Glycosylated engineered interferon beta proteins

Abstract

Disclosed herein are engineered, non-naturally occurring protein variants with de novo glycosylation sites having wildtype-like biological activity of wildtype interferon beta. Also disclosed herein are vectors encoding the non-naturally occurring protein variants described herein. Also disclosed herein are pharmaceutical compositions comprising the non-naturally occurring protein variants described herein or a vector encoding the non-naturally occurring protein variants described herein. Also disclosed herein are methods of treating a disease by administering to a subject the non-naturally occurring protein variants described herein, a vector encoding the non-naturally occurring protein variants described herein, or a pharmaceutical composition comprising the non-naturally occurring protein variants described herein or a vector described herein.

Description

Attorney Docket No.217863-712601 GLYCOSYLATED ENGINEERED INTERFERON BETA PROTEINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 from U.S. Provisional Application No.63 / 566,081 filed on March 15, 2024, the disclosure of which is incorporated herein by reference in their entirety. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Contract number 1R43HG012537-01A1 awarded by The National Institutes of Health. The government has certain rights in the invention. SUMMARY

[0003] Provided herein are engineered interferon beta (IFN-beta) proteins or a functional fragments thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the modification comprises an amino acid substitution, a deletion, or an insertion in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modification is an amino acid substitution, and the amino acid substitution comprises at least one amino acid substitution in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modification is a deletion of one or more amino acids in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modification comprises an insertion of one or more amino acids in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution results in an increase in a biological activity of the engineered IFN-beta protein as compared to a biological activity of the wildtype protein. In some embodiments, the at least one amino acid substitution results in a decrease in a biological activity of the engineered IFN-beta protein as compared to a biological activity of the wildtype protein. In some embodiments, the increase in the biological activity of biological activity comprises an increase in signaling, an increase in protein expression of one or more biological targets, an increase in mRNA expression, or any combination thereof, as measured by an in vitro assay. In some embodiments, the increase in signaling comprises an increase in IFNAR1-mediated signaling, as measured by an in vitro assay. In some embodiments, increase in signaling comprises an increase in IFNAR2-mediated signaling, as measured by an in vitro assay. In some embodiments, wherein the modification comprises: (a) an amino acid substitutionAttorney Docket No.217863-712601 that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a glycosylation motif into a glycosylation motif, thereby introducing a glycosylation motif into the engineered protein; (b) an amino acid substitution that results in a conversion of a glycosylation motif in the amino acid sequence of SEQ ID NO: 1 into a motif that is not a glycosylation motif, thereby eliminating a glycosylation motif from the engineered protein; or (c) a combination of (a) and (b). In some embodiments, the at least one amino acid substitution results in the conversion of 1, 2, 3, 4, or more motif(s) in the amino acid sequence of SEQ ID NO: 1 that are not glycosylation motifs into 1, 2, 3, 4, or more motif(s) that are glycosylation motif(s), thereby introducing 1, 2, 3, 4, or more glycosylation motif(s) into the engineered protein. In some embodiments, the at least one amino acid substitution results in the conversion of 1, 2, 3, 4, or more glycosylation motif(s) in the amino acid sequence of SEQ ID NO: 1 into 1, 2, 3, 4, or more motif(s) that are not glycosylation motifs, thereby eliminating 1, 2, 3, 4, or more glycosylation motif(s) into the engineered protein. In some embodiments, the glycosylation motif has a consensus sequence of N-X-S / T, wherein N is an Asparagine that is N-glycosylated at the amide bond in the side chain of the Asparagine, X is any amino acid except for proline, S is a serine, and T is a threonine. In some embodiments, the modification comprises an amino acid substitution that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a site for a post-translational modification into a post-translational modification consensus sequence motif, thereby introducing a site for a post translational modification into the engineered protein. In some embodiments, the post-translational modification consensus sequence motif is selected from the amino acid sequences in TABLE 5. In some embodiments, the amino acid substitution results in the addition of a post-translational modification on the engineered protein at the site of the post-translational modification consensus sequence motif that is not present in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the post-translational modification is selected from the group consisting of: glycosylation, phosphorylation, lipidation, glycation, proline isomerization, methylation, acetylation, hydroxylation