WO2025136986A2 - Il-12 loss of potency muteins - Google Patents

Abstract

The present invention provides pharmaceutical compositions comprising IL-12 muteins, isolated nucleic acids encoding the muteins, vectors, and host cells containing the same, and methods or uses of such compositions for the treatment of a disease or disorder.

Description

IL-12 LOSS OF POTENCY MUTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/611,673, filed December 18, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Cytokines can be used to treat a variety of diseases or conditions, such as cancer, inflammatory conditions, autoimmune conditions, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer’s disease, Schizophrenia, viral infections, (e.g., chronic hepatitis C, AIDS), allergic asthma, retinal neurodegenerative processes, metabolic disorder, insulin resistance, and diabetic cardiomyopathy. IL- 12 is a pleiotropic secreted cytokine composed of two subunits, namely p35 (IL-12p35), and p40 (IL-12p40). In the naturally occurring IL- 12, the two subunits are linked through a disulfide bond to form the bioactive IL-12p70 cytokine. IL- 12 is, inter alia, pro-inflammatory, and mediates its functions through binding to IL-12 receptors (IL-12R). The high affinity IL-12R is heterodimeric comprising a IL-12Rβ1 subunit and a IL-12Rβ32 subunit. IL- 12 is produced primarily by cells of the immune system to signal and organize an immune response. Binding of IL- 12 to IL-12R leads to activation of T- cells and NK cells, leading to increased production of IFN-y, proliferation, and cytotoxic potential. IL- 12 has been recognized as an extremely attractive anticancer candidate because it can activate innate and adaptive immunity, induce antiangiogenic chemokines, promote remodeling of the tumor extracellular matrix, and stimulate expression of MHC class I molecules.

[0003] However, the therapeutic utility of IL- 12 is limited due its cellular toxicity and the potential to elicit undesired immune responses in patients. This limits the practical applications of IL- 12 in a therapeutic setting. Although multiple clinical trials have been on-going since the first human clinical trial of IL- 12 in 1996, an FDA-approved IL- 12 product remains elusive.

[0004] This presents a significant unmet need for new strategies that can overcome the challenges for the therapeutic use of IL- 12. If the potency of cytokines like IL- 12 could be safely harnessed and the toxicity challenges could be controlled, these agents could serve as powerful therapeutics for potential use against a broad spectrum of cancers. SUMMARY

[0005] Disclosed herein is an interleukin- 12 (IL-12) mutein comprising a variant p35 subunit, a variant p40 subunit, or both a variant p35 subunit and a variant p40 subunit, wherein the variant p35 subunit comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 1; wherein the variant p40 subunit comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 2. In some embodiments, the IL-12 mutein comprises a variant p35 subunit, and wherein the variant p35 subunit comprises a substitution at amino acid residue L133 relative to SEQ ID NO: 1. In some embodiments, the LI 33 substitution of the variant p35 subunit is selected from the group consisting of L133A, L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133I, L133M, L133F, L133W, L133V, and L133Y. In some embodiments, the variant p35 subunit further comprises one or more amino acid substitutions relative to SEQ ID NO: 1.

[0006] In some embodiments, the variant p35 subunit comprises one, two, three, four, five, or more amino acid substitutions at a position selected from the group consisting of L37, F39, Y40, P41, D48, M125, D126, K128, R129, 1131, L133, P164, D165, F166, Y167, KI 70, L173, L177, Hl 78 relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37R, L37H, L37K, L37D, L37E, L37S, L37T, L37N, L37Q, L37G, L37P, L37A, L37V, L37I, L37M, L37F, L37Y, or L37W. In some embodiments, the variant p35 subunit comprises a substitution at Y40, optionally wherein the substitution is Y40R, Y40H, Y40K, Y40D, Y40E, Y40S, Y40T, Y40N, Y40Q, Y40G, Y40P, Y40A, Y40V, Y40I, Y40L, Y40M, Y40F, or Y40W. In some embodiments, the variant p35 subunit comprises a substitution at D48, optionally wherein the substitution is D48R, D48H, D48K, D48E, D48S, D48T, D48N, D48Q, D48G, D48P, D48A, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W. In some embodiments, the variant p35 subunit comprises a substitution at K128, optionally wherein the substitution is K128R, K128H, K128K, K128D, K128E, K128S, K128T, K128N, K128Q, K128G, K128P, K128A, K128V, K128I, K128L, K128M, K128F, K128Y, or K128W.

[0007] In some embodiments, the variant p35 subunit comprises a substitution at M125, optionally wherein the substitution is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125A, M125V, M125I, M125L, M125F, M125Y, or M125W. In some embodiments, the variant p35 subunit comprises a substitution at R129, optionally wherein the substitution is R129H, R129K, R129D, R129E, R129S, R129T, R129N, R129Q, R129G, R129P, R129A, R129V, R129I, R129L, R129M, R129F, R129Y, or R129W. In some embodiments, the variant p35 subunit comprises a substitution at LI 33, optionally wherein the substitution is L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133A, L133V, L133I, L133M, L133F, L133Y, or L133W. In some embodiments, the variant p35 subunit comprises a substitution at LI 77, optionally wherein the substitution is L177R, L177H, L177K, L177D, L177E, L177S, L177T, L177N, L177Q, L177G, L177P, L177A, LI 77V, LI 771, L177M, L177F, L177Y, or L177W. In some embodiments, the variant p35 subunit comprises a substitution at H 178, optionally wherein the substitution is H 178R, H178K, H178D, H178E, H178S, H178T, H178N, H178Q, H178G, H178P, H178A, H178V, H178I, H178L, H178M, H178F, H178Y, or H178W.

[0008] In some embodiments, the IL-12 mutein comprises a variant p35 subunit, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; and a substitution at Y40, optionally wherein the substitution is Y40S. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at L133, optionally wherein the substitution is L133A. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at KI 28, optionally wherein the substitution is K128N or K128Q. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; a substitution at KI 28, optionally wherein the substitution is K128N or K128Q; and a substitution at L133, optionally wherein the substitution is L133A. In some embodiments, the variant p35 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1.

[0009] In some embodiments, the IL-12 mutein comprises a variant p40 subunit, wherein the variant p40 subunit comprises one or more amino acid substitutions relative to SEQ ID NO: 2, and wherein the one or more amino acid substitutions comprises a substitution at amino acid residue Y16 of SEQ ID NO: 2. In some embodiments, the Y16 substitution of the variant p40 subunit is selected from the group consisting of Y16A, Y16R, Y16H, Y16K, Y16D, Y16E, Y16S, Y16T, Y16N, Y16Q, Y16G, Y16P, Y16I, Y16L, Y16M, Y16F, Y16W, and Y16V.

[0010] In some embodiments, the IL-12 mutein comprises a variant p40 subunit further comprising one or more amino acid substitutions relative to SEQ ID NO: 2. In some embodiments, the variant p40 subunit further comprises one or more, two or more, three or more, four or more, or five or more amino acid substitutions at a position selected from the group consisting of W15, P17, D18, E59, F60, K84, E86, D93, H194, K195, and K197 relative to SEQ ID NO: 2. In some embodiments, the variant p40 subunit further comprises one or more, two or more, or three or more amino acid substitutions at a position selected from E59, F60, K84, KI 95, and KI 97 relative to SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.

[0011] In some embodiments, the interleukin- 12 mutein comprising a variant p35 subunit and a variant p40 subunit, wherein the variant p35 subunit comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions are at a position selected from A33, R34, L37, E38, F39, Y40, P41, E45, E46, 147, D48, H49, E50, D51, 152, T53, K54, K56, T57, D108, L109, KI 10, Ml 11, Y112, QI 13, VI 14, El 15, Fl 16, KI 17, T118, A121, L123, M125, D126, R129, 1131, P156, S159, S160, L161, E162, E163, D165, F166, Y167, K168, T169, K170, 1171, KI 72, L173, 1175, H178, and R181 relative to SEQ ID NO: 1, and the variant p40 subunit comprises one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions are at a position selected from VI 0, VI 1, El 2, L13, D14, W15, Y16, P17, D18, A19, P20, G21, W38, T39, L40, D41, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, G64, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, 194, L95, D97, Q98, K99, E100, N103, KI 04, T105, F106, L107, L123, K135, S136, S137, R138, G139, S140, S141, D142, N162, E181, S183, L184, P185, 1186, E187, V188, V190, D191, H194, K195, L196, K197, Y198, E199, N200, T202, S203, S204, F206, and D209 relative to SEQ ID NO: 2.

[0012] In some embodiments, Y16 of SEQ ID NO: 2 is substituted, preferably with an alanine residue. In some embodiments, LI 33 of SEQ ID NO: 1 is substituted, preferably with an alanine residue. In some embodiments, the variant p35 further comprises one or more amino acid substitutions or substitutions at a position selected from: (a) wherein D126 of SEQ ID NO: 1 is substituted, preferably with an alanine residue; (b) wherein 1131 of SEQ ID NO: 1 is substituted, preferably with an alanine residue; (c) wherein Hl 78 of SEQ ID NO: 1 is substituted, preferably with an alanine residue; and (d) any combination thereof. In some embodiments, the IL- 12 mutein comprises a variant p35 subunit and a variant p40 subunit, wherein: (a) the variant p35 subunit comprises a substitution of one or more amino acids as shown in Table 1; (b) the p40 moiety comprises a substitution of one or more amino acids as shown in Table 4; or (c) the p35 moiety comprises a substitution of one or more amino acids as shown in Table 1 and the p40 moiety comprises a substitution of one or more amino acids as shown in Table 4. In some embodiments, the interleukin- 12 (IL- 12) mutein comprises a variant p35 subunit and a variant p40 subunit, wherein: (a) the variant p35 subunit comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 1 , wherein the substitution of one or more amino acids is a substitution listed in Table 1 ; (b) the variant p40 subunit comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 2, wherein the substitution of one or more amino acids is a substitution listed in Table 4; or (c) the variant p35 subunit comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 1, wherein the substitution of one or more amino acids is a substitution listed in Table I, and the p40 moiety comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 2, wherein the substitution of one or more amino acids is a substitution listed in Table 4.

[0013] In some embodiments, the IL- 12 mutein is a human IL- 12 mutein. In some embodiments, the variant p35 subunit, the variant p40 subunit, or the variant p35 and p40 subunit are variant of the respective human IL- 12 subunits

[0014] Disclosed herein is an interleukin- 12 (IL- 12) mutein comprising a variant p35 subunit, a variant p40 subunit, or both a variant p35 subunit and a variant p40 subunit, wherein the variant p35 subunit, if present, comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 1; and wherein the variant p40 subunit, if present, comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 2. In some embodiments, the IL-12 mutein comprises the variant p35 subunit.

[0015] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L133, optionally wherein the substitution is L133A, L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133I, L133M, L133F, L133W, L133V, or L133Y. In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L37, optionally wherein the substitution is L37R, L37H, L37K, L37D, L37E, L37S, L37T, L37N, L37Q, L37G, L37P, L37A, L37V, L37I, L37M, L37F, L37Y, or L37W. In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L37, wherein the substitution is L37A. In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L37, wherein the substitution at amino acid residue L37 is L37R, L37H, L37K, L37D, L37N, L37Q, L37P, L37A, L37V, L37M, L37F, L37Y, or L37W.

[0016] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue Y40, optionally wherein the substitution is Y40R, Y40H, Y40K, Y40D, Y40E, Y40S, Y40T, Y40N, Y40Q, Y40G, Y40P, Y40A, Y40V, Y40I, Y40L, Y40M, Y40F, or Y40W. In some embodiments, variant p35 subunit comprises a substitution at amino acid residue Y40, wherein the substitution at amino acid residue Y40 is Y40H, Y40E, Y40V, Y40I, Y40L, Y40M, or Y40F. In some embodiments, variant p35 subunit comprises a substitution at amino acid residue Y40, wherein the Y40 substitution is Y40S. [0017] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue D48, optionally wherein the substitution is D48R, D48H, D48K, D48E, D48S, D48T, D48N, D48Q, D48G, D48P, D48A, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W. In some embodiments, wherein the substitution at amino acid residue D48 is D48H, D48K, D48E, D48S, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W, optionally wherein the substitution at amino acid residue D48 is D48K, D48E, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, or D48F.

[0018] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue KI 28, optionally wherein the substitution is K128R, K128H, K128K, K128D, K128E, K128S, K128T, K128N, K128Q, K128G, K128P, K128A, K128V, K128I, K128L, K128M, K128F, K128Y, or K128W. In some embodiments the variant p35 subunit comprises a substitution at amino acid residue KI 28, wherein the substitution at amino acid residue K128 is K128N or K128Q.

[0019] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue M125, optionally wherein the substitution is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125A, M125V, M125I, M125L, M125F, M125Y, or M125W. In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue M125, wherein the substitution at amino acid residue M125 is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125L, M125F, M125Y, or M125W.

[0020] In some embodiments, wherein the variant p35 subunit comprises a substitution at amino acid residue R129, optionally wherein the substitution is R129H, R129K, R129D, R129E, R129S, R129T, R129N, R129Q, R129G, R129P, R129A, R129V, R129I, R129L, R129M, R129F, R129Y, or R129W.

[0021] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L177, optionally wherein the substitution is L177R, L177H, L177K, L177D, L177E, L177S, L177T, L177N, L177Q, L177G, L177P, L177A, L177V, L177I, L177M, L177F, L177Y, or L177W. In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue LI 77, wherein the substitution at amino acid residue LI 77 is L177D, L177S, L177T, L177N, L177G, L177P, L177V, L177I, L177M, or L177W.

[0022] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue H178, optionally wherein the substitution is H178R, H178K, H178D, H178E, H178S, H178T, H178N, H178Q, H178G, H178P, H178A, H178V, H178I, H178L, H178M, H178F, H178Y, or H178W. [0023] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue K56, optionally wherein the substitution at amino acid residue K56 is K56R, K56H, K56D, K56E, K56S, K56T, K56N, K56Q, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W. In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue K56, wherein the K56 substitution is K56R, K56H, K56D, K56E, K56S, K56T, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W, optionally wherein the substitution at amino acid residue K56 is K56E, K56S, K56T, K56G, K56A, K56V, K56I, or K56L. In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue K56, wherein the substitution at amino acid residue K56 is K56N.

[0024] In some embodiments, wherein the variant p35 subunit comprises a substitution at amino acid residue KI 70, optionally wherein the substitution at amino acid residue KI 70 is K170R, K170H, K170D, K170E, KI 70S, K170T, K170N, K170Q, K170G, K170P, K170A, KI 70V, KI 701, K170L, K170M, K170F, K170Y, or KI 70 W.

