Interleukin-12 variants and methods of use

Abstract

The present disclosure provides compositions and methods comprising variant polypeptides of IL-12 that have partial agonism relative to wild-type IL-12 for use in therapeutic and non-therapeutic applications.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 233,511, filed August 16, 2021, the disclosure of which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. U01 CA233096 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Interleukin-12 (IL-12) regulates Th1 immunity and potently induces the secretion of IFN-γ by NK cells and cytotoxic T cells, making it an attractive candidate for cancer immunotherapy. Administration of IL-12 has shown great promise in stimulating antitumor responses preclinically, but has not been translated into humans due to unacceptable toxicity and a narrow therapeutic index. The full therapeutic potential of IL-12 may be limited by its pleiotropic nature, resulting in the activation of multiple cell types that promote beneficial and detrimental effects.

[0004] Thus, there is a need in the art for improved compositions and methods of providing IL-12 that provide minimal toxicity and a wide therapeutic index for treating and preventing cancer and other diseases and disorders. This disclosure fulfills this unmet need. Summary of the Invention [Problem to be solved by the invention]

[0005] In one embodiment, the present disclosure relates to a composition comprising an IL-12 variant polypeptide, the IL-12 variant polypeptide having less than maximal signaling efficacy through its receptor relative to wild-type (WT) IL-12. In one embodiment, the IL-12 variant polypeptide comprises at least one mutation relative to WT IL-12. In one embodiment, the IL-12 variant polypeptide comprises a p35 subunit (IL-12p35) with or without a signal peptide and a p40 subunit (IL-12p40) with or without a signal peptide. In one embodiment, said IL-12p40 of said IL-12 variant polypeptide comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X with respect to SEQ ID NO:1. In one embodiment, said IL-12p40 of said IL-12 variant polypeptide comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A with respect to SEQ ID NO:1. In one embodiment, the above-mentioned IL-12p40 of the IL-12 variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:34.

[0006] In one embodiment of the composition of the present disclosure, the IL-12 variant polypeptide, IL-12p35 as described above, comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30 and SEQ ID NO:35.

[0007] In one embodiment of the composition of the present disclosure, the above-mentioned IL-12p40, the above-mentioned IL-12p35, or a combination thereof is fused to at least one in vivo half-life extending fusion selected from the group consisting of an IgG Fc domain, an IgG Fc variant domain, human serum albumin (HSA), polyethylene glycol (PEG), and an anti-HSA nanobody.

[0008] In one embodiment of the composition of the present disclosure, said IgG Fc domain comprises a human IgG1 domain comprising the amino acid sequence of SEQ ID NO: 10, and said IgG Fc variant domain comprises at least one selected from the group consisting of a human IgG1 Fc "knob" domain comprising the amino acid sequence of SEQ ID NO: 14, and a human IgG1 Fc "hole" domain comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the IL-12 variant polypeptide comprises a bivalent homodimeric IgG Fc comprising at least two human IgG1 Fc domains. In one embodiment, the IL-12 variant polypeptide comprises a bispecific heterodimeric IgG Fc comprising at least one human IgG1 Fc "knob" and at least one IgG Fc "hole". In one embodiment, the IL-12 variant polypeptide comprises said IL-12p40 fused via a linker to said IL-p35, and a single chain bivalent homodimeric IgG Fc comprising at least two human IgG1 Fc domains. In one embodiment, the IL-12 variant polypeptide comprises a single chain monomeric IL-12 comprising IL-12p40 fused to IL-p35 via a linker, and a bispecific heterodimeric IgG Fc comprising at least one human IgG1 Fc "knob" and at least one IgG Fc "hole." In one embodiment, the IL-12 variant polypeptide comprises a dimeric IL-12 comprising IL-12p40 as described above and IL-p35 as described above, and a bispecific heterodimeric IgG Fc comprising at least one human IgG1 Fc "knob" and at least one IgG Fc "hole."

[0009] In one embodiment, the disclosure relates to a composition comprising one or more nucleic acid molecules encoding at least one IL-12p40 peptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and SEQ ID NO:34. In one embodiment, the composition further comprises a nucleic acid molecule encoding an IL-12p35 peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35.

[0010] In one embodiment of the composition of the present disclosure, the aforementioned nucleic acid encoding the aforementioned IL-12p40, the aforementioned nucleic acid encoding the aforementioned IL-12p35, or a combination thereof, further encodes a nucleic acid sequence encoding an in vivo half-life extending fusion selected from the group consisting of an IgG Fc domain, an IgG Fc variant domain, human serum albumin (HSA), polyethylene glycol (PEG), and an anti-HSA nanobody.

[0011] In one embodiment, the present disclosure relates to a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising an IL-12 variant polypeptide, wherein said IL-12 variant polypeptide has less than maximal signaling efficacy through its receptor relative to WT IL-12.

[0012] In one embodiment of the disclosed method, the IL-12 variant polypeptide comprises a p35 subunit (IL-12p35) and a p40 subunit (IL-12p40). In one embodiment, the aforementioned IL-12p40 of the IL-12 variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34. In one embodiment, the aforementioned IL-12p35 of the IL-12 variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35. In one embodiment, the aforementioned IL-12p40, the aforementioned IL-12p35, or a combination thereof, is fused to at least one in vivo half-life extending fusion selected from the group consisting of an IgG Fc domain, an IgG Fc variant domain, human serum albumin (HSA), polyethylene glycol (PEG), and an anti-HSA nanobody. In one embodiment, the aforementioned disease or disorder is cancer.

[0013] In one embodiment, the method further comprises administering to the subject at least one additional agent selected from the group consisting of a chemical compound, a polypeptide, a peptide, a peptidomimetic, an antibody, a cytokine, a nucleic acid molecule (e.g., mRNA), a ribozyme, a small molecule chemical compound, and an antisense nucleic acid molecule. In one embodiment, the at least one additional agent comprises one or more selected from the group consisting of a cancer therapeutic agent or a cancer immunotherapeutic agent.

[0014] In one embodiment, the method of the disclosure comprises administering the above-mentioned IL-12 variant polypeptide via one or more mechanisms selected from the group consisting of: a) lipid nanoparticle-encapsulated mRNA molecules encoding the above-mentioned IL-12 variant polypeptide, b) a viral vector expressing the above-mentioned IL-12 variant polypeptide, and c) engineered immune cells expressing the above-mentioned IL-12 variant polypeptide. In one embodiment, the above-mentioned administration comprises one or more selected from the group consisting of: a) systemic administration, and b) local administration to at least one specific tissue.

[0015] The following detailed description of the embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings, in which it is understood that the present disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0016] [Figure 1A] Figure 1 shows a model predicting residues at the IL-12Rβ1:IL-12p40 interface. A zoomed-in model of IL-12p40 in complex with a neutralizing nanobody is shown. Residues predicted to mediate interactions with the nanobody and therefore with IL-12Rβ1 as well are shown in blue (see labels for colors), IL-12p40 is shown in green, and the nanobody is shown in light yellow. [Figure 1B]1 shows a model predicting residues at the IL-12Rβ1:IL-12p40 interface. A schematic of predicted interactions between IL-12, including IL-12p40 and IL-12p35 subunits, and IL-12Rβ2 and IL-12Rβ1 is shown. The grey "X" indicates the interface between IL-12Rβ1 and IL-12p40, which, if selectively disrupted, may reduce recruitment of IL-12Rβ1 to the IL-12:IL-12Rβ2 complex and attenuate downstream STAT4 signaling. [Figure 2A] Illustrated are exemplary results showing protein expression and purification of wild-type (WT) IL-12 and mutant variants. Illustrated are exemplary results showing equivalent expression and mobility of WT and mutant IL-12 proteins. Proteins were separated on SDS-PAGE gels and stained with Coomassie Brilliant Blue. [Figure 2B] Figure 1 shows exemplary results showing protein expression and purification of wild-type (WT) IL-12 and mutant variants. Figure 2 shows exemplary results showing purification of IL-12 and mutant variants. Proteins were isolated by nickel-NTA affinity chromatography and then further purified by size exclusion chromatography. [Figure 3A] Exemplary results are shown showing a graded response to WT depending on the number and nature of interface residues mutated to alanine residues. Exemplary results are shown showing reduced agonism of the H216A / K217A / K219A triple mutant (HKK) compared to WT IL-12. In all cases, human NK cells were stimulated with IL-12 mutant variants or left unstimulated and phosphorylated STAT4 was measured by flow cytometry as a measure of IL-12 agonism. [Figure 3B]Exemplary results are shown showing a graded response to WT depending on the number and nature of interface residues mutated to alanine residues. Exemplary results are shown showing that agonism is reduced to a lesser extent in the H216A / K219A double mutant compared to the WT. In all cases, human NK cells were stimulated with IL-12 mutant variants or left unstimulated and phosphorylated STAT4 was measured by flow cytometry as a measure of IL-12 agonism. [Figure 3C] Exemplary results are shown showing a graded response to WT depending on the number and nature of the interface residues mutated to alanine residues. Exemplary results of single point responses to all variants compared to WT IL-12 and unstimulated cells are shown, showing a graded response that can be tailored to a particular level of agonism desired. In all cases, human NK cells were stimulated with IL-12 mutant variants or left unstimulated, and phosphorylated STAT4 was measured by flow cytometry as a measure of IL-12 agonism. [Figure 3D] Exemplary results are shown showing a graded response to WT depending on the number and nature of interface residues mutated to alanine residues. Results from 3C are shown as a percentage of agonism relative to WT. In all cases, human NK cells were stimulated with IL-12 mutant variants or left unstimulated, and phosphorylated STAT4 was measured by flow cytometry as a measure of IL-12 agonism. [Figure 4A] 3A-3D show exemplary results of IL-12 expressed as a bispecific heterodimeric Fc fusion protein, a method to extend in vivo half-life. Schematic diagram of IL-12 bispecific heterodimeric Fc fusion proteins tested for IL-12 agonism. Phosphorylated STAT4 was measured by flow cytometry as in FIG. 3A-3D. [Figure 4B]Figure 3 shows exemplary results for IL-12 expressed as a bispecific heterodimeric Fc fusion protein, a method to extend in vivo half-life. Figure 3 shows exemplary results showing that a bispecific heterodimeric Fc fusion protein of IL-12 unexpectedly attenuated agonism compared to WT IL-12, but not as markedly as the HKK triple mutant described in Figures 3A-3D. Phosphorylated STAT4 was measured by flow cytometry as in Figures 3A-3D. [Figure 5A] 1 shows an exemplary method of using Fc-fused IL-12 variants to extend the half-life of partial IL-12 agonists of the present disclosure. A schematic diagram of a bivalent Fc fused to either p40 or p35 and co-expressed with the corresponding subunits to generate dimeric IL-12. [Figure 5B] 1 shows an exemplary method of using Fc-fused IL-12 variants to extend the half-life of partial IL-12 agonists of the present disclosure. A schematic diagram of a bivalent Fc fused to p35, which is then fused to p40 and expressed as a single chain construct to form dimeric IL-12. [Figure 5C] 1 shows an exemplary method of using Fc-fused IL-12 variants to extend the half-life of partial IL-12 agonists of the present disclosure. A schematic diagram of a bispecific Fc "knob" fused to p35, which is then fused to p40, expressed as a single chain construct, and co-expressed with the corresponding bispecific Fc "hole" to form monomeric IL-12. [Figure 5D] 1 shows an exemplary method of using Fc-fused IL-12 variants to extend the half-life of partial IL-12 agonists of the present disclosure. A schematic diagram of the bispecific Fc "knob" fused to either p40 or p35 and co-expressed with the corresponding subunit and the corresponding bispecific Fc "hole" to form monomeric IL-12. [Figure 6A]Figure 3 shows exemplary results showing further reduction in agonism of Fc fusions for selected Fc fusion variants. Figure 3 shows exemplary results of single point responses to Fc fusion variants compared to WT IL-12 and IL-12 H216A / K217A / K219A triple mutant (HKK), demonstrating that the graded responses shown in Figures 3A-3D can be further tailored via Fc fusions. Proteins were expressed in Expi293 cells and cell culture supernatants containing secreted proteins were used to stimulate NK cells. The x-axis shows titration of cell culture supernatants shown as a percentage of the total volume used to stimulate NK cells. [Figure 6B] Figure 1 shows exemplary results showing further reduction in agonism of Fc fusions for selected Fc fusion variants. Figure 2 shows the effect of WT IL-12, IL-12 HKK and Fc fusion variants on phosphorylation of STAT4 in human NK cells in response to titrated amounts of supernatant. Proteins were expressed in Expi293 cells and cell culture supernatants containing secreted proteins were used to stimulate NK cells. The x-axis shows titration of cell culture supernatants shown as a percentage of the total volume used to stimulate NK cells. [Figure 7] 1 shows a schematic diagram of additional exemplary methods of extending the in vivo half-life of partial IL-12 agonists of the present disclosure, including, but not limited to, fusion to human serum albumin (HSA), fusion to polyethylene glycol (PEG), or fusion to an anti-HSA nanobody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure generally relates to variants of IL-12 that have less than maximal signaling efficacy through its receptor relative to wild-type IL-12. In one aspect, the disclosure provided herein relates to one or more IL-12 variant polypeptides that specifically bind to IL-12Rβ2 with activity comparable to wild-type IL-12, but have reduced or abolished binding to IL-12Rβ1 relative to wild-type IL-12. In one aspect, the present disclosure relates to variants of one or more IL-12 p40 subunits (IL-12p40) that dimerize with the IL-12 p35 subunit to an extent comparable to wild-type IL-12, but have reduced or abolished binding to IL-12Rβ1 relative to wild-type IL-12.

[0018] In various embodiments, the disclosure provides nucleic acids encoding one or more variants of IL-12 that have less than maximal signaling efficacy through its receptor relative to wild-type IL-12. In other embodiments, the disclosure relates to methods of administering one or more variant polypeptides of IL-12 that have less than maximal signaling efficacy through its receptor relative to wild-type IL-12, or compositions comprising one or more nucleic acid molecules encoding one or more variants. In some embodiments, the disclosure relates to methods of treating or preventing one or more diseases or disorders by administering, alone or in combination with other therapeutic agents, one or more variants of IL-12, or compositions comprising one or more nucleic acid molecules encoding one or more variants of IL-12 that have less than maximal signaling efficacy through its receptor relative to wild-type IL-12.

[0019] definition 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 disclosure belongs.

[0020] As used herein, each of the following terms has the meaning associated with it in this section.

[0021] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0022] As used herein, "about" when referring to a measurable value, such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0023] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. An antibody may be an intact immunoglobulin derived from natural sources or from recombinant sources or may be an immunoreactive portion of an intact immunoglobulin. The antibodies of the present disclosure may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, and F(ab)2, as well as single chain antibodies (scFv), heavy chain antibodies such as camelid antibodies, synthetic antibodies, chimeric antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0024] "Cancer," as used herein, refers to the abnormal growth or division of cells. Generally, the growth and / or lifespan of cancer cells exceeds and is uncoordinated with the growth and / or lifespan of normal cells and surrounding tissues. Cancer can be benign, pre-malignant, or malignant. Cancers arise in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lungs, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous, meningioma, etc.), breast, reproductive system (e.g., uterus, ovaries, prostate, testes, etc.), urinary system (e.g., bladder, kidneys, ureters, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0025] The terms "co-administration," "co-administered," and "in combination with" include administration of two or more therapeutic agents (e.g., an IL-12 variant polypeptide in combination with an additional agent) simultaneously, concomitantly, or sequentially without specific time limitations. As used herein, the term "co-administration" is meant to encompass conjugated and unconjugated compounds.

[0026] A "disease" is a state of health of an animal in which the animal is unable to maintain homeostasis and if the disease is not ameliorated, then the animal's health will continue to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but the animal's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration of the animal's health.

