three or more amino acid substitutions include T43A / P127A / K170A. In some embodiments, three or more amino acid substitutions include T43A / R129A / K170A. In some embodiments, three or more amino acid substitutions include T43A / K168A / K170A. In some embodiments, three or more amino acid substitutions include D126A / P127A / K170A. In some embodiments, three or more amino acid substitutions include D126A / R129A / K170A. In some embodiments, three or more amino acid substitutions include D126A / K168A / K170A. In some embodiments, three or more amino acid substitutions include P127A / R129A / K170A. In some embodiments, three or more amino acid substitutions include P127A / K168A / K170A. In some embodiments, three or more amino acid substitutions include R129A / K168A / K170A.

[0430] 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, four or more amino acid substitutions comprise Y40A / T43A / D126A / P127A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / D126A / R129A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / D126A / K168A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / P127A / R129A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / P127A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / R129A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / P127A / R129A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / P127A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / R129A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / R129A / K168A. In some embodiments, four or more amino acid substitutions include T43A / D126A / P127A / R129A. In some embodiments, four or more amino acid substitutions include T43A / D126A / P127A / K168A. In some embodiments, four or more amino acid substitutions include T43A / D126A / R129A / K168A. In some embodiments, four or more amino acid substitutions include T43A / P127A / R129A / K168A. In some embodiments, four or more amino acid substitutions include D126A / P127A / R129A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / D126A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / P127A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / R129A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / K168A / K170A. In some embodiments, the four or more amino acid substitutions include Y40A / D126A / P127A / K170A.In some embodiments, four or more amino acid substitutions include Y40A / D126A / R129A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / K168A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / P127A / R129A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / P127A / K168A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / R129A / K168A / K170A. In some embodiments, four or more amino acid substitutions include T43A / D126A / P127A / K170A. In some embodiments, four or more amino acid substitutions include T43A / D126A / R129A / K170A. In some embodiments, four or more amino acid substitutions include T43A / D126A / K168A / K170A. In some embodiments, four or more amino acid substitutions include T43A / P127A / R129A / K170A. In some embodiments, four or more amino acid substitutions include T43A / P127A / K168A / K170A. In some embodiments, four or more amino acid substitutions include T43A / R129A / K168A / K170A. In some embodiments, four or more amino acid substitutions include D126A / P127A / R129A / K170A. In some embodiments, four or more amino acid substitutions include D126A / P127A / K168A / K170A. In some embodiments, four or more amino acid substitutions include D126A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions include P127A / R129A / K168A / K170A.

[0431] In some embodiments, 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. In some embodiments, five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A. In some embodiments, five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / K168A. In some embodiments, five or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A. In some embodiments, five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / R129A / K168A. In some embodiments, five or more amino acid substitutions include T43A / D126A / P127A / R129A / K168A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / P127A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / R129A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / P127A / R129A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / P127A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / D126A / P127A / R129A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / D126A / P127A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / D126A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / P127A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include T43A / D126A / P127A / R129A / K170A. In some embodiments, five or more amino acid substitutions include T43A / D126A / P127A / K168A / K170A.In some embodiments, five or more amino acid substitutions include T43A / D126A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include T43A / P127A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include D126A / P127A / R129A / K168A / K170A.

[0432] In some embodiments, the variant IL-12 p35 subunit comprises six or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A. In some embodiments, six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K168A. In some embodiments, six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K170A. In some embodiments, six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions include Y40A / T43A / P127A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions include Y40A / T43A / D126A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions include T43A / D126A / P127A / R129A / K168A / K170A.

[0433] In some embodiments, the variant IL-12 p35 subunit comprises seven or more amino acid substitutions selected from the group consisting of Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0434] In some embodiments, the variant IL-12 p35 subunit comprises an amino acid sequence 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 (T43A / R129A), SEQ ID NO: 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 IDNO: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 IDNO: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: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 IDNO: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 IDNO: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 IDNO:109(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 IDNO: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 / R129A / 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)、SEQID NO:132(Y40A / P127A / R129A / K170A)、SEQ ID NO:133(Y40A / P127A / K168A / K170A)、SEQ IDNO:134(Y40A / R129A / K168A / K170A)、SEQ ID NO:135(T43A / D126A / 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)、SEQID 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)、SE Q 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 IDNO:158(T43A / P127A / R129A / K168A / K170A), SEQ ID NO:159(D126A / P127A / R129A / K168A / K170A), 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(Y40A / T43A / D126A / R129A / K168A / K170A), SEQ ID NO: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) (such as Figure 5-9 , 78 and 80).

[0435] 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 NO: 24-86, SEQ ID NO: 103-166, and SEQ ID NO: 87 (C74S) (e.g. Figure 1 , 5 -9, 78 and 80).

[0436] 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 consisting of: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S and Y40T. In some embodiments, the variant IL-12 p35 subunit comprises any one of SEQ ID NOs: 177-187.

[0437] 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 consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

[0438] 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 one of SEQ ID NOs: 199-214.

[0439] 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 consisting of P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

[0440] 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 one of SEQ ID NOs: 279-290.

[0441] 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 consisting of R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W and R129Y.

[0442] 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 one of SEQ ID NOs: 215-231.

[0443] 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 consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

[0444] 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 one of SEQ ID NOs: 232-247.

[0445] 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 consisting of 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 one of SEQ ID NOs: 188-198 or 306-308.

[0446] 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. In some embodiments, the variant IL-12 p35 subunit comprises any one of SEQ ID NO: 248 or 249.

[0447] In some embodiments, variant IL-12 The 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) a second substitution mutation selected from the group consisting of D126A, D126C, D126E, D126F, D126G, 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, R129 Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K16 8N, K170P, K170Q, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

[0448] 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 one of SEQ ID NO: 250-253 or 311.

[0449] 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 one of SEQ ID NOs: 254-257 or 309.

[0450] 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 one of SEQ ID NO: 258-261 or 310.

[0451] 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. In some embodiments, the variant IL-12 p35 subunit comprises any one of SEQ ID NOs: 262-267.

[0452] 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. In some embodiments, the variant IL-12 p35 subunit comprises any one of SEQ ID NOs: 268-270.

[0453] 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. In some embodiments, the variant IL-12 p35 subunit comprises any one of SEQ ID NOs: 271-274.

[0454] 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 one of SEQ ID NOs: 275-278.

[0455] In some embodiments, the variant IL-12 p35 subunit may further comprise a C74S substitution mutation.

[0456] 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, L 197, M198, M199, M100, M101, M102, M103, M104, M105, M106, M107, M108, M109, M110, M111, M112, M113, M114, M115, M116, M117, M118, M119, M120, M121, M122, M123, M124, M125, K128, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, F166, Y167, 1171, R181, 1182, R183, V185, T186, D188, R189, V190, M191, S192, Y193, N195, A196, and S197. Figure 1 , 5 -9, 78, and 80 comprise one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more additional amino acid substitutions. In some embodiments, the above, below, or Figure 1 , 5-9, 78, and 80 comprise 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 (e.g., Fig. 9 ), and further comprising 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.

[0457] 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; however, changes in one or more of the foregoing parameters (such as, for example, binding affinity) are not necessarily associated with changes in one or more of the other foregoing parameters (such as, for example, potency or activity). In some embodiments, one or more amino acid substitutions in the variant IL-12 p35 subunit result in a change in binding affinity to IL-12Rβ2 compared to the binding affinity of the reference IL-12. In some embodiments, one or more amino acid substitutions reduce the binding affinity of the variant IL-12 p35 subunit to IL-12Rβ2 compared to the reference IL-12. In some embodiments, the reference IL-12 includes one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein. 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.

[0458] Various 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 immunoassay, immunoprecipitation, Biacore assay, KinExA assay, fluorescence activated cell sorting (FACS), Octet assay, Western blot analysis, etc. The binding activity of the non-naturally occurring IL-12 variants of the present disclosure can be determined by any suitable method known in the art, such as, for example, surface plasmon resonance (SPR) assay, enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Biacore assay, KinExA assay, etc.

[0459] One of ordinary skill in the art will appreciate that binding affinity can also be used as a measure of the "strength" of a non-covalent interaction between two binding partners (e.g., variant IL-12 p35 subunit and IL-12Rβ2). The binding affinity between two molecules can be determined by determining the dissociation constant (K D ). K, in turn, can be determined by measuring the kinetics of complex formation and dissociation using a suitable assay known in the art (such as, for example, an SPR assay). D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant k a (or k on ) and the dissociation rate constant k d (or k off ). D By the following equation and k a and k d Related: K D =k d / k a The value of the dissociation constant can be determined directly by well-known methods.

[0460] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, non-naturally occurring IL-12 variants) to elicit a response at a certain dose or concentration in a given biological system or experimental environment. The potency of the non-naturally occurring IL-12 variants disclosed herein can be determined by any suitable method known in the art, such as, for example, IL-12 HEK reporter gene assay (e.g., InvivoGen's IL-12 HEK reporter gene assay (Catalog No. hkb-il12)), ligand binding assay (e.g., ELISA or flow cytometry) and / or functional assay. Typically, the change in potency can be graphically displayed as a response curve moving to the left or right compared to the control. A response curve moving to the right generally indicates a decrease in potency, while a response curve moving to the left generally indicates an increase in potency. As used herein, the term "activity" refers to a specific response elicited by a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, non-naturally occurring IL-12 variants) at a given dose or concentration in a specific biological system or experimental environment. The activity of the non-naturally occurring IL-12 variants of the present disclosure can be determined by any suitable method known in the art, such as, for example, a ligand binding assay and / or a functional assay. Typically, a change in activity can be graphically displayed as an upward or downward shift in a response curve compared to a control. An upward shift in a response curve generally indicates an increase in activity, while a downward shift in a response curve generally indicates a decrease in activity.