[0025] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue 1171, optionally wherein the substitution at amino acid residue 1171 is I171R, I171K, I171H, I171D, I171E, 1171 S, I171T, I171N, 1171 Q, I171G, I171P, I171A, I171V, I171L, I171M, I171F, I171Y, or I171W.

[0026] In some embodiments, wherein the variant p35 subunit comprises a substitution at amino acid residue A33, R34, E38, F39, P41, E45, E46, 147, H49, E50, D51, 152, T53, K54, T57, D108, L109, KI 10, Mi l 1, Y112, QI 13, VI 14, El 15, Fl 16, KI 17, T118, A121, L123, D126, 1131, P156, S159, S160, L161, E162, E163, D165, F166, Y167, K168, T169, K172, L173, 1175, or R181.

[0027] In some embodiments, the variant p35 subunit comprises one or more amino acid substitutions selected from Table la, Table lb, or Table 1c. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; and a substitution at Y40, optionally wherein the substitution is Y40S. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at L133, optionally wherein the substitution is L133A. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at KI 28, optionally wherein the substitution is K128N or K128Q. In some embodiments, the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; a substitution at KI 28, optionally wherein the substitution is K128N or K128Q; and a substitution at L133, optionally wherein the substitution is L133A. [0028] In some embodiments, the variant p35 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1.

[0029] In some embodiments, the IL- 12 mutein of the disclosure comprises the variant p40 subunit.

[0030] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue VI 0, optionally wherein the substitution is V10A, VI OR, VI OH, VI OK, VI 0D, V10E, V10S, V10T, V10N, V10Q, V10G, V10P, V10I, VIOL, V10M, V10F, V10W, or V10Y.

[0031] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue 194, optionally wherein the substitution is I94A, I94R, I94H, I94K, I94D, I94E, I94S, I94T, I94N, I94Q, I94G, I94P, I94L, I94M, I94F, I94W, I94V, or I94Y.

[0032] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue L95, optionally wherein the substitution is L95A, L95R, L95H, L95K, L95D, L95E, L95S, L95T, L95N, L95Q, L95G, L95P, L95I, L95M, L95F, L95W, L95V, or L95Y.

[0033] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue G64, optionally wherein the substitution is G64A, G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue G64, wherein the G64 substitution is G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y, optionally wherein the G64 substitution is G64R, G64K, G64E, G64S, G64T, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y.

[0034] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue Y198, optionally wherein the substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198S, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue Y198, wherein the Y198 substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V, optionally wherein the Y 198 substitution is Y198G, Y198E, Y198K, Y198Q, Y198R, Y198W.

[0035] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue Y16, optionally wherein the substitution is Y16A, Y16R, Y16H, Y16K, Y16D, Y16E, Y16S, Y16T, Y16N, Y16Q, Y16G, Y16P, Y16I, Y16L, Y16M, Y16F, Y16W, or Y16V.

[0036] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue VI 1, E12, L13, D14, W15, P17, D18, A19, P20, G21, W38, T39, L40, D41, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, D97, Q98, K99, E100, N1O3, KI 04, T105, F106, L107, L123, K135, S136, S137, R138, G139, S140, S141, D142, N162, E181, S183, L184, P185, 1186, E187, V188, V190, D191, H194, K195, L196, K197, E199, N200, T202, S203, S204, F206, or D209. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue W15, P17, D18, E59, F60, K84, E86, D93, H194, K195, or K197, optionally wherein the variant p40 subunit comprises a substitution at amino acid residue E59, F60, K84, K195, or K197. In some embodiments, the variant p40 subunit comprises one or more amino acid substitutions selected from Table 4a, Table 4b, or Table 4c. In some embodiments, the variant p40 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.

[0037] In some embodiments, the invention comprises a fusion protein comprising any IL- 12 mutein disclosed herein. In some embodiments, the invention comprises pharmaceutical composition comprising any IL- 12 mutein disclosed herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the invention comprises a polynucleotide sequence that encodes any IL-12 mutein disclosed herein. In some embodiments, the invention comprises an expression vector comprising a polynucleotide encoding any of the IL- 12 muteins disclosed herein. In some embodiments, the invention comprises a host cell comprising an expression comprising a polynucleotide encoding any of the IL- 12 muteins disclosed herein.

[0038] In some embodiments, the invention comprises a kit comprising any IL- 12 mutein disclosed herein.

[0039] In some embodiments, the invention is directed to a method of producing any of the IL- 12 muteins disclosed herein, comprising culturing a host cell under conditions to express the IL- 12 mutein. In some embodiments, the method further comprises isolating the IL- 12 mutein from the host cell.

[0040] In some embodiments, the invention is directed to a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any IL- 12 mutein disclosed herein. In some embodiments, the cancer comprises a solid tumor.

INCORPORATION BY REFERENCE

[0041] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS The features and advantages of the invention may be further explained by reference to the following detailed description and accompanying drawings that sets forth illustrative embodiments.

[0042] FIG. 1 is a predicted model structure of IL- 12 generated by AlphaFold 2 (AF2) from an initial ten angstrom cryoEM map of the IL- 12 p35/p40 complex (grey cloud area), the crystal structure of the p40/IL-12Rbl complex (ID No: 6WDQ in the Protein Data Bank), and the crystal structure of the IL-12 p35/p40 complex (ID No: 1F45 in the Protein Data Bank).

[0043] FIG. 2 is a comparison of the starting model of IL-12/receptor interaction as generated by AlphaFold2 and the refined model after molecular dynamics simulation in the GROMACS system. The starting model is colored grey and the refined model yellow.

[0044] FIG. 3 is a structure model of IL- 12 (p35 and p40 subunits) interacting with the IL- 12 receptor (IL-12Rbl and IL-12Rb2) showing structure -predicted mutein effector regions.

[0045] FIG. 4 is a graph showing surface plasmon resonance (SPR) binding data for an IL- 12 mutein including single alanine mutants of the p35 subunit and the effect on binding to IL-12Rbl and IL-12Rb2.

[0046] FIG. 5 is a graph showing surface plasmon resonance (SPR) binding data for an IL- 12 mutein including single alanine mutants of the p40 subunit and the effect on binding to IL-12Rbl and IL-12Rb2.

DETAILED DESCRIPTION

[0047] While IL- 12 has a tremendous therapeutic potential, it can produce undesirable side effects even at low concentrations. When administered systemically, IL- 12 may activate immune cells in the bloodstream that express IL-12R, thereby creating a systemic inflammatory response that may elicit side effects. Thus, the practical application of IL- 12 in a clinical setting is limited. The present disclosure provides IL- 12 proteins including substitutions (e.g., substitution substitutions) within the p35 and/ or p40 subunits of IL- 12 to create mutant IL- 12 muteins with attenuated IL- 12 activity (e.g., decreased IL-12 receptor binding or activation relative to wild-type IL-12). Substitutions described herein limit IL- 12 mutein activity to reduce or eliminate unwanted systemic effects and toxicity. IL- 12 muteins and fusion proteins described herein are therefore candidates for therapeutic uses. The present disclosure provides, IL- 12 muteins and fusion proteins containing the same for use in therapeutic compositions for the treatment of diseases. The present disclosure provides, IL- 12 muteins and fusion proteins containing the same for use in therapeutic compositions for the prevention of diseases. [0048] Before the embodiments of the invention are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

[0050] IL-12 is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40), which are linked covalently to give rise to the so-called bioactive heterodimeric (p70) molecule. Apart from forming heterodimers (IL- 12 and IL-23), the p40 subunit is also secreted as a monomer (p40) and a homodimer (p4Cb). It is known in the art that synthesis of the heterodimer as a single chain with a linker connecting the p35 to the p40 subunit preserves the full biological activity of the heterodimer. IL- 12 plays a critical role in the early inflammatory response to infection and in the generation of Thl cells, which favor cell-mediated immunity. It has been found that overproduction of IL- 12 can be dangerous to the host because it is involved in the pathogenesis of a number of autoimmune inflammatory diseases (e.g. MS, arthritis, type 1 diabetes).

[0051] The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of IL-12Rβ 1 and IL- 12R[32 chains expressed on the surface of activated T-cells and natural killer cells. The IL- 12Rβ 1 chain binds to the IL-12p40 subunit, whereas IL-12p35 in association with IL-12R[32 confers an intracellular signaling ability. Signal transduction through IL-12R induces phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2), that phosphorylate and activate signal transducer and activator of transcription (STAT)l, STAT3, STAT4, and STATS. The specific cellular effects ofIL-12 are due mainly to activation of STAT4. IL-12 induces natural killer and T-cells to produce cytokines, in particular interferon (IFN)y, that mediate many of the proinflammatory activities of IL-12, including CD4+ T-cell differentiation toward the Thl phenotype.

[0052] IL- 12 is a pleiotropic cytokine, that creates an interconnection between the innate and adaptive immunity. IL- 12 was first described as a factor secreted from PMA-induced EBV- transformed B-cell lines. Based on its actions, IL- 12 has been designated as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor. Due to bridging the innate and adaptive immunity and potently stimulating the production of IFNy cytokine coordinating natural mechanisms of anticancer defense, IL- 12 seemed like a promising candidate for tumor immunotherapy in humans. However, severe side effects associated with systemic administration of IL- 12 in clinical investigations and the very narrow therapeutic index of this cytokine markedly hampered the use of this cytokine in cancer patients. Approaches to IL- 12 therapy in which delivery of the cytokine is tumor-targeted, which may diminish some of the previous issues with IL- 12 therapy, are currently in clinical trials for cancers.

[0053] The direct use of IL-12 as an agonist to bind the IL-12R and modulate immune responses therapeutically has been limited due its therapeutic risks, e.g., its short serum half-life and high toxicity. These risks have also limited the therapeutic development and use of other cytokines. New forms of cytokines that reduce these risks are needed. Disclosed herein are compositions and methods comprising IL- 12 muteins designed to address the risks associated with conventional cytokine therapy and provide much needed immunomodulatory therapeutics.

Definitions

[0054] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0055] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0056] The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0057] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0058] The term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable error range for that particular value or composition. In some embodiments, “about” refers to a value of +/- 10% when compared to a reference value (e.g., about 100 includes a range of 90 to 110).

[0059] The term “interleukin 12” or “IL-12” refers to a human IL-12 protein or polypeptide. The term encompasses functional IL- 12 protein complexes comprising an IL- 12 p35 subunit and an IL-12 p40 subunit. An IL-12 described herein may be an IL-12 mutein comprising a variant p35 subunit (e.g., paired with a wild-type p40 subunit or a variant p40 subunit) and/or a variant p40 subunit (e.g., paired with a wild-type p35 subunit or a variant p35 subunit).

[0060] The term “p35” " or “p35 subunit” as used, interchangeably, herein refers to the human alpha subunit of the heterodimeric human IL- 12 protein. The amino acid sequence of an exemplary reference IL- 12 p35 can be found under Uniprot Accession Number P29459, and herein set forth in SEQ ID NO: 1.

[0061] The term “p40” " or “p40 subunit” as used, interchangeably, herein refers to the human beta subunit of the heterodimeric human IL- 12 protein. The amino acid sequence of an exemplary reference IL- 12 p40 can be found under Uniprot Accession Number P29460, and herein set forth in SEQ ID NO: 2.

[0062] By “wild type” or “WT” or “native” herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A wild-type protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been modified by the hand of man.

[0063] As used herein, the term “variant” or “variants” is used to refer to a protein with a modified amino acid sequence when compared to a reference sequence. For example, a variant p35 subunit can refer to a p35 subunit with an amino acid sequence that has one or more substitutions or deletions when compared to a reference human p35 subunit protein sequence. Unless otherwise specified, variant proteins and subunits have an amino acid sequence modified when compared to a human amino acid sequence.

[0064] As used herein, the term “mutein” is used to refer to modified versions of wild type polypeptides comprising modifications to a recognized reference primary structure (i.e., amino acid sequence) of such polypeptide. The term mutein may refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence that encodes it. A mutein may be at least about 90, 91, 92, 94, 94, 95, 96, 97, 98, or 99% identical to the parent polypeptide. In some instances, as used herein, the compositions comprise a variant p40 subunit, wherein the p40 comprises an amino acid sequence that is at least about 90, 91, 92, 94, 94, 95, 96, 97, 98, or 99% identical to the wild type p40 polypeptide (SEQ ID NO: 2). In some instances, as used herein, the compositions comprise a variant p35 subunit, wherein the p35 comprises an amino acid sequence that is at least about 90, 91, 92, 94, 94, 95, 96, 97, 98, or 99% identical to the wild type p40 polypeptide (SEQ ID NO: 1). As used herein with respect to IL-12, the term “mutein” refers to an IL- 12 comprising a variant p35 subunit, variant p40 subunit, or both a variant p35 subunit and variant p40 subunit.

[0065] As used herein, the term “receptor” refers to a polypeptide having a domain that specifically binds a ligand that binding of the ligand results in a change to at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane associated protein that comprises an extracellular domain (ECD) and a membrane associated domain which serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a membrane spanning polypeptide comprising an intracellular domain (ICD) and extracellular domain (ECD) linked by a membrane spanning domain referred to as a transmembrane domain (TM). The binding of a ligand to the receptor results in a conformational change in the receptor resulting in a measurable biological effect. In some instances, where the receptor is a membrane spanning polypeptide comprising an ECD, TM and ICD, the binding of a ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the binding of the ligand to the ECD. In some embodiments, a receptor is a component of a multi- component complex to facilitate intracellular signaling. For example, the ligand may bind a cell surface receptor that is not associated with any intracellular signaling alone but upon ligand binding facilitates the formation of a heteromultimeric (including heterodimeric, heterotrimeric, etc.) or homomultimeric (including homodimeric, homotrimeric, homotetrameric, etc.) complex that results in a measurable biological effect in the cell such as activation of an intracellular signaling cascade (e.g., the Jak/STAT pathway). In some embodiments, a receptor is a membrane spanning single chain polypeptide comprising ECD, TM and ICD domains wherein the ECD, TM and ICD domains are derived from the same or differing naturally occurring receptor variants or synthetic functional equivalents thereof.

[0066] The term “response,” for example, of a cell, tissue, organ, or organism, encompasses a quantitative or qualitative change in a evaluable biochemical or physiological parameter, (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzymatic activity, level of gene expression, rate of gene expression, rate of energy consumption, level of or state of differentiation) where the change is correlated with the activation, stimulation, or treatment, with or contact with exogenous agents or internal mechanisms such as genetic programming. In certain contexts, the terms “activation”, “stimulation”, and the like refer to cell activation as regulated by internal mechanisms, as well as by external or environmental factors; whereas the terms “inhibition”, “down-regulation” and the like refer to the opposite effects. A “response” may be evaluated in vitro such as through the use of assay systems, surface plasmon resonance, enzymatic activity, mass spectroscopy, amino acid or protein sequencing technologies. A “response” may be evaluated in vivo quantitatively by evaluation of objective physiological parameters such as body temperature, bodyweight, tumor volume, blood pressure, results of X- ray or other imaging technology or qualitatively through changes in reported subjective feelings of well-being, depression, agitation, or pain. In some embodiments, the level of activation of T cells in response to the administration of a test agent may be determined by flow cytometric methods. In some methods, a response can be measured by determining the level of STAT (e.g., STAT3, STAT4) phosphorylation, or IFNy production, in accordance with methods well known in the art.