[0027] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0028] "Homologous," "identical," or "identity," as used herein in the context of two or more nucleic acid or polypeptide sequences, means that the sequences have a certain percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions at which identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths or the alignment results in one or more staggered ends and a particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0029] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0030] "Isolated nucleic acid" refers to a nucleic acid segment or fragment that is separated from sequences adjacent to it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment, e.g., sequences adjacent to the fragment in the genome in which it naturally occurs. The term also applies to a nucleic acid that has been substantially purified from other components that naturally accompany the nucleic acid, e.g., RNA or DNA or proteins that naturally accompany it in a cell. Thus, the term includes recombinant DNA that is, for example, incorporated into a vector, incorporated into an autonomously replicating plasmid or virus, or incorporated into the genomic DNA of a prokaryote or eukaryote, or exists as a separate molecule independent of other sequences (e.g., as a cDNA, or as a genomic or cDNA fragment produced by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0031] The term "modulate," as used herein, means to mediate a detectable increase or decrease in the activity and / or level of an mRNA, polypeptide, or response in a subject compared to the activity and / or level of the mRNA, polypeptide, or response in the subject in the absence of a treatment or compound, and / or compared to the activity and / or level of the mRNA, polypeptide, or response in an otherwise identical but untreated subject. The term encompasses mediating a beneficial therapeutic, prophylactic, or other desired response in a subject, e.g., a human, by activating, inhibiting, and / or otherwise affecting a natural signal or response.

[0032] The terms "multispecific" or "bispecific" are commonly used to refer to an agent (e.g., a ligand or antibody) that has at least one region specific for a first target (e.g., a ligand or Fab of a first antibody) and at least a second region specific for a second target (e.g., a ligand or Fab of a second antibody), thereby recognizing two or more different antigens. A bispecific agent specifically binds to two targets and is therefore a type of multispecific agent.

[0033] As used herein, "mutation," "mutant," or "variant" refers to an alteration in a nucleic acid or polypeptide sequence compared to a reference sequence (which may be a naturally occurring normal or "wild-type" sequence), including translocations, deletions, insertions, and substitutions / point mutations. As used herein, "mutant" or "variant" refers to either a nucleic acid or protein that contains a mutation.

[0034] "Nucleic acid" or "nucleic acid molecule" refers to polynucleotides, including polyribonucleotides and polydeoxyribonucleotides. Nucleic acids according to the present disclosure may contain any polymer or oligomer of pyrimidine and purine bases, such as cytosine, thymine, and uracil, and adenine and guanine, respectively. (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982), which is incorporated by reference in its entirety for all purposes.) Indeed, the present disclosure contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymers or oligomers may be heterogeneous or homogeneous in composition, isolated from natural sources, or artificially or synthetically produced. In addition, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single- or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states.

[0035] An "oligonucleotide" or "polynucleotide" is a nucleic acid ranging from at least 2, at least 8, at least 15, or at least 25 nucleotides in length, but may be up to 50, 100, 1000, or 5000 nucleotides in length, or a compound that specifically hybridizes to a polynucleotide. A polynucleotide includes sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or mimetics thereof, that may be isolated from natural sources, recombinantly produced, or artificially synthesized. A further example of a polynucleotide of the present disclosure may be a peptide nucleic acid (PNA). (See U.S. Patent No. 6,156,501, which is incorporated herein by reference in its entirety.) The present disclosure also encompasses situations in which non-traditional base pairing exists, such as Hoogsteen base pairs, identified in certain tRNA molecules and hypothesized to exist in triple helices. "Polynucleotide" and "oligonucleotide" are used interchangeably in this disclosure. When a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C), it will be understood that this also includes the corresponding RNA sequence in which "U" replaces "T" (e.g., A, U, G, C).

[0036] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof, whether in vivo, in vitro, or in a system, that can be subjected to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0037] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, with no limit on the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains (also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (commonly referred to in the art as proteins, of which there are many varieties). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, mutant polypeptides, variant polypeptides, or combinations thereof.

[0038] As used herein, the term "pharmaceutical acceptable carrier" refers to a chemical composition with which a composition of the present disclosure can be combined and, after combination, can be used to administer the appropriate composition to a subject.

[0039] As used herein, "polynucleotide" includes cDNA, RNA, DNA / RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to exhibit non-natural or derivatized, synthetic, or semi-synthetic nucleotide bases. Alterations of wild-type or synthetic genes are also contemplated, including, but not limited to, deletion, insertion, substitution of one or more nucleotides, or fusion to other polynucleotide sequences.

[0040] "Preventing" a disease or disorder, as that term is used herein, means reducing the severity or frequency of at least one sign or symptom of the disease or disorder experienced by a subject.

[0041] As used herein, "sample" or "biological sample" refers to biological material isolated from a subject. A biological sample may include any biological material suitable for detecting mRNA, polypeptides, or other markers of a physiological or pathological process in a subject, and may include fluids, tissues, cells, and / or non-cellular material obtained from an individual.

[0042] As used herein, the term "therapy" or "therapeutic regimen" refers to those activities undertaken to prevent, treat, or modify a disease or disorder, e.g., using pharmacological, surgical, dietary, and / or other techniques, the course of treatment intended to reduce or eliminate at least one sign or symptom of the disease or disorder. A therapeutic regimen may include one or more compounds or surgery at a prescribed dosage. Although therapy is most often beneficial and reduces or eliminates at least one sign or symptom of a disorder or disease state, in some cases, the effects of therapy may be undesirable or have side effects. The effects of therapy are also influenced by the physiological state of the subject, e.g., age, sex, genetics, weight, other disease states, etc.

[0043] The term "therapeutically effective amount" refers to an amount of a subject compound or composition that will elicit the biological, physiological, clinical, or medical response of a cell, tissue, organ, system, or subject that is being sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound or composition that is sufficient, when administered, to prevent the onset of, or treat to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound or composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0044] "Treating" a disease or disorder, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject. The terms "treatment," "treating," "treat," and the like are used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that the disease or its symptom(s) is / are completely or partially prevented, and / or therapeutic, in that the disease and / or side effects resulting from the disease are partially or completely stabilised or cured. The term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease and / or condition(s) from occurring in a subject who may be predisposed to, but has not yet been diagnosed as having, the disease or condition; (b) inhibiting the disease and / or condition(s), e.g., delaying or preventing the onset of the disease and / or condition(s) (e.g., halting the growth of a tumor, slowing the rate of growth of a tumor, halting the rate of proliferation of cancer cells, etc.); or (c) alleviating the disease symptom(s), i.e., causing regression of the disease and / or condition(s) (e.g., causing a reduction in tumor size, reducing the number of cancer cells present, etc.). Those in need of treatment include those already with an illness (e.g., those with cancer, those with an infection, those with a metabolic disorder, those with macular degeneration, etc.) and those for whom prevention is desired (e.g., those with an increased susceptibility to cancer, those at high risk of infection, those suspected of having cancer, those suspected of having an infection, those with an increased susceptibility to metabolic disease, those with an increased susceptibility to macular degeneration, etc.).

[0045] As used herein, the term "wild type" refers to a gene or gene product isolated from a natural source. A wild type gene is that gene most frequently observed in a population and thus arbitrarily designates the "normal" or "wild type" form of the gene. In contrast, the terms "modified," "variant," or "mutant" refer to a gene or gene product that has alterations in sequence and / or functional properties (i.e., altered properties) when compared to the wild type gene or gene product.

[0046] Ranges: Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0047] As used herein with respect to IL-12 variant polypeptides, the term "specifically binds" refers to an IL-12 variant polypeptide that recognizes and binds to a specific receptor, such as IL-12Rβ2 or IL-12Rβ1. In some cases, the IL-12 variant polypeptide has substantially reduced binding to IL-12Rβ1 relative to wild-type IL-12. For example, an IL-12 variant polypeptide that specifically binds to a receptor from one species may also bind to that receptor from one or more species. However, such cross-species reactivity in itself does not change the classification of the IL-12 variant polypeptide as specific. In another example, an IL-12 variant polypeptide that specifically binds to a receptor may also bind to different allelic forms of that receptor. However, such cross-reactivity in itself does not change the classification of the IL-12 variant polypeptide as specific. In some instances, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species, e.g., an IL-12 variant polypeptide recognizes and binds to a particular protein structure, rather than proteins in general.

[0048] composition In some embodiments, the compositions of the present disclosure comprise one or more IL-12 variant polypeptide molecules, where the variants enhance immune responses but have relatively low toxicity and a relatively wide therapeutic index. In some embodiments, the compositions comprise a partial inducer of IL-12 activity by reducing recruitment of IL-12Rβ1 to the active receptor signaling complex consisting of both IL-12Rβ2 and IL-12Rβ1. In some embodiments, the compositions comprise one or more molecules that can bind to IL-12Rβ2 but reduce or abolish binding via IL-12Rβ1. In some embodiments, the compositions comprise one or more IL-12 variant polypeptides. In some embodiments, the compositions comprise one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides.

[0049] Polypeptides In some embodiments, the disclosure includes one or more IL-12 variant polypeptides, or fragments thereof, that specifically bind to IL-12Rβ2 but have reduced binding to IL-12Rβ1. In some embodiments, the one or more IL-12 variant polypeptides exhibit increased binding affinity to IL-12Rβ2 compared to wild-type (WT) IL-12. In some embodiments, the one or more IL-12 variant polypeptides exhibit similar binding affinity to IL-12Rβ2 compared to WT IL-12. In some embodiments, the one or more IL-12 variant polypeptides exhibit reduced binding affinity to IL-12Rβ1 compared to WT IL-12.

[0050] In some embodiments, one or more IL-12 variant polypeptides are useful for treating or preventing a disease or disorder. In some embodiments, the disease of the disorder is cancer. In some embodiments, one or more IL-12 variant polypeptides are useful for treating or preventing a disease or disorder, alone or in combination with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is a cancer immunotherapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In any such embodiment, the disease or disorder may include cancer, e.g., acute myeloma leukemia, anaplastic lymphoma, astrocytoma, B cell cancer, breast cancer, colon cancer, ependymoma, esophageal cancer, glioblastoma, glioma, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, mantle cell lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, ovarian cancer, pancreatic cancer, peripheral T cell lymphoma, renal cancer, sarcoma, gastric cancer, carcinoma, mesothelioma, or sarcoma.

[0051] In some embodiments, one or more IL-12 variant polypeptides bind to IL-12Rβ2 and exhibit substantially reduced binding to IL-12Rβ1. In some embodiments, the IL-12 variant polypeptides bind to IL-12Rβ1 with a binding affinity that is between about 0.000000000001% and about 95% of the binding affinity of wild-type IL-12 to IL-12Rβ1. In some embodiments, the IL-12 variant polypeptide has about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55% of the binding affinity of wild-type IL-12 to IL-12Rβ1. ,About 54%,About 53%,About 52%,About 51%,About 50%,About 49%,About 48%,About 47%,About 46%,About 45%,About 44%,About 43%,About 42%,About 41%,About 40%,About 39%,About 38%,About 37%,About 36%,About 35%,About 30%,About 29%,About 28%,About 27%,About 26%,About 25%,About 24%,About 30 ... %, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or about 0%.

[0052] In some embodiments, the one or more IL-12 variant polypeptides comprise an IL-12 variant polypeptide and an IL-12Rβ2 K D A dissociation constant (K D In some embodiments, the IL-12 variant polypeptide binds to IL-12Rβ1 with a K of WT IL-12 and IL-12Rβ2 (a higher K indicates a lower binding affinity). D Substantially the same as or lower than K D It binds to IL-12Rβ2.

[0053] In some embodiments, the one or more IL-12 variant polypeptides have a K D Substantially higher K D In some embodiments, the IL-12 variant polypeptide binds to IL-12Rβ1 at the K D at least 10 times, at least 100 times, at least 1,000 times, at least 10,000 times, at least 100,000 times, at least 1,000,000 times, at least 10,000,000 times, or at least 100,000,000 times greater than D In some embodiments, the IL-12 variant polypeptide binds to IL-12Rβ1 with a K of 10 nM or more, 15 nM or more, 20 nM or more, 25 nM or more, 30 nM or more, 35 nM or more, 40 nM or more, 45 nM or more, 50 nM or more, 55 nM or more, 60 nM or more, 65 nM or more, 70 nM or more, 75 nM or more, 80 nM or more, 85 nM or more, 90 nM or more, 95 nM or more, 100 nM or more, 200 nM or more, 300 nM or more, 400 nM or more, 500 nM or more, or 1 μM or more. D It binds to IL-12Rβ1.

[0054] In some embodiments, one or more IL-12 variant polypeptides exhibit substantially reduced agonism at IL-12Rβ1 and IL-12Rβ2. In some embodiments, the IL-12 variant polypeptides exhibit substantially reduced agonism at IL-12Rβ1 and IL-12Rβ2. In some embodiments, the IL-12 variant polypeptides exhibit approximately 95%, approximately 94%, approximately 93%, approximately 92%, approximately 91%, approximately 90%, approximately 89%, approximately 88%, approximately 87%, approximately 86%, approximately 85%, approximately 84%, approximately 83%, approximately 82%, approximately 81%, approximately 80%, approximately 79%, approximately 78%, approximately 77%, approximately 76%, approximately 75%, approximately 74%, approximately 78%, approximately 79%, approximately 79%, approximately 78%, approximately 77%, approximately 76%, approximately 75%, approximately 74%, approximately 78 ... 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49%, about In some embodiments, the IL-12Rβ1 and IL-12Rβ2 agonism is about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or about 0%.

[0055] In some embodiments, the one or more IL-12 variant polypeptides have 50% of maximal agonism (EC 50 In some embodiments, the IL-12 variant requires substantially higher effective concentrations to reach the EC of wild-type IL-12 for IL-12Rβ1 and IL-12Rβ2. 50of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 41 EC for IL-12Rβ1 and IL-12Rβ2 that is 0%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1100%, about 1200%, about 1300%, about 1400%, about 1500%, about 1600%, about 1700%, about 1800%, about 1900%, about 2000%, about 2100%, or about 2200%. 50 In some embodiments, the IL-12 variant has an EC of WT IL-12 for IL-12Rβ1 and IL-12Rβ2. 50 EC for IL-12Rβ1 and IL-12Rβ2 that is at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 50-fold higher, at least 100-fold higher, at least 200-fold higher, at least 500-fold higher, or at least 1000-fold higher than 50 has.

[0056] In various embodiments, the one or more IL-12 variant polypeptides comprise one or more mutations relative to a WT IL-12 polypeptide. In some embodiments, the WT IL-12 polypeptide comprises human WT IL-12. In some embodiments, the one or more IL-12 variant polypeptides and the WT IL-12 polypeptide both comprise a p35 subunit (IL-12p35) and a p40 subunit (IL-12p40). In some embodiments, the IL-12p40 of the WT IL-12 comprises the amino acid sequence of SEQ ID NO: 1 (see Table 1 in Example 1 below for the sequence). In some embodiments, the IL-12p35 of the WT IL-12 comprises the amino acid sequence of SEQ ID NO: 2.

[0057] In one embodiment, the IL-12p35 of the one or more IL-12 variant polypeptides comprises the amino acid sequence of WT IL-12p35, with or without a signal peptide. In one embodiment, the IL-12p35 of the one or more IL-12 variant polypeptides comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:35. In some embodiments, the IL-12p35 of the one or more IL-12 variant polypeptides further comprises a purification tag. In some embodiments, the purification tag is a polyhistidine tag. In some embodiments, the polyhistidine tag comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 histidine residues. In one embodiment, the IL-12p35 of the one or more IL-12 variant polypeptides further comprising a purification tag comprises the amino acid sequence of SEQ ID NO:30. Unless otherwise specified, the term "X" is used below to represent any amino acid.

[0058] In one embodiment, the IL-12p40 of the one or more IL-12 variant polypeptides comprises the amino acid sequence of WT IL-12p40, with or without the signal peptide. In one embodiment, the IL-12p40 of the one or more IL-12 variant polypeptides comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In some embodiments, the IL-12p40 of the one or more IL-12 variant polypeptides comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the one or more IL-12 variant polypeptides comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the IL-12p40 of the one or more IL-12 variant polypeptides comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1. In one embodiment, the IL-12 variant polypeptide IL-12p40 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, with or without a signal peptide. In one embodiment, the IL-12 variant polypeptide IL-12p40 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0059] In some embodiments, one or more IL-12 variant polypeptides, or fragments thereof, comprise an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12. In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12, and (ii) contains at least one, at least two, or at least three mutations relative to WT IL-12.