[0461] As used herein, the term "manufacturability" refers to any property that can affect the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, etc. at a scale and quantity sufficient for administration to an individual. Examples of properties that affect manufacturability include, but are not limited to, stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, 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. (such as, for example, a non-naturally occurring IL-12 variant) to retain the same properties and characteristics that it had when manufactured within specific limits and / or storage and / or use parameters. The stability of the non-naturally occurring IL-12 variants of the present disclosure can be determined by any suitable method known in the art, such as, for example, ELISA, Western blotting, 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 quantity of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, 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 art. Any suitable method can be used to quantify or determine expression yield, such as, for example, UV absorption measurements, colorimetric assays (e.g., Bradford assays, BCA assays, and Lowry assays), and fluorescence assays.

[0462] In some embodiments, the binding affinity of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit to IL-12Rβ2 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 the reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0463] In some embodiments, the binding affinity of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit to IL-12Rβ2 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 the reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0464] In some embodiments, the binding affinity of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit to IL-12Rβ2 is below the lowest detectable level of the assay, and the binding affinity of the reference IL-12 to IL-12Rβ2 is between or equivalent to the lowest detectable level or the highest detectable level of the assay (i.e., the binding affinity of the reference IL-12 may be equal to the lowest detectable level, the highest detectable level, or a value between the lowest detectable level and the highest detectable level; 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 the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12Fc fusion protein.

[0465] In some embodiments, the potency of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit is reduced by 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, 3.1-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, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times, 11.0 times, 12.0 times, 13.0 times times, 14.0 times, 15.0 times, 16.0 times, 17.0 times, 18.0 times, 19.0 times, 20.0 times, 21.0 times, 22.0 times, 23.0 times, 24.0 times, 25.0 times, 30.0 times, 35.0 times, 40.0 times, 45.0 times, 50.0 times, 100.0 times, 150.0 times, 200.0 times, 250.0 times, 300.0 times, 350.0 times, 400.0 times, 450.0 times, 50 0.0 times, 550.0 times, 600.0 times, 650.0 times, 700.0 times, 750.0 times, 800.0 times, 850.0 times, 900.0 times, 950.0 times, 1000.0 times, 2000.0 times, 3000.0 times, 4000.0 times, 5000.0 times, 6000.0 times, 7000.0 times, 8000.0 times, 9000.0 times, 10,000.0 times or more, as determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter gene assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0466] In some embodiments, the potency of the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit is reduced by 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 50.0-fold, or about 50.0-fold to about 50.0-fold, compared to the potency of the reference IL-12. about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more, as determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter gene assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0467] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit has reduced ability to stimulate STAT4 signaling compared to a reference IL-12, as determined by an assay. Reduced ability to stimulate STAT4 signaling can refer to a decrease in the maximum response observed and / or EC 50In 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 the reference IL-12, as determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter gene assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0468] 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 determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter gene assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0469] In some embodiments, the non-naturally occurring IL-12 variant and / or variant IL-12 p35 subunit has reduced ability to stimulate IFNγ production compared to a reference IL-12, as determined by an assay. Reduced ability to stimulate IFNγ production can refer to a decrease in the maximum response observed and / or 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 is reduced by 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, 3.1-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, 1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times, 11.0 times, 12.0 times, 13 .0 times, 14.0 times, 15.0 times, 16.0 times, 17.0 times, 18.0 times, 19.0 times, 20.0 times, 21.0 times, 22.0 times, 23.0 times, 24.0 times, 25.0 times, 30.0 times, 35.0 times, 40.0 times, 45.0 times, 50.0 times, 100.0 times, 150.0 times, 200.0 times, 250.0 times, 300.0 times, 350.0 times, 400.0 times, 450.0 times, 5 fold, 500.0 fold, 600.0 fold, 650.0 fold, 700.0 fold, 750.0 fold, 800.0 fold, 850.0 fold, 900.0 fold, 950.0 fold, 1000.0 fold, 2000.0 fold, 3000.0 fold, 4000.0 fold, 5000.0 fold, 6000.0 fold, 7000.0 fold, 8000.0 fold, 9000.0 fold, 10,000.0 fold or more, as determined by an assay. In some embodiments, the assay comprises an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA or ELISpot assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0470] 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 reduced by 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 50.0-fold, about 5 ... .0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 80 0.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times , about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more, as determined by an assay. In some embodiments, the assay comprises an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA or ELISpot assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0471] 2. p40 subunit :

[0472] 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, wherein the 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) an IL-12p40 subunit.

[0473] In some embodiments, the IL-12 p40 subunit comprises a variant IL-12 p40 subunit.

[0474] 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 comprising: C177S, C252S, and C177S / C252S.

[0475] 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) (e.g. Figure 1 and 10 ).

[0476] 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 (e.g., Figure 1 and 10 ).

[0477] In addition to the novel, non-naturally occurring IL-12 p35 variants described above, various modifications to 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, E 166, S168, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, M18 9. H194, K195, L196, K197, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, In some embodiments, the above, below, or Figure 1 and 10 53, 54, 55, 56, 57, 58, 59, 60 or more additional amino acid substitutions. In some embodiments, the (variant) IL-12 p40 subunit described above, below, or in any of the above, below, or Figure 1 and 10The (variant) IL-12 p40 subunit described in any of the foregoing 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.

[0478] 3. Single-chain IL-12 complex and domain linker :

[0479] 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.

[0480] In some embodiments, the non-naturally occurring IL-12 variant comprises a single-chain IL-12 complex (sc-IL-12) in which the variant IL-12 p35 subunit and the (variant) IL-12 p40 subunit are linked. In further embodiments, the subunits are linked together using a domain linker (also referred to herein as a "linker" or "linker domain"). In yet further 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 (e.g. Figure 4 ).

[0481] In some embodiments, the non-naturally occurring IL-12 variant comprises a single-chain IL-12 complex (sc-IL-12), wherein the variant IL-12 p35 subunit and the (variant) IL-12 p40 subunit are connected. In a further embodiment, the C-terminus of the IL-12 p35 subunit is connected to the N-terminus of the (variant) IL-12 p40 subunit (sc-IL-12 (p35 / p40)). In a further embodiment, the sc-IL-12 (p35 / p40) further comprises a linker domain, wherein the C-terminus of the IL-12 p35 subunit is connected to the N-terminus of the linker domain, and the C-terminus of the linker domain is connected to the N-terminus of the (variant) IL-12 p40 subunit. In even further 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 (e.g. Figure 4 ).

[0482] In some embodiments, the non-naturally occurring IL-12 variant comprises a single-chain IL-12 complex (sc-IL-12), wherein the variant IL-12 p35 subunit and the (variant) IL-12 p40 subunit are connected. In some embodiments, the C-terminus of the (variant) IL-12 p40 subunit is connected to the N-terminus of the IL-12 p35 subunit (sc-IL-12 (p40 / p35)). In yet further embodiments, sc-IL-12 (p40 / p35) further comprises a linker domain, wherein the C-terminus of the (variant) IL-12 p40 subunit is connected to the N-terminus of the linker domain, and the C-terminus of the linker domain is connected to the N-terminus of the IL-12 p35 subunit. In even further embodiments, the linker domain comprises an amino acid 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 (e.g. Figure 4 ).

[0483] In some embodiments, the subunits are not attached via a linker domain.

[0484] Any of the sc-IL-12 complexes described herein can be utilized to produce heterodimeric IL-12 Fc fusion proteins and / or homodimeric IL-12 Fc fusion proteins, as discussed in further detail below.

[0485] 4. Compositions with improved half-life :

[0486] 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.

[0487] In some embodiments, a non-naturally occurring IL-12 variant is provided, wherein one or more amino acid substitutions of 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 increased by 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, 3.1-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, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold, fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 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 comprises 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.

[0488] Various methods for determining the half-life of an agent (such as, for example, IL-12) are known in the art. Those skilled in the art will be able to easily determine and adopt any number of such methods to determine the change in half-life. In some embodiments, half-life is measured using at least one sample selected from the group comprising: (i) one or more blood samples, (ii) one or more plasma samples, (iii) one or more serum samples, (iv) one or more tissue samples, and (v) any combination thereof. In order to measure half-life, one, two, three, four, five, six, seven, eight, nine, 10 or more samples (as described above) can be used to measure half-life.

[0489] 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, wherein the Fc domain comprises 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 (e.g. Figure 3 ), (ii) albumin, (iii) one or more unstructured biodegradable polypeptides ("XTEN"), or (iv) polyethylene glycol (PEG).

[0490] 5. Nucleic acids and vectors :

[0491] According to any of the aspects and embodiments described herein, the present disclosure provides a nucleic acid composition encoding: (i) a non-naturally occurring IL-12 variant, (ii) a variant IL-12 p35 subunit, (iii) a (variant) IL-12 p40 subunit, (iv) a sc-IL-12 complex, and (v) a domain linker, as described above.