[0067] Where proteins and/or polypeptides are described herein, it is understood that polynucleotides (e.g., RNA (e.g., mRNA) or DNA polynucleotides) encoding the protein or polypeptide are also provided herein.

[0068] Where proteins, polypeptides, polynucleotides, cells, expression vectors, etc. are described herein, it is understood that isolated forms of the proteins, polypeptides, polynucleotides, cells, expression vectors, etc. are also provided herein.

[0069] The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0070] As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.

[0071] The term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline

(P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine

(Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T). [0072] The term “non-naturally occurring,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.

[0073] The terms “hydrophilic” and “hydrophobic” refer to the degree of affinity that a substance has with water. A hydrophilic substance has a strong affinity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water. Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is a scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, which is listed in Hopp, TP, et al., Proc Natl Acad Sci U S A (1981) 78:3824. Examples of “hydrophilic amino acids” are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of “hydrophobic amino acids” are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.

[0074] As used herein in the context of the structure of a polypeptide, “N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N-terminus and the C- terminus, respectively, and can include the residues at the N-terminus and C-terminus, respectively. “Immediately N-terminal” refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the N-terminus of the polypeptide. “Immediately C-terminal” refers to the position of a first amino ac-id residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the C-terminus of the polypeptide. As used herein in the context of nucleic acids, the “5'-terminus” (or “five-prime terminus”) and “3'-terminus” (or “carboxyl terminus”) refer to the extreme ends of the nucleic acid sequence, respectively, while the terms “5” and “3” refer to relative positions in the nucleic acid sequence of the polypeptide toward the 5'-terminus and the 3'-terminus, respectively, and can include the residues at the 5'- terminus and 3 '-terminus, respectively.

[0075] A “fragment” is a truncated form of a native biologically active protein that retains at least a portion of the therapeutic and/or biological activity. As used herein, the term “biologically active protein moiety” includes proteins modified deliberately, as for example, by site directed mutagenesis, insertions, or accidentally through substitutions.

[0076] A “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate substitution. A host cell includes cells transfected in vivo with a vector of this invention.

[0077] “Isolated,” when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.”

[0078] An “isolated” polynucleotide or polypeptide-encoding nucleic acid or other polypeptide- encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.

[0079] A “fusion protein” contains at least one polypeptide comprising regions in a different position in the sequence than that which occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion protein; or they may normally exist in the same protein but are placed in a new arrangement in the fusion protein. A fusion protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0080] “Conjugated”, “linked,” “fused,” and “fusion” are used interchangeably herein. These terms refer to the joining together of two more chemical elements or components, by whatever means including chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and in reading phase or in- frame. An “in- frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature). The terms “link,” “linked,” and “linking” are used in the broadest sense, and are specifically intended to include both covalent and non- covalent attachment of a moiety of the therapeutic agent to another moiety of the therapeutic agent in a direct or indirect way. The term “linked directly,” as used herein in the context of a therapeutic agent, generally refers to a structure in which a moiety is connected with or attached to another moiety without an intervening tether. The term “linked indirectly,” as used herein in the context of a therapeutic agent, generally refers to a structure in which a moiety of the therapeutic agent is connected with, or attached to, another moiety of the therapeutic agent via an intervening tether. [0081] In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.

[0082] “Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

[0083] The terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown. The following are non- limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0084] The term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.

[0085] “Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.

[0086] The terms “gene” or “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.

[0087] “Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity to those sequences.

[0088] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0089] “Percent (%) amino acid sequence identity,” with respect to the polypeptide sequences identified herein, is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0090] A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

[0091] “Physiological conditions” refer to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject. A host of physiologically relevant conditions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers is listed in Sambrook et al. (1989). Physiologically relevant temperature ranges from about 25°C to about 38°C, and preferably from about 35°C to about 37°C.

[0092] The terms “antigen”, “target antigen” or “immunogen” are used interchangeably herein to refer to the structure or binding determinant that an antibody fragment or an antibody fragmentbased therapeutic binds to or has specificity against.

[0093] The term “antagonist”, as used herein, includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. Methods for identifying antagonists of a polypeptide may comprise contacting a native polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide. In the context of the present invention, antagonists may include proteins, nucleic acids, carbohydrates, antibodies or any other molecules that decrease the effect of a biologically active protein.

[0094] The term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native polypeptide may comprise contacting a native polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.

[0095] “Activity” for the purposes herein refers to an action or effect of a component of a protein consistent with that of the corresponding native biologically active protein, wherein “biological activity” refers to an in vitro or in vivo biological function or effect, including but not limited to receptor binding, antagonist activity, agonist activity, or a cellular or physiologic response.

[0096] As used herein, “treatment” or “treating,” refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit. By therapeutic benefit is meant eradication, amelioration, or a delay in the progression of the underlying disorder being treated. Thus, for example, treatment refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the disclosed method, treatment can refer to at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0097] A “therapeutic effect”, as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or delay in progression of a disease or condition in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, caused by a polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. Determination of a therapeutically effective amount is within the capability of those skilled in the art.

[0098] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refers to an amount of a biologically active protein, either alone, as a part of a fusion protein composition, or as part of a therapeutic regimen, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. The disease or condition can refer to a disorder or a disease.

[0099] The term “therapeutically effective dose regimen”, as used herein, refers to a schedule for consecutively administered doses of a biologically active protein, either alone or as a part of a fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.

[0100] As used herein, the terms “prevent”, “preventing”, and “prevention” of a disease or disorder refers to an action, for example, administration of the fusion protein or nucleic acid sequence encoding the fusion protein, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.

[0101] As used herein, references to “decreasing”, “reducing”, or “inhibiting” include a change of at least about 10%, of at least about 20%, of at least about 30%, of at least about 40%, of at least about 50%, of at least about 60%, of at least about 70%, of at least about 80%, of at least about 90% or greater as compared to a suitable control level. Such terms can include but do not necessarily include complete elimination of a function or property, such as agonist activity.

[0102] The terms “cancer” and “tumor” are used interchangeably herein and refer to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that can invade neighboring tissues and may also metastasize to distant parts of the body through, e.g., the lymphatic system or bloodstream.

IL-12 Protein

[0103] IL-12 is a covalently disulfide linked heterodimeric protein comprising two subunits, p35 and p40. In humans, the naturally occurring form of IL- 12 comprises an interchain disulfide linkage between residue C74 of p35 (numbered in accordance with SEQ ID NO:1, Uniprot ID: P29459) and residue C177 of p40 (numbered in accordance with SEQ ID NO:2, Uniprot ID: P29459).

[0104] In some embodiments, the IL-12 mutein comprises a heterodimer comprising p35 and p40 subunits. In some embodiments the two subunits of the IL- 12 heterodimer are covalently linked. In some embodiments, the covalent linkage is a disulfide bond. Alternatively, the IL- 12 mutein may be single chain IL- 12 comprising a single polypeptide comprising both the p35 and p40 subunit moieties. In some embodiments, the single chain IL- 12 may further comprise additional domains, for example, a half-life extending moiety.

[0105] In some embodiments, the present disclosure provides IL-12 muteins with one or more amino acid substitutions in p35. In some embodiments, IL-12 muteins comprise one or more amino acid substitutions in p35. In some embodiments, IL- 12 muteins comprise one more amino acid substitutions in both p35 and p40.

[0106] In some embodiments, the IL-12 mutein has decreased affinity for an IL-12 receptor (e.g., relative to wild- type human IL- 12 having a p35 comprising SEQ ID NO:1 and a p40 comprising SEQ ID NO: 2). In some embodiments, the IL-12 mutein has a decreased affinity for the IL-12 receptor beta 1. In some embodiments, the IL- 12 mutein has a decreased affinity for the IL- 12 receptor beta 2. In some embodiments, decreased affinity is measured by surface plasmon resonance (SPR).

[0107] In some embodiments, the IL-12 protein has a 10% decrease in affinity for IL-12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 15% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 20% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL-12 protein has a 25% decrease in affinity for IL-12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 30% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL-12 protein has a 35% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 40% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 45% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 50% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 55% decrease in affinity for IL-12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 60% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL-12 protein has a 65% decrease in affinity for IL-12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 70% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 75% decrease in affinity for IL-12 receptor beta 1 when measured by SPR. In some embodiments, the IL- 12 protein has a 80% decrease in affinity for IL- 12 receptor beta 1 when measured by SPR. In some embodiments, the IL-12 protein has a 85% decrease in affinity for IL-12 receptor beta 1 when measured by SPR.

[0108] In some embodiments, the IL-12 protein has a 10% decrease in affinity for IL-12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 15% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 20% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL-12 protein has a 25% decrease in affinity for IL-12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 30% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL-12 protein has a 35% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 40% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 45% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 50% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 55% decrease in affinity for IL-12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 60% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL-12 protein has a 65% decrease in affinity for IL-12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 70% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 75% decrease in affinity for IL-12 receptor beta 2 when measured by SPR. In some embodiments, the IL- 12 protein has a 80% decrease in affinity for IL- 12 receptor beta 2 when measured by SPR. In some embodiments, the IL-12 protein has a 85% decrease in affinity for IL-12 receptor beta 1 when measured by SPR.

[0109] The IL-12 protein of the invention can comprise mutein-effector regions. Mutein-effector regions are regions that may interact with other proteins and in which a substitution can alter a proteins interaction with IL- 12. The IL- 12 protein of the invention can comprise a mutein-effector region in the p35 subunit, the p40 subunit, or in both the p35 and p40 subunits. In some embodiments, a mutein effector region comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions such as substitutions or deletions (e.g., at least one, at least two, at least three, at least four, or at least five amino acid residues within the mutein effector region are substituted or deleted). In some embodiments, the IL- 12 protein comprises multiple mutein effector regions, wherein each mutein effector region comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions such as substitutions or deletions (e.g., at least one, at least two, at least three, at least four, or at least five amino acid residues within each mutein effector region are substituted or deleted).

[0110] In some embodiments, the IL-12 protein comprises a p35 subunit which comprises a mutein-effector region. In some embodiments, the variant p35 subunit- effector region comprises amino acid residues 36-52, 124-133, or 163-178 of SEQ ID NO: 1. In some embodiments, the variant p35 subunit-effector region comprises amino acid residues 36-52 of SEQ ID NO: 1. In some embodiments, the variant p35 subunit-effector region comprises amino acid residues 37-48 of SEQ ID NO: 1. In some embodiments, the variant p35 subunit-effector region comprises amino acid residues 37-41 of SEQ ID NO: Lin some embodiments, the variant p35 subunit-effector region comprises amino acid residues 124-133 of SEQ ID NO: 1. In some embodiments, the variant p35 subunit-effector region comprises amino acid residues 163-178 of SEQ ID NO: 1. In some embodiments, the variant p35 subunit-effector region comprises amino acid residues 164- 178 of SEQ ID NO: 1. [0111] In some embodiments, the IL- 12 mutein comprises a p40 subunit which comprises a mutein-effector region. In some embodiments, the variant p40 subunit- effector region comprises amino acid residues 14-18, 41-45, 58-62, 84-107, 195-197, 201-202 of SEQ ID NO: 2. In some embodiments, the variant p40 subunit-effector region comprises amino acid residues 14-18 of SEQ ID NO: 2. In some embodiments, the variant p35 subunit-effector region comprises amino acid residues 41-45 of SEQ ID NO: 2. In some embodiments, the variant p40 subunit-effector region comprises amino acid residues 58-62 of SEQ ID NO: 2. In some embodiments, the variant p40 subunit-effector region comprises amino acid residues 84-107 of SEQ ID NO: 2. In some embodiments, the variant p40 subunit-effector region comprises amino acid residues 195-197 of SEQ ID NO: 2. In some embodiments, the variant p40 subunit-effector region comprises amino acid residues 201-202 of SEQ ID NO: 2.

[0112] A number of IL- 12 mutants have been produced, such as those disclosed in patent publications W02020072821, WO2021189139, WO2021212083, WO2022140797,

WO2022192898, W02023004282, WO2023043978, W02023050006, W02023070038, W02023070056, WO2023133540, U.S. Patent No. 8,114,839, WO2016172703, W02022094046, WO2022155263, WO2023279085, W02023004282, and WO2023133540, each of which is incorporated herein by reference. In some embodiments, the IL- 12 muteins described herein comprise one or more IL- 12 substitutions described in the patent publications above.

IL 12 p35 Subunit and Muteins

[0113] A reference p35 monomer (p35) is expressed as a 219 amino acid pro-protein (SEQ ID NO: 3) comprising a 22 amino acid signal sequence which is post-translationally removed to render a 197 amino acid mature protein (SEQ ID NO:1).

[0114] In some embodiments, the IL- 12 muteins of the present disclosure comprises a modified p35 sequence comprising one or more amino acid substitutions and/or deletions at the interface with the extracellular domain of IL-12R[32. In one aspect, provided herein are variant p35 subunits comprising or consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0115] In some embodiments, the variant p35 subunit comprises one or more amino acid substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises one amino acid substitution/substitution or deletion relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises two amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises three amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises four amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises five amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises six amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises seven amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises eight amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises nine amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises ten amino acid substitutions/substitutions and/or deletions relative to SEQ ID NO: 1.

[0116] In some embodiments, the variant p35 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1.

[0117] In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue A33, R34, L37, E38, F39, Y40, P41, T43, S44, E45, E46, 147, D48, H49, E50, D51, 152, T53, K54, K56, T57, D108, L109, K110, Mi l l, Y112, QI 13, V114, E115, F116, K117, T118, A121, L123, M125, D126, K128, R129, Q130, 1131, L133, P156, S159, S160, L161, E162, E163, P164, D165, F166, Y167, K168, T169, K170, 1171, K172, L173, 1175, L177, H178, or R181 with respect to the position in the sequence of mature p35 (SEQ ID NO: 1). In some embodiments, the variant p35 subunit comprises one, two, three, four, five, or more substitutions selected from the positions of the foregoing list. In some embodiments, the variant p35 subunit comprises an amino acid substitution, wherein the amino acid substitution is any naturally occurring amino acid other than the corresponding wildtype residue in SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution, wherein the amino acid substitution is any naturally occurring amino acid other than the corresponding wild-type residue in SEQ ID NO: 1 or cysteine.