sites, and proteolysis. In some embodiments, the engineered protein is glycosylated. In some embodiments, the engineered protein is aglycosylated. In some embodiments, the engineered protein binds to an interferon-α / β receptor-1(IFNAR1) or an interferon-α / β receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by enzyme-linked immunosorbent assay (ELISA), or by measuring signaling activity of an Interferon Stimulated Response Element (ISRE)-Green fluorescent protein (GFP) reporter in a live human cell in vitro assay. In some embodiments, the engineered protein binds to and activates an IFNAR1 or an IFNAR2 heterodimer subunits at a level greater than or equal to theAttorney Docket No.217863-712601 wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA, or by measuring signaling activity of an ISRE-GFP reporter in a live human cell in vitro assay. In some embodiments, the engineered protein binds to and inhibits an activity of an IFNAR1 or an IFNAR2 heterodimer subunits at a level greater than or equal to the wildtype IFN- beta protein as measured by ELISA, or by measuring signaling activity of an ISRE-GFP reporter in a live human cell in vitro assay. In some embodiments, the engineered protein is an agonist of IFNAR1 or IFNAR2 subunits. In some embodiments, the engineered protein binds and activates IFNAR1 or IFNAR2 at a similar level than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE-GFP reporter assay or an ISRE luciferase reporter assay. In some embodiments, the engineered protein reduces an extracellular level of a pro-inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA. In some embodiments, the pro- inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof. In some embodiments, the pro- inflammatory cytokine is IL-1β, IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, TNF-β, or any combination thereof. In some embodiments, the engineered protein increases an extracellular level of an anti-inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA. In some embodiments, the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof. In some embodiments, the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN-α, TGF-β, or any combination thereof. In some embodiments, the engineered protein binds to IFNAR1 or IFNAR2 subunits at a level less than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA. In some embodiments, the engineered protein is an antagonist of IFNAR1 or IFNAR2 subunits. In some embodiments, the engineered protein binds and inhibits an activity of the IFNAR1 or IFNAR2 at a level greater than wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE GFP reporter assay or an ISRE luciferase reporter assay. In some embodiments, the engineered protein is a recombinant IFN-beta or a portion thereof. In some embodiments, the engineered protein is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, conjugated to a polyethylene glycol (PEG) moiety, or any combination thereof. In some embodiments, a glycosylation site is introduced in the engineered protein that results in diminished binding of an antibody to the engineered protein, relative to an amount of binding of the antibody to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modification results in a reduction of immunogenicity levels when administered to a subject, as compared to anAttorney Docket No.217863-712601 immunogenicity level when the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 is administered to a subject. In some embodiments, the reduction of immunogenicity levels comprises a reduced level of anti-drug antibody in the subject when the engineered protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN- beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject. In some embodiments, the engineered protein, when administered to a subject in need thereof results in a reduction in a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering. In some embodiments, the reduction in the level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA. In some embodiments, the reduction in the level of inflammation in the subject results in a reduction in serum levels of C reactive protein as measured by high-sensitivity C-reactive protein (hs-CRP) test. In some embodiments, the reduction in serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg / dL to about 2.9 mg / dL. In some embodiments, the administering to the subject results in a reduction in blood levels of a pro- inflammatory cytokine as compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA. In some embodiments, the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence length to any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the engineered protein comprises an amino acid sequence that comprises a natural amino acid, or a non-natural amino acid.

[0004] Also provided herein are methods of making a engineered IFN-beta protein in a cell, comprising introducing into the cell a vector encoding the engineered IFN-beta protein disclosed herein. In some embodiments, the introducing results in expression of the engineered IFN-beta protein in the cell. In some embodiments, the engineered protein is isolated from the cell. In some embodiments, the vector comprises a mammalian expression plasmid. In some embodiments, the engineered IFN-beta protein comprises a tag. In some embodiments, the tag is CBP, FLAG, GST, Myc, poly-His, or V5. In some embodiments, the tag comprises a cleavable tag or a non-cleavable tag.Attorney Docket No.217863-712601