[0492] In some embodiments, one or more nucleic acids encoding components of the present invention are incorporated into expression cassettes or expression vectors. It will be appreciated by those skilled in the art that expression cassettes typically include constructs of genetic material containing coding sequences and sufficient regulatory information to guide the appropriate transcription and / or translation of coding sequences in recipient cells (in vivo and / or in vitro). Typically, expression cassettes can be inserted into vectors to target desired host cells and / or individuals. Therefore, in some embodiments, expression cassettes of the present disclosure include coding sequences of polypeptides as disclosed herein, and the coding sequences are operably linked to expression control elements, such as promoters, and optionally, any other nucleic acid sequences or combinations thereof (such as, for example, replication origins, selectable markers, ribosome binding sites, inducers, etc.) that affect transcription or translation of coding sequences.

[0493] In some embodiments, the nucleotide sequence is incorporated into an expression vector. It will be understood by those skilled in the art that the term "vector" generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and it can be used for the purpose of transformation (e.g., introducing heterologous DNA into a host cell). Therefore, in some embodiments, the vector can be a replicon (such as, for example, a plasmid, a phage, or a cosmid) into which another DNA segment can be inserted to cause replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0494] In some embodiments, the expression vector can be a viral vector. It will be appreciated by those skilled in the art that the term "viral vector" is widely used to refer to nucleic acid molecules (e.g., transfer plasmids) including virus-derived nucleic acid elements, which generally promote the transfer or integration of nucleic acid molecules into the cell genome, or refer to viral particles mediating nucleic acid transfer, and may further include oncolytic viruses (naturally occurring viruses and viruses recombinantly produced or modified in a laboratory or clinical setting). In addition to the nucleic acid of interest, viral particles generally include various viral components, and sometimes also host cell components. The term viral vector may refer to a virus or viral particle capable of transferring nucleic acid to a cell, or to the nucleic acid itself being transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. In some embodiments, viral vectors are baculovirus vectors, retroviral vectors, or lentiviral vectors. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements primarily derived from retroviruses or parts thereof. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements derived primarily from lentiviruses (a genus of retroviruses), or portions thereof, including the long terminal repeat (LTR).

[0495] Thus, also provided herein are vectors, plasmids or viruses containing one or more 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 acid may be contained within said vector capable of directing its expression in, for example, cells transformed / transduced with the vector. Vectors suitable for use in prokaryotic and eukaryotic cells are known in the art and are commercially available or can be readily prepared by a skilled artisan.

[0496] Those skilled in the art will appreciate that the nucleic acid composition will depend on the configuration of the non-naturally occurring IL-12 variant. Thus, for example, when the configuration requires three amino acid sequences (e.g., a non-naturally occurring IL-12 variant in which a domain linker is used to connect the variant IL-12 p35 and (variant) IL-12 p40 subunits), the three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, for other configurations, when only two nucleic acids are required, they can be incorporated into one or two expression vectors.

[0497] The DNA vector can be introduced into a host cell (such as, for example, a eukaryotic cell or a prokaryotic cell) via 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, scratch loading, ballistic introduction, nuclear poration, hydrodynamic shock, infection, and the like.

[0498] Viral vectors that can 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 virus, simian virus 40 (SV40), bovine papilloma virus vectors, and the like.

[0499] 6. Recombinant cells and cell culture :

[0500] 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 culture medium suitable for culturing the cells described herein. Techniques for transforming a variety of the above-mentioned cells and species are known in the art. Therefore, a cell culture comprising at least one recombinant cell as disclosed herein is also within the scope of the present application. Methods and systems suitable for producing and maintaining cell cultures are known in the art.

[0501] Host cells can be used for production purposes to propagate one or more nucleic acids encoding: (i) non-naturally occurring IL-12 variants, (ii) variant IL-12 p35 subunits, (iii) (variant) IL-12 p40 subunits, (iv) sc-IL-12 complexes, and (v) domain linkers, as well as combinations and / or components thereof. Host cells can include prokaryotic or eukaryotic cells, where production of non-naturally occurring IL-12 variants is particularly intended. Non-limiting examples of host cells include bacterial cells (such as, for example, cells of Gram-positive bacteria (e.g., species of Bacillus, Streptomyces, and Staphylococcus) or Gram-negative bacterial cells (e.g., cells of Escherichia coli and Pseudomonas)), fungal cells or yeast cells (e.g., Saccharomyces, Pichia pastoris, and Hansenula), 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 cancers, glial cells, hepatocyte cell lines, Chinese hamster ovary (CHO) cells, COS cells, BHK cells, HeLa cells, D3 cells of a mouse embryonic stem cell (mESC) line, 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.), and the like.

[0502] B. Heterodimeric IL-12 Fc fusion protein

[0503] In one aspect, 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 C-terminus of the variant IL-12 p35 subunit domain is covalently attached 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 attached to the N-terminus of the second Fc domain.

[0504] In another aspect, 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 N-terminus of the variant IL-12 p35 subunit domain is covalently attached 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 attached to the C-terminus of the second Fc domain.

[0505] The heterodimeric Fc fusion proteins and various configurations of variant IL-12 p35 subunit domain, IL-12 p40 subunit domain and Fc domain are described in detail below.

[0506] 1. p35 subunit domain :

[0507] 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 attached to the N-terminus or 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 IL-12 p40 subunit domain is covalently attached to the N-terminus or the C-terminus of the second Fc domain.

[0508] In some embodiments, the 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. In some embodiments, one or more amino acid substitutions include Y40A. In some embodiments, one or more amino acid substitutions include T43A. In some embodiments, one or more amino acid substitutions include D126A. In some embodiments, one or more amino acid substitutions include P127A. In some embodiments, one or more amino acid substitutions include R129A. In some embodiments, one or more amino acid substitutions include K168A. In some embodiments, one or more amino acid substitutions include K170A.

[0509] 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, two or more amino acid substitutions include Y40A / T43A. In some embodiments, two or more amino acid substitutions include Y40A / D126A. In some embodiments, two or more amino acid substitutions include Y40A / P127A. In some embodiments, two or more amino acid substitutions include Y40A / R129A. In some embodiments, two or more amino acid substitutions include Y40A / K168A. In some embodiments, two or more amino acid substitutions include T43A / D126A. In some embodiments, two or more amino acid substitutions include T43A / P127A. In some embodiments, two or more amino acid substitutions include T43A / R129A. In some embodiments, two or more amino acid substitutions include T43A / K168A. In some embodiments, two or more amino acid substitutions include D126A / P127A. In some embodiments, two or more amino acid substitutions include D126A / R129A. In some embodiments, two or more amino acid substitutions include D126A / K168A. In some embodiments, two or more amino acid substitutions include P127A / R129A. In some embodiments, two or more amino acid substitutions include P127A / K168A. In some embodiments, two or more amino acid substitutions include R129A / K168A. In some embodiments, two or more amino acid substitutions include Y40A / K170A. In some embodiments, two or more amino acid substitutions include T43A / K170A. In some embodiments, two or more amino acid substitutions include D126A / K170A. In some embodiments, two or more amino acid substitutions include P127A / K170A. In some embodiments, the two or more amino acid substitutions include R129A / K170A. In some embodiments, the two or more amino acid substitutions include K168A / K170A.

[0510] 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, three or more amino acid substitutions include Y40A / T43A / D126A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / P127A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / R129A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / P127A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / R129A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / P127A / R129A. In some embodiments, three or more amino acid substitutions include Y40A / P127A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / R129A / K168A. In some embodiments, three or more amino acid substitutions include T43A / D126A / P127A. In some embodiments, three or more amino acid substitutions include T43A / D126A / R129A. In some embodiments, three or more amino acid substitutions include T43A / D126A / K168A. In some embodiments, three or more amino acid substitutions include T43A / P127A / R129A. In some embodiments, three or more amino acid substitutions include T43A / P127A / K168A. In some embodiments, three or more amino acid substitutions include T43A / R129A / K168A. In some embodiments, three or more amino acid substitutions include D126A / P127A / R129A. In some embodiments, three or more amino acid substitutions include D126A / P127A / K168A. In some embodiments, three or more amino acid substitutions include D126A / R129A / K168A. In some embodiments, three or more amino acid substitutions include P127A / R129A / K168A. In some embodiments, three or more amino acid substitutions include Y40A / T43A / K170A. In some embodiments, three or more amino acid substitutions include Y40A / D126A / K170A. In some embodiments, three or more amino acid substitutions include Y40A / P127A / K170A.In some embodiments, three or more amino acid substitutions include Y40A / R129A / K170A. In some embodiments, three or more amino acid substitutions include Y40A / K168A / K170A. In some embodiments, three or more amino acid substitutions include T43A / D126A / K170A. In some embodiments, three or more amino acid substitutions include T43A / P127A / K170A. In some embodiments, three or more amino acid substitutions include T43A / R129A / K170A. In some embodiments, three or more amino acid substitutions include T43A / K168A / K170A. In some embodiments, three or more amino acid substitutions include D126A / P127A / K170A. In some embodiments, three or more amino acid substitutions include D126A / R129A / K170A. In some embodiments, three or more amino acid substitutions include D126A / K168A / K170A. In some embodiments, three or more amino acid substitutions include P127A / R129A / K170A. In some embodiments, three or more amino acid substitutions include P127A / K168A / K170A. In some embodiments, three or more amino acid substitutions include R129A / K168A / K170A.