[0118] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L133, optionally wherein the substitution is L133A, L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133I, L133M, L133F, L133W, L133V, or L133Y.

[0119] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L37, optionally wherein the substitution is L37R, L37H, L37K, L37D, L37E, L37S, L37T, L37N, L37Q, L37G, L37P, L37A, L37V, L37I, L37M, L37F, L37Y, or L37W. In some embodiments, the substitution at amino acid residue L37 is L37R, L37H, L37K, L37D, L37N, L37Q, L37P, L37A, L37V, L37M, L37F, L37Y, or L37W. In some embodiments, the L37 substitution is L37A.

[0120] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue Y40, optionally wherein the substitution is Y40R, Y40H, Y40K, Y40D, Y40E, Y40S, Y40T, Y40N, Y40Q, Y40G, Y40P, Y40A, Y40V, Y40I, Y40L, Y40M, Y40F, or Y40W. In some embodiments, the substitution at amino acid residue Y40 is Y40H, Y40E, Y40V, Y40I, Y40L, Y40M, or Y40F. In some embodiments, the substitution at amino acid residue Y40 is Y40S.

[0121] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue D48, optionally wherein the substitution is D48R, D48H, D48K, D48E, D48S, D48T, D48N, D48Q, D48G, D48P, D48A, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W. In some embodiments, the substitution at amino acid residue D48 is D48H, D48K, D48E, D48S, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W, optionally wherein the substitution at amino acid residue D48 is D48K, D48E, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, or D48F.

[0122] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue M125, optionally wherein the substitution is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125A, M125V, M125I, M125L, M125F, M125Y, or M125W. In some embodiments, the substitution at amino acid residue M125 is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125L, M125F, M125Y, or M125W.

[0123] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue KI 28, optionally wherein the substitution is K128R, K128H, K128K, K128D, K128E, K128S, K128T, K128N, K128Q, K128G, K128P, K128A, K128V, K128I, K128L, K128M, K128F, K128Y, or K128W. In some embodiments, the substitution at amino acid residue K128 is K128N or K128Q.

[0124] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue R129, optionally wherein the substitution is R129H, R129K, R129D, R129E, R129S, R129T, R129N, R129Q, R129G, R129P, R129A, R129V, R129I, R129L, R129M, R129F, R129Y, or R129W.

[0125] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue L177, optionally wherein the substitution is L177R, L177H, L177K, L177D, L177E, L177S, L177T, L177N, L177Q, L177G, L177P, L177A, L177V, L177I, L177M, L177F, L177Y, or L177W. In some embodiments, the substitution at amino acid residue LI 77 is L177D, L177S, L177T, L177N, L177G, L177P, L177V, L177I, L177M, or L177W. [0126] In some embodiments, wherein the variant p35 subunit comprises a substitution at amino acid residue Hl 78, optionally wherein the substitution is H178R, H178K, H178D, H178E, H178S, H178T, H178N, H178Q, H178G, H178P, H178A, H178V, H178I, H178L, H178M, H178F, H178Y, or H178W.

[0127] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue K56, optionally wherein the substitution at amino acid residue K56 is K56R, K56H, K56D, K56E, K56S, K56T, K56N, K56Q, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W. In some embodiments, the K56 substitution is K56R, K56H, K56D, K56E, K56S, K56T, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W, optionally wherein the substitution at amino acid residue K56 is K56E, K56S, K56T, K56G, K56A, K56V, K56I, or K56L. In some embodiments, the substitution at amino acid residue K56 is K56N. [0128] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue KI 70, optionally wherein the substitution at amino acid residue KI 70 is K170R, K170H, K170D, K170E, KI 70S, K170T, K170N, K170Q, K170G, K170P, K170A, KI 70V, KI 701, K170L, K170M, K170F, K170Y, or K170W.

[0129] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue 1171, optionally wherein the substitution at amino acid residue 1171 is I171R, I171K, I171H, I171D, I171E, 1171 S, I171T, I171N, 1171 Q, I171G, I171P, I171A, I171V, I171L, I171M, I171F, I171Y, or I171W.

[0130] In some embodiments, the variant p35 subunit comprises a substitution at amino acid residue A33, R34, E38, F39, P41, E45, E46, 147, H49, E50, D51, 152, T53, K54, T57, D108, L109, KI 10, Ml 11, Y112, QI 13, VI 14, El 15, Fl 16, KI 17, T118, A121, L123, D126, 1131, P156, S159, S160, L161, E162, E163, D165, F166, Y167, K168, T169, KI 72, L173, 1175, or R181.

[0131] In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 33 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 34 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 37 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 38 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 39 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 40 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 41 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 43 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 44 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 45 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 46 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 47 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 48 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 49 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 50 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 51 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 52 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 54 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 56 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 57 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 108 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 109 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 110 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 111 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 112 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 113 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 114 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 115 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 116 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 117 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 118 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 121 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 123 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 125 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 126 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 128 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 129 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 130 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 131 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 133 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 156 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 159 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 160 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 161 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 162 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 163 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 164 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 165 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 166 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 167 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 168 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 169 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 170 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 171 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 172 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 173 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 175 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 177 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 178 with respect to the position in mature p35 sequence of SEQ ID NO: 1. In some embodiments, the variant p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 181 with respect to the position in mature p35 sequence of SEQ ID NO: 1. Exemplary p35 substitution substitutions are provided in Table la, Table lb, and Table 1c. In some embodiments, the variant p35 subunit comprises one or more amino acid substitutions (e.g., one, two, three, four, five, or more amino acid substitutions) selected from Table la, Table lb, or Table 1c.

Table la. p35 Substitutions

Table lb. p35 Substitutions

Table 1c. p35 Substitutions

[0132] In some embodiments, the IL- 12 mutein comprises a substitution or deletion of an amino acid in the p35 subunit corresponding to an amino acid shown in the first column of the Table 2 below, where the amino acid residue to be mutated corresponds to an amino acid residue in SEQ ID NO: 1. In some embodiments, the IL-12 mutein comprises a substitution or deletion of an amino acid in the p35 subunit corresponding to the amino acids shown in the first column of the Table 2 below, in combination with one, two, three, four, five or more additional p35 substitutions shown in the corresponding second column in Table 2 below.

Table 2. p35 Substitution Combinations

[0133] In some embodiments, the modified IL-12 protein comprises a p35 subunit comprising a substitution at an amino acid corresponding to an amino acid at position LI 33 of SEQ ID NO: 1. In some embodiments, the modified IL- 12 protein comprises a p35 subunit comprising a substitution or deletion of an amino acid corresponding to amino acid L133 of SEQ ID NO: 1, and the modified IL- 12 protein further comprises a p40 subunit with one more substitutions. For exemplary combinations, see Table 3.

Table 3. Exemplary Combinations

IL-12 p40

[0134] A reference p40 monomer (p40) is expressed as a 328 amino acid pro-protein (SEQ ID NO: 4) comprising a 22 amino acid signal sequence which is post-translationally removed to render a 306 amino acid mature protein (SEQ ID NO:2). [0135] The IL- 12 muteins of the present disclosure comprise a modified p40 comprising one or more amino acid substitutions and/or deletions at the interface with the extracellular domain of IL-12Rβ 1. In one aspect, provided herein are variant p40 subunits comprising or consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0136] In some embodiments, the variant p40 subunit comprises one or more amino acid substitutions and/or deletions (e.g., one, two, three, four, five, or more substitutions and/or deletions). In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at amino position VI 0, VI 1, E12, L13, D14, W15, Y16, Pl 7, DI 8, Al 9, P20, G21, W38, T39, L40, D41, Q42, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, G64, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, 194, L95, D97, Q98, K99, E100, P101, N103, KI 04, T105, F106, L107, L123, 1126, T128, L130, K135, S136, S137, R138, G139, S140, S141, D142, R157, R159, D161, N162, K163, E181, S183, L184, P185, 1186, E187, V188, V190, D191, K195, L196, K197, Y198, E199, N200, T202, S203, S204, F206, or D209 with respect to the position in mature p40 (SEQ ID NO: 2). In some embodiments, the variant p40 subunit comprises one, two, three, four, five, or more substitutions selected from the positions of the foregoing list. In some embodiments, the variant p40 subunit comprises an amino acid substitution, wherein the amino acid substitution is any naturally occurring amino acid other than the corresponding wild-type residue in SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution, wherein the amino acid substitution is any naturally occurring amino acid other than the corresponding wild-type residue in SEQ ID NO: 2 or cysteine. [0137] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue VI 0, optionally wherein the substitution is V10A, VI OR, VI OH, VI OK, VI 0D, V10E, V10S, V10T, V10N, V10Q, V10G, V10P, V10I, VIOL, V10M, V10F, V10W, or V10Y.

[0138] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue 194, optionally wherein the substitution is I94A, I94R, I94H, I94K, I94D, I94E, I94S, I94T, I94N, I94Q, I94G, I94P, I94L, I94M, I94F, I94W, I94V, or I94Y. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue L95, optionally wherein the substitution is L95A, L95R, L95H, L95K, L95D, L95E, L95S, L95T, L95N, L95Q, L95G, L95P, L95I, L95M, L95F, L95W, L95V, or L95Y.

[0139] In some embodiments, variant p40 subunit comprises a substitution at amino acid residue G64, optionally wherein the substitution is G64A, G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y. In some embodiments, variant p40 subunit comprises a substitution at amino acid residue G64, wherein the G64 substitution is G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y, optionally wherein the G64 substitution is G64R, G64K, G64E, G64S, G64T, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y.

[0140] In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue W15, P17, D18, E59, F60, K84, E86, D93, H194, K195, or K197, optionally wherein the variant p40 subunit comprises a substitution at amino acid residue E59, F60, K84, K195, or K197. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue VI 1 , E12, L13, D14, W15, P17, D18, A19, P20, G21, W38, T39, L40, D41, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, D97, Q98, K99, E100, N103, KI 04, T105, F106, L107, L123, K135, S136, S137, R138, G139, S140, S141, D142, N162, E181, S183, L184, P185, 1186, E187, V188, V190, D191, H194, K195, L196, K197, E199, N200, T202, S203, S204, F206, or D209. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue Y198, optionally wherein the substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198S, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue Y198, wherein the Y198 substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V, optionally wherein the Y198 substitution is Y198G, Y198E, Y198K, Y198Q, Y198R, Y198W. In some embodiments, the variant p40 subunit comprises a substitution at amino acid residue Y16, optionally wherein the substitution is Y16A, Y16R, Y16H, Y16K, Y16D, Y16E, Y16S, Y16T, Y16N, Y16Q, Y16G, Y16P, Y16I, Y16L, Y16M, Y16F, Y16W, or Y16V.

[0141] In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 10 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 11 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 12 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 13 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 14 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 16 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 17 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 18 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 19 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 20 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 21 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 38 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 39 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 40 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 41 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 42 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 43 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 44 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 45 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 46 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 47 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 55 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 57 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 58 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 59 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 60 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 62 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 63 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 64 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 65 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 66 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 81 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 82 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 83 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 84 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 85 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 86 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 88 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 89 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 90 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 91 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 92 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 93 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 94 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 95 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 97 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 98 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 99 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 100 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 101 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 103 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 104 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 105 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 106 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 107 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 123 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 126 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 128 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 130 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 135 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 136 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 137 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 138 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 139 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 140 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 141 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 142 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 157 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 159 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 161 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 162 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 163 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 181 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 183 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 184 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 185 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 186 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 187 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 188 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 190 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 191 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 195 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 196 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 197 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 198 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 199 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 200 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 202 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 203 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 204 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 206 with respect to the position in mature p40 sequence of SEQ ID NO: 2. In some embodiments, the variant p40 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue 209 with respect to the position in mature p40 sequence of SEQ ID NO: 2. Exemplary p40 substitution substitutions are provided in Table 4a, Table 4c, and Table 4c. In some embodiments, the variant p40 subunit comprises one or more amino acid substitutions (e.g., one, two, three, four, five, or more amino acid substitutions) selected from Table 4a, Table 4b, or Table 4c.

Table 4a. p40 Substitutions

Table 4b. p40 Substitutions

Table 4c. p40 Substitutions

[0142] In some embodiments, IL- 12 muteins comprise one or more p40 substitutions shown in the first column of the Table 5 below. In some embodiments, IL-12 muteins comprise a substitution at an amino acid residue selected from the left column of Table 5 below in combination with one, two, three, four, five or more additional p40 substitutions shown in the corresponding second column in Table 5 below.

Table 5 - p40 Mutant Combinations

[0143] In one aspect of the invention, the modified IL- 12 protein comprises a p40 subunit with a substitution or deletion at an amino acid residue corresponding to amino acid Y16 of SEQ ID NO: 2. In some embodiments, the modified IL- 12 protein comprises a p40 subunit comprising a substitution at an amino acid corresponding to amino acid residue position Y16 of SEQ ID NO: 2 and a p35 subunit with one more substitutions. For exemplary combinations, see Table 6.

Table 6. Exemplary Combinations

IL-12 Mutant Combinations

In some embodiments, the IL- 12 mutein comprises one or more substitutions in the p35 subunit and one or more substitutions in the p40 subunit. In some embodiments, the IL- 12 mutein comprises a p35 subunit and a p40 subunit, wherein the p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue A33, R34, L37, E38, F39, Y40, P41, T43, S44, E45, E46, 147, D48, H49, E50, D51, 152, T53, K54, K56, T57, D108, L109, KI 10, Mi l l, Y112, QI 13, VI 14, El 15, F116, KI 17, T118, A121, L123, M125, D126, K128, R129, Q130, 1131, L133, P156, S159, S160, L161, E162, E163, P164, D165, F166, Y167, K168, T169, KI 70, 1171, KI 72, L173, 1175, L177, H178, or R181 with respect to the position in the sequence of mature p35 (SEQ ID NO: 1), and wherein the p40 subunit comprises one substitution selected from the p40 substitution column of Table 5, and optionally wherein the p40 subunit comprises two or more substitutions selected from the corresponding additional substitutions column of Table 5. In some embodiments, the IL- 12 mutein comprises a p35 subunit and a p40 subunit, wherein the p40 subunit comprises an amino acid substitution and/or deletion at amino position VI 0, VI 1, E12, L13, D14, W15, Y16, Pl 7, DI 8, Al 9, P20, G21, W38, T39, L40, D41, Q42, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, G64, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, 194, L95, D97, Q98, K99, E100, P1O1, N1O3, KI 04, T105, F106, L107, L123, 1126, T128, L130, K135, S136, S137, R138, G139, S140, S141, D142, R157, R159, D161, N162, K163, E181, S183, L184, P185, 1186, E187, V188, V190, D191, K195, L196, K197, Y198, E199, N200, T202, S203, S204, F206, and/or D209 with respect to the position in mature p40 (SEQ ID NO: 2), and wherein the p35 subunit comprises one substitution selected from the p35 substitution column of Table 2, and optionally wherein the p35 subunit comprises two or more substitutions selected from the corresponding additional substitutions column of Table 2.