[0005] Provided herein are polynucleotides encoding an engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the polynucleotide comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 209 – SEQ ID NO: 342. In some embodiments, the polynucleotide comprises any one of SEQ ID NO: 209 – SEQ ID NO: 342. In some embodiments, the polynucleotide comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence length to any one of SEQ ID NO: 209 – SEQ ID NO: 342. In some embodiments, each codon in the polynucleotide can independently be replaced by a degenerate codon. In some embodiments, the polynucleotide comprise one or more degenerate codons. In some embodiments, the polynucleotide comprises a nucleic acid mimic. In some embodiments, the nucleic acid mimic comprises a phosphorothioate nucleic acid, a phosphoramidate nucleic acid, a morpholino nucleic acid, a hexitol nucleic acid (HNA), a peptide nucleic acid (PNA), or a locked nucleic acid (LNA). In some embodiments, one or more codons in the polynucleotide is modified. In some embodiments, one or more residues of an amino acid in the engineered IFN-beta protein is substituted for a non-natural amino acid. Also provided herein are vectors comprising the polynucleotides disclosed herein.

[0006] Also provided herein are pharmaceutical compositions in unit dose form comprising: (a) the engineered proteins or the polynucleotides disclosed herein; and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is encapsulated. In some embodiments, the pharmaceutical composition is in the form of a liquid. In some embodiments, the pharmaceutical composition is formulated for local, systemic, or topical administration. In some embodiments, the pharmaceutical composition is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parental, intravenous, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for controlled-release. In some embodiments, the pharmaceutical composition is formulated for single-dosage administration.

[0007] Also provided herein are methods of treating a disease in a subject, the methods comprising: administering to the subject a therapeutically effective amount of a composition comprising (a) an engineered interferon beta (IFN-beta) protein or a functional fragment thereof; and (b) a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the engineered IFN-beta protein comprises a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta proteinAttorney Docket No.217863-712601 comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the disease is an infection. In some embodiments, the infection is a viral infection. In some embodiments, the viral infection is chronic Hepatitis B infection, or a chronic Hepatitis C infection. In some embodiments, the disease is a demyelinating disorder. In some embodiments, the demyelinating disorder is selected from the group consisting of Multiple Sclerosis (MS), Acute Disseminated Encephalomyelitis (ADEM), Acute Hemorrhagic Leucoencephalitis (AHLE), Balo’s disease (Concentric Sclerosis), Charcot-Marie-Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease). In some embodiments, the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms. In some embodiments, the subject is a human subject. In some embodiments, the administering is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra-cisternal injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, a surgical route, or any combination thereof. In some embodiments, the administering occurs from about once a day, about twice a day, about once a week, about twice a week, about once every two weeks, about once a month, about once every 3 months, about once every 6 months, about once every 9 months, to about once a year. In some embodiments, the administering is at a dose selected from the group consisting of about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, or about 2000 mg. In some embodiments, the administering is at a dose selected from the group consisting of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, and 10 mg / kg. In some embodiments, the administering reduces a volume of a tumor in the subject. In some embodiments, the administering inhibits or diminishes the growth a tumor in the subject. In some embodiments, the methods further comprise administering a second therapeutic. In some embodiments, the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof. In some embodiments, the methods further comprise administering an immunotherapy. In some embodiments, the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1, a PD-L1, CTLA-4 inhibitor. In some embodiments, the administering induces activation of an IFN-stimulated response element (ISRE) or a Gamma interferon activation site (GAS) element, wherein the activation of the ISRE or GAS induces an interferon-like activity as measured by a luciferase reporter assay.

[0008] Also provided herein are methods of treating a disease or a condition in a subject, the methods comprising: administering to the subject a therapeutically effective amount ofAttorney Docket No.217863-712601 engineered proteins disclosed herein, the polynucleotides disclosed herein, or the pharmaceutical composition disclosed herein, thereby treating the subject.

[0009] Provided herein are methods of treating a demyelinating disorder, the methods comprising: administering to the subject a therapeutically effective amount of a composition comprising: (a) an engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208; and (b) a pharmaceutically acceptable excipient, carrier or diluent. In some embodiments, the demyelinating disorder is selected from the group consisting of Multiple Sclerosis (MS), Acute Disseminated Encephalomyelitis (ADEM), Acute Hemorrhagic Leucoencephalitis (AHLE), Balo’s disease (Concentric Sclerosis), Charcot-Marie- Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease). In some embodiments, the demyelinating disorder is a MS, wherein the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.

[0010] Also provided herein are kits comprising the engineered proteins disclosed herein, the polynucleotides disclosed herein, or the pharmaceutical composition disclosed herein,, and a container. INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0013] FIG.1 depicts the design and application of the high-throughput molecular function assay system for assessing mutational effects on gene activity (MEGA) and to produce MEGA- maps.