[0511] 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, four or more amino acid substitutions comprise Y40A / T43A / D126A / P127A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / D126A / R129A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / D126A / K168A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / P127A / R129A. In some embodiments, four or more amino acid substitutions comprise Y40A / T43A / P127A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / R129A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / P127A / R129A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / P127A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / R129A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / R129A / K168A. In some embodiments, four or more amino acid substitutions include T43A / D126A / P127A / R129A. In some embodiments, four or more amino acid substitutions include T43A / D126A / P127A / K168A. In some embodiments, four or more amino acid substitutions include T43A / D126A / R129A / K168A. In some embodiments, four or more amino acid substitutions include T43A / P127A / R129A / K168A. In some embodiments, four or more amino acid substitutions include D126A / P127A / R129A / K168A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / D126A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / P127A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / R129A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / T43A / K168A / K170A. In some embodiments, the four or more amino acid substitutions include Y40A / D126A / P127A / K170A.In some embodiments, four or more amino acid substitutions include Y40A / D126A / R129A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / D126A / K168A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / P127A / R129A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / P127A / K168A / K170A. In some embodiments, four or more amino acid substitutions include Y40A / R129A / K168A / K170A. In some embodiments, four or more amino acid substitutions include T43A / D126A / P127A / K170A. In some embodiments, four or more amino acid substitutions include T43A / D126A / R129A / K170A. In some embodiments, four or more amino acid substitutions include T43A / D126A / K168A / K170A. In some embodiments, four or more amino acid substitutions include T43A / P127A / R129A / K170A. In some embodiments, four or more amino acid substitutions include T43A / P127A / K168A / K170A. In some embodiments, four or more amino acid substitutions include T43A / R129A / K168A / K170A. In some embodiments, four or more amino acid substitutions include D126A / P127A / R129A / K170A. In some embodiments, four or more amino acid substitutions include D126A / P127A / K168A / K170A. In some embodiments, four or more amino acid substitutions include D126A / R129A / K168A / K170A. In some embodiments, the four or more amino acid substitutions include P127A / R129A / K168A / K170A.

[0512] 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, five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A. In some embodiments, five or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / K168A. In some embodiments, five or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A. In some embodiments, five or more amino acid substitutions comprise Y40A / T43A / P127A / R129A / K168A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / R129A / K168A. In some embodiments, five or more amino acid substitutions include T43A / D126A / P127A / R129A / K168A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / P127A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / R129A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / D126A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / P127A / R129A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / P127A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / T43A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / D126A / P127A / R129A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / D126A / P127A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / D126A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include Y40A / P127A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include T43A / D126A / P127A / R129A / K170A. In some embodiments, five or more amino acid substitutions include T43A / D126A / P127A / K168A / K170A.In some embodiments, five or more amino acid substitutions include T43A / D126A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include T43A / P127A / R129A / K168A / K170A. In some embodiments, five or more amino acid substitutions include D126A / P127A / R129A / K168A / K170A.

[0513] 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, six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K168A. In some embodiments, six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K170A. In some embodiments, six or more amino acid substitutions comprise Y40A / T43A / D126A / P127A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions comprise Y40A / D126A / P127A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions include Y40A / T43A / P127A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions include Y40A / T43A / D126A / R129A / K168A / K170A. In some embodiments, six or more amino acid substitutions include T43A / D126A / P127A / R129A / K168A / K170A.

[0514] In some embodiments, the variant IL-12 p35 subunit domain comprises seven or more amino acid substitutions selected from the group consisting of: Y40A, T43A, D126A, P127A, R129A, K168A, and K170A.

[0515] 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 SEQ ID NOs: 103-166 (eg, Figure 5-9 , 78 and 80).

[0516] 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 NO: 24-87, and SEQ ID NO: 103-166 (e.g., Figure 1 , 5 -9, 78 and 80).

[0517] 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 consisting of: Y40C, Y40D, Y40E, Y40G, Y40K, Y40N, Y40P, Y40Q, Y40R, Y40S, and Y40T. In some embodiments, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 177-187.

[0518] 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 consisting of D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, and D126W.

[0519] 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 one of SEQ ID NOs: 199-214.

[0520] 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 consisting of P127C, P127D, P127E, P127F, P127G, P127H, P127K, P127M, P127N, P127Q, P127R, and P127S.

[0521] 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 one of SEQ ID NOs: 279-290.

[0522] 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 consisting of R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W and R129Y.

[0523] 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 one of SEQ ID NOs: 215-231.

[0524] 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 consisting of K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K168N, K168P, K168Q, K168S, K168T, K168W, and K168Y.

[0525] 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 one of SEQ ID NOs: 232-247.

[0526] 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 consisting of 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 one of SEQ ID NOs: 188-198 or 306-308.

[0527] 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. In some embodiments, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NO: 248 or 249.

[0528] In some embodiments, variant IL-12 The 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) a second substitution mutation selected from the group consisting of D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126P, D126Q, D126R, D126S, D126T, D126V, D126W, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129 I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129T, R129V, R129W, R12 9Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K168L, K168M, K1 70S, K170T, K170V, K170F, K170L, K170N, and K170W.

[0529] 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 one of SEQ ID NO: 250-253 or 311.

[0530] 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 one of SEQ ID NOs: 254-257 or 309.

[0531] 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 one of SEQ ID NOs: 258-261 or 310.

[0532] 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. In some embodiments, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 262-267.

[0533] 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. In some embodiments, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 268-270.

[0534] 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. In some embodiments, the variant IL-12 p35 subunit domain comprises any one of SEQ ID NOs: 271-274.

[0535] 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 one of SEQ ID NOs: 275-278.

[0536] In some embodiments, the variant IL-12 p35 subunit domain may further comprise a C74S substitution mutation.

[0537] In addition to the novel, heterodimeric Fc fusion proteins described above, additional modifications to various residues are known in the art. 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, L 197, M198, M199, M100, M101, M102, M103, M104, M105, M106, M107, M108, M109, M110, M111, M112, M113, M114, M115, M116, M117, M118, M119, M120, M121, M122, M123, M124, M125, K128, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, F166, Y167, 1171, R181, 1182, R183, V185, T186, D188, R189, V190, M191, S192, Y193, N195, A196, and S197. Figure 1 , 5 -9, 78, and 80 comprises one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more additional amino acid substitutions. In some embodiments, the above, below, or Figure 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 (e.g., Fig. 9 ), and further comprising 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.

[0538] 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; however, changes in one or more of the foregoing parameters (such as, for example, binding affinity) are not necessarily associated with changes in one or more of the other foregoing parameters (such as, for example, potency or activity). In some embodiments, one or more amino acid substitutions in the variant IL-12 p35 subunit domain result in a change in binding affinity to IL-12Rβ2 compared to the binding affinity of the reference IL-12. In some embodiments, one or more amino acid substitutions reduce the binding affinity of the variant IL-12 p35 subunit domain to IL-12Rβ2 compared to the reference IL-12. In some embodiments, the reference IL-12 includes one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, IL-12 Fc fusion proteins, 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.

[0539] 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 immunoassay, immunoprecipitation, Biacore assay, KinExA assay, fluorescence activated cell sorting (FACS), Octet assay, Western blot analysis, etc. The binding activity of the heterodimeric Fc fusion proteins of the present disclosure can be determined by any suitable method known in the art, such as, for example, surface plasmon resonance (SPR) assay, enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Biacore assay, KinExA assay, etc.

[0540] One of ordinary skill in the art will appreciate that binding affinity can also be used as a measure of the "strength" of a non-covalent interaction between two binding partners (e.g., a variant IL-12 p35 subunit domain and IL-12Rβ2). The binding affinity between two molecules can be determined by determining the dissociation constant (K D ). K, in turn, can be determined by measuring the kinetics of complex formation and dissociation using a suitable assay known in the art (such as, for example, an SPR assay). D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant k a (or k on ) and the dissociation rate constant kd (or k off ). D By the following equation and k a and k d Related: K D =k d / k a The value of the dissociation constant can be determined directly by well-known methods.

[0541] As used herein, the term "potency" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a heterodimeric Fc fusion protein) to elicit a response at a certain dose or concentration in a given biological system or experimental environment. The potency of the heterodimeric Fc fusion protein disclosed herein can be determined by any suitable method known in the art, such as, for example, an IL-12 HEK reporter gene assay (e.g., InvivoGen's IL-12 HEK reporter gene assay (Catalog No. hkb-il12)), a ligand binding assay (e.g., ELISA or flow cytometry) and / or a functional assay. Typically, the change in potency can be graphically displayed as a response curve moving to the left or right compared to a control. A response curve moving to the right generally indicates a decrease in potency, while a response curve moving to the left generally indicates an increase in potency. As used herein, the term "activity" refers to a specific response elicited by a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a heterodimeric Fc fusion protein) at a given dose or concentration in a specific biological system or experimental environment. The activity of the heterodimeric Fc fusion proteins disclosed herein can be determined by any suitable method known in the art, such as, for example, ligand binding assays and / or functional assays. Typically, changes in activity can be graphically displayed as a response curve moving up or down compared to a control. An upward movement of the response curve typically indicates an increase in activity, while a downward movement of the response curve typically indicates a decrease in activity.