Fusion Proteins and Methods of use

[0144] In certain embodiments, it is contemplated that the IL- 12 muteins of the present invention also may be fused with other proteins or peptides to create fusion proteins. In some embodiments, an IL- 12 mutein may be fused to a half-life extending moiety.

[0145] In certain embodiments, it is contemplated that the IL- 12 muteins of the present invention may be used in a method for preventing a disease or disorder.

Polynucleotides, Vectors, and Host Cells

[0146] Disclosed herein are also polynucleotides, vectors, and host cells encoding any IL- 12 mutein disclosed herein, and/or the reverse complements of such polynucleotides. A polynucleotide of the invention comprises a nucleic acid encoding an IL- 12 mutein comprising a p35 subunit and a p40 subunit, wherein the p35 subunit comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 1, and wherein the p40 subunit comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 2. In some embodiments, the nucleic acid encodes an IL- 12 mutein comprising a p35 subunit and a p40 subunit, wherein the p35 subunit comprises an amino acid substitution and/or deletion at an amino acid residue corresponding to amino acid residue A33, R34, L37, E38, F39, Y40, P41, T43, S44, E45, E46, 147, D48, H49, E50, D51, 152, T53, K54, K56, T57, D108, L109, KI 10, Mi l l, Y112, QI 13, VI 14, El 15, Fl 16, KI 17, T118, A121, L123, M125, D126, K128, R129, Q130, 1131, L133, P156, S159, S160, L161, E162, E163, P164, D165, F166, Y167, K168, T169, KI 70, 1171, KI 72, L173, 1175, L177, H178, or R181 with respect to the position in the sequence of mature p35 (SEQ ID NO: 1), and wherein the p40 subunit comprises two or more substitutions selected from Table 5. In some embodiments, the nucleic acid encodes an IL- 12 mutein comprising a p35 subunit and a p40 subunit, wherein the p40 subunit comprises an amino acid substitution and/or deletion at amino position V10, VI 1, E12, L13, D14, W15, Y16, P17, D18, A19, P20, G21, W38, T39, L40, D41, Q42, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, G64, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, 194, L95, D97, Q98, K99, E100, P1O1, N1O3, KI 04, T105, F106, L107, L123, 1126, T128, L130, K135, S136, S137, R138, G139, S140, S 141, D142, R157, R159, D161, N162, K163, E181, S183, L184, P185, 1186, E187, V188, V190, D191, K195, L196, K197, Y198, El 99, N200, T202, S203, S204, F206, and/or D209with respect to the position in mature p40 (SEQ ID NO: 2), and wherein the p35 subunit comprises two or more substitutions selected from Table 2.

[0147] Expression vectors of the invention comprise a nucleic acid or a set of nucleic acids encoding any IL- 12 mutein disclosed herein, and a regulatory sequence operably linked to the nucleic acid or the set of nucleic acids.

[0148] Host cells of the invention may be prokaryotic or eukaryotic in origin. In some embodiments, IL- 12 muteins of the instant invention are produced in eukaryotic cells. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is a HEK293 cell, a CHO cell, or a genetic variant thereof. In some embodiments, IL- 12 muteins of the instant invention are produced in prokaryotic cells. In some embodiments, the prokaryotic cell is E. coli or a genetic variant thereof.

EXAMPLES

General Techniques

[0149] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al. , “Molecular Cloning: A Laboratory Manual,” 3^rd edition, Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology”, F. M. Ausubel, et al. eds.,1987; the series “Methods in Enzymology,” Academic Press, San Diego, CA.; “PCR 2: a practical approach”, M.J. MacPherson, B.D. Hames and G.R. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman’s The Pharmacological Basis of Therapeutics,” 11^th Edition, McGraw-Hill, 2005; and Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” 4^th edition, John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated in their entirety herein by reference.

Example 1: Structure Modeling of IL-12 Interactions

IL- 12 interacts with IL-12Rbl and IL-12Rb2. To identify amino acid residues that, when mutated, may result in an altered binding affinity with these two receptors, a predictive model was developed. To obtain high resolution information, AlphaFold2 and molecular dynamic (MD) simulation techniques were used to model the IL-12-IL-12R interactions from the low-resolution (~10A) cryoEM map. Firstly, the structure of IL-12Rb2 was modeled by AlphaFold2 from its primary sequences. Secondly, the crystal structure of IL- 12 (PDB 1F45), the crystal structure of IL-12 p40- IL-12-Rbl (PDB 6WDQ), and the AlphaFold2 model of IL-12Rb2 were rigid-body fitted into the cryoEM map (EMD-21645) by Chimera (Pettersen EF, et al. J Comput Chem. 2004 Oct;25(13):1605-12.) (Fig. 1).

Thirdly, the missing loops in IL-12 p35 were modeled by AlphaFold2. Finally, the resulting model was subjected to 200ns MD simulation by GROMACS to optimize and stabilize the structure and interactions. The IL-12-IL-12R complex remains stable during the 200 ns simulation time, and only minor conformational changes happened comparing to the starting model (Fig. 2). The structure model at the 200ns snapshot was used for downstream design. Based on above, region of putative receptor interaction region were identified (Table 7 and Fig. 3).

TABLE 7. IL-12 / IL-12R Interaction Regions Identified by Structure Modeling

Example 2: In Silico Screening of IL-12 Single Mutants

[0150] In addition to the structure modeling described above, IL- 12 p35 and p40 single mutants were screened in silico using a Molecular Operating Environment (MOE) modeling system using the structure model described above to predict change in affinity (MOE SAffinity) and change in stability (MOE SStability) relative to wild-type IL-12. In total, (306 p40 residues + 178 p35 residues) x 19 amino acid substitutions = 9196 single point substitutions were modeled and then computed for both affinity changes for binding IL- 12 receptors, and stability changes. The substitution sites identified by this in silico screen are shown in Table 8 and were selected according to the following criteria: MOE SAffinity of > 0.05 kcal/mol (where an increase in MOE SAffinity predicts a decrease in receptor affinity) and MOE SStability of < 6 kcal/mol (where an increase in MOE (/Stability predicts a decrease in stability). Thus the substitutions in Table 8 predict IL- 12 having decreased affinity relative to wild-type IL- 12, while maintain the stability of IL- 12. The predicted immunogenicity of the single mutants according to a proprietary putative T cell epitope score algorithm was also considered in the selection of IL- 12 mutants. The MOE 6 Affinity and MOE 6Stability values of selected p35 mutants are provided in Table 9 and of selected p40 mutants are provided in Table 10.

Table 8. Identified Substitution Sites

Table 9. Predicted Binding Dynamics for Additional Amino Acid Substitutions in p35

Subunit

Table 10. Predicted Binding Dynamics for Additional Amino Acid Substitutions in p40

Subunit

Example 3: Alanine-Scanning Analysis of Structure-Predicted Mutein Effector Regions

[0151] Based on the structural analysis of predicted mutein effector regions, the effect of substitution of single sites was determined by alanine-scanning substitution and analyzed by Surface Plasmon Resonance (SPR). Analysis of IL- 12 variant binding to recombinant human IL12R was performed on a Biacore 8K instrument using Fc capture. A series S CM5 chip was directly immobilized with anti-human IgG (Fc specific) according to the protocol provided by GE Healthcare in the Human Antibody Capture Kit. Recombinant human IL12RP1 Fc and IL12RP2 Fc (R&D Systems) were diluted in PBSP pH 7.4 running buffer and captured to the anti-huFc surface for 36 at 10 pL/min flowrate to obtain capture levels between 90 and 125 RU. Wild-type and IL 12 variants were diluted to 500 nM in running buffer and injected over the captured receptors for 150 sec followed by 300 sec dissociation in buffer at 30 pL/min at 22°C. The surface was regenerated with 10 mM glycine pH 1.5 for 30 sec. Sensorgrams were processed using the Insight Evaluation Software (GE Healthcare). A response point was taken at the end of IL12 association phase and normalized to the capture level of IL12RP1 Fc or IL12RP2 Fc for the comparison plots. Results of the SPR analysis assay in WT and mutated IL- 12 variants is shown in Table 11, Fig. 4 (p35) and Fig. 5 (p40).

Table 11.

Example 4: Analysis of Mutant Combinations

[0152] Following identification of potential substitution sites within the p35 and p40 subunits, a number of combination mutants were designed and tested. An IL- 12 fusion protein including the below-described mutant combinations was expressed and purified. Combinations were evaluated in an IL-12 activity reporter assay for monitoring IL-12 triggered STAT4 activation. HEK-Blue IL- 12 reporter cells (InvivoGen) were generated by stably introducing the human genes for the IL- 12 receptor and the genes of the IL- 12 signaling pathway into HEK293 cells. These cells express a STAT4-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were cultured and frozen stocks were prepared according to the protocol described by InvivoGen. Frozen stocks with 1E+07 /ml /vial were prepared as Thaw-And-Use cells for the assay. At Dayl frozen cells were thawed and adjusted to 6E+06 cells/ml with DMEM w/o phenol red supplemented with 8% of heat-inactivated fetal bovine serum (FBS). Cells were seeded at 15000 cells/well/25 pl into 384-well tissue culture-treated white/flat bottom tissue culture plate (# 781080, Greiner Bio-One Germany) by using a Multidrop Combi. For dose response a series of 3-fold dilutions of test samples were prepared in DMEM containing 1% of heat-inactivated FBS in 384-well plate with non-binding surface (#3640, Coming). 5 pl/well of the diluted samples were transferred into the test plate containing seeded cells. The final sample concentrations in the wells were in the range from lOOnM to 0.005 InM with assays run in 10- point concentration response in duplicate. The assay plates were incubated for 24 h at 37 °C/5%CO2/95% humidity. For signal detection 5 pkwell of cells supernatant were transferred into a new 384-well plate, followed by addition of 20 pl of QUANTI-Bhie solution (InvivoGen) prepared as manufacture’s description. SEAP levels were determined by absorption at OD 620nm after 2 h incubation at 37°C using a PheraStar plate reader (BMG Labtech, Germany). As for IL- 12 reporter assay data normalization, the readings of wells without any test samples were used as 0% activation (E%).

The results of the IL- 12 reporter assay for panels of mutant combinations are provided in Table 12 and Table 13.

Table 12. IL-12 Reporter assay of combination mutants (panel 1)

Table 13. IL-12 Reporter assay of combination mutants (panel 2)

Table 15. IL-12 Reporter assay of combination mutants (panel 3)

Example 5: Analysis of IL-12 muteins

[0153] Potential substitution sites within the p35 and p40 subunits were identified by structural analysis of IL- 12 in complex with IL-12R as described in Example 1 and in silico screening as described in Example 2. Of the full single-mutant substitutional space (9196 possible single mutants across p35 and p40), 965 amino acid positions corresponding to 368 p35 single mutants (Table 9) and 597 p40 single mutants (Table 10) were selected and produced. Among those, 905 IL- 12 fusion proteins were successfully expressed, purified, and tested in the IL- 12 reporter assay (InvivoGen), as described in Example 4. The following selection criteria were used to select mutants of interest: protein mass concentration > 0.1 mg/mL, good protein quality (as assessed by LabChip QC) and a fold-change EC50 >50 over WT IL-12 controls. Selected mutants were tested in an IL12RB1/IL12RB2 dimerization assay. Briefly, PathHunter® U2OS IL12RB1/IL12RB2 cells (Discover X, Eurofins) were transduced to overexpress human IL12RB1 and IL12RB2 genes. Binding of IL-12 (or IL-12 fusion protein) to one receptor subunit and subsequent dimerization with the other receptor subunit, leads to the complementation of two enzyme fragments, resulting in the formation of a functional enzyme that hydrolyzes a substrate to generate a chemiluminescent signal. Cells were thawed and cultured following recommendations and reagents provided by the manufacturer. For the assay, cells were seeded at 10000 cells/well/90 pl into 96-well white flat bottom tissue culture-treated plates and incubated at 37 °C/5%CO2/95% humidity for 4h before treatment. For dose response, a series of 5 -fold dilutions of compounds were prepared in test medium and 20 phwell of the diluted compounds were transferred into the test plate containing seeded cells. The final sample concentrations in the wells were in the range from 100 nM to 0.0013 nM with assays run in 8- point concentration response in triplicate. The assay plates were incubated for 18h to 24h at 37 °C/5%CO2/95% humidity. For signal detection, 110 pl/well of PathHunter Flash Detection Reagent (prepared following manufacturer’s protocol) were added directly onto the cells. After Ih incubation at room temperature in the dark, luminescence values were determined using a Spark® microplate reader (Tecan). For data normalization, the readings of wells without any compounds were used as 0% activation (E%). Results (%Emax and EC50) obtained for 93 selected single mutants are provided in Tables 16, 17 and 18.

Table 16. IL-12RB1/B2 dimerization assay of single mutants (assay 1)

Table 17. IL-12RB1/B2 dimerization assay of single mutants (assay 2)

Table 18. IL-12RB1/B2 dimerization assay of single mutants (assay 3)

[0154] 21 mutants of interest were tested again in the IL-12 activity reporter assay (Invivogen) for monitoring IL- 12 triggered STAT4 activation. Cells were cultured and frozen stocks were prepared according to the protocol described by InvivoGen. Cells were thawed and passaged twice in complete growth medium (DMEM 4.5 g/L glucose, 2 mM L-glutamine, 10% heat- inactivated fetal bovine serum (FBS)) before adding selection antibiotics (blasticidin, hygromycin B and Zeocin™) at concentrations recommended by Invivogen. After a minimum of two passages in the selection medium, cells were prepared for the assay as follows: cells were resuspended in test medium (complete medium without selection antibiotics) and seeded at 50000 cells/well/160 pl into 96-well clear flat bottom tissue culture-treated plates (# 353072, Falcon®). For dose response, a series of 5-fold dilutions of compounds were prepared in test medium and 40 pkwell of the diluted compounds were transferred into the test plate containing seeded cells. The final sample concentrations in the wells were in the range from 100 nM to 0.0013 nM with assays run in 8-point concentration response in triplicate. The assay plates were incubated for 18h to 24h at 37 °C/5%CO2/95% humidity. For signal detection, 20 pkwell of cells supernatant were transferred into a new 96-well plate, followed by addition of 180 pl of QUANTI-Blue solution (InvivoGen) prepared as manufacturer’s description. SEAP levels were determined by absorption at OD 630nm after 3h incubation at 37°C using a Spark® microplate reader (Tecan). As for IL-12 reporter assay data normalization, the readings of wells without any compounds were used as 0% activation (E%). Results (%Emax and EC50) are provided in Table 19. Table 19. IL-12 Reporter assay of single mutants

EMBODIMENTS

El. An interleukin- 12 (IL12) mutein comprising a variant p35 subunit, a variant p40 subunit, or both a variant p35 subunit and a variant p40 subunit, wherein the variant p35 subunit comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 1; wherein the variant p40 subunit comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 2.