[0060] In some embodiments, the IL-12p35 of one or more IL-12 variant polypeptides, or fragments thereof, comprises an amino acid sequence that has 100% sequence identity to WT IL-12p35. In some embodiments, the IL-12p35 of one or more IL-12 variant polypeptides, or fragments thereof, comprises an amino acid sequence that (i) has 100% sequence identity to WT IL-12p35, and (ii) does not contain a mutation relative to WT IL-12p35.

[0061] In some embodiments, one or more IL-12 variant polypeptides of IL-12p40, or fragments thereof, comprise an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12p40. In some embodiments, one or more IL-12 variant polypeptides of IL-12p40, or fragments thereof, comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12p40, and (ii) contains at least one, at least two, or at least three mutations relative to WT IL-12p40.

[0062] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, comprise: (i) an IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12p40 and comprising at least one, at least two, or at least three mutations relative to WT IL-12p40; and (ii) an IL-12p35 comprising an amino acid sequence having 100% sequence identity to WT IL-12p35.

[0063] In some embodiments, one or more IL-12 variant polypeptides IL-12p35, or fragments thereof, comprise an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35. In some embodiments, one or more IL-12 variant polypeptides IL-12p35, or fragments thereof, comprise an amino acid sequence that (i) has 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35, and (ii) does not contain a mutation relative to SEQ ID NO:2 or SEQ ID NO:35.

[0064] In some embodiments, one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1. In some embodiments, one or more of the IL-12 variant polypeptides IL-12p40, or fragments thereof, comprises an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1, and (ii) contains at least one, at least two, or at least three mutations relative to SEQ ID NO:1.

[0065] In some embodiments, one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34. In some embodiments, one or more of the IL-12 variant polypeptides IL-12p40, or fragments thereof, comprises an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34, and (ii) contains at least one, at least two, or at least three mutations relative to SEQ ID NO:34.

[0066] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, include (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1 and comprising at least one, at least two, or at least three mutations to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0067] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, include (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34 and comprising at least one, at least two, or at least three mutations to SEQ ID NO:34; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0068] In some embodiments, the one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1, and (ii) comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X to SEQ ID NO:1.

[0069] In some embodiments, the one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34, and (ii) comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO:1.

[0070] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, comprise: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1, and comprising at least one mutation selected from the group consisting of H216X, K217X, and K219X, relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0071] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, comprise: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34 and comprising at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0072] In some embodiments, the one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1, and (ii) comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A to SEQ ID NO:1.

[0073] In some embodiments, the one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34, and (ii) comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:1.

[0074] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, comprise: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1, and comprising at least one mutation selected from the group consisting of H216A, K217A, and K219A, relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0075] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, comprise: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34 and comprising at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0076] In some embodiments, one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, with or without a signal peptide.

[0077] In some embodiments, one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0078] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, include (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, with or without a signal peptide; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0079] In some embodiments, the one or more IL-12 variant polypeptides, or fragments thereof, comprise: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0080] In some cases, as described above, it may be desirable to extend the in vivo half-life of one or more IL-12 variant polypeptides of the present disclosure. Various techniques for extending the in vivo half-life of a polypeptide are known in the art, including fusing the polypeptide to one or more stable proteins or protein domains. Exemplary stabilized fusion proteins / domains include, but are not limited to, IgG Fc domains (i.e., capable of forming homodimeric IgG Fc), IgG Fc variant domains (i.e., capable of forming heterodimeric IgG Fc), human serum albumin (HSA), polyethylene glycol (PEG), and anti-HSA nanobodies.

[0081] In one embodiment, the IL-12 variant polypeptide(s) is fused to a peptide that enhances the stability or half-life of the fusion protein as described herein. In one embodiment, the fusion peptide comprises at least one region of an immunoglobulin, or a variant or fragment thereof. In one embodiment, the peptide comprises an Fc domain of an immunoglobulin. In one embodiment, the fusion peptide comprises an Fc domain of a human IgG1. In one embodiment, the fusion peptide comprises an Fc domain of an immunoglobulin that comprises one or more mutations to eliminate Fc effector functions via the Fc receptor or complement. In one embodiment, the fusion peptide comprises an Fc domain of a human IgG1 that comprises a mutation at residue N297 relative to wild type human IgG1 to deglycosylate the Fc domain.

[0082] Heterodimeric Fc fusion constructs are based on the self-assembly of two Fc domains of the heavy chains of an antibody, e.g., two "monomers" that assemble into a "dimer." Heterodimeric Fc fusions are created by varying the amino acid sequence of each monomer, as described in WO2018071919A1, which is incorporated herein by reference in its entirety. The generation of heterodimeric Fc relies on amino acid variants in the constant regions that differ in each chain to promote heterodimer formation and / or to facilitate purification of the heterodimer over homodimers. Thus, in some embodiments, the present disclosure relates to compositions and methods using the IL-12 variant polypeptide(s) described herein fused to a heterodimeric Fc, thereby extending the half-life of the IL-12 variant polypeptide(s).

[0083] In one embodiment, one or more IL-12 variant polypeptides comprise a bivalent homodimeric Fc. In one embodiment, the bivalent homodimeric Fc comprises at least two IgG Fc domains. In one embodiment, the IgG is human IgG. In one embodiment, the human IgG is human IgG1. In one embodiment, the human IgG1 Fc domain comprises the amino acid sequence of SEQ ID NO: 10.

[0084] In one embodiment, the IL-12p40 of the bivalent homodimeric Fc comprises IL-12p40 of WT IL-12. In one embodiment, the IL-12p40 of the bivalent homodimeric Fc comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the bivalent homodimeric Fc comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the bivalent homodimeric Fc comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the IL-12p40 of the bivalent homodimeric Fc comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0085] In one embodiment, the IL-12p35 of the bivalent homodimeric Fc comprises IL-12p35 of WT IL-12. In one embodiment, the IL-12p35 of the bivalent homodimeric Fc comprises a purification tag. In one embodiment, the IL-12p35 of the bivalent homodimeric Fc comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30, or SEQ ID NO:35.

[0086] In one embodiment, the IL-12p40 of the bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, the IL-12p35 of the bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:11.

[0087] In one embodiment, the IL-12p40 of the bivalent homodimeric Fc fused to an IgG Fc domain via a linker comprises the amino acid sequence of SEQ ID NO: 12. In one embodiment, the IL-12p40 of the bivalent homodimeric Fc fused to an IgG Fc domain via a linker comprises the amino acid sequence of SEQ ID NO: 12, but with one or more mutations in the IL-12p40 portion. In one embodiment, the IL-12p40 of the bivalent homodimeric Fc fused to an IgG Fc domain via a linker comprises the amino acid sequence of SEQ ID NO: 12, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the bivalent homodimeric Fc IL-12p40 fused via a linker to an IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 12, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1. In one embodiment, the bivalent homodimeric Fc IL-12p35 fused via a linker to an IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 13.

[0088] In one embodiment, the one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprise (i) IL-12p40 fused to an IgG Fc domain via a linker, and (ii) IL-12p35 in a bivalent homodimeric Fc. In one embodiment, the one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprise (i) IL-12p35 fused to an IgG Fc domain via a linker, and (ii) IL-12p40 in a bivalent homodimeric Fc.

[0089] In one embodiment, the one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprise (i) IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12, and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 35. In one embodiment, the one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprise (i) IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12, but with one or more mutations in the IL-12p40 portion, and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 35. In one embodiment, the one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprise: (i) IL-12p40 comprising the amino acid sequence of SEQ ID NO: 12 fused to an IgG Fc domain via a linker having at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1; and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 35. In one embodiment, the one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprise: (i) IL-12p40 comprising the amino acid sequence of SEQ ID NO: 12 fused to an IgG Fc domain via a linker having at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1; and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 35.

[0090] In one embodiment, the one or more IL-12 variant polypeptides comprise (i) IL-12p35 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO:13, and (ii) IL-12p40, a bivalent homodimeric Fc comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0091] In one embodiment, the bivalent homodimeric Fc IL-12p40 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0092] One of skill in the art will recognize that co-expression of either IL-12p40 or IL-12p35 fused to IgG Fc by a linker and the corresponding subunit (IL-12p35 or IL-12p40, respectively) results in dimeric IL-12 (i.e., a tetrameric structure comprising a homodimer of a heterodimer stabilized by a bivalent IgG Fc domain, see Example 1 and FIG. 5A).

[0093] In one embodiment, one or more IL-12 variant polypeptides comprise a bispecific heterodimeric Fc. In one embodiment, the bispecific heterodimeric Fc comprises an IgG Fc "knob" and an IgG Fc "hole". In one embodiment, the IgG Fc "knob" and the IgG Fc "hole" are variants of IgG Fc. In one embodiment, the IgG Fc comprises a human IgG Fc. In one embodiment, the human IgG Fc comprises a human IgG1 Fc. In one embodiment, the IgG Fc "knob" comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the IgG Fc "hole" comprises the amino acid sequence of SEQ ID NO: 15.

[0094] In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc comprises the IL-12p40 of WT IL-12. In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0095] In one embodiment, the bispecific heterodimeric Fc IL-12p40 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0096] In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0097] In one embodiment, the IL-12p35 of the bispecific heterodimeric Fc comprises IL-12p35 of WT IL-12. In one embodiment, the IL-12p35 of the bispecific heterodimeric Fc comprises a purification tag. In one embodiment, the IL-12p35 of the bispecific heterodimeric Fc comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30 or SEQ ID NO:35.

[0098] In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc is fused to the IgG Fc "knob" via a linker. In one embodiment, the IL-12p40 of the bispecific heterodimeric Fc is fused to the IgG Fc "hole" via a linker. In one embodiment, the IL-12p35 of the bispecific heterodimeric Fc is fused to the IgG Fc "knob" via a linker. In one embodiment, the IL-12p35 of the bispecific heterodimeric Fc is fused to the IgG Fc "hole" via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 11.

[0099] In one embodiment, a bispecific heterodimeric Fc comprising IL-12p40 fused to an IgG Fc "knob" via a linker comprises the amino acid sequence of SEQ ID NO: 16. In one embodiment, a bispecific heterodimeric Fc comprising IL-12p40 fused to an IgG Fc "knob" via a linker comprises the amino acid sequence of SEQ ID NO: 16, but with one or more mutations in the IL-12p40 portion. In one embodiment, a bispecific heterodimeric Fc comprising IL-12p40 fused to an IgG Fc "knob" via a linker comprises the amino acid sequence of SEQ ID NO: 16, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the bispecific heterodimeric Fc comprising IL-12p40 fused via a linker to an IgG Fc "knob" comprises the amino acid sequence of SEQ ID NO: 16, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0100] In one embodiment, the bispecific heterodimeric Fc comprising IL-12p35 fused via a linker to an IgG Fc "hole" comprises the amino acid sequence of SEQ ID NO:17.

[0101] In one embodiment, a bispecific heterodimeric Fc comprising IL-12p40 fused to an IgG Fc "hole" via a linker comprises the amino acid sequence of SEQ ID NO: 18. In one embodiment, a bispecific heterodimeric Fc comprising IL-12p40 fused to an IgG Fc "hole" via a linker comprises the amino acid sequence of SEQ ID NO: 18, but with one or more mutations in the IL-12p40 portion. In one embodiment, a bispecific heterodimeric Fc comprising IL-12p40 fused to an IgG Fc "hole" via a linker comprises the amino acid sequence of SEQ ID NO: 18, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the bispecific heterodimeric Fc comprising IL-12p40 fused via a linker to an IgG Fc "hole" comprises the amino acid sequence of SEQ ID NO: 18, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0102] In one embodiment, the bispecific heterodimeric Fc comprising IL-12p35 fused via a linker to an IgG Fc "knob" comprises the amino acid sequence of SEQ ID NO:19.

[0103] In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p40 fused to an IgG Fc "knob" via a linker, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker. In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p35 fused to an IgG Fc "knob" via a linker, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker.

[0104] In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but with one or more mutations in the IL-12p40 portion, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17.

[0105] In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but with one or more mutations in the IL-12p40 portion. In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but with at least one mutation selected from the group consisting of H216X, K217X, and K219X. In one embodiment, the bispecific heterodimeric Fc comprises (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but with at least one mutation selected from the group consisting of H216A, K217A, and K219A.

[0106] Those skilled in the art will recognize that co-expression of either the IL-12p40 "knob" or IL-12p35 "hole" with the corresponding subunit (IL-12p40 "hole" or IL-12p35 "knob," respectively) results in a modified dimeric IL-12 that is stabilized by the interaction between the "hole" and "knob" IgG Fc domains (see Example 1, Figures 4A and 5B).

[0107] In one embodiment, one or more IL-12 variant polypeptides comprise a single chain bivalent homodimeric Fc. In one embodiment, the single chain bivalent homodimeric Fc comprises at least two IgG Fc domains. In one embodiment, the IgG is human IgG. In one embodiment, the human IgG is human IgG1. In one embodiment, the human IgG1 Fc domain comprises the amino acid sequence of SEQ ID NO: 10.

[0108] In one embodiment, the IL-12p40 of the single chain bivalent homodimeric Fc comprises the IL-12p40 of WT IL-12. In one embodiment, the IL-12p40 of the single chain bivalent homodimeric Fc comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the single chain bivalent homodimeric Fc comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the single chain bivalent homodimeric Fc comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the IL-12p40 of the single chain bivalent homodimeric Fc comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0109] In one embodiment, the single chain bivalent homodimeric Fc of IL-12p40 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0110] In one embodiment, the single chain bivalent homodimeric Fc IL-12p40 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0111] In one embodiment, the IL-12p35 of the single chain bivalent homodimeric Fc comprises IL-12p35 of WT IL-12. In one embodiment, the IL-12p35 of the single chain bivalent homodimeric Fc comprises a purification tag. In one embodiment, the IL-12p35 of the single chain bivalent homodimeric Fc comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30 or SEQ ID NO:35.

[0112] In one embodiment, the IL-12p40 of the single chain bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, the IL-12p35 of the single chain bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, the IL-12p40 of the single chain bivalent homodimeric Fc is fused to an IL-12p35 of the single chain bivalent homodimeric Fc via a linker.

[0113] In one embodiment, (i) IL-12p40 of the single chain bivalent homodimeric Fc is fused to IL-12p35 of the single chain bivalent homodimeric Fc via a linker, and (ii) IL-12p35 of the single chain bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, (i) IL-12p40 of the single chain bivalent homodimeric Fc is fused to IL-12p35 of the single chain bivalent homodimeric Fc via a linker, and (ii) IL-12p40 of the single chain bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:20 and SEQ ID NO:21.

[0114] In one embodiment, the one or more IL-12 variant polypeptides comprising a single-chain bivalent homodimeric Fc comprise the amino acid sequence of SEQ ID NO: 22. In one embodiment, the one or more IL-12 variant polypeptides comprising a single-chain bivalent homodimeric Fc comprise the amino acid sequence of SEQ ID NO: 23. In one embodiment, the one or more IL-12 variant polypeptides comprise a single-chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 22, but with one more mutation in the IL-12p40 portion of the single-chain bivalent homodimeric Fc. In one embodiment, the one or more IL-12 variant polypeptides comprise a single-chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 22, but with at least one mutation selected from the group consisting of H216X, K217X, and K219X, relative to SEQ ID NO: 1, in the IL-12p40 portion of the single-chain bivalent homodimeric Fc. In one embodiment, the one or more IL-12 variant polypeptides comprise a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 22, but with at least one mutation in the IL-12p40 portion of the single chain bivalent homodimeric Fc selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1. In one embodiment, the one or more IL-12 variant polypeptides comprise a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 23, but with one more mutation in the IL-12p40 portion of the single chain bivalent homodimeric Fc. In one embodiment, the one or more IL-12 variant polypeptides comprise a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 23, but with at least one mutation in the IL-12p40 portion of the single chain bivalent homodimeric Fc selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the one or more IL-12 variant polypeptides comprise a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO:23, but with at least one mutation selected from the group consisting of H216A, K217A, and K219A in the IL-12p40 portion of the single chain bivalent homodimeric Fc, relative to SEQ ID NO:1.