[0542] As used herein, the term "manufacturability" refers to any property that can affect the process of producing and / or storing a given protein, cytokine, fusion protein, antibody, etc. on a scale and quantity sufficient for administration to an individual. Examples of properties that affect manufacturability include, but are not limited to, the stability, purity, aggregation level, and / or expression yield of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a heterodimeric Fc fusion protein). As used herein, the term "stability" refers to the ability of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a heterodimeric Fc fusion protein) to retain the same properties and characteristics it had when manufactured within specific limits and / or storage and / or use parameters. The stability of the heterodimeric Fc fusion protein disclosed herein can be determined by any suitable method known in the art, such as, for example, ELISA, Western blotting, 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 quantity of a given protein, cytokine, fusion protein, antibody, etc. (such as, for example, a 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 art. Any suitable method can be used to quantify or determine expression yield, such as, for example, UV absorption measurements, colorimetric assays (e.g., Bradford assays, BCA assays, and Lowry assays), and fluorescence assays.

[0543] In some embodiments, the binding affinity of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain to IL-12Rβ2 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 determined by an SPR assay. In some embodiments, the binding affinity of the heterodimeric Fc fusion protein and / or variant IL-12p35 subunit domain to IL-12Rβ2 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 the reference IL-12, as determined by an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0544] In some embodiments, the binding affinity of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain to IL-12Rβ2 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 the reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0545] In some embodiments, the binding affinity of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain to IL-12Rβ2 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 the reference IL-12, as determined by an assay. In some embodiments, the assay comprises an SPR assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0546] In some embodiments, the binding affinity of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain to IL-12Rβ2 is below the lowest detectable level of the assay, and the binding affinity of the reference IL-12 to IL-12Rβ2 is between or equivalent to the lowest detectable level or the highest detectable level of the assay (i.e., the binding affinity of the reference IL-12 may be equal to the lowest detectable level, the highest detectable level, or a value between the lowest detectable level and the highest detectable level; 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 the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0547] In some embodiments, the potency of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain is reduced by 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, 3.1-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 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times, 11.0 times, 12.0 times, 13. 0 times, 14.0 times, 15.0 times, 16.0 times, 17.0 times, 18.0 times, 19.0 times, 20.0 times, 21.0 times, 22.0 times, 23.0 times, 24.0 times, 25.0 times, 30.0 times, 35.0 times, 40.0 times, 45.0 times, 50.0 times, 100.0 times, 150.0 times, 200.0 times, 250.0 times, 300.0 times, 350.0 times, 400.0 times, 450.0 times, 5 In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, commercially available IL-12 molecules, or IL-12 Fc fusion proteins.

[0548] In some embodiments, the potency of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain is reduced by 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 50.0-fold, about 5 ... 0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times .0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, About 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more, as determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter gene assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0549] In some embodiments, the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain has reduced ability to stimulate STAT4 signaling compared to a reference IL-12, as determined by an assay. Reduced ability to stimulate STAT4 signaling can refer to a decrease in the maximum response observed and / or EC 50In 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 determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter gene assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0550] 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 determined by an assay. In some embodiments, the assay comprises an IL-12 HEK reporter gene assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0551] In some embodiments, the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain has reduced ability to stimulate IFNγ production compared to a reference IL-12, as determined by an assay. Reduced ability to stimulate IFNγ production can refer to a decrease in the maximum response observed and / or EC 50In some embodiments, the ability of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain to stimulate IFNγ production is reduced by 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 as compared to a reference IL-12. , 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times, 11.0 times, 12.0 times, 13.0 times, 14.0 times, 15.0 times, 16.0 times, 17.0 times, 18.0 times, 19.0 times, 20.0 times, 21.0 times, 22.0 times, 23.0 times, 24.0 times, 25.0 times, 30.0 times, 35.0 times, 40.0 times, 45.0 times, 50.0 times, 100.0 times, 150.0 times, 200.0 times, 250.0 times, 300.0 times, 350.0 times, 400.0 times, 450.0 times, 500.0 times, 550.0 times, 600.0 times, 650.0 times, 700.0 times, 750.0 times, 800.0 times, 850.0 times, 900.0 times, 950.0 times, 1000.0 times, 2000.0 times, 3000.0 times, 4000.0 times, 5000.0 times, 6000.0 times, 7000.0 times, 8000.0 times, 9000.0 times, 10,000.0 times or more, as determined by an assay. In some embodiments, the assay comprises an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA or ELISpot assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0552] In some embodiments, the ability of the heterodimeric Fc fusion protein and / or variant IL-12 p35 subunit domain to stimulate IFNγ production is reduced by 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 45.0-fold to about 50.0-fold, about 40.0-fold to about 55.0-fold, about 45.0-fold to about 50.0-fold, about 45 ... 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 8 0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times , about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more, as determined by an assay. In some embodiments, the assay comprises an intracellular cytokine staining assay, a Luminex bead-based cytokine release assay, an ELISA or ELISpot assay. In some embodiments, the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein.

[0553] In some embodiments, heterodimeric Fc fusion proteins and / or variant IL-12 p35 subunit domains are provided, wherein one or more amino acid substitutions of the variant IL-12 p35 subunit domain improve the half-life compared to the half-life of a reference IL-12. In some embodiments, the fusion of the variant IL-12 p35 subunit domain and / or (variant) IL-12 p40 subunit domain improves the half-life compared to the half-life of a reference. In some embodiments, heterodimeric Fc fusion proteins and / or variant IL-12 The half-life of the p35 subunit domain is decreased or increased by 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, 3.1-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, fold, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times or more. In some embodiments, the reference IL-12 includes one or more of the following: 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.

[0554] Various methods for determining the half-life of an agent (such as, for example, IL-12) are known in the art. Those skilled in the art will be able to easily determine and adopt any number of such methods to determine the change in half-life. In some embodiments, half-life is measured using at least one sample selected from the group comprising: (i) one or more blood samples, (ii) one or more plasma samples, (iii) one or more serum samples, (iv) one or more tissue samples, and (v) any combination thereof. In order to measure half-life, one, two, three, four, five, six, seven, eight, nine, 10 or more samples (as described above) can be used to measure half-life.

[0555] 2. p40 subunit domain :

[0556] 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 attached to the N-terminus or 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 IL-12 p40 subunit domain is covalently attached to the N-terminus or the C-terminus of the second Fc domain.

[0557] In some embodiments, the IL-12 p40 subunit domain comprises a variant IL-12 p40 subunit domain.

[0558] 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 comprising: C177S, C252S, and C177S / C252S.

[0559] In some embodiments, the (variant) 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 (e.g. Figure 1 and 10 ).

[0560] 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 (e.g. Figure 1 and 10 ).

[0561] In addition to the novel, heterodimeric Fc fusion proteins described above, various modifications to the IL-12 p40 subunit domain are known in the art and can be further included in the heterodimeric Fc fusion proteins described herein. Non-limiting examples of residues that can 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, E 166, S168, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, M18 9. H194, K195, L196, K197, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, In some embodiments, the above, below, or Figure 1 and 10 53, 54, 55, 56, 57, 58, 59, 60 or more additional amino acid substitutions. In some embodiments, the (variant) IL-12 p40 subunit domain described above, below, or in any of the above, below, or Figure 1 and 10The (variant) IL-12 p40 subunit domain described in any of the foregoing 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.

[0562] 3. Fc domain :

[0563] 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 attached to the N-terminus or the C-terminus of the first Fc domain, and b) a second fusion construct comprising: a (variant) IL-12 p40 subunit domain and a second Fc domain, wherein the (variant) IL-12 p40 subunit domain is covalently attached to the N-terminus or the C-terminus of the second Fc domain.

[0564] In some embodiments, the first Fc domain and / or the second Fc domain comprises the CH2-CH3 domain of human immunoglobulin G1 (IgG1) and optionally all or part of the hinge of human IgG1, and fragments thereof. In EU numbering, the CH2-CH3 domain of human IgG1 comprises amino acids 231 to 447, and the hinge comprises amino acids 216 to 230. An "Fc fragment" may contain one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or fewer amino acids from the N-terminus or the C-terminus or both, but still retains the ability to form a dimer with another Fc domain or Fc fragment, which can be detected using standard methods (such as, for example, non-denaturing chromatography, size exclusion chromatography, etc.).

[0565] In some embodiments, the first Fc domain and / or 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 (e.g. Figure 3). In some embodiments, the first Fc domain comprises SEQ ID NO: 9, and the second Fc domain comprises SEQ ID NO: 9. In some embodiments, the first Fc domain comprises SEQ ID NO: 10, and the second Fc domain comprises SEQ ID NO: 11. In some embodiments, the first Fc domain comprises SEQ ID NO 11, and the second Fc domain comprises SEQ ID NO: 10. In some embodiments, the first Fc domain comprises SEQ ID NO: 12, and the second Fc domain comprises SEQ ID NO: 13. In some embodiments, the first Fc domain comprises SEQ ID NO: 13, and the second Fc domain comprises SEQ ID NO: 12.