E2. The IL-12 mutein of embodiment El, comprising a variant p35 subunit, wherein the variant p35 subunit comprises a substitution at amino acid residue LI 33 relative to SEQ ID NO: 1.

E3. The IL-12 mutein of embodiment E2, wherein the L133 substitution of the variant p35 subunit is selected from the group consisting ofL133A, L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133I, L133M, L133F, L133W, L133V, and L133Y.

E4. The IL-12 mutein of embodiment E2 or E3, wherein the variant p35 subunit further comprises one or more amino acid substitutions relative to SEQ ID NO: 1.

E5. The IL- 12 mutein of embodiment E4, wherein the variant p35 subunit comprises one, two, three, four, five, or more amino acid substitutions at a position selected from the group consisting of L37, F39, Y40, P41, D48, M125, D126, K128, R129, 1131, L133, P164, D165, F166, Y167, K170, L173, L177, H178 relative to SEQ ID NO: 1.

E6. The IL-12 mutein of embodiment E4 or E5, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37R, L37H, L37K, L37D, L37E, L37S, L37T, L37N, L37Q, L37G, L37P, L37A, L37V, L37I, L37M, L37F, L37Y, or L37W. E7. The IL-12 mutein of any one of embodiments E4 to E6, wherein the variant p35 subunit comprises a substitution at Y40, optionally wherein the substitution is Y40R, Y40H, Y40K, Y40D, Y40E, Y40S, Y40T, Y40N, Y40Q, Y40G, Y40P, Y40A, Y40V, Y40I, Y40L, Y40M, Y40F, or Y40W. E8. The IL- 12 mutein of any one of embodiments E4 to E7, wherein the variant p35 subunit comprises a substitution at D48, optionally wherein the substitution is D48R, D48H, D48K, D48E, D48S, D48T, D48N, D48Q, D48G, D48P, D48A, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W.

E9. The IL-12 mutein of any one of embodiments E4 to E8, wherein the variant p35 subunit comprises a substitution at KI 28, optionally wherein the substitution is K128R, K128H, K128K, K128D, K128E, K128S, K128T, K128N, K128Q, K128G, K128P, K128A, K128V, K128I, K128L, K128M, K128F, K128Y, or K128W.

E10. The IL-12 mutein of any one of embodiments E4 to E9, wherein the variant p35 subunit comprises a substitution at M125, optionally wherein the substitution is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125A, M125V, M125I, M125L, M125F, M125Y, or M125W.

El l. The IL- 12 mutein of any one of embodiments E4 to E10, wherein the variant p35 subunit comprises a substitution at R129, optionally wherein the substitution is R129H, R129K, R129D, R129E, R129S, R129T, R129N, R129Q, R129G, R129P, R129A, R129V, R129I, R129L, R129M, R129F, R129Y, or R129W.

E12. The IL-12 mutein of any one of embodiments E4 to El 1, wherein the variant p35 subunit comprises a substitution at L133, optionally wherein the substitution is L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133A, L133V, L133I, L133M, L133F, L133Y, or L133W.

El 3. The IL- 12 mutein of any one of embodiments E4 to El 2, wherein the variant p35 subunit comprises a substitution at L177, optionally wherein the substitution is L177R, L177H, L177K, L177D, L177E, L177S, L177T, L177N, L177Q, L177G, L177P, L177A, L177V, L177I, L177M, L177F, L177Y, or L177W.

E14. The IL-12 mutein of any one of embodiments E4 to E13, wherein the variant p35 subunit comprises a substitution at Hl 78, optionally wherein the substitution is H178R, H178K, H178D, H178E, H178S, H178T, H178N, H178Q, H178G, H178P, H178A, H178V, H178I, H178L, H178M, H178F, H178Y, or H178W.

El 5. The IL- 12 mutein of embodiment El, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; and a substitution at Y40, optionally wherein the substitution is Y40S.

E16. The IL-12 mutein of embodiment El, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at LI 33, optionally wherein the substitution is LI 33 A.

E17. The IL-12 mutein of embodiment El, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at KI 28, optionally wherein the substitution is K128N or K128Q.

El 8. The IL- 12 mutein of embodiment El, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; a substitution at KI 28, optionally wherein the substitution is K128N or K128Q; and a substitution at LI 33, optionally wherein the substitution is LI 33 A.

E19. The IL-12 mutein of any one of embodiments El to E18, wherein the variant p35 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1.

E20. The IL-12 mutein of any one of embodiments El to E19, comprising a variant p40 subunit, wherein the variant p40 subunit comprises a substitution at amino acid residue Y16 relative to SEQ ID NO: 2.

E21. The IL- 12 mutein of embodiment E20, wherein the Y16 substitution of the variant p40 subunit is selected from the group consisting of Y16A, Y16R, Y16H, Y16K, Y16D, Y16E, Y16S, Y16T, Y16N, Y16Q, Y16G, Y16P, Y16I, Y16L, Y16M, Y16F, Y16W, and Y16V. E22. The IL- 12 mutein of embodiment E20 or E21, wherein the variant p40 subunit further comprises one or more amino acid substitutions relative to SEQ ID NO: 2.

E23. The IL- 12 mutein of any one of embodiments El to E22, wherein the variant p40 subunit comprises one, two, three, four, five, or more amino acid substitutions at a position selected from the group consisting of W15, P17, D18, E59, F60, K84, E86, D93, H194, K195, and K197 relative to SEQ ID NO: 2.

E24. The IL-12 mutein of any one of embodiments El to E23, wherein the variant p40 subunit further comprises one or more, two or more, or three or more amino acid substitutions at a position selected from E59, F60, K84, K195, and K197 relative to SEQ ID NO: 2.

E25. The IL- 12 mutein of any one of embodiments El to E24, wherein the variant p40 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.

E26. An interleukin- 12 (IL- 12) mutein comprising a variant p35 subunit and a variant p40 subunit, wherein: the variant p35 subunit comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions are at a position selected from A33, R34, L37, E38, F39, Y40, P41, E45, E46, 147, D48, H49, E50, D51, 152, T53, K54, K56, T57, D108, L109, KI 10, Ml 11, Y112, QI 13, VI 14, El 15, Fl 16, KI 17, T118, A121, L123, M125, D126, R129, 1131, P156, S159, S160, L161, E162, E163, D165, F166, Y167, K168, T169, KI 70, 1171, KI 72, L173, 1175, H178, and R181 relative to SEQ ID NO: 1, and the variant p40 subunit comprises one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions are at a position selected from V10, VI 1, E12, L13, D14, W15, Y16, P17, D18, A19, P20, G21, W38, T39, L40, D41, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, G64, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, 194, L95, D97, Q98, K99, E100, N103, KI 04, T105, F106, L107, L123, K135, S136, S137, R138, G139, S140, S141, D142, N162, E181, S183, L184, P185, 1186, E187, V188, V190, D191, H194, K195, L196, K197, Y198, E199, N200, T202, S203, S204, F206, and D209 relative to SEQ ID NO: 2.

E27. The IL-12 mutein of embodiment E26, wherein Y16 of SEQ ID NO: 2 is substituted, preferably with an alanine residue.

E28. The IL-12 mutein of embodiment E26 or E27, wherein L133 of SEQ ID NO: 1 is substituted, preferably with an alanine residue.

E29. The IL-12 mutein of any one of embodiment E26 to E28, wherein the variant p35 further comprises one or more amino acid substitutions or substitutions at a position selected from:

(a) wherein DI 26 of SEQ ID NO: 1 is substituted, preferably with an alanine residue;

(b) wherein 1131 of SEQ ID NO: 1 is substituted, preferably with an alanine residue;

(c) wherein Hl 78 of SEQ ID NO: 1 is substituted, preferably with an alanine residue; and

(d) any combination thereof.

E30. An interleukin- 12 (IL-12) mutein comprising a variant p35 subunit and a variant p40 subunit, wherein:

(a) the variant p35 subunit comprises a substitution of one or more amino acids as shown in Table 1;

(b) the p40 moiety comprises a substitution of one or more amino acids as shown in Table 4; or

(c) the p35 moiety comprises a substitution of one or more amino acids as shown in Table 1 and the p40 moiety comprises a substitution of one or more amino acids as shown in Table 4. E31. An interleukin- 12 (IL- 12) mutein comprising a variant p35 subunit and a variant p40 subunit, wherein:

(a) the variant p35 subunit comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 1, wherein the substitution of one or more amino acids is a substitution listed in Table I;

(b) the variant p40 subunit comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 2, wherein the substitution of one or more amino acids is a substitution listed in Table 4; or

(c) the variant p35 subunit comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 1, wherein the substitution of one or more amino acids is a substitution listed in Table I, and the p40 moiety comprises a substitution of one or more amino acid residues corresponding to an amino acid residue in SEQ ID NO: 2, wherein the substitution of one or more amino acids is a substitution listed in Table 4.

E32. The IL-12 mutein of any one of embodiments E1-E31, wherein the IL-12 mutein is a human IL- 12 mutein.

E33. A fusion protein comprising the IL-12 mutein of any one of embodiments E1-E32.

E34. A pharmaceutical composition comprising the IL-12 mutein of any one of embodiment El to E32 or the fusion protein of embodiment E33, and a pharmaceutically acceptable carrier or excipient.

E35. A polynucleotide sequence that encodes the IL- 12 mutein according to any one of embodiments El to E32 or the fusion protein of embodiment E33.

E36. An expression vector comprising the polynucleotide sequence of embodiment E35.

E37. A host cell comprising the expression vector of embodiment E36.

E38. A kit comprising the IL-12 mutein according to any one of c embodiments El to E32 or the fusion protein of embodiment E33 and optionally packaging material.

E39. A method of producing the IL-12 mutein of any one of embodiments El to E32 or the fusion protein of embodiment E33, comprising culturing the host cell of embodiment E35 under conditions to express the IL- 12 mutein or the fusion protein.

E40. The method of embodiment E39, further comprising isolating the IL-12 mutein from the host cell.

E41. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the IL-12 mutein of any one of embodiments El to E32, the fusion protein of embodiment E33, or the pharmaceutical composition of embodiment E34. E42. The method of embodiment E41, wherein the cancer comprises a solid tumor.

E43. An interleukin- 12 (IL- 12) mutein comprising a variant p35 subunit, a variant p40 subunit, or both a variant p35 subunit and a variant p40 subunit, wherein the variant p35 subunit, if present, comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 1; and wherein the variant p40 subunit, if present, comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 2.

E44. The IL-12 mutein of embodiment E43, comprising the variant p35 subunit.

E45. The IL-12 mutein of embodiment E44, wherein the variant p35 subunit comprises a substitution at amino acid residue L133, optionally wherein the substitution is L133A, L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133I, L133M, L133F, L133W, L133V, or L133Y.

E46. The IL- 12 mutein of embodiment E44 or E45, wherein the variant p35 subunit comprises a substitution at amino acid residue L37, optionally wherein the substitution is L37R, L37H, L37K, L37D, L37E, L37S, L37T, L37N, L37Q, L37G, L37P, L37A, L37V, L37I, L37M, L37F, L37Y, or L37W.

E47. The IL- 12 mutein of embodiment E46, wherein, the substitution at amino acid residue L37 is L37R, L37H, L37K, L37D, L37N, L37Q, L37P, L37A, L37V, L37M, L37F, L37Y, or L37W. E48. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37A.

E49. The IL-12 mutein of any one of embodiments E44 to E48, wherein the variant p35 subunit comprises a substitution at amino acid residue Y40, optionally wherein the substitution is Y40R, Y40H, Y40K, Y40D, Y40E, Y40S, Y40T, Y40N, Y40Q, Y40G, Y40P, Y40A, Y40V, Y40I, Y40L, Y40M, Y40F, or Y40W.

E50. The IL-12 mutein of embodiment E49, wherein the substitution at amino acid residue Y40 is Y40H, Y40E, Y40V, Y40I, Y40L, Y40M, or Y40F.

E51. The IL-12 mutein of embodiment E49, wherein the Y40 substitution is Y40S.

E52. The IL-12 mutein of any one of embodiments E44 to E51, wherein the variant p35 subunit comprises a substitution at amino acid residue D48, optionally wherein the substitution is D48R, D48H, D48K, D48E, D48S, D48T, D48N, D48Q, D48G, D48P, D48A, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W.

E53. The IL-12 mutein of embodiment E52, wherein the D48 substitution is D48H, D48K, D48E, D48S, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W, optionally wherein the D48 substitution is D48K, D48E, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, or D48F. E54. The IL-12 mutein of any one of embodiments E44 to E53, wherein the variant p35 subunit comprises a substitution at amino acid residue KI 28, optionally wherein the substitution is K128R, K128H, K128K, K128D, K128E, K128S, K128T, K128N, K128Q, K128G, K128P, K128A, K128V, K128I, K128L, K128M, K128F, K128Y, or K128W.

E55. The IL-12 mutein of embodiment E54, wherein the K128 substitution is K128N or K128Q. E56. The IL-12 mutein of any one of embodiments E44 to E55, wherein the variant p35 subunit comprises a substitution at amino acid residue M125, optionally wherein the substitution is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125A, M125V, M125I, M125L, M125F, M125Y, or M125W.

E57. The IL-12 mutein of embodiment E56, wherein the M125 substitution is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125L, M125F, M125Y, or M125W.

E58. The IL- 12 mutein of any one of embodiments E44 to E57, wherein the variant p35 subunit comprises a substitution at amino acid residue R129, optionally wherein the substitution is R129H, R129K, R129D, R129E, R129S, R129T, R129N, R129Q, R129G, R129P, R129A, R129V, R129I, R129L, R129M, R129F, R129Y, or R129W.

E59. The IL-12 mutein of any one of embodiments E44 to E58, wherein the variant p35 subunit comprises a substitution at amino acid residue LI 77, optionally wherein the substitution is L177R, L177H, L177K, L177D, L177E, L177S, L177T, L177N, L177Q, L177G, L177P, L177A, L177V, L177I, L177M, L177F, L177Y, or L177W.

E60. The IL-12 mutein of embodiment E59, wherein the L177 substitution is L177D, L177S, L177T, L177N, L177G, L177P, L177V, L177I, L177M, or L177W.