[0115] One of skill in the art will recognize that expression of either configuration of the single chain bivalent homodimeric Fc results in dimeric IL-12 (i.e., a dimer of a fusion dimer stabilized by a bivalent IgG Fc domain, see Example 1 and FIG. 5B).

[0116] In one embodiment, the one or more IL-12 variant polypeptides comprise a single chain monomeric IL-12 and a bispecific heterodimeric Fc.

[0117] In one embodiment, the bispecific heterodimeric Fc comprises an IgG Fc "knob" and an IgG Fc "hole". In one embodiment, the IgG Fc "knob" and the IgG Fc "hole" are variants of IgG Fc. In one embodiment, the IgG Fc comprises a human IgG Fc. In one embodiment, the human IgG Fc comprises a human IgG1 Fc. In one embodiment, the IgG Fc "hole" is fused to a signal peptide via a linker. In one embodiment, the IgG Fc "knob" is fused to a signal peptide via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 11.

[0118] In one embodiment, the IL-12p40 of the single chain monomeric IL-12 comprises the IL-12p40 of WT IL-12. In one embodiment, the IL-12p40 of the single chain monomeric IL-12 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the single chain monomeric IL-12 comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the single chain monomeric IL-12 comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the IL-12p40 of the single chain monomeric IL-12 comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0119] In one embodiment, the single chain monomeric IL-12, IL-12p40, comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0120] In one embodiment, IL-12p40 of single chain monomeric IL-12 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0121] In one embodiment, the IL-12p35 of the single chain monomeric IL-12 comprises IL-12p35 of WT IL-12. In one embodiment, the IL-12p35 of the single chain monomeric IL-12 comprises a purification tag. In one embodiment, the IL-12p35 of the single chain monomeric IL-12 comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30 or SEQ ID NO:35.

[0122] In one embodiment, the single chain monomeric IL-12 comprises IL-12p40 fused to IL-12p35 via a linker. In one embodiment, the single chain monomeric IL-12 comprises (i) IL-12p40 fused to IL-12p35 via a linker, and (ii) IL-12p35 fused to an IgG Fc "knob" via a linker. In one embodiment, the single chain monomeric IL-12 comprises (i) IL-12p35 fused to IL-12p40 via a linker, and (ii) IL-12p40 fused to an IgG Fc "knob" via a linker. In one embodiment, the single chain monomeric IL-12 comprises (i) IL-12p40 fused to IL-12p35 via a linker, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker. In one embodiment, the single chain monomeric IL-12 comprises (i) IL-12p35 fused to IL-12p40 via a linker, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker. In one embodiment, the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:20 and SEQ ID NO:21.

[0123] In one embodiment, the single chain monomeric IL-12 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0124] In one embodiment, the single chain monomeric IL-12 comprises an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. In one embodiment, the single chain monomeric IL-12 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, but with one more mutation in the IL-12p40 portion of the single chain monomeric IL-12. In one embodiment, the single chain monomeric IL-12 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, but with at least one mutation selected from the group consisting of H216X, K217X, and K219X in the IL-12p40 portion of the single chain monomeric IL-12 relative to SEQ ID NO: 1. In one embodiment, the single chain monomeric IL-12 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, but with at least one mutation selected from the group consisting of H216A, K217A, and K219A in the IL-12p40 portion of the single chain monomeric IL-12 relative to SEQ ID NO: 1.

[0125] In one embodiment, the one or more IL-12 variant polypeptides comprising a single chain monomeric IL-12 and a bispecific heterodimeric Fc comprise (i) a single chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:26, and (ii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0126] In one embodiment, the one or more IL-12 variant polypeptides comprising a single chain monomeric IL-12 and a bispecific heterodimeric Fc comprise (i) a single chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:27, and (ii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0127] In one embodiment, the one or more IL-12 variant polypeptides comprising a single chain monomeric IL-12 and a bispecific heterodimeric Fc comprise (i) a single chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:28, and (ii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0128] In one embodiment, the one or more IL-12 variant polypeptides comprising a single chain monomeric IL-12 and a bispecific heterodimeric Fc comprise (i) a single chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:27, and (ii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0129] Those skilled in the art will recognize that co-expression of an IgG Fc "knob" or "hole" with a single-chain IL-12 fused to the corresponding IgG Fc "hole" or "knob", respectively, results in monomeric IL-12 stabilized by the heterodimeric Fc (see Example 1 and FIG. 5C).

[0130] In one embodiment, the one or more IL-12 variant polypeptides comprise a dimeric IL-12 and a bispecific heterodimeric Fc.

[0131] In one embodiment, the bispecific heterodimeric Fc comprises an IgG Fc "knob" and an IgG Fc "hole". In one embodiment, the IgG Fc "knob" and the IgG Fc "hole" are variants of IgG Fc. In one embodiment, the IgG Fc comprises a human IgG Fc. In one embodiment, the human IgG Fc comprises a human IgG1 Fc. In one embodiment, the IgG Fc "hole" is fused to a signal peptide via a linker. In one embodiment, the IgG Fc "knob" is fused to a signal peptide via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 11.

[0132] In one embodiment, the IL-12p40 of the dimeric IL-12 comprises the IL-12p40 of WT IL-12. In one embodiment, the IL-12p40 of the dimeric IL-12 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the dimeric IL-12 comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the IL-12p40 of the dimeric IL-12 comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the IL-12p40 of the dimeric IL-12 comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0133] In one embodiment, IL-12p40 of the dimeric IL-12 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0134] In one embodiment, IL-12p40 of dimeric IL-12 comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0135] In one embodiment, the IL-12p35 of the dimeric IL-12 comprises IL-12p35 of WT IL-12. In one embodiment, the IL-12p35 of the dimeric IL-12 comprises a purification tag. In one embodiment, the IL-12p35 of the dimeric IL-12 comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30, or SEQ ID NO:35.

[0136] In one embodiment, the dimeric IL-12 comprises (i) IL-12p40 of the dimeric IL-12 and (ii) IL-12p35 fused to the IgG Fc "knob" via a linker. In one embodiment, the dimeric IL-12 comprises (i) IL-12p40 of the dimeric IL-12 and (ii) IL-12p35 fused to the IgG Fc "hole" via a linker. In one embodiment, the dimeric IL-12 comprises (i) IL-12p35 of the dimeric IL-12 and (ii) IL-12p40 fused to the IgG Fc "knob" via a linker. In one embodiment, the dimeric IL-12 comprises (i) IL-12p35 of the dimeric IL-12 and (ii) IL-12p40 fused to the IgG Fc "hole" via a linker.

[0137] In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) IL-12p40 of the dimeric IL-12, (ii) IL-12p35 fused to an IgG Fc "knob" via a linker, and (iii) an IgG Fc "hole." In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) IL-12p35 of the dimeric IL-12, (ii) IL-12p40 fused to an IgG Fc "knob" via a linker, and (iii) an IgG Fc "hole." In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) IL-12p40 of the dimeric IL-12, (ii) IL-12p35 fused to an IgG Fc "hole" via a linker, and (iii) an IgG Fc "knob." In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) IL-12p35 of the dimeric IL-12, (ii) IL-12p40 fused to an IgG Fc "hole" via a linker, and (iii) an IgG Fc "knob."

[0138] In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34; (ii) IL-12p35 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:19; and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0139] In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33; (ii) IL-12p35 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:19; and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0140] In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16, and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25. In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16 but with one or more mutations in the IL-12p40 portion, and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25. In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216X, K217X, and K219X; and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25. In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) an IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216A, K217A, and K219A; and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0141] In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34; (ii) IL-12p35 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:17; and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0142] In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33; (ii) IL-12p35 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:17; and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0143] In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18, and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24. In one embodiment, the one or more IL-12 variant polypeptides comprising a dimeric IL-12 and a bispecific heterodimeric Fc comprise (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18 but with at least one mutation in the IL-12p40 portion, and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24. In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216X, K217X, and K219X; and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24. In one embodiment, the one or more IL-12 variant polypeptides comprising dimeric IL-12 and a bispecific heterodimeric Fc comprise: (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216A, K217A, and K219A; and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0144] One of skill in the art will recognize that (i) the corresponding subunits fused to the IgG Fc "hole" or "knob", and (ii) co-expression of IL-12p40 or IL-12p35 with the corresponding IgG Fc "knob" or "hole", results in dimeric IL-12 stabilized by the heterodimeric IgG Fc (see Example 1 and FIG. 5D).

[0145] Human serum albumin (HSA) has been genetically fused to therapeutically beneficial peptides (WO2001079271A and WO2003059934A, which are incorporated herein by reference in their entireties), with the typical result that the fusion has the activity of the therapeutically beneficial peptide and has a plasma half-life significantly longer than the plasma half-life of the therapeutically beneficial peptide alone. Thus, in some embodiments, the present disclosure relates to compositions and methods using the IL-12 variant polypeptide(s) described herein that are fused to HSA, thereby extending the half-life of the IL-12 variant polypeptide(s).

[0146] One of the most widely used methods to improve protein stability is the chemical modification of polypeptides with highly soluble macromolecules such as polyethylene glycol ("PEG"), which prevents the polypeptide from coming into contact with proteases. PEG is a highly flexible, uncharged, mostly non-immunogenic, hydrophilic, non-biodegradable molecule that produces a larger hydrodynamic radius than proteins of comparable size. It is also well known that when specifically or non-specifically bound to a polypeptide drug, PEG increases the solubility of the polypeptide drug and prevents its hydrolysis, thereby increasing the serum stability of the polypeptide drug without eliciting any immune response due to its low antigenicity (Sada et al, J. Fermentation Bioengineering, 1991, 71:137-139). Thus, in some embodiments, the present disclosure relates to compositions and methods using the IL-12 variant polypeptide(s) described herein fused to PEG, thereby extending the half-life of the IL-12 variant polypeptide(s).

[0147] Another method for improving the in vivo half-life of a protein includes fusion to a single domain antibody as described in WO2004041865, which is incorporated herein by reference in its entirety. A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any of the art or any future single domain antibody. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, cow. According to one aspect of the present disclosure, the single domain antibody used herein is a naturally occurring single domain antibody known as a heavy chain antibody devoid of light chains. For obvious reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules or nanobodies may be derived from antibodies raised in Camelidae species, such as camel, dromedary, alpaca, and guanaco. Thus, in some embodiments, the present disclosure relates to compositions and methods using the IL-12 variant polypeptide(s) described herein fused to a nanobody, thereby extending the half-life of the IL-12 variant polypeptide(s). In one embodiment, the nanobody comprises an anti-HSA nanobody.

[0148] In some embodiments, the one or more IL-12 variant polypeptides further comprise one or more signal peptides. In one embodiment, the one or more signal peptides facilitate extracellular secretion of the one or more IL-12 variant polypeptides. In one embodiment, the one or more signal peptides comprise one or more amino acid sequences selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.

[0149] Those skilled in the art will recognize that any known method of producing a polypeptide can be used to generate the polypeptide(s) of the present disclosure. The polypeptide(s) of the present disclosure can be made using chemical methods. For example, the polypeptide(s) can be synthesized by solid-phase techniques (Roberge JY et al (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be achieved, for example, using an ABI 431 A Peptide Synthesizer (Perkin Elmer) following the instructions provided by the manufacturer.

[0150] The polypeptide(s) of the present disclosure can be synthesized by conventional techniques. For example, the peptide or chimeric protein can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods use either solid-phase or solution-phase synthesis methods (for example, for solid-phase synthesis techniques, see JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford Ill. (1984), and G. Barany and RB Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254, and for classical solution synthesis, see M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1). By way of example, peptides of the present disclosure may be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0151] The polypeptide(s) may alternatively be produced by recombinant means or by cleavage from one or more longer polypeptides. The composition of the polypeptide(s) may be confirmed by amino acid analysis or sequencing.

[0152] Variants of the polypeptide(s) according to the present disclosure may be (i) those in which one or more of the amino acid residues are replaced with a conserved or non-conserved amino acid residue, such replaced amino acid residues being encoded by the genetic code or not, (ii) those in which one or more modified amino acid residues are present, e.g., those residues modified by attachment of a substituent group, (iii) those in which the peptide is an alternative splice variant of the polypeptide(s) of the present disclosure, (iv) fragments of the polypeptide(s) and / or (v) those in which the polypeptide(s) are / are fused to another peptide, such as a leader or secretory sequence, or a sequence used for purification (e.g., His-tag) or detection (e.g., Sv5 epitope tag). Fragments include polypeptide(s) generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally or chemically modified. Such variants are considered to be within the scope of one of skill in the art from the teachings herein.

[0153] The polypeptide(s) of the present disclosure can be post-translationally modified. For example, post-translational modifications within the scope of the present disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation are examined by adding dog microsomal membranes or Xenopus egg extracts (US Pat. No. 6,103,489) to a standard translation reaction.

[0154] The polypeptide(s) of the disclosure can include unnatural amino acids, formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[0155] The polypeptide(s) of the present disclosure may be phosphorylated using conventional methods, such as those described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0156] The polypeptide(s) of the present disclosure may be converted into a pharmaceutical salt by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like, or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0157] In some other embodiments, the IL-12 variant polypeptides of the present disclosure include further modified agents to improve resistance to proteolysis, or optimize solubility properties, or to make them more suitable as therapeutics. For example, the variants of the present disclosure further include residues other than naturally occurring L-amino acids, such as D-amino acids or analogs that include non-naturally occurring synthetic amino acids. D-amino acids can be substituted for some or all of the amino acid residues.

[0158] In some embodiments, one or more IL-12 variant polypeptides may be fused to another protein (i.e., a "second polypeptide"). In some embodiments, the second polypeptide specifically binds to a target molecule other than the target molecule bound by the one or more IL-12 variant polypeptides (e.g., other than IL-12Rβ1 and / or IL-12Rβ2). Thus, in some embodiments, one or more IL-12 variant polypeptides are multispecific (e.g., bispecific), such that a first region of the polypeptide comprises an IL-12 variant polypeptide sequence (i.e., the first region comprises an IL-12 variant polypeptide) and a second region that specifically binds to another target molecule (e.g., an antigen). For example, in some cases, an IL-12 variant polypeptide is fused to a second polypeptide that specifically binds to a target molecule other than the target molecule bound by the IL-12 variant polypeptide.

[0159] In some embodiments, one or more IL-12 variant polypeptides include a linker (e.g., a linker polypeptide). For example, in some embodiments, one or more IL-12 variant polypeptides and a fusion partner (i.e., a second polypeptide) are separated by a linker (e.g., a linker polypeptide). The linker polypeptide may have any of a variety of amino acid sequences. The proteins may be linked by a linker polypeptide (e.g., a flexible linker polypeptide) that may be of a flexible nature, although other chemical bonds are not excluded. Suitable linkers include polypeptides from about 6 amino acids to about 40 amino acids in length, or from about 6 amino acids to about 25 amino acids in length. These linkers may be generated by coupling to the proteins using synthetic oligonucleotides that code for the linker. Peptide linkers with some degree of flexibility may be used. The linking peptide may have virtually any amino acid sequence, with the understanding that in some cases the linker has a sequence that results in a generally flexible peptide. The use of small amino acids such as glycine and alanine is useful for generating flexible peptides. The generation of such sequences is routine for those skilled in the art. A variety of different linkers are commercially available and are deemed suitable for use. In some embodiments, the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:11, SEQ ID NO:20, and SEQ ID NO:21.

[0160] nucleic acid In some embodiments, the disclosure includes one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides of the disclosure, as described above. In some embodiments, the disclosure includes at least two nucleic acid molecules encoding one or more IL-12 variant polypeptides described above.

[0161] In some embodiments, the disclosure includes one or more nucleic acid molecules encoding IL-12p40 of one or more IL-12 variant polypeptides of the disclosure, as described above. In some embodiments, the disclosure includes one or more nucleic acid molecules encoding IL-12p35 of one or more IL-12 variant polypeptides of the disclosure, as described above.

[0162] In some embodiments, the disclosure includes one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides of the disclosure, IL-12p40 and IL-12p35, as described above. In some embodiments, the disclosure includes at least two nucleic acid molecules encoding one or more IL-12 variant polypeptides, IL-12p40 and IL-12p35, as described above.