[0566] In some embodiments, the first Fc domain and / or the second Fc domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to 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 (e.g., Figure 3In some embodiments, the first Fc domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:9, and the second Fc domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:9. In some embodiments, the first Fc domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:10, and the second Fc domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:11. In some embodiments, the first Fc domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:11, and the second Fc domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:10. In some embodiments, the first Fc domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:12, and the second Fc domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:13. In some embodiments, the first Fc domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:13, and the second Fc domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:12.

[0567] In some embodiments, the first Fc domain and / or the second Fc domain comprises one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid substitutions.

[0568] In some embodiments, the first Fc domain and the second Fc domain comprise modifications that promote heterodimerization of the first Fc domain and the second Fc domain. In further embodiments, the first Fc domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 10 and SEQ ID NO: 12, and the second Fc domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11 and SEQ ID NO: 13. In other embodiments, the first Fc domain and the second Fc domain do not comprise modifications that promote heterodimerization of the first Fc domain and the second Fc domain.

[0569] 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 do...

Claims

1. A heterodimeric Fc fusion protein comprising: a) a first fusion construct, comprising: a variant IL-12p35 subunit domain, a first linker domain and a first Fc domain, wherein: i) the C-terminus of the variant IL-12p35 subunit domain is covalently attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached 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-12p35 subunit domain comprises one or more amino acid substitutions selected from the group consisting of: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170E, 0L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129 A. Y40G, Y40G / K170A, Y40G / K168A, Y40G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y 40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, 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-12p40 subunit domain, a second linker domain, and a second Fc domain, wherein: i) the C-terminus of the IL-12p40 subunit domain is covalently attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached 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, and iv) the IL-12p40 subunit domain comprises SEQ ID NO: 89, Optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

2. A heterodimeric Fc fusion protein comprising: a) a first fusion construct, comprising: a variant IL-12p35 subunit domain, a first linker domain and a first Fc domain, wherein: i) the C-terminus of the variant IL-12p35 subunit domain is covalently attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached 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-12p35 subunit domain comprises one or more amino acid substitutions selected from the group consisting of: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170E, 0L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129 A. Y40G, Y40G / K170A, Y40G / K168A, Y40G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y 40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, 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-12p40 subunit domain, a second linker domain, and a second Fc domain, wherein: i) the C-terminus of the IL-12p40 subunit domain is covalently attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached 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, and iv) the IL-12p40 subunit domain comprises SEQ ID NO: 89, Optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

3. A heterodimeric Fc fusion protein comprising: a) a first fusion construct, comprising: a variant IL-12p35 subunit domain, a first linker domain and a first Fc domain, wherein: i) the C-terminus of the first Fc domain is covalently attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached to the N-terminus of the variant IL-12p35 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-12p35 subunit domain comprises one or more amino acid substitutions selected from the group consisting of: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170E, 0L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129 A. Y40G, Y40G / K170A, Y40G / K168A, Y40G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y 40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, 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-12p40 subunit domain, a second linker domain, and a second Fc domain, wherein: i) the C-terminus of the second Fc domain is covalently attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached to the N-terminus of the IL-12p40 subunit domain, ii) the second linker domain comprises SEQ ID NO: 15, iii) the second Fc domain comprises SEQ ID NO: 13, and iv) the IL-12p40 subunit domain comprises SEQ ID NO: 89, Optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

4. A heterodimeric Fc fusion protein comprising: a) a first fusion construct, comprising: a variant IL-12p35 subunit domain, a first linker domain and a first Fc domain, wherein: i) the C-terminus of the first Fc domain is covalently attached to the N-terminus of the first linker domain, and the C-terminus of the first linker domain is covalently attached to the N-terminus of the variant IL-12p35 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-12p35 subunit domain comprises one or more amino acid substitutions selected from the group consisting of: Y40A, Y40A / K168A, Y40A / K168D, Y40A / K168E, Y40A / K168I, Y40A / K168M, Y40A / K168Q, Y40A / K168T, Y40A / K170A, Y40A / K170E, 0L, Y40A / K170T, Y40E, Y40E / K170A, Y40E / K168A, Y40E / K168I, Y40E / K168T, Y40E / R129 A. Y40G, Y40G / K170A, Y40G / K168A, Y40G / K168I, Y40G / K168T, Y40G / R129A, Y40P, Y40P / K170A, Y40P / K168A, Y40P / K168D, Y40P / K168I, Y40P / K168T, Y40R, Y40S, Y40S / K168I, Y 40S / K168T, Y40S / K170A, Y40S / K170L, Y40S / K170T, Y40S / R129A, K170A, K170A / K168A, K170A / K168I, K170A / K168T, K170A / R129E, 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-12p40 subunit domain, a second linker domain, and a second Fc domain, wherein: i) the C-terminus of the second Fc domain is covalently attached to the N-terminus of the second linker domain, and the C-terminus of the second linker domain is covalently attached to the N-terminus of the IL-12p40 subunit 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-12p40 subunit domain comprises SEQ ID NO: 89, Optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

5. The heterodimeric Fc fusion protein of any one of claims 1 to 4, wherein the variant IL-12p35 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-12p40 subunit domain further comprises a C177S substitution mutation.

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

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

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

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

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

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

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

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

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

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

18. The non-naturally occurring IL-12 variant of any one of claims 7-17, wherein the IL-12p40 subunit comprises a variant IL-12p40 subunit, wherein the variant IL-12p40 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 7-18, wherein the IL-12p40 subunit comprises any one of SEQ ID NOs: 4, 88, 89, and 90.

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-12p35 subunit and / or the IL-12p40 subunit: (i) an Fc domain, wherein the Fc domain comprises 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. The non-naturally occurring IL-12 variant of any one of claims 7-20, wherein the C-terminus of the variant IL-12p35 subunit is covalently attached to the N-terminus of the IL-12p40 subunit.

22. The non-naturally occurring IL-12 variant of claim 21, wherein the non-naturally occurring IL-12 variant further comprises 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, and wherein the C-terminus of the variant IL-12p35 subunit is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the IL-12p40 subunit.

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

24. The non-naturally occurring IL-12 variant of claim 23, wherein the non-naturally occurring IL-12 variant further comprises 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, and wherein the C-terminus of the IL-12p40 subunit is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the variant IL-12p35 subunit.

25. The non-naturally occurring IL-12 variant of any one of claims 7-24, 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 7-25 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 7-25.

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

29. A method for producing a non-naturally occurring IL-12 variant, the method comprising: The host cell is cultured with one or more nucleic acids or vectors under conditions that produce the non-naturally occurring variant, wherein: i) the one or more nucleic acids or vectors comprise one or more nucleic acids as claimed in claim 27, and ii) at least one substitution mutation of the variant IL-12 p35 subunit improves the half-life compared to the half-life of a reference IL-12.

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

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-12p35 subunit and / or the IL-12p40 subunit: (i) an Fc domain, wherein the Fc domain comprises 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. The method of any one of claims 29-31, wherein the non-naturally occurring IL-12 variant produced has an altered binding affinity for interleukin 12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

33. The method of claim 32, wherein the non-naturally occurring IL-12 variant produced 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 determined by an assay.

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

35. The method of claim 32, wherein the binding affinity of the non-naturally occurring IL-12 variant produced for IL-12Rβ2 is below the minimum detectable level of the assay and the binding affinity of the reference IL-12 is detectable as determined by the assay.

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

37. The method of any one of claims 29-31, wherein the potency of the non-naturally occurring IL-12 variant produced is reduced by 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 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times .0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more as determined by an assay.

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

39. The method of any one of claims 29-31, wherein the ability of the produced non-naturally occurring IL-12 variant to stimulate IFNγ production is reduced by 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 times, about 30.0 times to about 35.0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times. 0.0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about .0-fold to about 8000.0-fold, about 8000.0-fold to about 9000.0-fold, about 9000.0-fold to about 10,000.0-fold, about 10,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more as determined by an assay.

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

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

43. The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid is substituted to Y40A; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L and K170T.

44. The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid substitution mutation is Y40E; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K170A, K168A, K168I, K168T and R129A.

45. The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid substitution mutation is Y40G; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K170A, K168A, K168I, K168T and R129A.

46. ​​The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K170A, K168A, K168D, K168I and K168T.

47. The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid substitution mutation is Y40S; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K168I, K168T, K170A, K170L, K170T and R129A.

48. The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid substitution mutation is K170A; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K168A, K168I, K168T and R129E.

49. The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid substitution mutation is K170P; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K168A, K168I, K168T and R129E.

50. The heterodimeric Fc fusion protein of claim 42, wherein: i) the first amino acid substitution mutation is K170T; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K168A, K168I, K168T and R129E.

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

53. A heterodimeric Fc fusion protein as described in any one of claims 42-51, wherein the IL-12p40 subunit domain comprises a variant IL-12p40 subunit domain, wherein the variant IL-12p40 subunit domain comprises one or more amino acid substitutions selected from the group consisting of: C177S, C252S and C177S / C252S.

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

55. The heterodimeric Fc fusion protein of any one of claims 42-54, wherein: i) the first Fc domain comprises an amino acid 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 ii) the second Fc domain comprises an amino acid 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. The heterodimeric Fc fusion protein of any one of claims 42-55, wherein: 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-12p35 subunit domain is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the first Fc domain.

57. The heterodimeric Fc fusion protein of any one of claims 42-56, wherein: 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-12p40 subunit domain is covalently attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the second Fc domain.