[0014] FIG.2 illustrates the application of the high-throughput molecular function assay system to screen variants of IFN-beta for biosimilar activity.Attorney Docket No.217863-712601

[0015] FIG.3 depicts an overview of the high-throughput molecular function assay system to assess tethered IFN-beta autocrine signaling.

[0016] FIG.4 depicts a graph flow-cytometry profile of ISRE reporter activity in IFN-beta treated cells vs. untreated. Untreated HEK-293 interferon-responsive reporter cells is used as control (grey; untreated) to compare with HEK-293 cells treated with purified wildtype IFNβ ligand (dark grey; treated). The x-axis depicts relative level fluorescence of ZsGreen and the y- axis depicts the number events (cell counts).

[0017] FIG.5 depicts a schematic of the purification of HIS-tagged engineered protein as described herein.

[0018] FIGs.6A-6B depicts a schematic for determination of EC50 of IFN-beta signaling in engineered protein as described herein. FIG 6A. depicts a 96-well plate assay for measuring EC50 of IFN signaling for comparing IFN chimera. FIG 6B. Dose response curve output from the 96-well ISRE reporter assay.

[0019] FIG.7 depicts an assay to determine immunogenicity of IFN leads using Peripheral Blood Mononuclear Cells (PBMCs).

[0020] FIG.8A-8B depicts a schematic of the purification of un-tagged engineered protein as described herein. FIG.8A depicts a schematic of the purification. FIG.8B depicts amino acid sequence of 6xHIS and the Enterokinase Light Chain Cleavage Site (EKCS) protease site upstream of the engineered protein described herein, with the cleavage position of enterokinase indicated by a dashed vertical line.

[0021] FIG.9 depicts the dose response of the engineered proteins described herein to determine the EC50 values in an antiviral Cytopathic Effect (CPE) assay.

[0022] FIG.10 illustrates a schematic to determine biomarker expression in human peripheral blood mononuclear cells (PBMCs) after treatment with the engineered proteins described herein.

[0023] FIG.11 depicts the prophylactic assessment of engineered proteins in Experimental Autoimmune Encephalomyelitis (EAE) mouse model for Multiple Sclerosis (MS).

[0024] FIG.12 depicts the liver toxicity assessment of engineered proteins described herein in mice.

[0025] FIG.13 depicts the pharmacokinetic study of the engineered proteins described herein in humans.

[0026] FIG.14 depicts the immunogenicity study of the engineered proteins described herein in humans.

[0027] FIGs.15A-15N depict the verification of the engineered proteins described herein on SDS-PAGE.Attorney Docket No.217863-712601

[0028] FIGs.16A-16L depict the plate reader output after cells were treated with exemplary engineered HIS-tagged engineered IFN-beta proteins or HIS-tagged wildtype IFN-beta protein. The x-axis depicts the ligand concentration, and the y-axis depicts the ISRE reporter activity.

[0029] FIGs.17A-17G depict the flow cytometry measurements in cells treated with exemplary HIS-tagged engineered IFN-beta proteins increased activity (GOF variant) as compared to wildtype at different concentrations. The x-axis depicts the ISRE reporter activity, and the y-axis depicts the cell counts.

[0030] FIGs.18A-18F depict quantitative RT-PCR determination of OAS1 mRNA expression in cells in expressing exemplary HIS-tagged engineered IFN-beta proteins as compared to HIS- tagged wildtype IFN beta protein. The x-axis ligand concentration (ng / mL), and the y-axis depicts OAS1 mRNA (normalized to HPRT). DETAILED DESCRIPTION OF THE INVENTION

[0031] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0032] Throughout this application, various embodiments may be presented in a range format. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0033] The singular forms “a”, “an”, and “the” are used herein to include plural references unless the context clearly dictates otherwise. Accordingly, unless the contrary is indicated, the numerical parameters set forth in this application are approximations that can vary depending upon the desired properties sought to be obtained.

[0034] The terms “determining”, “measuring”, “evaluating”, “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement and include determining if an element may be present or not (for example, detection), or the amount of an element. These terms can include quantitative and qualitative determinations. Assessing can beAttorney Docket No.217863-712601 alternatively relative or absolute. “Detecting the presence of” includes determining the amount of something present, as well as determining whether it may be present or absent.