E61. The IL- 12 mutein of any one of embodiments E44 to E60, wherein the variant p35 subunit comprises a substitution at amino acid residue Hl 78, optionally wherein the substitution is H178R, H178K, H178D, H178E, H178S, H178T, H178N, H178Q, H178G, H178P, H178A, H178V, H178I, H178L, H178M, H178F, H178Y, or H178W.

E62. The IL-12 mutein of any one of embodiments E44 to E61, wherein the variant p35 subunit comprises a substitution at amino acid residue K56, optionally wherein the K56 substitution is K56R, K56H, K56D, K56E, K56S, K56T, K56N, K56Q, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W.

E63. The IL-12 mutein of embodiment E62, wherein the K56 substitution is K56R, K56H, K56D, K56E, K56S, K56T, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W, optionally wherein the K56 substitution is K56E, K56S, K56T, K56G, K56A, K56V, K56I, or K56L. E64. The IL- 12 mutein of embodiment E62, wherein the K56 substitution is K56N.

E65. The IL- 12 mutein of any one of embodiments E44 to E64, wherein the variant p35 subunit comprises a substitution at amino acid residue KI 70, optionally wherein the KI 70 substitution is K170R, K170H, K170D, K170E, KI 70S, K170T, K170N, K170Q, K170G, K170P, K170A, KI 70V, KI 701, K170L, K170M, K170F, K170Y, or K170W.

E66. The IL-12 mutein of any one of embodiments E44 to E65, wherein the variant p35 subunit comprises a substitution at amino acid residue 1171, optionally wherein the 1171 substitution is I171R, I171K, I171H, I171D, I171E, 1171 S, I171T, I171N, I171Q, I171G, I171P, I171A, I171V, I171L, I171M, I171F, I171Y, or I171W.

E67. The IL-12 mutein of any one of embodiments E44 to E66, wherein the variant p35 subunit comprises a substitution at amino acid residue A33, R34, E38, F39, P41, E45, E46, 147, H49, E50, D51, 152, T53, K54, T57, D108, L109, KI 10, Mi l l, Y112, QI 13, VI 14, El 15, Fl 16, K117, T118, A121, L123, D126, 1131, P156, S159, S160, L161, E162, E163, D165, F166, Y167, K168, T169, KI 72, L173, 1175, and R181.

E68. The IL-12 mutein of embodiment E44, wherein the variant p35 subunit comprises one or more amino acid substitutions selected from Table la, Table lb, or Table 1c.

E69. The IL-12 mutein of embodiment E44, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; and a substitution at Y40, optionally wherein the substitution is Y40S.

E70. The IL-12 mutein of embodiment E44, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at LI 33, optionally wherein the substitution is LI 33 A.

E71. The IL- 12 mutein of embodiment E44, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at KI 28, optionally wherein the substitution is K128N or K128Q.

E72. The IL- 12 mutein of embodiment E44, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; a substitution at KI 28, optionally wherein the substitution is K128N or K128Q; and a substitution at LI 33, optionally wherein the substitution is LI 33 A. E73. The IL-12 mutein of any one of embodiments E44 to E72, wherein the variant p35 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1.

E74. The IL-12 mutein of any one of embodiments E43 to E73, comprising the variant p40 subunit.

E75. The IL-12 mutein of embodiment E74, wherein the variant p40 subunit comprises a substitution at amino acid residue V10, optionally wherein the substitution is V10A, VI OR, V10H, V10K, V10D, V10E, V10S, V10T, V10N, V10Q, V10G, V10P, V10I, VIOL, V10M, VI OF, VI 0W, or VlOY.

E76. The IL-12 mutein of embodiment E74 or E75, wherein the variant p40 subunit comprises a substitution at amino acid residue 194, optionally wherein the substitution is I94A, I94R, I94H, I94K, I94D, I94E, I94S, I94T, I94N, I94Q, I94G, I94P, I94L, I94M, I94F, I94W, I94V, or I94Y. E77. The IL-12 mutein of any one of embodiments E74 to E76, wherein the variant p40 subunit comprises a substitution at amino acid residue L95, optionally wherein the substitution is L95A, L95R, L95H, L95K, L95D, L95E, L95S, L95T, L95N, L95Q, L95G, L95P, L95I, L95M, L95F, L95W, L95V, or L95Y.

E78. The IL-12 mutein of any one of embodiments E74 to E77, wherein the variant p40 subunit comprises a substitution at amino acid residue G64, optionally wherein the substitution is G64A, G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y.

E79. The IL-12 mutein of embodiment E78, wherein the G64 substitution is G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y, optionally wherein the G64 substitution is G64R, G64K, G64E, G64S, G64T, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y.

E80. The IL-12 mutein of any one of embodiments E74 to E79, wherein the variant p40 subunit comprises a substitution at amino acid residue Y198, optionally wherein the substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198S, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V.

E81. The IL- 12 mutein of embodiment E80, wherein the Y198 substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V, optionally wherein the Y198 substitution is Y198G, Y198E, Y198K, Y198Q, Y198R, Y198W.

E82. The IL- 12 mutein of any one of embodiments E74 to E81, wherein the variant p40 subunit comprises a substitution at amino acid residue Y16, optionally wherein the substitution is Y16A, Y16R, Y16H, Y16K, Y16D, Y16E, Y16S, Y16T, Y16N, Y16Q, Y16G, Y16P, Y16I, Y16L, Y16M, Y16F, Y16W, or Y16V.

E83. The IL- 12 mutein of any one of embodiments E74 to E82, wherein the variant p40 subunit comprises a substitution at amino acid residue VI 1, E12, L13, D14, W15, P17, D18, A19, P20, G21, W38, T39, L40, D41, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, D97, Q98, K99, E100, N103, KI 04, T105, F106, L107, L123, K135, S136, S137, R138, G139, S140, S141, D142, N162, E181, S183, L184, P185, 1186, E187, V188, V190, D191, H194, K195, L196, K197, El 99, N200, T202, S203, S204, F206, or D209.

E84. The IL-12 mutein of embodiment E83, wherein the variant p40 subunit comprises a substitution at amino acid residue W15, P17, D18, E59, F60, K84, E86, D93, H194, K195, or KI 97, optionally wherein the variant p40 subunit comprises a substitution at amino acid residue E59, F60, K84, K195, or K197.

E85. The IL-12 mutein of embodiment E74, wherein the variant p40 subunit comprises one or more amino acid substitutions selected from Table 4a, Table 4b, or Table 4c.

E86. The IL- 12 mutein of any one of embodiments E74 to E85, wherein the variant p40 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.

E87. The IL- 12 mutein of any one of embodiments E43 to E86, wherein the IL- 12 mutein is a human IL- 12 mutein.

E88. A fusion protein comprising the IL-12 mutein of any one of embodiments E43- E87.

E89. A pharmaceutical composition comprising the IL- 12 mutein of any one of embodiments E43 to E87 or the fusion protein of embodiment E88, and a pharmaceutically acceptable carrier or excipient.

E90. A polynucleotide sequence that encodes the IL- 12 mutein according to any one of embodiments E43 to E87 or the fusion protein of embodiment E88.

E91. An expression vector comprising the polynucleotide sequence of embodiment E90.

E92. A host cell comprising the expression vector of embodiment E91.

E93. A kit comprising the IL-12 mutein according to any one of embodiments E43 to E87 or the fusion protein of embodiment E88 and optionally packaging material.

E94. A method of producing the IL- 12 mutein of any one of embodiments E43 to E87 or the fusion protein of embodiment E88, comprising culturing the host cell of embodiment E92 under conditions to express the IL- 12 mutein or the fusion protein. E95. The method of embodiment E94, further comprising isolating the IL- 12 mutein from the host cell.

E96. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the IL- 12 mutein of any one of embodiments E43 to E87, the fusion protein of embodiment E88, or the pharmaceutical composition of embodiment E89.

E97. The method of embodiment E96, wherein the cancer comprises a solid tumor.

E98. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37R.

E99. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37H.

El 00. The IL- 12 mutein of embodiment E46, wherein the L37 substitution is L37K. E101. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37D.

El 02. The IL- 12 mutein of embodiment E46, wherein the L37 substitution is L37N.

E103. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37Q.

E104. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37P.

E105. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37V.

El 06. The IL- 12 mutein of embodiment E46, wherein the L37 substitution is L37M.

El 07. The IL- 12 mutein of embodiment E46, wherein the L37 substitution is L37L.

E108. The IL-12 mutein of embodiment E46, wherein the L37 substitution is L37Y.

El 09. The IL- 12 mutein of embodiment E46, wherein the L37 substitution is L37W.

E110. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133A.

El 11. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133R.

El 12. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133H.

El 13. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133K.

El 14. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133D.

El 15. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133E.

El 16. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133S.

El 17. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133T.

El 18. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133N.

El 19. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133Q.

E120. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133G.

E121. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133P.

E122. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133I.

E123. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133M.

E124. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133L. E125. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133W. E126. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133V. E127. The IL-12 mutein of embodiment E45, wherein the L133 substitution is L133Y. E128. The IL-12 mutein of embodiment E50, wherein the Y40 substitution is Y40H. El 29. The IL- 12 mutein of embodiment E50, wherein the Y40 substitution is Y40E. El 30. The IL- 12 mutein of embodiment E50, wherein the Y40 substitution is Y40V. E131. The IL- 12 mutein of embodiment E50, wherein the Y40 substitution is Y40I. E132. The IL-12 mutein of embodiment E50, wherein the Y40 substitution is Y40L. El 33. The IL- 12 mutein of embodiment E50, wherein the Y40 substitution is Y40M. E134. The IL-12 mutein of embodiment E50, wherein the Y40 substitution is Y40E. E135. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48K. E136. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48E. E137. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48T. E138. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48N. E139. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48G. E140. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48P. E141. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48V. E142. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48I. E143. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48L. E144. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48M. E145. The IL-12 mutein of embodiment E53, wherein the D48 substitution is D48L. E146. The IL-12 mutein of embodiment E55, wherein the K128 substitution is K128N. E147. The IL-12 mutein of embodiment E55, wherein the K128 substitution is K128Q. E148. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125R. E149. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125H. E150. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125K. E151. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125D. E152. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125E. E153. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125S. E154. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125T. E155. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125N. E156. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125Q. E157. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125G. E158. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125P. E159. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125L. E160. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125F. E161. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125Y. E162. The IL-12 mutein of embodiment E57, wherein the M125 substitution is M125W. E163. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129H. E164. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129K. E165. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129D. E166. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129E. E167. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129S. E168. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129T. E169. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129N. E170. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129Q. E171. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129G. E172. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129P. E173. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129A. E174. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129V. E175. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129I. E176. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129L. E177. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129M. E178. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129F. E179. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129Y. El 80. The IL-12 mutein of embodiment E58, wherein the R129 substitution is R129W. E181. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177D. E182. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177S. E183. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177T. E184. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177N. E185. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177G. E186. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177P. E187. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177V. E188. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177I. E189. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177M. E190. The IL-12 mutein of embodiment E60, wherein the L177 substitution is L177W. El 91. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178R. El 92. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178K. E193. The IL-12 mutein of embodiment E61, wherein the H178 substitution is H178D. El 94. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178E. E195. The IL-12 mutein of embodiment E61, wherein the H178 substitution is H178S. El 96. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178T. El 97. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178N. E198. The IL-12 mutein of embodiment E61, wherein the H178 substitution is H178Q. El 99. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178G. E200. The IL-12 mutein of embodiment E61, wherein the H178 substitution is H178P. E201. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178A. E202. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is Hl 78V. E203. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is Hl 781. E204. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178L. E205. The IL-12 mutein of embodiment E61, wherein the H178 substitution is H178M. E206. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178L. E207. The IL- 12 mutein of embodiment E61, wherein the Hl 78 substitution is H178Y. E208. The IL-12 mutein of embodiment E61, wherein the H178 substitution is H178W. E209. The IL-12 mutein of embodiment E64, wherein the K56 substitution is K56N. E210. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170R. E211. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170H. E212. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170D. E213. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170E. E214. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is KI 70S. E215. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170T. E216. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170N. E217. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170Q. E218. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170G. E219. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170P. E220. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170A. E221. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170V. E222. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170I. E223. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170L. E224. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170M. E225. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170L E226. The IL-12 mutein of embodiment E65, wherein the KI 70 substitution is K170Y. E227. The IL-12 mutein of embodiment E65, wherein the K170 substitution is K170W. E228. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171R. E229. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171K. E230. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171H. E231. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171D. E232. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is II 71 E. E233. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is II 71 S. E234. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is II 71 T. E235. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171N. E236. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171Q. E237. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171G. E238. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is II 71 P. E239. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171A. E240. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is 1171 V. E241. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is II 71 L. E242. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is I171M. E243. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is II 71 F E244. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is 1171 Y. E245. The IL-12 mutein of embodiment E66, wherein the 1171 substitution is 1171 W. E246. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10A. E247. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10R. E248. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10H. E249. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10K. E250. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10D. E251. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10E. E252. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10S. E253. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10T. E254. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10N. E255. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10Q. E256. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10G. E257. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10P. E258. The IL-12 mutein of embodiment E75, wherein the V10 substitution is VIOL E259. The IL-12 mutein of embodiment E75, wherein the V10 substitution is VIOL. E260. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10M. E261. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10F. E262. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10W. E263. The IL-12 mutein of embodiment E75, wherein the V10 substitution is V10Y. E264. The IL- 12 mutein of embodiment E76, wherein the 194 substitution is I94A. E265. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94R. E266. The IL- 12 mutein of embodiment E76, wherein the 194 substitution is I94H. E267. The IL- 12 mutein of embodiment E76, wherein the 194 substitution is I94K. E268. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94D. E269. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94E. E270. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94S. E271. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94T. E272. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94N. E273. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94Q. E274. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94G. E275. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94P. E276. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94L. E277. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94M.