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

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

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

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

62. A method for producing a heterodimeric Fc fusion protein, the method comprising: The host cell is cultured with one or more nucleic acids or vectors under conditions that produce the heterodimeric Fc fusion protein, wherein: i) the one or more nucleic acids or vectors comprise one or more nucleic acids as claimed in claim 60, 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.

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

64. The method of claim 62 or 63, wherein the resulting heterodimeric Fc fusion protein has an altered binding affinity for interleukin 12 receptor β2 (IL-12Rβ2) compared to the binding affinity of a reference IL-12.

65. The method of claim 64, wherein the binding affinity of the produced heterodimeric Fc fusion protein to IL-12Rβ2 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 determined by an assay.

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

67. The method of claim 64, wherein the binding affinity of the produced heterodimeric Fc fusion protein to IL-12Rβ2 is below the minimum detectable level of the assay and the binding affinity of the reference IL-12 is detectable as determined by the assay.

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

69. The method of claim 62 or 63, wherein the potency of the produced heterodimeric Fc fusion protein is reduced by 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 compared to the potency of a reference IL-12. , about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times From about 800.0 times, from about 800.0 times to about 900.0 times, from about 900.0 times to about 1000.0 times, from about 1000.0 times to about 2000.0 times, from about 2000.0 times to about 3000.0 times, from about 3000.0 times to about 4000.0 times, from about 4000.0 times to about 5000.0 times, from about 5000.0 times to about 6000.0 times, from about 6000.0 times to about 7000.0 times, from about 7000.0 times to about 8 , about 10,000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more as determined by the assay.

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

71. The method of claim 62 or 63, wherein the ability of the produced heterodimeric Fc fusion protein to stimulate IFNγ production is reduced by 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 compared to the ability of a reference IL-12 to stimulate IFNγ production , about 30.0 times to about 35.0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times. times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about fold to about 8000.0 fold, about 8000.0 fold to about 9000.0 fold, about 9000.0 fold to about 10,000.0 fold, about 10,000.0 fold to about 50,000.0 fold, about 50,000.0 fold to about 100,000.0 fold, about 100,000.0 fold to about 200,000.0 fold, about 200,000.0 fold to about 300,000.0 fold, about 300,000.0 fold or more as determined by an assay.

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

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

75. The heterodimeric Fc fusion protein of claim 74, wherein: i) the first amino acid is substituted to Y40A; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of K168A, K168D, K168E, K168I, K168M, K168Q, K168T, K170A, K170L and K170T.

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

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

78. The heterodimeric Fc fusion protein of claim 74, wherein: i) the first amino acid substitution mutation is Y40P; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K170A, K168A, K168D, K168I and K168T.

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

80. The heterodimeric Fc fusion protein of claim 74, wherein: i) the first amino acid substitution mutation is K170A; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K168A, K168I, K168T and R129E.

81. The heterodimeric Fc fusion protein of claim 74, wherein: i) the first amino acid substitution mutation is K170P; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K168A, K168I, K168T and R129E.

82. The heterodimeric Fc fusion protein of claim 74, wherein: i) the first amino acid substitution mutation is K170T; ii) the variant IL-12p35 subunit domain further comprises a second substitution mutation; and iii) the second substitution mutation is selected from the group consisting of: K168A, K168I, K168T and R129E.

83. A heterodimeric Fc fusion protein as described in claim 74, wherein the variant IL-12p35 subunit domain comprises any one of SEQ ID NO: 24, 34, 103, 104, 109, 179, 180, 183, 185, 186, 194, 196, 233, 234, 238, 240, 243, 245 and 248-278.

84. A heterodimeric Fc fusion protein as described in any one of claims 74-83, wherein the variant IL-12p35 subunit domain further comprises a C74S substitution mutation.

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

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

87. The heterodimeric Fc fusion protein of any one of claims 74-86, wherein: i) the first Fc domain comprises an amino acid 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 ii) the second Fc domain comprises an amino acid 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. The heterodimeric Fc fusion protein of any one of claims 74-87, wherein: 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 attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the variant IL-12p35 subunit domain.

89. The heterodimeric Fc fusion protein of any one of claims 74-88, wherein: 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 attached to the N-terminus of the linker domain, and the C-terminus of the linker domain is covalently attached to the N-terminus of the IL-12p40 subunit domain.

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

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

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

93. A host cell comprising one or more nucleic acids as described in claim 92.

94. A method for producing a heterodimeric Fc fusion protein, the method comprising: The host cell is cultured with one or more nucleic acids or vectors under conditions that produce the heterodimeric Fc fusion protein, wherein: i) the one or more nucleic acids or vectors comprise one or more nucleic acids as described in claim 92, 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.

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

96. The method of claim 94 or 95, wherein the binding affinity of the produced heterodimeric Fc fusion protein to interleukin 12 receptor β2 (IL-12Rβ2) is altered compared to the binding affinity of a reference IL-12.

97. The method of claim 96, wherein the binding affinity of the produced heterodimeric Fc fusion protein to IL-12Rβ2 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 determined by an assay.

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

99. The method of claim 96, wherein the binding affinity of the produced heterodimeric Fc fusion protein to IL-12Rβ2 is below the minimum detectable level of the assay and the binding affinity of the reference IL-12 is detectable as determined by the assay.

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

101. The method of claim 94 or 95, wherein the potency of the produced heterodimeric Fc fusion protein is reduced by 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, or about 30.0-fold to about 35.0-fold compared to the potency of a reference IL-12. times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times From about 800.0 times, from about 800.0 times to about 900.0 times, from about 900.0 times to about 1000.0 times, from about 1000.0 times to about 2000.0 times, from about 2000.0 times to about 3000.0 times, from about 3000.0 times to about 4000.0 times, from about 4000.0 times to about 5000.0 times, from about 5000.0 times to about 6000.0 times, from about 6000.0 times to about 7000.0 times, from about 7000.0 times to about 8 , about 10,000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more as determined by the assay.

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

103. The method of claim 94 or 95, wherein the ability of the produced heterodimeric Fc fusion protein to stimulate IFNγ production is reduced by 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 compared to the ability of a reference IL-12 to stimulate IFNγ production. , about 30.0 times to about 35.0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times. times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about fold to about 8000.0 fold, about 8000.0 fold to about 9000.0 fold, about 9000.0 fold to about 10,000.0 fold, about 10,000.0 fold to about 50,000.0 fold, about 50,000.0 fold to about 100,000.0 fold, about 100,000.0 fold to about 200,000.0 fold, about 200,000.0 fold to about 300,000.0 fold, about 300,000.0 fold or more as determined by an assay.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

119. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12p35 subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and SEQ ID NOs: 103-166, and the IL-12p40 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. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12p35 subunit comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, and SEQ ID NO: 103-166, and the IL-12p40 subunit comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO:

90.

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

90.

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

90.

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

90.

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

90.

125. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12p35 subunit consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:24-86 and SEQ ID NO:103-166, and the IL-12p40 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. The non-naturally occurring IL-12 variant of claim 106, wherein the variant IL-12p35 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. The non-naturally occurring IL-12 variant of any one of claims 106-126, wherein the one or more amino acid substitutions of the variant IL-12p35 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 the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12Fc fusion protein.

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-12p35 subunit and / or the IL-12p40 subunit: (i) an Fc domain, wherein the Fc domain comprises 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. The non-naturally occurring IL-12 variant of any one of claims 106-128, wherein the C-terminus of the variant IL-12p35 subunit is covalently attached to the N-terminus of the IL-12p40 subunit.

130. The non-naturally occurring IL-12 variant of claim 129, wherein the non-naturally occurring IL-12 variant further comprises 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, and wherein the C-terminus of the variant IL-12p35 subunit is covalently attached to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently attached to the N-terminus of the IL-12p40 subunit.

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

132. The non-naturally occurring IL-12 variant of claim 131, wherein the non-naturally occurring IL-12 variant further comprises 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, and wherein the C-terminus of the IL-12p40 subunit is covalently attached to the N-terminus of the linker domain and the C-terminus of the linker domain is covalently attached to the N-terminus of the variant IL-12p35 subunit.

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

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

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

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

137. A method of producing a non-naturally occurring IL-12 variant, the method comprising: The host cell is cultured with one or more nucleic acids or vectors under conditions whereby 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. The method of claim 137, further comprising isolating and / or purifying the produced non-naturally occurring IL-12 variant.

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-12p35 subunit and / or the IL-12p40 subunit: (i) an Fc domain, wherein the Fc domain comprises 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. The method of any one of claims 137-139, 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, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12Fc fusion protein.

141. The method of claim 140, wherein the non-naturally occurring IL-12 variant produced 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 determined by an assay.

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

143. The method of claim 140, wherein the binding affinity of the non-naturally occurring IL-12 variant produced for IL-12Rβ2 is below the minimum detectable level of the assay and the binding affinity of the reference IL-12 is detectable as determined by the assay.

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

145. The method of any one of claims 137-139, wherein the potency of the non-naturally occurring IL-12 variant produced is reduced by 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 36.0-fold, about 37.0-fold to about 38.0-fold, about 39.0-fold to about 40.0-fold, about 41.0-fold to about 42.0-fold, about 43.0-fold to about 44.0-fold, about 45.0-fold to about 46.0-fold, about 47.0-fold to about 48.0-fold, about 49.0-fold to about 50.0-fold, about 50.0-fold to about 51.0-fold, about 51.0-fold to about 52.0-fold, about 52.0-fold to about 53.0-fold about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 9 fold, about 1000.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 10,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by an assay.