[0035] The term “substantially” refers to a qualitative condition that exhibits at least 70 % of a total range or degree of a feature or characteristic of interest.

[0036] The term “operably coupled” refers to functional linkage between a regulatory sequence and a nucleic acid sequence resulting in expression of the latter.

[0037] Unless otherwise indicated, open terms for example “contain,” “containing,” “include,” “including,” and the like mean comprising.

[0038] As used herein, the term, “about” or “approximately,” means within an acceptable error range for the particular value and includes a range of up to 10% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0039] When used herein, a percentage of a material (e.g., a biological material, an excipient, a compound) of a composition is with respect to a total weight of a composition. In some cases, a percentage of a material of a composition is with respect to a total volume of a composition. In some cases, “Percentage by weight” or “w / w” means ratio of the mass of the specified ingredient verses the mass of the entire composition (e.g., dosage unit).

[0040] The term “homology” can refer to the relationship among sequences whereby there is some extent of likeness, typically due to descent from a common ancestral sequence. Homologous sequences can share homology based on genic, structural, functional and / or behavioral properties. Similarity of a polypeptide to a reference polypeptide is generally determined based on conservation of certain properties of amino acids (such as conservative substitutions), and the degree of similarity can be determined using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis.53711).

[0041] The term “native” or “wildtype,” in reference to interferon beta (IFN-beta), refers to biologically active, naturally-occurring IFN-beta, including biologically active, naturally- occurring IFN-beta variants. As used herein, the terms “interferon beta,” “IFN-beta,” “IFN-β” and “IFNβ” are used interchangeably, and refer to a polypeptide or protein with at least 95% sequence identity to SEQ ID NO: 1.

[0042] The term includes the 166 amino acid human IFN-beta mature sequence (SEQ ID NO: 1).

[0043] The term "glycosylation" refers to post-translational protein modification in which carbohydrates, particularly glycans, are attached to functional groups of amino acids. Glycosylation is the process by which carbohydrates are converted to nitrogen on the side chain of asparagine or arginine (N-glycosylation); hydroxyl oxygens of serine, threonine, tyrosine,Attorney Docket No.217863-712601 hydroxylysine or hydroxyproline side chains (O-glycosylation); phosphate of phosphoserine (phosphoglycosylation); or attached to a carbon of the tryptophan side chain (C-glycosylation).

[0044] The terms “non-naturally occurring protein,” “engineered protein,” and “engineered IFN- beta protein” as used herein are used interchangeably. As used herein, the terms “engineered IFN-beta protein,” “non-naturally occurring IFN-beta polypeptides” or “non-naturally occurring IFN-beta proteins” is used interchangeably and refers to an engineered interferon-beta polypeptide or protein as disclosed herein.

[0045] The term "glycosylation motif" refers to an amino-acid residue sequence that tends to be post-translationally modified by glycosylation (i.e., enzyme-catalyzed covalent linkage of one or more carbohydrate moieties) during protein biosynthesis. In some instances, “glycosylation motif" as used herein refers to a consensus sequence motif that contains a site for glycosylation. In some embodiments, the site for glycosylation comprises an amino acid residue that is capable of forming a covalent linkage, wherein the covalent linkage is to a carbohydrate moiety. In some embodiments, the covalent linkage occurs via a nitrogen atom. In some embodiments, the nitrogen atom is in a sidechain amide group of an asparagine [Asn] residue).

[0046] The term “aglycosylate” or “aglycosylated” or “aglycosylation” as used herein, refers to a protein that bypass glycosylation, thereby preventing glycosyl groups from being added to the protein.

[0047] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to an animal, typically mammalian animals. Any suitable mammal can be administered a composition as described herein or be treated by a method as described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). Mammals can be any age or at any stage of development, for example a mammal can be neonatal, infant, adolescent, adult or in utero. In some embodiments, a subject is a human. Humans can be more than about: 1, 2, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or about 120 years of age. Humans can be less than about: 1, 2, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or about 120 years of age. In some cases, a human can be less than about 18 years of age. In some cases, human is from about 1 week to about 5 weeks old, 1 month to about 12 months old, from about 1 year to about 20 years, from about 15 years to about 50 years, from about 40 years to about 80 years, or from about 60 years to about 110 years. In some cases, a human is more than about 18 years of age. A human may be a pediatric subject. A human may be an adult subject. A human is a child subject. A mammal such as a human is born a male or a female. In some embodiments, a subject has or is suspectedAttorney Docket No.217863-712601 of having a disease or condition, such as a cancer. The subject is a patient, such as a patient being treated for a condition or a disease, such as a cancer. In some cases, a subject is a responder to cancer therapy. In some cases, a subject is a non-responder to a cancer therapy. A subject is predisposed to a risk of developing a condition or a disease. A subject is in remission from a condition or a disease. In some instances, a subject is healthy. A subject may be a subject in need thereof. A subject may have received a positive diagnosis of the cancer. A subject may have received a cancer therapy that failed to treat a cancer.