E278. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94F. E279. The IL- 12 mutein of embodiment E76, wherein the 194 substitution is 194 W. E280. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94V. E281. The IL-12 mutein of embodiment E76, wherein the 194 substitution is I94Y. E282. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95A. E283. The IL-12 mutein of embodiment E77, wherein the L95 substitution is L95R. E284. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95H. E285. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95K. E286. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95D. E287. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95E. E288. The IL-12 mutein of embodiment E77, wherein the L95 substitution is L95S. E289. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95T. E290. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95N. E291. The IL-12 mutein of embodiment E77, wherein the L95 substitution is L95Q. E292. The IL-12 mutein of embodiment E77, wherein the L95 substitution is L95G. E293. The IL-12 mutein of embodiment E77, wherein the L95 substitution is L95P. E294. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95I. E295. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L9M. E296. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95F. E297. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95W. E298. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95V. E299. The IL- 12 mutein of embodiment E77, wherein the L95 substitution is L95Y. E300. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64R. E301. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64K. E302. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64E. E303. The IL-12 mutein of embodiment E79, wherein the G64 substitution is G64S. E304. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64T. E305. The IL-12 mutein of embodiment E79, wherein the G64 substitution is G64Q. E306. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64P. E307. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64I. E308. The IL-12 mutein of embodiment E79, wherein the G64 substitution is G64L. E309. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64M. E310. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64F. E311. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64W. E312. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64V. E313. The IL- 12 mutein of embodiment E79, wherein the G64 substitution is G64Y. E314. The IL- 12 mutein of embodiment E81, wherein the Y198 substitution is Y198G. E315. The IL- 12 mutein of embodiment E81, wherein the Y198 substitution is Y198E. E316. The IL- 12 mutein of embodiment E81, wherein the Y198 substitution is Y198K. E317. The IL- 12 mutein of embodiment E81, wherein the Y198 substitution is Y198Q. E318. The IL- 12 mutein of embodiment E81, wherein the Y198 substitution is Y198R. E319. The IL- 12 mutein of embodiment E81, wherein the Y198 substitution is Y198W. E320. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16A. E321. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16R. E322. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16H. E323. The IL-12 mutein of embodiment E82, wherein the Y16 substitution is Y16K. E324. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16D. E325. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16E. E326. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16S. E327. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16T. E328. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16N. E329. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16Q.

E330. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16G.

E331. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16P.

E332. The IL-12 mutein of embodiment E82, wherein the Y16 substitution is Y16I.

E333. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16L.

E334. The IL-12 mutein of embodiment E82, wherein the Y16 substitution is Y16M.

E335. The IL-12 mutein of embodiment E82, wherein the Y16 substitution is Y16F.

E336. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16W.

E337. The IL- 12 mutein of embodiment E82, wherein the Y16 substitution is Y16V.

Table 20: Polypeptide sequences of subunits of wild-type IL-12 and IL-12 receptor

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An interleukin- 12 (IL- 12) mutein comprising a variant p35 subunit, a variant p40 subunit, or both a variant p35 subunit and a variant p40 subunit, wherein the variant p35 subunit, if present, comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 1; and wherein the variant p40 subunit, if present, comprises an amino acid sequence with one or more substitutions or deletions relative to SEQ ID NO: 2.

2. The IL-12 mutein of claim 1, comprising the variant p35 subunit.

3. The IL- 12 mutein of claim 2, wherein the variant p35 subunit comprises a substitution at amino acid residue L133, optionally wherein the substitution is L133A, L133R, L133H, L133K, L133D, L133E, L133S, L133T, L133N, L133Q, L133G, L133P, L133I, L133M, L133F, L133W, L133V, or L133Y.

4. The IL-12 mutein of claim 2 or 3, wherein the variant p35 subunit comprises a substitution at amino acid residue L37, optionally wherein the substitution is L37R, L37H, L37K, L37D, L37E, L37S, L37T, L37N, L37Q, L37G, L37P, L37A, L37V, L37I, L37M, L37F, L37Y, or L37W.

5. The IL- 12 mutein of claim 4, wherein the substitution at amino acid residue L37 is L37R, L37H, L37K, L37D, L37N, L37Q, L37P, L37A, L37V, L37M, L37F, L37Y, or L37W.

6. The IL-12 mutein of claim 4, wherein the L37 substitution is L37A.

7. The IL-12 mutein of any one of claims 2 to 6, wherein the variant p35 subunit comprises a substitution at amino acid residue Y40, optionally wherein the substitution is Y40R, Y40H, Y40K, Y40D, Y40E, Y40S, Y40T, Y40N, Y40Q, Y40G, Y40P, Y40A, Y40V, Y40I, Y40L, Y40M, Y40F, or Y40W.

8. The IL- 12 mutein of claim 7, wherein the substitution at amino acid residue Y40 is Y40H, Y40E, Y40V, Y40I, Y40L, Y40M, or Y40F.

9. The IL-12 mutein of claim 7, wherein the substitution at amino acid residue Y40 is Y40S.

10. The IL-12 mutein of any one of claims 2 to 9, wherein the variant p35 subunit comprises a substitution at amino acid residue D48, optionally wherein the substitution is D48R, D48H, D48K, D48E, D48S, D48T, D48N, D48Q, D48G, D48P, D48A, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W.

11. The IL- 12 mutein of claim 10, wherein the substitution at amino acid residue D48 is D48H, D48K, D48E, D48S, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, D48F, D48Y, or D48W, optionally wherein the substitution at amino acid residue D48 is D48K, D48E, D48T, D48N, D48G, D48P, D48V, D48I, D48L, D48M, or D48F.

12. The IL-12 mutein of any one of claims 2 to 11, wherein the variant p35 subunit comprises a substitution at amino acid residue K128, optionally wherein the substitution is K128R, K128H, K128K, K128D, K128E, K128S, K128T, K128N, K128Q, K128G, K128P, K128A, K128V, K128I, K128L, K128M, K128F, K128Y, or K128W.

13. The IL-12 mutein of claim 12, wherein the substitution at amino acid residue K128 is K128N or K128Q.

14. The IL-12 mutein of any one of claims 2 to 13, wherein the variant p35 subunit comprises a substitution at amino acid residue M125, optionally wherein the substitution is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125A, M125V, M125I, M125L, M125F, M125Y, or M125W.

15. The IL- 12 mutein of claim 14, wherein the substitution at amino acid residue Ml 25 is M125R, M125H, M125K, M125D, M125E, M125S, M125T, M125N, M125Q, M125G, M125P, M125L, M125F, M125Y, or M125W.

16. The IL-12 mutein of any one of claims 2 to 15, wherein the variant p35 subunit comprises a substitution at amino acid residue R129, optionally wherein the substitution is R129H, R129K, R129D, R129E, R129S, R129T, R129N, R129Q, R129G, R129P, R129A, R129V, R129I, R129L, R129M, R129F, R129Y, or R129W.

17. The IL- 12 mutein of any one of claims 2 to 16, wherein the variant p35 subunit comprises a substitution at amino acid residue LI 77, optionally wherein the substitution is L177R, L177H, L177K, L177D, L177E, L177S, L177T, L177N, L177Q, L177G, L177P, L177A, L177V, L177I, L177M, L177F, L177Y, or L177W.

18. The IL-12 mutein of claim 17, wherein the substitution at amino acid residue L177 is L177D, L177S, L177T, L177N, L177G, L177P, L177V, L177I, L177M, or L177W.

19. The IL- 12 mutein of any one of claims 2 to 18, wherein the variant p35 subunit comprises a substitution at amino acid residue H178, optionally wherein the substitution is H178R, H178K, H178D, H178E, H178S, H178T, H178N, H178Q, H178G, H178P, H178A, H178V, H178I, H178L, H178M, H178F, H178Y, or H178W.

20. The IL-12 mutein of any one of claims 2 to 19, wherein the variant p35 subunit comprises a substitution at amino acid residue K56, optionally wherein the substitution at amino acid residue K56 is K56R, K56H, K56D, K56E, K56S, K56T, K56N, K56Q, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W.

21. The IL-12 mutein of claim 20, wherein the K56 substitution is K56R, K56H, K56D, K56E, K56S, K56T, K56G, K56P, K56A, K56V, K56I, K56L, K56M, K56F, K56Y, or K56W, optionally wherein the substitution at amino acid residue K56 is K56E, K56S, K56T, K56G, K56A, K56V, K56I, or K56L.

22. The IL-12 mutein of claim 20, wherein the substitution at amino acid residue K56 is K56N.

23. The IL- 12 mutein of any one of claims 2 to 22, wherein the variant p35 subunit comprises a substitution at amino acid residue KI 70, optionally wherein the substitution at amino acid residue KI 70 is K170R, K170H, K170D, K170E, KI 70S, K170T, K170N, K170Q, K170G, K170P, K170A, KI 70V, KI 701, K170L, K170M, K170F, K170Y, or K170W.

24. The IL-12 mutein of any one of claims 2 to 23, wherein the variant p35 subunit comprises a substitution at amino acid residue 1171, optionally wherein the substitution at amino acid residue 1171 is I171R, I171K, I171H, I171D, I171E, 1171 S, I171T, I171N, I171Q, I171G, I171P, I171A, I171V, I171L, I171M, I171F, I171Y, or I171W.

25. The IL- 12 mutein of any one of claims 2 to 24, wherein the variant p35 subunit comprises a substitution at amino acid residue A33, R34, E38, F39, P41, E45, E46, 147, H49, E50, D51, 152, T53, K54, T57, D108, L109, K110, Mi l l, Y112, QI 13, V114, E115, F116, K117, T118, A121, L123, D126, 1131, P156, S159, S160, L161, E162, E163, D165, F166, Y167, K168, T169,

KI 72, L173, 1175, or R181.

26. The IL-12 mutein of claim 2, wherein the variant p35 subunit comprises one or more amino acid substitutions selected from Table la, Table lb, or Table 1c.

27. The IL- 12 mutein of claim 2, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; and a substitution at Y40, optionally wherein the substitution is Y40S.

28. The IL-12 mutein of claim 2, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at LI 33, optionally wherein the substitution is LI 33 A.

29. The IL-12 mutein of claim 2, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; and a substitution at KI 28, optionally wherein the substitution is K128N or K128Q.

30. The IL-12 mutein of claim 2, wherein the variant p35 subunit comprises a substitution at L37, optionally wherein the substitution is L37A; a substitution at Y40, optionally wherein the substitution is Y40S; a substitution at KI 28, optionally wherein the substitution is K128N or K128Q; and a substitution at LI 33, optionally wherein the substitution is LI 33 A.

31. The IL- 12 mutein of any one of claims 2 to 30, wherein the variant p35 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1.

32. The IL- 12 mutein of any one of claims 1 to 31, comprising the variant p40 subunit.

33. The IL- 12 mutein of claim 32, wherein the variant p40 subunit comprises a substitution at amino acid residue VI 0, optionally wherein the substitution is V10A, VI OR, VI OH, VI OK, V10D, V10E, V10S, V10T, V10N, V10Q, V10G, V10P, V10I, VIOL, V10M, V10F, V10W, or V10Y.

34. The IL-12 mutein of claim 32 or 33, wherein the variant p40 subunit comprises a substitution at amino acid residue 194, optionally wherein the substitution is I94A, I94R, I94H, I94K, I94D, I94E, I94S, I94T, I94N, I94Q, I94G, I94P, I94L, I94M, I94F, I94W, I94V, or I94Y.

35. The IL-12 mutein of any one of claims 32 to 34, wherein the variant p40 subunit comprises a substitution at amino acid residue L95, optionally wherein the substitution is L95A, L95R, L95H, L95K, L95D, L95E, L95S, L95T, L95N, L95Q, L95G, L95P, L95I, L95M, L95F, L95W, L95V, or L95Y.

36. The IL-12 mutein of any one of claims 32 to 35, wherein the variant p40 subunit comprises a substitution at amino acid residue G64, optionally wherein the substitution is G64A, G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y.

37. The IL-12 mutein of claim 36, wherein the G64 substitution is G64R, G64H, G64K, G64D, G64E, G64S, G64T, G64N, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y, optionally wherein the G64 substitution is G64R, G64K, G64E, G64S, G64T, G64Q, G64P, G64I, G64L, G64M, G64F, G64W, G64V, or G64Y.

38. The IL-12 mutein of any one of claims 32 to 37, wherein the variant p40 subunit comprises a substitution at amino acid residue Y198, optionally wherein the substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198S, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V.

39. The IL-12 mutein of claim 38, wherein the Y198 substitution is Y198A, Y198R, Y198H, Y198K, Y198D, Y198E, Y198T, Y198N, Y198Q, Y198G, Y198P, Y198I, Y198L, Y198M, Y198F, Y198W, or Y198V, optionally wherein the Y198 substitution is Y198G, Y198E, Y198K, Y198Q, Y198R, Y198W.

40. The IL-12 mutein of any one of claims 32 to 39, wherein the variant p40 subunit comprises a substitution at amino acid residue Y16, optionally wherein the substitution is Y16A, Y16R, Y16H, Y16K, Y16D, Y16E, Y16S, Y16T, Y16N, Y16Q, Y16G, Y16P, Y16I, Y16L, Y16M, Y16F, Y16W, or Y16V.

41. The IL- 12 mutein of any one of claims 32 to 40, wherein the variant p40 subunit comprises a substitution at amino acid residue Vl l, E12, L13, D14, W15, P17, D18, A19, P20, G21, W38, T39, L40, D41, S43, S44, E45, V46, L47, 155, V57, K58, E59, F60, D62, A63, Q65, Y66, L81, L82, H83, K84, K85, E86, G88, 189, W90, S91, T92, D93, D97, Q98, K99, E100, N103, KI 04, T105, F106, L107, L123, K135, S136, S137, R138, G139, S140, S141, D142, N162, E181, S183, L184, P185, 1186, E187, V188, V190, D191, H194, K195, L196, K197, E199, N200, T202, S203, S204, F206, or D209.

42. The IL-12 mutein of claim 41, wherein the variant p40 subunit comprises a substitution at amino acid residue W15, P17, D18, E59, F60, K84, E86, D93, H194, K195, or K197, optionally wherein the variant p40 subunit comprises a substitution at amino acid residue E59, F60, K84, KI 95, or KI 97.

43. The IL- 12 mutein of claim 32, wherein the variant p40 subunit comprises one or more amino acid substitutions selected from Table 4a, Table 4b, or Table 4c.

44. The IL-12 mutein of any one of claims 32 to 43, wherein the variant p40 subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.

45. The IL- 12 mutein of any one of claims 1 to 44, wherein the IL- 12 mutein is a mutein of the human IL- 12 sequence.

46. A fusion protein comprising the IL-12 mutein of any one of claim 1 to 45.

47. A pharmaceutical composition comprising the IL- 12 mutein of any one of claims 1 to 45 or the fusion protein of claim 46, and a pharmaceutically acceptable carrier or excipient.

48. A polynucleotide sequence that encodes the IL-12 mutein according to any one of claims 1 to 45 or the fusion protein of claim 46.

49. An expression vector comprising the polynucleotide sequence of claim 48.

50. A host cell comprising the expression vector of claim 49.

51. A kit comprising the IL- 12 mutein according to any one of claims 1 to 45 or the fusion protein of claim 46 and optionally packaging material.

52. A method of producing the IL- 12 mutein of any one of claims 1 to 45 or the fusion protein of claim 46, comprising culturing the host cell of claim 50 under conditions to express the IL- 12 mutein or the fusion protein.

53. The method of claim 52, further comprising isolating the IL-12 mutein from the host cell.

54. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the IL- 12 mutein of any one of claims 1 to 45, the fusion protein of claim 46, or the pharmaceutical composition of claim 47.

55. The method of claim 54, wherein the cancer comprises a solid tumor.