[0163] In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, wherein the IL-12p40 comprises the amino acid sequence of WT IL-12p40, with or without a signal peptide. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, wherein the IL-12p40 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, wherein the IL-12p40 comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, wherein the IL-12p40 comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, a nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, wherein IL-12p40 comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1. In one embodiment, a nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, wherein IL-12p40 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, with or without a signal peptide. In one embodiment, a nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, wherein IL-12p40 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, with or without a signal peptide, in which amino acid residues 1-22, comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 31), are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0164] In some embodiments, the nucleic acid molecules encode one or more IL-12 variant polypeptides, or fragments thereof, comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12. In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, that comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12, and (ii) contains at least one, at least two, or at least three mutations relative to WT IL-12.

[0165] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p35, or fragments thereof, that comprise an amino acid sequence that has 100% sequence identity to WT IL-12p35. In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p35, or fragments thereof, that comprise an amino acid sequence that (i) has 100% sequence identity to WT IL-12p35 and (ii) does not contain a mutation relative to WT IL-12p35.

[0166] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, IL-12p40, or fragments thereof, wherein IL-12p40 comprises an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12p40. In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, IL-12p40, or fragments thereof, wherein IL-12p40 comprises an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12p40, and (ii) contains at least one, at least two, or at least three mutations relative to WT IL-12p40.

[0167] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) an IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to WT IL-12p40 and comprising at least one, at least two, or at least three mutations relative to WT IL-12p40; and (ii) an IL-12p35 comprising an amino acid sequence having 100% sequence identity to WT IL-12p35.

[0168] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p35, or fragments thereof, that comprise an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35. In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p35, or fragments thereof, that comprise an amino acid sequence that (i) has 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35, and (ii) does not contain a mutation relative to SEQ ID NO:2 or SEQ ID NO:35.

[0169] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprising an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1; and (ii) contains at least one, at least two, or at least three mutations relative to SEQ ID NO:1.

[0170] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34. In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprising an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34, and (ii) contains at least one, at least two, or at least three mutations relative to SEQ ID NO:34.

[0171] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1 and comprising at least one, at least two, or at least three mutations to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0172] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34 and comprising at least one, at least two, or at least three mutations relative to SEQ ID NO:34; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0173] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprising an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1; and (ii) comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X, relative to SEQ ID NO:1.

[0174] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or a fragment thereof, comprising an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34, and (ii) comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X, relative to SEQ ID NO:1.

[0175] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1 and comprising at least one mutation selected from the group consisting of H216X, K217X, and K219X, relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0176] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34 and comprising at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0177] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprising an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1, and (ii) comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A, relative to SEQ ID NO:1.

[0178] In some embodiments, the one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprise an amino acid sequence that (i) has 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34, and (ii) comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:1.

[0179] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:1 and comprising at least one mutation selected from the group consisting of H216A, K217A, and K219A, relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0180] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO:34 and comprising at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0181] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, with or without a signal peptide.

[0182] In some embodiments, the nucleic acid molecule comprises an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and encodes one or more IL-12 variant polypeptides IL-12p40, or fragments thereof, in which amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0183] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, including: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, with or without a signal peptide; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0184] In some embodiments, the nucleic acid molecule encodes one or more IL-12 variant polypeptides, or fragments thereof, comprising: (i) IL-12p40 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33; and (ii) IL-12p35 comprising an amino acid sequence having 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:35.

[0185] In one embodiment, the nucleic acid molecule encodes an IL-12 variant polypeptide(s) as described herein, fused to a peptide that enhances the stability or half-life of the fusion protein. In one embodiment, the nucleic acid molecule encodes a fusion peptide comprising at least one region of an immunoglobulin, or a variant or fragment thereof. In one embodiment, the nucleic acid molecule encodes a peptide comprising an Fc domain of an immunoglobulin. In one embodiment, the nucleic acid molecule encodes a fusion peptide comprising an Fc domain of a human IgG1. In one embodiment, the nucleic acid molecule encodes a fusion peptide comprising an Fc domain of an immunoglobulin comprising one or more mutations to eliminate Fc effector functions via the Fc receptor or complement. In one embodiment, the nucleic acid molecule encodes a fusion peptide comprising an Fc domain of a human IgG1 comprising a mutation at residue N297 relative to wild type human IgG1, to deglycosylate the Fc domain.

[0186] In some embodiments, the nucleic acid molecule encodes an IL-12 variant polypeptide(s) described herein fused to a heterodimeric Fc, thereby extending the half-life of the IL-12 variant polypeptide(s).

[0187] In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc comprising at least two IgG Fc domains. In one embodiment, the nucleic acid molecule encodes an IgG, wherein the IgG is a human IgG. In one embodiment, the nucleic acid molecule encodes a human IgG, wherein the human IgG is a human IgG1. In one embodiment, the nucleic acid molecule encodes a human IgG1 Fc domain comprising the amino acid sequence of SEQ ID NO: 10.

[0188] In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 comprising IL-12p40 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 comprising at least one, at least two, or at least three mutations relative to WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 comprising at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 comprising at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0189] In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p35 comprising IL-12p35 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p35 comprising a purification tag. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p35 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30, or SEQ ID NO:35.

[0190] In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc in which IL-12p40 is fused to an IgG Fc domain via a linker. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc in which IL-12p35 is fused to an IgG Fc domain via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:11.

[0191] In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12, but having one or more mutations in the IL-12p40 portion. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc IL-12p35 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 13.

[0192] In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprising (i) IL-12p40 fused via a linker to an IgG Fc domain, and (ii) IL-12p35 in the bivalent homodimeric Fc. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprising (i) IL-12p35 fused via a linker to an IgG Fc domain, and (ii) IL-12p40 in the bivalent homodimeric Fc.

[0193] In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprising (i) IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12, and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 35. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprising (i) IL-12p40 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 12, but with one or more mutations in the IL-12p40 portion, and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 35. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprising: (i) IL-12p40 comprising the amino acid sequence of SEQ ID NO: 12 fused to an IgG Fc domain via a linker having at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1; and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 35. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a bivalent homodimeric Fc comprising: (i) IL-12p40 comprising the amino acid sequence of SEQ ID NO:12 fused to an IgG Fc domain via a linker having at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:1; and (ii) IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35.

[0194] In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising: (i) IL-12p35 fused to an IgG Fc domain via a linker comprising the amino acid sequence of SEQ ID NO:13; and (ii) IL-12p40, a bivalent homodimeric Fc comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0195] In one embodiment, the nucleic acid molecule encodes a bivalent homodimeric Fc of IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, in which amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0196] One of skill in the art will recognize that co-expression of either IL-12p40 or IL-12p35 fused to IgG Fc by a linker and the corresponding subunit (IL-12p35 or IL-12p40, respectively) results in dimeric IL-12 (i.e., a tetrameric structure comprising a homodimer of a heterodimer stabilized by a bivalent IgG Fc domain, see Example 1 and FIG. 5A).

[0197] In one embodiment, the nucleic acid molecule encodes an IgG Fc "knob" or an IgG Fc "hole". In one embodiment, the IgG Fc "knob" and the IgG Fc "hole" are variants of IgG Fc. In one embodiment, the nucleic acid molecule encodes an IgG Fc, wherein the IgG Fc comprises a human IgG Fc. In one embodiment, the nucleic acid molecule encodes a human IgG Fc, wherein the human IgG Fc comprises a human IgG1 Fc. In one embodiment, the nucleic acid molecule encodes an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the nucleic acid molecule encodes an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO: 15.

[0198] In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 comprising IL-12p40 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 comprising at least one, at least two, or at least three mutations relative to WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 comprising at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 comprising at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0199] In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0200] In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0201] In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p35 comprising IL-12p35 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p35 comprising a purification tag. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p35 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30 or SEQ ID NO:35.

[0202] In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 fused to an IgG Fc "knob" via a linker. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p40 fused to an IgG Fc "hole" via a linker. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p35 fused to an IgG Fc "knob" via a linker. In one embodiment, the nucleic acid molecule encodes a bispecific heterodimeric Fc of IL-12p35 fused to an IgG Fc "hole" via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:11.

[0203] In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but with one or more mutations in the IL-12p40 portion. In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but with at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0204] In one embodiment, the nucleic acid molecule encodes IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO:17.

[0205] In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but having one or more mutations in the IL-12p40 portion. In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0206] In one embodiment, the nucleic acid molecule encodes IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO:19.

[0207] In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p40 fused to an IgG Fc "knob" via a linker, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker. In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p35 fused to an IgG Fc "knob" via a linker, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker.

[0208] In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but with one or more mutations in the IL-12p40 portion, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising: (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1; and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising: (i) IL-12p40 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 16, but having at least one mutation in the IL-12p40 portion selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1; and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 17.

[0209] In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but with one or more mutations in the IL-12p40 portion. In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but with at least one mutation selected from the group consisting of H216X, K217X, and K219X. In one embodiment, the one or more nucleic acid molecules encode a bispecific heterodimeric Fc comprising (i) IL-12p35 fused to an IgG Fc "knob" via a linker comprising the amino acid sequence of SEQ ID NO: 19, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker comprising the amino acid sequence of SEQ ID NO: 18, but with at least one mutation selected from the group consisting of H216A, K217A, and K219A.

[0210] Those skilled in the art will recognize that co-expression of either the IL-12p40 "knob" or IL-12p35 "hole" with the corresponding subunit (IL-12p40 "hole" or IL-12p35 "knob," respectively) results in a modified dimeric IL-12 that is stabilized by the interaction between the "hole" and "knob" IgG Fc domains (see Example 1, Figures 4A and 5B).

[0211] In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc comprising at least two IgG Fc domains. In one embodiment, the IgG is human IgG. In one embodiment, the human IgG is human IgG1. In one embodiment, the nucleic acid molecule encodes a human IgG1 Fc domain comprising the amino acid sequence of SEQ ID NO: 10.

[0212] In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p40 comprising IL-12p40 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p40 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p40 comprising at least one, at least two, or at least three mutations relative to WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p40 comprising at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc IL-12p40 comprising at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:1.

[0213] In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0214] In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0215] In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p35 comprising IL-12p35 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p35 comprising a purification tag. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc of IL-12p35 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30 or SEQ ID NO:35.

[0216] In one embodiment, the nucleic acid molecule encodes IL-12p40 in a single chain bivalent homodimeric Fc fused via a linker to an IgG Fc domain. In one embodiment, the nucleic acid molecule encodes IL-12p35 in a single chain bivalent homodimeric Fc fused via a linker to an IgG Fc domain. In one embodiment, the nucleic acid molecule encodes IL-12p40 in a single chain bivalent homodimeric Fc fused via a linker to an IL-12p35 in a single chain bivalent homodimeric Fc.

[0217] In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc, (i) IL-12p40 of the single chain bivalent homodimeric Fc is fused to IL-12p35 of the single chain bivalent homodimeric Fc via a linker, and (ii) IL-12p35 of the single chain bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, the nucleic acid molecule encodes a single chain bivalent homodimeric Fc, (i) IL-12p40 of the single chain bivalent homodimeric Fc is fused to IL-12p35 of the single chain bivalent homodimeric Fc via a linker, and (ii) IL-12p40 of the single chain bivalent homodimeric Fc is fused to an IgG Fc domain via a linker. In one embodiment, the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:20 and SEQ ID NO:21.

[0218] In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single-chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single-chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 23. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single-chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 22, but with one more mutation in the IL-12p40 portion of the single-chain bivalent homodimeric Fc. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single-chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 22, but with at least one mutation selected from the group consisting of H216X, K217X, and K219X in the IL-12p40 portion of the single-chain bivalent homodimeric Fc relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 22, but with at least one mutation in the IL-12p40 portion of the single chain bivalent homodimeric Fc selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO: 23, but with one more mutation in the IL-12p40 portion of the single chain bivalent homodimeric Fc. In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO:23, but having at least one mutation selected from the group consisting of H216X, K217X, and K219X in the IL-12p40 portion of the single chain bivalent homodimeric Fc relative to SEQ ID NO:1.In one embodiment, the nucleic acid molecule encodes one or more IL-12 variant polypeptides comprising a single chain bivalent homodimeric Fc comprising the amino acid sequence of SEQ ID NO:23, but having at least one mutation selected from the group consisting of H216A, K217A, and K219A in the IL-12p40 portion of the single chain bivalent homodimeric Fc relative to SEQ ID NO:1.

[0219] One of skill in the art will recognize that expression of either configuration of the single chain bivalent homodimeric Fc results in dimeric IL-12 (i.e., a dimer of a fusion dimer stabilized by a bivalent IgG Fc domain, see Example 1 and FIG. 5B).

[0220] In one embodiment, the disclosure provides one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides comprising a single chain monomeric IL-12 and a bispecific heterodimeric Fc.

[0221] In one embodiment, the bispecific heterodimeric Fc comprises an IgG Fc "knob" and an IgG Fc "hole". In one embodiment, the IgG Fc "knob" and the IgG Fc "hole" are variants of IgG Fc. In one embodiment, the IgG Fc comprises a human IgG Fc. In one embodiment, the human IgG Fc comprises a human IgG1 Fc. In one embodiment, the nucleic acid molecule encodes the IgG Fc "hole" fused to a signal peptide via a linker. In one embodiment, the nucleic acid molecule encodes the IgG Fc "knob" fused to a signal peptide via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 11.

[0222] In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 IL-12p40 that comprises IL-12p40 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 IL-12p40 that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 IL-12p40 that comprises at least one, at least two, or at least three mutations relative to WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 IL-12p40 that comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes IL-12p40, a single chain monomeric IL-12 that contains at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:1.

[0223] In one embodiment, the nucleic acid molecule encodes IL-12p40, a single chain monomeric IL-12 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0224] In one embodiment, the nucleic acid molecule comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and encodes IL-12p40, a single chain monomeric IL-12, in which amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0225] In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12p35 that comprises IL-12p35 of WT IL-12. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12p35 that comprises a purification tag. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12p35 that comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30 or SEQ ID NO:35.

[0226] In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 comprising IL-12p40 fused to IL-12p35 via a linker. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 comprising (i) IL-12p40 fused to IL-12p35 via a linker, and (ii) IL-12p35 fused to an IgG Fc "knob" via a linker. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 comprising (i) IL-12p35 fused to IL-12p40 via a linker, and (ii) IL-12p40 fused to an IgG Fc "knob" via a linker. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 comprising (i) IL-12p40 fused to IL-12p35 via a linker, and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker. In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 comprising (i) IL-12p35 fused to IL-12p40 via a linker, and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker. In one embodiment, the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:20 and SEQ ID NO:21.

[0227] In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. In one embodiment, the nucleic acid molecule encodes one or more single chain monomeric IL-12 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, but with one more mutation in the IL-12p40 portion of the single chain monomeric IL-12. In one embodiment, the nucleic acid molecule encodes one or more single chain monomeric IL-12 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, but with at least one mutation in the IL-12p40 portion of the single chain monomeric IL-12, relative to SEQ ID NO:1, selected from the group consisting of H216X, K217X, and K219X. In one embodiment, the nucleic acid molecule encodes one or more single chain monomeric IL-12 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, but having at least one mutation in the IL-12p40 portion of the single chain monomeric IL-12, selected from the group consisting of H216A, K217A, and K219A, relative to SEQ ID NO:1.

[0228] In one embodiment, the nucleic acid molecule encodes a single chain monomeric IL-12 comprising an amino acid sequence having 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to at least one selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0229] In one embodiment, the one or more nucleic acid molecules encode (i) a single chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:26, and (ii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0230] In one embodiment, the one or more nucleic acid molecules encode (i) a single chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:27, and (ii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0231] In one embodiment, the one or more nucleic acid molecules encode (i) a single-chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:28, and (ii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0232] In one embodiment, the one or more nucleic acid molecules encode (i) a single-chain monomeric IL-12 comprising the amino acid sequence of SEQ ID NO:27, and (ii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0233] Those skilled in the art will recognize that co-expression of an IgG Fc "knob" or "hole" with a single-chain IL-12 fused to the corresponding IgG Fc "hole" or "knob", respectively, results in monomeric IL-12 stabilized by the heterodimeric Fc (see Example 1 and FIG. 5C).