[0048] A “therapeutically effective amount” refers to an amount of a composition as disclosed herein with or without additional agents that is effective to achieve its intended purpose, for example to treat a disease. Individual patient needs may vary. Generally, the dosage required to provide an effective amount of the composition will vary, depending on the age, health, physical condition, sex, weight, extent of the disease of the recipient, frequency of treatment and the nature and scope of the disease or condition.

[0049] As used herein, the terms “treatment” or “treating” refers to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit refers to eradication or amelioration of one or more symptoms of an underlying disorder being treated. For example, a therapeutic benefit can comprise shrinking a tumor, at least partially inhibiting a tumor to spread, reducing the size of a tumor, inhibiting a tumor to grow, slowing tumor growth, or any combination thereof. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement may be observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For a prophylactic benefit, a subject at risk of developing a particular disease, or a subject reporting one or more of the physiological symptoms of a disease may undergo a treatment disclosed herein, even though a diagnosis of this disease may not have been made.

[0050] As used herein, the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.Attorney Docket No.217863-712601

[0051] Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0052] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having 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 if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, is referred to as encoding the protein or other product of that gene or cDNA.

[0053] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0054] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0055] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0056] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression is supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0057] A “vector” is a composition of matter which comprises an isolated nucleic acid and which is used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non- plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.Attorney Docket No.217863-712601

[0058] “Sequence identity” as used herein, refers to the similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are the same at that position. The sequence identity between two sequences is a direct function of the number of matching positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are similar, the two sequences have 50% sequence identity, if 90% of the positions (e.g., 9 of 10), are matched, the two sequences have 90% sequence identity. In some instances, the phrase “sequence identity” means the percentage of identical subunits at corresponding positions in two sequences (e.g., nucleic acid sequences, amino acid sequences) when the two sequences are aligned to maximize subunit matching, therefore, taking into account gaps and insertions resulting from indels.

[0059] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0060] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0061] The term “linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0062] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which is hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides is hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available inAttorney Docket No.217863-712601 the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0063] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0064] Ranges: throughout this disclosure, various aspects of the invention are 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 invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 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.

[0065] Although this disclosure has been described in terms of certain implementations and uses, other implementations and other uses, including implementations and uses which do not provide all of the features and advantages set forth herein, are also within the scope of this disclosure. Components, elements, features, acts, or steps is arranged or performed differently than described and components, elements, features, acts, or steps is combined, merged, added, or left out in various implementations. All possible combinations and sub combinations of elements and components described herein are intended to be included in this disclosure. No single feature or group of features is necessary or indispensable.

[0066] Certain features that are described in this disclosure in the context of separate embodiments are implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single implementation is also implemented inAttorney Docket No.217863-712601 multiple embodiments separately or in any suitable sub combination. Moreover, although features may be described above as acting in certain combinations, one or more features from a claimed combination can in some cases be excised from the combination, and the combination may be claimed as a sub combination or variation of a sub combination.

[0067] While operations may be described in the specification in a particular order, such operations need not be performed in the particular order described or in sequential order, or that all operations be performed, to achieve desirable results. Other operations that are not depicted or described is incorporated in the example methods and processes. For example, one or more additional operations can be performed before, after, simultaneously, or between any of the described operations. Additionally, the operations may be rearranged or reordered in some implementations. Also, the separation of various components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products. Additionally, some implementations are within the scope of this disclosure.