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

147. The method of any one of claims 137-139, wherein the ability of the produced non-naturally occurring IL-12 variant to stimulate IFNγ production is reduced by 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 fold, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000. .0 times, about 10,000.0 times to about 50,000.0 times, about 50,000.0 times to about 100,000.0 times, about 100,000.0 times to about 200,000.0 times, about 200,000.0 times to about 300,000.0 times, about 300,000.0 times or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by an assay.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

171. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A and D126A.

172. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A and P127A.

173. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A and T43A.

174. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A, D126A and P127A.

175. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A, T43A, D126A and P127A.

176. A heterodimeric Fc fusion protein as described in any of claims 164-175, wherein the IL-12p40 subunit domain comprises a variant IL-12p40 subunit domain.

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

178. A heterodimeric Fc fusion protein as described in any one of claims 164-177, wherein 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, and 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.

179. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 24-86 and SEQ ID NOs: 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-12p40 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; and 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.

180. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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 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. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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; and 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. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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; and 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. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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; and 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. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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 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. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 subunit domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and SEQ ID NOs: 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-12p40 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; and 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.

186. The heterodimeric Fc fusion protein of claim 164, wherein: i) the variant IL-12p35 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-12p40 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 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. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises seven or more amino acid substitutions consisting of: Y40A, T43A, D126A, P127A, R129A, K168A and K170A.

188. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A and R129A.

189. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A and K168A.

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

191. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A, P127A and R129A.

192. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A, P127A and K168A.

193. A heterodimeric Fc fusion protein as described in claim 164, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A, P127A and K170A.

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

195. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. The heterodimeric Fc fusion protein of claim 194, wherein the variant IL-12p35 subunit domain comprises any one of SEQ ID NOs: 177-198.

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

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

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

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

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

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

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

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

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

211. A heterodimeric Fc fusion protein as described in claim 203, wherein the variant IL-12p35 subunit domain comprises amino acid substitutions Y40A and P127A.

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

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

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

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

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

217. A heterodimeric Fc fusion protein as described in any one of claims 203-216, wherein 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, and 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.

218. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 24-86 and SEQ ID NOs: 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-12p40 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; and 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.

219. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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 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. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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; and 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. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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; and 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. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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; and 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. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 subunit domain comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 24-87, or SEQ ID NO: 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-12p40 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 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. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 subunit domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-86 and SEQ ID NOs: 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-12p40 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; and 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.

225. The heterodimeric Fc fusion protein of claim 203, wherein: i) the variant IL-12p35 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-12p40 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 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. A heterodimeric Fc fusion protein as described in claim 203, wherein the variant IL-12p35 subunit domain comprises seven or more amino acid substitutions consisting of: Y40A, T43A, D126A, P127A, R129A, K168A and K170A.

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

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

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

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

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

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

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

234. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. A heterodimeric Fc fusion protein as described in 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. The heterodimeric Fc fusion protein of claim 233, wherein the variant IL-12p35 subunit domain comprises any one of SEQ ID NOs: 177-198.

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

242. A composition comprising a heterodimeric Fc fusion protein as described in any one of claims 164-241, wherein the composition is used to treat cancer in a subject.

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

244. A host cell comprising one or more nucleic acids as described in claim 243.

245. A method for producing a heterodimeric Fc fusion protein, the method comprising: The host cell is cultured with one or more nucleic acids or vectors under conditions that produce the heterodimeric Fc fusion protein, wherein the nucleic acid or vector comprises one or more nucleic acids as described in claim 243, The generated 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 the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12Fc fusion protein.

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

247. The method of any one of claims 245 or 246, wherein the binding affinity of the produced heterodimeric Fc fusion protein to interleukin 12 receptor β2 (IL-12Rβ2) is altered compared to the binding affinity of a reference IL-12.

248. The method of claim 247, wherein the binding affinity of the produced heterodimeric Fc fusion protein to IL-12Rβ2 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 determined by an assay.

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

250. The method of claim 247, wherein the binding affinity of the produced heterodimeric Fc fusion protein to IL-12Rβ2 is below the minimum detectable level of the assay and the binding affinity of the reference IL-12 is detectable as determined by the assay.

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

252. The method of any one of claims 245 or 246, wherein the potency of the produced heterodimeric Fc fusion protein is reduced by 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, .0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 900.0 times, about 900.0 times .0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000.0 times, about 1 0,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by an assay.

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

254. The method of any one of claims 245 or 246, wherein the ability of the produced heterodimeric Fc fusion protein to stimulate IFNγ production is reduced by 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, or about 35.0-fold to about 35.0-fold compared to the ability of a reference IL-12 to stimulate IFNγ production. .0 times, about 35.0 times to about 40.0 times, about 40.0 times to about 45.0 times, about 45.0 times to about 50.0 times, about 50.0 times to about 100.0 times, about 100.0 times to about 200.0 times, about 200.0 times to about 300.0 times, about 300.0 times to about 400.0 times, about 400.0 times to about 500.0 times, about 500.0 times to about 600.0 times, about 600.0 times to about 700.0 times, about 700.0 times to about 800.0 times, about 800.0 times to about 90 0.0 times, about 900.0 times to about 1000.0 times, about 1000.0 times to about 2000.0 times, about 2000.0 times to about 3000.0 times, about 3000.0 times to about 4000.0 times, about 4000.0 times to about 5000.0 times, about 5000.0 times to about 6000.0 times, about 6000.0 times to about 7000.0 times, about 7000.0 times to about 8000.0 times, about 8000.0 times to about 9000.0 times, about 9000.0 times to about 10,000. 0-fold, about 10,000.0-fold to about 50,000.0-fold, about 50,000.0-fold to about 100,000.0-fold, about 100,000.0-fold to about 200,000.0-fold, about 200,000.0-fold to about 300,000.0-fold, about 300,000.0-fold or more, wherein the reference IL-12 comprises one or more of the following: wild-type IL-12, human wild-type IL-12, a commercially available IL-12 molecule, or an IL-12 Fc fusion protein, as determined by an assay.

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 non-naturally occurring IL-12 variant comprising: a) a variant IL-12p35 subunit, wherein the variant IL-12p35 subunit 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; and b) IL-12p40 subunit.

257. The non-naturally occurring IL-12 variant of claim 256, wherein the variant IL-12p35 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, D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126 6P, D126Q, D126R, D126S, D126T, D126V, D126W, P127A, P127C, P127D, P127E, P127F, P 127G, P127H, P127K, P127M, P127N, P127Q, P127R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129 T, R129V, R129W, R129Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K16 8L, K170M, K170N, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

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

259. A heterodimeric Fc fusion protein comprising: a) a first fusion construct comprising: a variant IL-12p35 subunit domain and a first Fc domain, wherein the C-terminus of the variant IL-12p35 subunit domain is covalently attached to the N-terminus of the first Fc domain; and the variant IL-12p35 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; and b) a second fusion construct comprising an IL-12p40 subunit domain and a second Fc domain, wherein the C-terminus of the IL-12p40 subunit domain is covalently attached to the N-terminus of the second Fc domain, Optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

260. The heterodimeric Fc fusion protein of claim 259, wherein the variant IL-12p35 subunit domain 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, D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126 6P, D126Q, D126R, D126S, D126T, D126V, D126W, P127A, P127C, P127D, P127E, P127F, P 127G, P127H, P127K, P127M, P127N, P127Q, P127R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129 T, R129V, R129W, R129Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K16 8L, K170M, K170N, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

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

262. A heterodimeric Fc fusion protein comprising: a) a first fusion construct comprising: a variant IL-12p35 subunit domain and a first Fc domain, wherein the N-terminus of the variant IL-12p35 subunit domain is covalently attached to the C-terminus of the first Fc domain; and the variant IL-12p35 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; and b) a second fusion construct comprising: an IL-12p40 subunit domain and a second Fc domain, wherein the N-terminus of the IL-12p40 subunit domain is covalently attached to the C-terminus of the second Fc domain, Optionally wherein the first Fc domain and the second Fc domain comprise modifications that (i) promote heterodimerization of the first Fc domain and the second Fc domain and / or (ii) silence or inhibit effector function.

263. The heterodimeric Fc fusion protein of claim 262, wherein the variant IL-12p35 subunit domain 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, D126A, D126C, D126E, D126F, D126G, D126I, D126K, D126L, D126M, D126N, D126 6P, D126Q, D126R, D126S, D126T, D126V, D126W, P127A, P127C, P127D, P127E, P127F, P 127G, P127H, P127K, P127M, P127N, P127Q, P127R, P127S, R129A, R129C, R129D, R129E, R129F, R129G, R129H, R129I, R129K, R129L, R129M, R129N, R129P, R129Q, R129S, R129 T, R129V, R129W, R129Y, K168A, K168C, K168D, K168E, K168F, K168G, K168H, K168I, K16 8L, K170M, K170N, K170P, K170S, K170T, K170V, K170F, K170L, K170N, and K170W.

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

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

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

267. A heterodimeric Fc fusion protein as described in any one of claims 164-241 or 259-264, wherein the variant IL-12p35 subunit domain further comprises a C74S substitution mutation.

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

Images