[0234] In one embodiment, the one or more nucleic acid molecules encode a dimeric IL-12 and a bispecific heterodimeric Fc.

[0235] In one embodiment, the bispecific heterodimeric Fc comprises an IgG Fc "knob" and an IgG Fc "hole". In one embodiment, the IgG Fc "knob" and the IgG Fc "hole" are variants of IgG Fc. In one embodiment, the IgG Fc comprises a human IgG Fc. In one embodiment, the human IgG Fc comprises a human IgG1 Fc. In one embodiment, the IgG Fc "hole" is fused to a signal peptide via a linker. In one embodiment, the IgG Fc "knob" is fused to a signal peptide via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 11.

[0236] In one embodiment, the nucleic acid molecule encodes an IL-12p40 of a dimeric IL-12 that comprises an IL-12p40 of a WT IL-12. In one embodiment, the nucleic acid molecule encodes an IL-12p40 of a dimeric IL-12 that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes an IL-12p40 of a dimeric IL-12 that comprises at least one, at least two, or at least three mutations relative to the WT IL-12p40 of SEQ ID NO: 1 or SEQ ID NO: 34. In one embodiment, the nucleic acid molecule encodes an IL-12p40 of a dimeric IL-12 that comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encodes an IL-12p40 of a dimeric IL-12 that comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO: 1.

[0237] In one embodiment, the nucleic acid molecule encodes IL-12p40 of dimeric IL-12 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34.

[0238] In one embodiment, the nucleic acid molecule comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and encodes IL-12p40 of dimeric IL-12, in which amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides. In one embodiment, the one or more different signal peptides are selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33.

[0239] In one embodiment, the nucleic acid molecule encodes IL-12p35 of a dimeric IL-12 that comprises IL-12p35 of WT IL-12. In one embodiment, the nucleic acid molecule encodes IL-12p35 of a dimeric IL-12 that comprises a purification tag. In one embodiment, the nucleic acid molecule encodes IL-12p35 of a dimeric IL-12 that comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:30, or SEQ ID NO:35.

[0240] In one embodiment, the nucleic acid molecule encodes a dimeric IL-12 comprising (i) IL-12p40 of the dimeric IL-12 and (ii) IL-12p35 fused to an IgG Fc "knob" via a linker. In one embodiment, the nucleic acid molecule encodes a dimeric IL-12 comprising (i) IL-12p40 of the dimeric IL-12 and (ii) IL-12p35 fused to an IgG Fc "hole" via a linker. In one embodiment, the nucleic acid molecule encodes a dimeric IL-12 comprising (i) IL-12p35 of the dimeric IL-12 and (ii) IL-12p40 fused to an IgG Fc "knob" via a linker. In one embodiment, the nucleic acid molecule encodes a dimeric IL-12 comprising (i) IL-12p35 of the dimeric IL-12 and (ii) IL-12p40 fused to an IgG Fc "hole" via a linker.

[0241] In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p40 of dimeric IL-12, (ii) IL-12p35 fused to the IgG Fc "knob" via a linker, and (iii) the IgG Fc "hole". In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p35 of dimeric IL-12, (ii) IL-12p40 fused to the IgG Fc "knob" via a linker, and (iii) the IgG Fc "hole". In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p40 of dimeric IL-12, (ii) IL-12p35 fused to the IgG Fc "hole" via a linker, and (iii) the IgG Fc "knob". In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p35 of dimeric IL-12, (ii) IL-12p40 fused to an IgG Fc "hole" via a linker, and (iii) an IgG Fc "knob."

[0242] In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34, (ii) IL-12p35 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:19, and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0243] In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33; (ii) IL-12p35 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:19; and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0244] In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16, and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25. In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16, but with one or more mutations in the IL-12p40 portion, and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25. In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) an IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216X, K217X, and K219X; and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25. In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) an IL-12p40 fused via a linker to an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:16, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216A, K217A, and K219A; and (iii) an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:25.

[0245] In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:34, (ii) IL-12p35 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:17, and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0246] In one embodiment, the one or more nucleic acid molecules encode (i) IL-12p40 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein amino acid residues 1-22 comprising the sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO:31) are replaced with one or more different signal peptides selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:33; (ii) IL-12p35 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:17; and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0247] In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18, and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24. In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18, but with at least one mutation in the IL-12p40 portion, and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24. In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216X, K217X, and K219X; and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24. In one embodiment, the one or more nucleic acid molecules encode (i) an IL-12p35 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35; (ii) an IL-12p40 fused via a linker to an IgG Fc "hole" comprising the amino acid sequence of SEQ ID NO:18, but having at least one mutation in the IL-12p40 portion relative to SEQ ID NO:1 selected from the group consisting of H216A, K217A, and K219A; and (iii) an IgG Fc "knob" comprising the amino acid sequence of SEQ ID NO:24.

[0248] One of skill in the art will recognize that (i) the corresponding subunits fused to the IgG Fc "hole" or "knob", and (ii) co-expression of IL-12p40 or IL-12p35 with the corresponding IgG Fc "knob" or "hole", results in dimeric IL-12 stabilized by the heterodimeric IgG Fc (see Example 1 and FIG. 5D).

[0249] Nucleic acid molecule(s) encoding the polypeptide(s) of the present disclosure can be obtained using a number of recombinant methods known in the art using standard techniques, such as, for example, screening libraries from cells expressing the gene, inducing the gene from a vector known to contain the gene, or isolating it directly from cells and tissues that contain them. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0250] The nucleic acid molecule(s) may comprise any type of nucleic acid, including, but not limited to, DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including, e.g., an isolated cDNA molecule, that encodes a polypeptide(s) of the present disclosure. In one embodiment, the composition comprises an isolated RNA molecule that encodes a polypeptide(s) of the present disclosure or a functional fragment thereof.

[0251] The nucleic acid molecule(s) of the present disclosure may be modified to improve stability in serum or growth medium for cell culture. Modifications may be added to enhance stability, functionality, and / or specificity and minimize the immunostimulatory properties of the nucleic acid molecules of the present disclosure. For example, to enhance stability, the 3'-residues may be stabilized against degradation and may be selected to consist of, for example, purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogs, for example, substitution of uridine by 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0252] In one embodiment of the present disclosure, the nucleic acid molecule(s) may contain at least one modified nucleotide analogue. For example, the termini may be stabilized by incorporating modified nucleotide analogues.

[0253] Non-limiting examples of nucleotide analogs include sugar and / or backbone modified ribonucleotides (i.e., modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA can be modified to include at least one of a nitrogen or sulfur heteroatom. In an exemplary backbone modified ribonucleotide, the phosphoester group connecting adjacent ribonucleotides is replaced by a modified group, e.g., a phosphothioate group. In an exemplary sugar modified ribonucleotide, the 2'OH group is replaced with a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0254] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide, which contains at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine, deazanucleotides, such as 7-deaza-adenosine, O- and N-alkylated nucleotides, such as N6-methyladenosine. It should be noted that the above modifications may be combined.

[0255] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modification of one or more nucleotides. In certain embodiments, the nucleic acid molecule of the present disclosure can have enhanced resistance to nucleases. For increased nuclease resistance, the nucleic acid molecule can comprise, for example, 2' modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced by many different "oxy" or "deoxy" substituents. For increased nuclease resistance, the nucleic acid molecule of the present disclosure can comprise 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. The inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene bridged nucleic acids, and certain nucleobase modifications, such as 2-amino-A, 2-thio (e.g., 2-thio-U), and G-clamp modifications can also increase binding affinity to targets.

[0256] In one embodiment, the nucleic acid molecule includes a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0257] In certain embodiments, the nucleic acid molecule(s) of the present disclosure have one or more of the following properties: The nucleic acid agents discussed herein include otherwise unmodified RNA and DNA, as well as RNA and DNA modified, for example, to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to molecules in which the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those naturally occurring or naturally occurring in the human body. The art refers to rare or unusual but naturally occurring RNA as modified RNA, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs are often referred to as modified RNAs, typically the result of post-transcriptional modifications, and are within the scope of the term unmodified RNA as used herein. As used herein, modified RNA refers to molecules in which one or more of the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those naturally occurring or those occurring in the human body. Although they are referred to as "modified RNAs", it will of course include molecules that are not, strictly speaking, RNAs, due to the modifications. A nucleoside surrogate is a molecule in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen in a ribophosphate backbone, e.g., an uncharged mimic of the ribophosphate backbone.

[0258] Modifications of the nucleic acids of this disclosure can be at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0259] The present disclosure also includes vectors into which the nucleic acid molecule(s) of the present disclosure are inserted. The art is replete with suitable vectors useful in the present disclosure.

[0260] Briefly, expression of a natural or synthetic nucleic acid encoding a fusion protein of the present disclosure is typically achieved by operably linking the nucleic acid encoding the fusion protein of the present disclosure or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and optional integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0261] The vector of the present disclosure can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols.Methods for gene delivery are known in the art.See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.In another embodiment, the present disclosure provides a gene therapy vector.

[0262] The isolated nucleic acids of the present disclosure can be cloned into several types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0263] Furthermore, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0264] method In various embodiments, the disclosure relates to methods of administering to a subject one or more IL-12 variant polypeptides of the disclosure, or one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides of the disclosure, as described above. In some embodiments, the disclosure relates to methods of treating or preventing one or more diseases or disorders in a subject, comprising administering to the subject one or more IL-12 variant polypeptides of the disclosure, or one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides of the disclosure, as described above.

[0265] Method of administration In one embodiment, the disclosure includes a method of administering to a subject one or more compositions of the disclosure, as described above. In one embodiment, the composition comprises one or more IL-12 variant polypeptides, wherein the one or more IL-12 variant polypeptides specifically bind to IL-12 receptor beta2 (IL-12Rβ2), and wherein the one or more IL-12 variant polypeptides exhibit substantially reduced binding to IL-12 receptor beta1 (IL-12Rβ1). In one embodiment, the composition comprises one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides, wherein the one or more IL-12 variant polypeptides specifically bind to IL-12 receptor beta2 (IL-12Rβ2), and wherein the one or more IL-12 variant polypeptides exhibit substantially reduced binding to IL-12 receptor beta1 (IL-12Rβ1).

[0266] In some embodiments, the subject has a disease or disorder that can be treated by decreasing the maximum level of agonism via the IL-12 receptor. In some embodiments, the subject is at risk of developing a disease or disorder that can be prevented by decreasing the maximum level of agonism via the IL-12 receptor. In some embodiments, the disease or disorder is cancer. Non-limiting examples of types of cancer that can benefit from administration of one or more compositions of the present disclosure are disclosed elsewhere herein.

[0267] The present disclosure encompasses the preparation and use of pharmaceutical compositions for administration comprising the compositions of the present disclosure disclosed herein as an active ingredient. Such pharmaceutical compositions may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharma- ceutically acceptable carriers, one or more additional ingredients, or some combination thereof. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable salt or ester, e.g., in combination with a physiologically acceptable cation or anion, as is well known in the art. In various embodiments, the active ingredient is one or more nucleic acid molecules, one or more polypeptides, or combinations thereof, as described elsewhere herein. The relative amounts of the active ingredient, pharma- ceutically acceptable carrier, and / or any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the compositions may comprise 0.1% to 100% (weight / weight) active ingredient.

[0268] In some embodiments, pharmaceutical compositions can include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex-functionalized Sepharose™, agarose, cellulose), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0269] Pharmaceutical compositions useful in practicing the present disclosure may be administered to deliver doses of about 0.1 ng / kg / day to 100 mg / kg / day, or more.

[0270] In various embodiments, pharmaceutical compositions useful in the methods of the present disclosure can be administered systemically, parenterally, or locally, such as oral formulations, inhalation formulations (including solids or aerosols), topical formulations, or other similar formulations, for example. In addition to the appropriate therapeutic composition, such pharmaceutical compositions can contain pharma- ceutically acceptable carriers and other components known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, other preparations containing active ingredients, and immunologically-based systems, can also be used to administer the appropriate regulator according to the methods of the present disclosure.

[0271] Carriers may carry the subject agents (e.g., one or more IL-12 variant polypeptides) in a variety of ways, including covalent attachment, either directly or via a linker group, and non-covalent attachment. Suitable covalent carriers include proteins such as albumin, peptides, and polysaccharides such as aminodextran, each of which has multiple sites for attachment of moieties. Carriers may also carry one or more IL-12 variant polypeptides by non-covalent association, such as non-covalent bonding, or encapsulation. The nature of the carrier, for purposes of this disclosure, may be soluble or insoluble.

[0272] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, zeolite, glycerol ... The in vivo formulations may include proteins such as saccharides, saccharides, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., zinc-protein complexes), and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0273] These active ingredients may also be encapsulated in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0274] The composition is prepared as an injectable, either as a liquid solution or suspension, but solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.The preparation can also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolide, or copolymers, for enhanced adjuvant effect, as discussed above.Langer, Science 249:1527,1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119,1997.The medicament of the present disclosure can be administered in the form of a depot injection or implant preparation, which can be formulated in such a way that it allows sustained or pulsatile release of active ingredient.The pharmaceutical composition is generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

[0275] As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of an active ingredient that is not harmful to the subject to which the composition is administered and that is compatible with any other ingredients of the pharmaceutical composition.

[0276] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the step of bringing into association the active ingredient with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single or multiple dosage unit.

[0277] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to a variety of animals are well understood, and a veterinarian of ordinary skill in the art can design and implement such modifications, if any, with no more than routine experimentation.

[0278] Pharmaceutical compositions useful in the methods of the present disclosure may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, transdermal, intralesional, subcutaneous, intramuscular, ophthalmic, intrathecal, and other known routes of administration. Other contemplated formulations include projected nanoparticles, liposomal formulations, other preparations containing the active ingredient, and immunologically-based formulations.

[0279] The pharmaceutical composition of the present disclosure can be prepared, packaged, and / or sold in bulk as a single unit dose or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of active ingredient that would be administered to a subject, or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0280] The relative amounts of the active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the present disclosure will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the compositions may contain from 0.1% to 100% (weight / weight) of the active ingredient.

[0281] In addition to the active ingredient, the pharmaceutical compositions of the present disclosure may further comprise one or more additional pharma- ceutical active agents.

[0282] Controlled or sustained release formulations of the pharmaceutical compositions of the disclosure may be made using conventional techniques.

[0283] Formulations of the pharmaceutical compositions of the present disclosure suitable for oral administration may be prepared, packaged, or sold in the form of discrete solid dosage units, including, but not limited to, tablets, hard or soft capsules, cachets, troches, or lozenges, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, or emulsions.

[0284] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface-active agents include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, acacia, pregelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0285] Liquid formulations of the pharmaceutical compositions of the disclosure may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended to be reconstituted with water or another suitable vehicle before use.

[0286] Liquid suspensions can be prepared using conventional methods to achieve suspension of active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions can further include one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweetening agents. Oily suspensions can further include thickening agents.

[0287] Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives, such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of alkylene oxides with fatty acids, with long chain aliphatic alcohols, with partial esters derived from fatty acids and hexitols, or with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0288] Liquid solutions of active ingredients in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, with the main difference being that the active ingredients are dissolved in the solvent rather than suspended.Liquid solutions of pharmaceutical compositions of the present disclosure may contain each of the ingredients described for liquid suspensions, but it is understood that the suspending agent does not necessarily aid in dissolving the active ingredients in the solvent.Aqueous solvents include, for example, water and isotonic saline.Oil solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0289] Powder and granular formulations of the pharmaceutical preparations of the present disclosure can be prepared using known methods. Such formulations can be administered directly to a subject, or can be used, for example, to form tablets, to fill capsules, or to prepare aqueous or oily suspensions or solutions by adding aqueous or oily vehicles thereto. Each of these formulations can further include one or more of dispersing or wetting agents, suspending agents, and preservatives. Additional excipients, such as fillers and sweeteners, flavoring agents, or coloring agents, can also be included in these formulations.