[0068] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Overview

[0069] Disclosed herein are engineered interferon (IFN)-beta proteins, where the engineered proteins described herein are single glycosylated, double glycosylated, multi-glycosylated or aglycosylated. The engineered proteins as described herein have binding affinity to an interferon- α / β receptor-1 (IFNAR1) or interferon-α / β receptor-2 (IFNAR2) heterodimer subunits, which is substantially similar or greater than the binding activity of the wildtype IFN-beta protein. IFN- beta is a biologic used to reduce inflammation and treat inflammatory conditions, such as Multiple Sclerosis. These novel engineered proteins are designed to have increased serum half- life, increased solubility, increased signaling, and reduced immunogenicity.

[0070] Also disclosed herein are compositions comprising non-naturally occurring protein, methods of making the engineered protein, and methods for treating disease. In some embodiments, the engineered protein as described herein is a recombinant IFN-beta protein. In some embodiments, the engineered protein can act as a signaling agent. In some embodiments, the biological activity of the engineered IFN-beta protein is identical to wildtype IFN-beta. Glycosylated engineered proteinsAttorney Docket No.217863-712601

[0071] Disclosed herein are engineered interferon beta (IFN-beta) proteins or functional fragments thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. Also disclosed herein are engineered, non-naturally occurring interferon (IFN)-beta protein variants comprising an amino acid substitution that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a glycosylation motif into a glycosylation motif, thereby introducing a glycosylation motif into the engineered protein. In some embodiments, the amino acid substitution results in the conversion of a glycosylation motif in the amino acid sequence of SEQ ID NO: 1 into a motif that is not a glycosylation motif, thereby eliminating a glycosylation motif from the engineered protein. In some embodiments, the amino acid substitution introduces, eliminates or both introduces and eliminates one or more glycosylation sites in the amino acid sequence of SEQ ID NO: 1. The engineered proteins has substantially wildtype IFN-beta like biological activity. In some embodiments, the engineered proteins described herein comprise a polypeptide sequence having a sequence with at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 2 – SEQ ID NO: 208. Such variants can be used as a treatment for inflammatory diseases or conditions such as multiple sclerosis and cancer.

[0072] In some embodiments, the engineered protein has an amino acid substitution (e.g., one or more amino acid substitutions) recited in TABLE 1 below, relative to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1 (MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALT IYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMS SLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN). An engineered, non-naturally occurring protein variant having an amino acid substitution recited in TABLE 1 which results in similar biological activity (e.g., binding activity) to a wildtype IFN-beta protein. TABLE 1. Exemplary IFN-beta amino acid substitutions Amino Acid Substitution – Mutated CodonAttorney Docket No.217863-712601 D54N-AAC & A56T-ACC D73N-AACAttorney Docket No.217863-712601 L47N-AAC & Q49T-ACC L57N-AAC & I59T-ACC & N80Q-CAGAttorney Docket No.217863-712601 R27N-AAC & E29T-ACC R27T-ACC

[0003] n some embod men s, e am no ac d subs u on results in the conversion of 1, 2, 3, 4, or more motif(s) in the amino acid sequence of SEQ ID NO: 1 that are not glycosylation motifs into 1, 2, 3, 4, or more motif(s). In some embodiments, the conversion results in the introduction of a glycosylation motif into the non-naturally occurring protein. In other embodiments, the amino acid substitution results in the conversion of 1, 2, 3, 4, or more glycosylation motif(s) in the amino acid sequence of SEQ ID NO: 1 into 1, 2, 3, 4, or more motif(s) that are not glycosylation motifs. In some embodiments, the conversion results in the elimination of a glycosylation motif into the non-naturally occurring protein. In some embodiments, a single glycosylation site is introduced. In other instances, a double-glycosylation site is introduced. In some instances, the double-glycosylation site contains a native glycosylation site of wildtype IFN-beta (Asn 80) protein. In other embodiments, the native glycosylation site of wildtype IFN- beta protein is eliminated in the non-naturally occurring protein. In some embodiments, the non- naturally occurring protein is aglycosylated.