[0290] The pharmaceutical compositions of the present disclosure can also be prepared, packaged, or sold in the form of oil-in-water emulsion or water-in-oil emulsion. The oil phase can be vegetable oil such as olive oil or peanut oil, mineral oil such as liquid paraffin, or a combination thereof. Such compositions can further comprise one or more emulsifiers, such as naturally occurring gums, such as gum acacia or gum tragacanth, naturally occurring phospholipids, such as soybean or lecithin phosphamide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides, such as sorbitan monooleate, and the condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions can also contain additional ingredients, including, for example, sweeteners or flavorings.

[0291] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods in which the chemical composition is deposited or attached onto a surface, methods in which the chemical composition is incorporated into the structure of the material during its synthesis (i.e., as in physiologically degradable materials), and methods in which an aqueous or oily solution or suspension is absorbed into an absorbent material with or without subsequent drying.

[0292] As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by the administration of the pharmaceutical composition through physical disruption of the tissue of a subject and disruption in the tissue. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-permeable non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, cutaneous, subcutaneous, intraperitoneal, intravenous, intramuscular, intrasternal injection, and kidney dialysis infusion techniques.

[0293] A formulation of a pharmaceutical composition suitable for parenteral administration comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, implantable sustained-release formulations, or biodegradable formulations, and the like. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0294] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations may be prepared using, for example, a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0295] Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions, e.g., creams, ointments or pastes, and solutions or suspensions. Topically administrable formulations may contain, for example, about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the topical solvent formulation, and may further include one or more of the additional ingredients described herein.

[0296] The pharmaceutical compositions of the present disclosure may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles comprising the active ingredient and having diameters in the range of about 0.5 to about 7 nanometers, or in certain embodiments, in the range of about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder, or using a self-propelling solvent / powder dispensing container, such as a device comprising the active ingredient dissolved or suspended in a low boiling propellant in a closed container. In certain embodiments, such powders comprise particles, at least 98% of the particles by weight have a diameter greater than 0.5 nanometers, and at least 95% of the particles by number have a diameter less than 7 nanometers. In certain embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer, and at least 90% of the particles by number have a diameter less than 6 nanometers. In certain embodiments, the dry powder composition comprises a solid fine powder diluent, such as sugar, and is conveniently provided in a unit dosage form.

[0297] Low boiling propellants generally include liquid propellants having a boiling point below 65° F. at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid nonionic or solid anionic surfactant or a solid diluent (in some cases having a particle size on the same order as the particles containing the active ingredient).

[0298] Pharmaceutical compositions of the present disclosure formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as an optionally sterile, aqueous or dilute alcoholic solution or suspension containing the active ingredient, and may be conveniently administered using any spray or atomizing device. Such formulations may further comprise one or more additional ingredients, including, but not limited to, a flavoring agent, such as sodium saccharin, a volatile oil, a buffering agent, a surfactant, or a preservative, such as methyl hydroxybenzoate. In certain embodiments, the droplets provided by this route of administration have an average diameter in the range of about 0.1 to about 200 nanometers. The formulations described herein as useful for pulmonary delivery are also useful for intranasal delivery of pharmaceutical compositions of the present disclosure. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 micrometers.

[0299] Such formulations are administered in a nasal manner, i.e., by rapid inhalation through the nasal passages from a container of the powder held close to the nasal cavity. Formulations suitable for nasal administration may, for example, contain as little as 0.1% (w / w) and as much as 100% (w / w) active ingredient, and may further include one or more of the additional ingredients described herein.

[0300] Pharmaceutical compositions of the present disclosure may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may be, for example, in the form of tablets or lozenges made using conventional methods and may contain, for example, 0.1 to 20% (w / w) of the active ingredient, with the remainder comprising an orally dissolvable or degradable composition, and optionally one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may include a powder or a spray or atomized solution or suspension comprising the active ingredient. In certain embodiments, such powder, spray, or atomized formulations, when dispersed, have an average particle or droplet size in the range of about 0.1 to about 200 nanometers, and may further include one or more of the additional ingredients described herein.

[0301] Pharmaceutical compositions of the disclosure may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may be in the form of, for example, eye drops comprising, for example, a 0.1 to 1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such eye drops may further comprise a buffer, salt, or one or more other of the additional ingredients described herein. Other ophthalmically administrable formulations that are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

[0302] As used herein, "additional ingredients" include, but are not limited to, one or more of excipients, surface active agents, dispersing agents, inert diluents, granulating and disintegrating agents, binders, lubricants, sweeteners, flavoring agents, coloring agents, preservatives, physiologically degradable compositions such as gelatin, aqueous vehicles and solvents, oily vehicles and solvents, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifiers, antioxidants, antibiotics, antifungal agents, stabilizers, and pharma- ceutically acceptable polymers or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa, incorporated herein by reference.

[0303] Typical dosages of the compounds of the present disclosure that may be administered to an animal (e.g., a human) range from about 0.001 mg to about 1000 mg per kg of the animal's body weight. The exact dosage administered will vary depending on any number of factors, including, but not limited to, the type of animal and the type of disease or disorder being treated, the age of the animal, and the route of administration. In some embodiments, the dosage of the compound varies from about 0.1 mg to about 10 mg per kg of the animal's body weight. The compound may be administered to the animal several times per day, or less frequently, such as once per day, once per week, once every two weeks, once per month, or even less frequently, such as once every few months, or once per year or less. The frequency of administration will be readily apparent to one of skill in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease or disorder being treated, the type and age of the animal.

[0304] Treatment / Prevention Methods In one embodiment, the disclosure includes a method of treating or preventing one or more diseases or disorders in a subject in need thereof, comprising administering one or more compositions of the disclosure, as described above. In one embodiment, the method comprises administering to the subject a composition comprising one or more IL-12 variant polypeptides, wherein the one or more IL-12 variant polypeptides specifically bind to IL-12 receptor beta2 (IL-12Rβ2), and wherein the one or more IL-12 variant polypeptides exhibit substantially reduced binding to IL-12 receptor beta1 (IL-12Rβ1), as described above. In one embodiment, the method comprises administering to the subject a composition comprising one or more nucleic acid molecules encoding one or more IL-12 variant polypeptides, wherein the one or more IL-12 variant polypeptides specifically bind to IL-12 receptor beta2 (IL-12Rβ2), and wherein the one or more IL-12 variant polypeptides exhibit substantially reduced binding to IL-12 receptor beta1 (IL-12Rβ1), as described above.

[0305] In some embodiments, the compositions of the present disclosure are administered to a cell, tissue, organ, system, or subject to treat or prevent a disease or disorder, as described above. Based on the disclosure provided herein, one of skill in the art will understand that the present disclosure is useful in a subject, either in whole (e.g., systemically) or in part (e.g., locally, cell, tissue, organ), that is being or will be treated for a disease or disorder for which a reduction in maximum IL-12 signaling activity is beneficial. Based on the teachings provided herein, one of skill in the art will understand that the diseases and disorders treatable by the compositions and methods described herein include any disease or disorder in which a reduction in maximum IL-12 signaling activity promotes a positive biological, physiological, clinical, or therapeutic outcome. One of skill in the art will also understand that administration can be acute (e.g., over a short period of time, such as a day, a week, or a month) or chronic (e.g., over an extended period of time, such as several months or a year or more).

[0306] Given the present disclosure, including the methods detailed herein, it will be understood by those skilled in the art that the present disclosure is not limited to the treatment of a disease or disorder once established. In particular, symptoms of the disease or disorder need not manifest to the point of being detrimental to the subject, and in fact the disease or disorder need not be detected in the subject before treatment is administered. That is, significant pathology from the disease or disorder need not occur before the present disclosure can provide benefits. Thus, as described more fully herein, the present disclosure includes methods for preventing disease and disorders in a subject, in which one or more IL-12 variant polypeptides that specifically bind to IL-12Rβ2, but exhibit substantially reduced binding to IL-12Rβ1, as discussed elsewhere herein, can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the onset of the disease or disorder.

[0307] In one embodiment, the one or more diseases or disorders include cancer. Those skilled in the art will recognize that the compositions of the present disclosure can be administered to subjects with cancer to treat cancer or to subjects at risk of developing cancer to prevent cancer. Non-limiting examples of types of cancer that can be treated or prevented by the methods and compositions of the present disclosure include solid tumor cancer, liquid cancer, blood cancer, teratoma, sarcoma, and carcinoma. The following are non-limiting examples of cancer that can be treated or prevented by the methods and compositions of the present disclosure: Acute lymphoblastic leukemia, acute myeloid leukemia, adrenal cortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma / malignant glioma, brain astrocytoma / malignant glioma, cervical cancer, pediatric visual pathway Tumors, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family tumors, extracranial cancer, extraglandular germ cell tumors, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, Gastrointestinal stromal tumor (gist), germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, renal (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity Cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders,Nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovary, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant ovarian potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma and primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, nut gene on chromosome 15 Respiratory cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineal tumor, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0308] In some embodiments, the methods of the present disclosure are useful for treating or preventing tumors or cancers resistant to immune checkpoint inhibitors (ICIs). Exemplary immune checkpoint inhibitors include, but are not limited to, anti-PD1 (e.g., nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-TIM3, anti-TIGIT, anti-LAG3, anti-B7H3, anti-B7H4, anti-VISTA, anti-ICOS, anti-GITR, anti-41BB, anti-OX40, and anti-CD40. Examples of targets of immune checkpoint inhibitors include, but are not limited to, PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, and CD40. Thus, examples of immune checkpoint inhibitors include agents that inhibit proteins such as: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40. In some cases, one or more IL-12 variant polypeptides are administered with an immune checkpoint inhibitor (e.g., an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40, or any combination thereof).

[0309] In some embodiments, the method includes administering one or more compositions of the present disclosure together with one or more additional agents. In some embodiments, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effects at the same time. In some embodiments, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, the first agent may be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the second therapeutic agent.

[0310] In some embodiments, the one or more additional agents include one or more selected from the group consisting of chemical compounds, polypeptides, peptides, peptidomimetics, antibodies, cytokines, nucleic acid molecules, ribozymes, small molecule chemical compounds, and antisense nucleic acid molecules. In some embodiments, the one or more additional agents include one or more cancer therapeutics (e.g., chemotherapeutic agents), or cancer immunotherapeutics (e.g., cancer-specific antibodies). Such administration may include simultaneous (i.e., at the same time), prior, or subsequent administration of the drug / antibody with respect to administration of one or more agents of the present disclosure. One of ordinary skill in the art would have no difficulty in determining the appropriate timing, sequence, and dosage of administration of particular drugs and compositions of the present disclosure.

[0311] In some embodiments, the cancer therapy comprises a chemotherapeutic agent. Exemplary chemotherapeutic agents that can be administered in conjunction with a composition of the present disclosure to treat or prevent cancer include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granulocytoma ... setron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine, prochlorperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine and vinorelbine tartrate.

[0312] In some embodiments, the cancer immunotherapy includes an agent that opsonizes a target cell. An agent that opsonizes a target cell ("opsonizing agent") is any agent that can bind to a target cell (e.g., a cancer cell) and opsonize the target cell (e.g., exhibit phagocytosis and / or antibody-dependent cell-mediated cytotoxicity (ADCC) to the target cell). For example, an antibody that can bind to a target cell (e.g., a cancer cell such as a tumor cell) and has an FC region is considered to be an agent that opsonizes the target cell. In some cases, an agent that opsonizes a target cell is an antibody that binds to the target cell (e.g., an anti-tumor antibody, an anti-cancer antibody, etc.). In one embodiment, the agent that opsonizes a target cell is rituximab. Rituximab is a chimeric, unconjugated monoclonal antibody directed to the CD20 antigen. CD20 has an important functional role in B cell activation, proliferation, and differentiation. In one embodiment, the agent that opsonizes a target cell is cetuximab. Cetuximab binds to the epidermal growth factor receptor (EGFR) and has been used to treat solid tumors, including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).

[0313] In some embodiments, the cancer immunotherapeutic comprises a specific antibody. For exemplary antibodies selective for tumor cell markers, radiation, surgery, and / or hormone deprivation, see Kwon et al., Proc. Natl. Acad. Sci USA, 96:15074-9, 1999. Angiogenesis inhibitors can also be combined with the methods of the present disclosure. Several antibodies are currently in clinical use for the treatment of cancer, and others are in various stages of clinical development. For example, there are several antigens and corresponding monoclonal antibodies for the treatment of B-cell malignancies. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for the treatment of chronic lymphocytic leukemia. CD22 is targeted by several antibodies, which have recently shown efficacy in combination with toxins in chemotherapy-resistant hairy cell leukemia. Two new monoclonal antibodies targeting CD20, tositumomab and ibritumomab, have been submitted to the Food and Drug Administration (FDA). These antibodies are conjugated with radioisotopes. Alemtuzumab (Campath) is used to treat chronic lymphocytic leukemia. Gemtuzumab (Mylotarg) finds use in the treatment of acute myeloid leukemia. Ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma. Panitumumab (Vectibix) finds use in the treatment of colon cancer.

[0314] Monoclonal antibodies useful in the methods of the present disclosure that have been used in solid tumors include, but are not limited to, edrecolomab and trastuzumab (Herceptin). Edrecolomab targets the 17-1A antigen found in colon and rectal cancer and is approved for use in Europe for these indications. Trastuzumab targets the HER-2 / neu antigen.

[0315] In one embodiment, the cancer immunotherapy is one or more selected from the group consisting of cetuximab (binds to EGFR), panitumumab (binds to EGFR), rituximab (binds to CD20), trastuzumab (binds to HER2), pertuzumab (binds to HER2), alemtuzumab (binds to CD52), brentuximab (binds to CD30), tositumomab, ibritumomab, gemtuzumab, ibritumomab, and edrecolomab (binds to 17-1A), and combinations thereof.

[0316] In one embodiment, the cancer immunotherapy comprises an antigen-binding region that targets one or more selected from the group consisting of: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD 62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152(CTLA-4), CD200, CD213A2, CD221, CD248, CD276(B7-H3), B7-H4, CD279(PD-1), CD274(PD-L1), CD319, EGFR, EP CAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2(TIM3 ), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin αvβ3, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, Tenascin-3, STEAP1, and NRP1.

[0317] In one embodiment, the cancer immunotherapeutic comprises an antigen binding region that targets one or more selected from the group consisting of CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

[0318] In some embodiments, the cancer immunotherapy comprises an immunomodulatory agent. In some embodiments, the immunomodulatory agent includes, but is not limited to, an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, a TIGIT antibody, a TIM3 antibody, a LAG3 antibody, a VISTA antibody, a B7H3 antibody, a B7H4 antibody, a CD40 agonist, a 4-1BB modulator (e.g., a 41BB-agonist), an OX-40 modulator (e.g., an OX-40 agonist), a GITR modulator (e.g., a GITR agonist), a CD47 binder, e.g., an anti-CD47 antibody or a high affinity CD47 binder, a SIRPA binder, e.g., an anti-SIRPA antibody or a high affinity SIRPA binder, a TGF beta antagonist, e.g., an anti-TGF beta antibody, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, an IL-13, an IL-14, an IL-15, an IL-16, an IL-17, an IL-18, an IL-19, an IL-20, an IL-21, an IL-22, an IL-23, an IL-24, an IL-25, an IL-26, an IL-27, an IL-28, an IL-30, an IL-31, an IL-32, an IL-33, an IL-34, an IL-35, an IL-36, an IL-37, an IL-38, an IL-40, an IL-41, an IL 10, cytokines or cytokine variants including IL-15, IL-18, IL-21, IL-33, interferon alpha, interferon beta, interferon gamma, TNF, TRAIL, lymphotoxin, LIGHT / TNSF14, or agonists of Toll-like receptors including TLR2, TLR4, TLR5, TLR7, TLR9, agonists of the inflammasome, agonists of the STING / cGAS pathway, agonists of the RIG-I pathway, antagonists of the adenosine receptors A2aR / A2bR, antagonists of the aryl hydrocarbon receptor, antagonists of IDO and / or TDO, or oncolytic viruses.

[0319] The IL-12 variant polypeptides can be administered in combination with or fused to immune checkpoint inhibitors and / or tumor opsonizing antibodies. The subject polypeptides can be administered in combination with cell therapy, such as chimeric antigen receptor T cell (CAR-T cell), TCR-T cell, chimeric antigen receptor NK cell (CAR-NK cell), and tumor infiltrating lymphocyte (TIL) therapy. The subject polypeptides can also be administered as part of cell therapy, e.g., proteins secreted by cells, e.g., TRUCK cells ("T cells redirected for antigen-non-restricted cytokine-initiated killing"), which are an aspect of CAR-T cells, CAR-NK cells, TIL cells, or T or NK cells transduced with engineered T cell receptors. In other embodiments, one or more IL-12 variant polypeptides are administered with an oncolytic virus.

[0320] In some embodiments, one or more nucleic acids encoding one or more IL-12 variant polypeptides are included in an engineered ("modified") immune cell, e.g., a CAR-T or CAR-NK cell, or a T cell or NK cell transduced with an engineered T cell receptor. In this case, the engineered cell (e.g., modified T cell, modified NK cell) secretes one or more IL-12 variant polypeptides. The ability to secrete one or more IL-12 variant peptides can be regulated in a context-dependent manner (e.g., turned on in the tumor microenvironment), e.g., by a synthetic NOTCH receptor.

[0321] In some embodiments, one or more nucleic acids encoding one or more IL-12 variant polypeptides are contained within an oncolytic virus, in which case cells infected with the oncolytic virus secrete one or more IL-12 variant polypeptides.

[0322] In some embodiments, one or more nucleic acids encoding one or more IL-12 variant polypeptides are formulated and administered systemically or locally (e.g., intratumorally) as lipid nanoparticles. In other embodiments, one or more nucleic acids encoding one or more IL-12 variant polypeptides are electroporated into the tumor. In these scenarios, the IL-12 variant polypeptides are produced endogenously by the patient's own cells.

[0323] In some embodiments, the methods of the disclosure are useful for treating or preventing tumors or cancer tumors that have lost surface expression of MHC class I, e.g., tumors that have lost the B2m MHC locus, or tumors that have mutations in other members of the antigen-presenting and / or antigen-carrying complex, such as tapasin. EXAMPLES

[0324] Experimental Example The present disclosure will be described in more detail by referring to the following experimental examples.These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified.Therefore, the present disclosure should not be construed as being limited to the following examples in any manner, but rather as encompassing any and all variations that become evident as a result of the teachings provided herein.

[0325] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present disclosure and practice the claimed methods. Thus, the following working examples are not to be construed as limiting in any way the remainder of the disclosure.

[0326] Example 1: IL-12 partial agonists Cytokine partial agonists can selectively bias the activation of cell populations and signaling pathways with different activation thresholds. Human IL-12 partial agonists were generated by mutating the cytokine to reduce its affinity for its signaling receptor. These IL-12 variants generate a range of signaling amplitudes that are less than maximum for the wild-type molecule. Without being bound by scientific theory, it is believed that these partial agonists may have the effect of selectively activating the desired cell populations that promote anti-tumor immunity while avoiding induced toxicity.

[0327] Figure 1A and Figure 1B show models predicting residues at the IL-12Rβ1:IL-12p40 interface. Figure 1A shows a zoomed-in model of IL-12p40 in complex with a neutralizing nanobody. Residues predicted to mediate interactions with the nanobody and therefore with IL-12Rβ1 as well are shown in blue (see labels described for color), IL-12p40 is shown in green, and the nanobody is shown in light yellow. Figure 1B shows a schematic of predicted interactions between IL-12p40 and IL-12p35 subunits, as well as IL-12Rβ2 and IL-12Rβ1. The grey "X" indicates the interface between IL-12Rβ1 and IL-12p40, which, if selectively disrupted, may reduce recruitment of IL-12Rβ1 to the IL-12:IL-12Rβ2 complex and attenuate downstream STAT4 signaling. Figures 2A and 2B provide results showing protein expression and purification of wild-type (WT) IL-12 and mutant variants. Figure 2A shows exemplary results showing comparable expression and mobility of WT and mutant IL-12 proteins. Proteins were separated on SDS-PAGE gels and stained with Coomassie Brilliant Blue. Figure 2B shows exemplary results showing purification of IL-12 and mutant variants. Proteins were isolated by nickel-NTA affinity chromatography and then further purified by size exclusion chromatography.

[0328] Based on the existing protein structure of the IL-12p40 subunit bound to a neutralizing nanobody (PDB: 5MZV, Figure 1A), we investigated whether the surface-exposed amino acids His216, Lys217, and Lys219 of IL-12p40 mediate the interaction with IL-12Rβ1 (Figure 1B). To test this hypothesis, each residue was mutated to alanine in each possible unique combination (i.e., three single mutants, three double mutants, and one triple mutant), expressed in Expi293 cells (Figure 2A), and purified using nickel-NTA affinity chromatography followed by size-exclusion chromatography (Figure 2B). Then, to test the agonism of IL-12Rβ1, phosphorylation of signal transducer and activator of transcription 4 (STAT4) in human NK cells was measured by flow cytometry in the presence of increasing concentrations of WT IL-12 or various mutants. 3A-3D show exemplary results showing a graded response to WT depending on the number and nature of the interface residues mutated to alanine residues. FIG. 3A provides exemplary results showing reduced agonism of the H216A / K217A / K219A triple mutant (HKK) compared to WT IL-12. FIG. 3B provides exemplary results showing that agonism is reduced to a lesser extent in the H216A / K219A double mutant compared to WT. FIG. 3C shows exemplary results of single point responses to all variants compared to WT IL-12 and unstimulated cells. This suggests a graded response that can be tailored to a particular level of agonism desired. FIG. 3D shows the results of 3C as a percentage of agonism relative to WT. In all cases, human NK cells were stimulated with IL-12 mutant variants or left unstimulated and phosphorylated STAT4 was measured by flow cytometry as a measure of IL-12 agonism.

[0329] As shown in Figure 3A, mutation of all three residues (HKK) reduced the expression of the wild type (EC 50 = 40.8ng / mL, E max ≒406) compared to maximum agonism (E maxThe effective concentration of IL-12 required to reach 50% of the EC 50 = 839 ng / mL), and maximal agonism was reduced by approximately 40-50%. Interestingly, the response can be graded depending on the number and nature of the mutations. As shown in Figure 3B, H216A / K219A IL-12 inhibited IL-12 more effectively than wild-type (EC 50 = 57.4ng / mL, E max 267) had a reduction in maximal agonism of approximately 70–75% (EC 50 = 290ng / mL, E max ≈191). Similarly, in single-point screening of all variants, the trend of graded responses was fairly consistent, with the triple mutant having the most reduced response, the double mutant having a slightly greater response, and the single mutants having a similar response to the wild type (Figure 3C-D). One exception appeared for H216A / K217A, which had a similar, if not greater, response than K219A. K219A also had a lesser response compared to H216A and K217A individually, suggesting that K219 may play a more important role in mediating IL-12Rβ1:IL-12p40 interaction than H216 and K217.

[0330] Existing approaches for delivering IL-12, such as intratumoral injection, antibody fusion, and adoptive transfer of IL-12-expressing cells, use wild-type protein and therefore are likely to still be toxic.This approach is unique in that it utilizes attenuated IL-12, which may be safer than wild-type when administered in tumor-targeted or systemic delivery approaches due to the inherent differences in the cell types it can activate.Furthermore, to extend the half-life and improve efficacy of these variants, fusion to a range of stabilizing proteins, including heterodimeric Fc fusions, can be used.

[0331] Figures 4A-4B show exemplary results of IL-12 expressed as a bispecific heterodimeric Fc fusion protein, a method to extend in vivo half-life. Figure 4A shows a schematic of the bispecific heterodimeric Fc fusion protein of IL-12 tested for IL-12 agonism. Interestingly, the bispecific heterodimeric Fc fusion protein of IL-12 unexpectedly attenuated agonism compared to WT IL-12, but not as significantly as the HKK triple mutant (Figures 4A and 4B). Figure 4B shows exemplary results showing that the bispecific heterodimeric Fc fusion protein of IL-12 unexpectedly attenuated agonism compared to WT IL-12, but not as significantly as the HKK triple mutant described in Figures 3A-3D. Phosphorylated STAT4 was measured by flow cytometry as in Figures 3A-3D.

[0332] Figure 5A shows a schematic of a bivalent Fc fused to either p40 or p35 and co-expressed with the corresponding subunit to generate dimeric IL-12. Figure 5B shows a schematic of a bivalent Fc fused to p35 which is then fused to p40 and expressed as a single chain construct to form dimeric IL-12. Figure 5C shows a schematic of a bispecific Fc "knob" fused to p35 which is then fused to p40, expressed as a single chain construct and co-expressed with the corresponding bispecific Fc "hole" to form monomeric IL-12. Figure 5D shows a schematic of a bispecific Fc "knob" fused to either p40 or p35 and co-expressed with the corresponding subunit and the corresponding bispecific Fc "hole" to form monomeric IL-12.

[0333] FIG. 6A shows exemplary results of single point responses to Fc fusion variants compared to WT IL-12 and IL-12 H216A / K217A / K219A triple mutant (HKK), demonstrating that the graded responses shown in FIGS. 3A-3D can be further tailored via Fc fusions. FIG. 6B shows the effect of WT IL-12, IL-12 HKK and Fc fusion variants on phosphorylation of STAT4 in human NK cells in response to titrated amounts of supernatant. Proteins were expressed in Expi293 cells and cell culture supernatants containing secreted proteins were used to stimulate NK cells. The x-axis shows titrations of cell culture supernatants shown as a percentage of the total volume used to stimulate NK cells.

[0334] As mentioned above, the bispecific heterodimeric Fc fusion proteins of IL-12 unexpectedly attenuated agonism compared to WT IL-12, but not as significantly as the HKK triple mutant (Figures 4A and 4B). Thus, incorporating mutations at the IL-12Rβ1:IL-12p40 interface into the bispecific heterodimeric Fc fusion proteins of IL-12 (Figures 4A-4B) and / or any number of additional Fc-fused IL-12 variants (Figures 5A-5D) would result in further attenuation in agonism. Indeed, Figures 6A and 6B show that the HKK bispecific heterodimeric Fc fusion IL-12 is further attenuated compared to WT bispecific heterodimeric Fc fusion IL-12 or IL-12 HKK.

[0335] As shown diagrammatically in Figures 5A-5D, the additional Fc variants are also attenuated compared to WT IL-12 and WT bispecific heterodimeric Fc fusion IL-12. Thus, incorporating mutations into these additional Fc fusion variants would further attenuate agonism. Finally, additional strategies could be employed to extend the in vivo half-life of these variants and improve efficacy, including but not limited to fusion to human serum albumin (HSA), fusion to polyethylene glycol (PEG), or fusion to anti-HSA nanobodies (Figure 7). For example, Figure 7 provides a schematic providing additional exemplary methods of extending the in vivo half-life of partial IL-12 agonists of the present disclosure. Thus, these partial agonists of IL-12 may represent a novel and safe approach to treat various types of cancer, alone or in combination with other immunotherapies. [Table 1-1] TIFF2024534041000003.tif211170TIFF2024534041000004.tif94170 [Table 1-2] TIFF2024534041000006.tif237170TIFF2024534041000007.tif74170 [Table 1-3] TIFF2024534041000009.tif202170TIFF2024534041000010.tif124170 [Table 1-4] TIFF2024534041000012.tif152170

[0336] The disclosures of each and every patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. Although the present disclosure has been disclosed with reference to certain embodiments, it is apparent that other embodiments and modifications of the present disclosure may be devised by those skilled in the art without departing from the true spirit and scope of the present disclosure. It is intended that the appended claims be construed to include all such embodiments and equivalent modifications.

Claims

1. 1. A composition comprising an IL-12 variant polypeptide, the IL-12 variant polypeptide has less than maximal signaling efficacy through its receptor relative to wild-type (WT) IL-12; the IL-12 variant polypeptide comprises a p35 subunit (IL-12p35), with or without a signal peptide, and a p40 subunit (IL-12p40), with or without a signal peptide; and 10. The composition of claim 1, wherein the IL-12 variant polypeptide IL-12p40 comprises at least one mutation selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1, where X represents any amino acid.

2. The composition of claim 1, wherein the IL-12 variant polypeptide IL-12p40 comprises at least two mutations selected from the group consisting of H216X, K217X, and K219X relative to SEQ ID NO: 1 (X represents any amino acid).

3. The composition of claim 1, wherein the IL-12 variant polypeptide IL-12p40 comprises H216X, K217X, and K219X mutations relative to SEQ ID NO: 1 (X represents any amino acid).

4. 4. The composition of claim 1, wherein the IL-12 variant polypeptide IL-12p40 comprises at least one mutation selected from the group consisting of H216A, K217A, and K219A relative to SEQ ID NO:

1.

5. A composition described in any one of claims 1 to 4, wherein the IL-12 variant polypeptide IL-12p40 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

6. The composition of claim 4 or 5, wherein the IL-12 variant polypeptide, IL-12p40, comprises the amino acid sequence of SEQ ID NO:

9.

7. 7. The composition of any one of claims 1 to 6, wherein the IL-12 variant polypeptide IL-12p35 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:

35.

8. 8. The composition of any one of claims 1 to 7, further comprising at least one of the IL-12p40 and the IL-12p35 fused to at least one in vivo half-life extending fusion selected from the group consisting of an IgG Fc domain, an IgG Fc variant domain, human serum albumin (HSA), polyethylene glycol (PEG), and an anti-HSA nanobody.

9. A method for treating IL-12p40 and IL-12p35 comprising: fusion of at least one of the IL-12p40 and IL-12p35 with at least one of the IgG Fc domains; and 9. The composition of claim 8, wherein the IgG Fc domain comprises a human IgG1 domain comprising the amino acid sequence of SEQ ID NO: 10, and the IgG Fc variant domain comprises at least one selected from the group consisting of a human IgG1 Fc "knob" domain comprising the amino acid sequence of SEQ ID NO: 14, and a human IgG1 Fc "hole" domain comprising the amino acid sequence of SEQ ID NO:

15.

10. the IL-12 variant polypeptide is (a) a bivalent homodimeric IgG Fc comprising at least two human IgG1 Fc domains; (b) a bispecific heterodimeric IgG Fc comprising at least one human IgG1 Fc "knob" and at least one IgG Fc "hole"; (c) a single-chain bivalent homodimeric IgG Fc comprising said IL-12p40 fused to said IL-12p35 via a linker and at least two human IgG1 Fc domains; (d) a single-chain monomeric IL-12 comprising IL-12p40 fused to IL-12p35 via a linker, and a bispecific heterodimeric IgG Fc comprising at least one human IgG1 Fc "knob" and at least one IgG Fc "hole"; or (e) a dimeric IL-12 comprising said IL-12p40 and said IL-12p35, and a bispecific heterodimeric IgG Fc comprising at least one human IgG1 Fc "knob" and at least one IgG Fc "hole"; 10. The composition of claim 9, comprising:

11. A composition comprising a nucleic acid molecule encoding the IL-12p40 described in any one of claims 1 to 10.

12. 12. The composition of claim 11, further comprising a nucleic acid molecule encoding a p35 subunit (IL-12p35), with or without a signal peptide, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:30, and SEQ ID NO:35, or wherein the nucleic acid molecule encoding the IL-12p40 further encodes the IL-12p35.

13. A composition according to any one of claims 1 to 12 for use in treating or preventing a disease or disorder in a subject in need thereof.

14. The composition of claim 13 , wherein the disease or disorder is cancer.

15. a) a lipid nanoparticle-encapsulated mRNA molecule encoding said IL-12 variant polypeptide; b) a viral vector expressing said IL-12 variant polypeptide, or c) administering said IL-12 variant polypeptide via a population of engineered immune cells that express said IL-12 variant polypeptide.

16. Use of a p40 subunit (IL-12p40) with or without a signal peptide, or a nucleic acid encoding said IL-12p40, in the manufacture of a medicament for treating or preventing cancer, comprising: The IL-12p40 comprises the mutations H216X, K217X, and K219X (X represents any amino acid) relative to SEQ ID NO: 1.

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