[0074] In some embodiments, the non-naturally occurring protein as disclosed herein having an amino acid substitution as recited in TABLE 1 can be present as a salt, such as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable salt canAttorney Docket No.217863-712601 include: acetate, acrylate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, bisulfite, bitartrate, bromide, butyrate, butyn-1,4-dioate, camphorate, camphorsulfonate, caproate, caprylate, chlorobenzoate, chloride, citrate, cyclopentanepropionate, decanoate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hexyne-1,6-dioate, hydroxybenzoate, γ-hydroxybutyrate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isobutyrate, lactate, maleate, malonate, methanesulfonate, mandelate. metaphosphate, methanesulfonate, methoxybenzoate, methylbenzoate, monohydrogenphosphate, 1-napthalenesulfonate, 2-napthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, pyrosulfate, pyrophosphate, propiolate, phthalate, phenylacetate, phenylbutyrate, propanesulfonate, salicylate, succinate, sulfate, sulfite, succinate, suberate, sebacate, sulfonate, tartrate, thiocyanate, tosylate, undeconate and xylenesulfonate.

[0075] In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1 can be encoded by a vector (e.g., a DNA vector). In some cases, the vector can be a viral vector such as a lentiviral vector or an adeno-associated viral vector.

[0076] In some embodiments, the non-naturally occurring protein variant has an amino acid substitution (e.g., one or more amino acid substitutions) recited in TABLE 2 below, relative to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1. A non-naturally occurring protein variant having an amino acid substitution where the amino acid substitution introduces a N-linked glycosylation site recited in TABLE 2 has similar biological activity (e.g., binding activity) to a wildtype IFN-beta protein. TABLE 2. Exemplary IFN-beta amino acid substitutions Amino Acid Substitution – Mutated CodonAttorney Docket No.217863-712601 F50N-AAC & K52T-ACC G26N-AAC & L28T-ACCAttorney Docket No.217863-712601 S13N-AAC & F15T-ACC S2N-AAC & N4T-ACCally occurring protein having an amino acid substitution as recited in TABLE 2 introduces a glycosylation motif. In some embodiments, the non-naturally occurring IFN-beta is modified to comprise 1, 2, 3, 4, or more glycosylation sites than wildtype IFN-beta protein. In some embodiments, introduction of glycosylation site is accomplished by insertion of a consensus sequence motif. In some embodiments, the consensus sequence motif is a post-translational modification (PTM) consensus sequence motif as recited in TABLE 5 below. In some embodiments, the glycosylation motif has a consensus sequence motif. In some embodiments, the consensus motif is (i) N-X-S / T, where X is independently selected from any amino acid except proline; (ii) D / E-X-N-Z-S / T, where X and Z are independently selected from any amino acid except proline; or (iii) N-X-C, where X is independently selected from any amino acid except proline.

[0078] In some embodiments, the engineered, non-naturally occurring protein having an amino acid substitution as recited in TABLE 2 introduces an amino acid substitution and can be further post-translationally modified. In some embodiments, the modifications can include: glycosylation, phosphorylation, lipidation, glycation, proline isomerization, methylation, acetylation, hydroxylation sites, and proteolysis. In some embodiments, the non-naturally occurring protein is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, conjugated to a polyethylene glycol (PEG) moiety, or any combination thereof. In some embodiments, the engineered IFN-beta protein or the functional fragment thereof is glycosylated. In some embodiments, the engineered IFN-beta protein or the functional fragment thereof is double glycosylated. In some embodiments, the engineered IFN-beta protein or the functional fragment thereof is multi-glycosylated. In some embodiments, the engineered IFN- beta protein or the functional fragment thereof is aglycosylated.

[0079] In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises at least one amino acid substitutions. In some embodiments, the at least one amino acid substitution is selected from TABLE 1 or TABLE 2. In some embodiments, the engineeredAttorney Docket No.217863-712601 IFN-beta protein comprises an amino acid sequence having at least about: 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 2 – SEQ ID NO: 136. TABLE 3. Exemplary engineered IFN-beta protein sequences SEQ ID Mutant Amino Acid Sequence NO: L I A L I A L I L I A L I A L I A L I A L I A L I AAttorney Docket No.217863-712601 11 S12N- MSYNLLGFLQRNSTFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL AAC|N14T- QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI ACC NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I A L I A L I A L I A L I A L I A L I AAttorney Docket No.217863-712601 22 Y3N-AAC|L5T- MSNNTLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL ACC QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I A L I A L I A L I A Q Q C L I A L I AAttorney Docket No.217863-712601 33 K45N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEINQT AAC|L47T- QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI ACC NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A N I A L I A L I A L I A L I A L I A L I A L I A L I AAttorney Docket No.217863-712601 44 L6N-AAC|F8T- MSYNLNGTLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL ACC QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I A L I A L I A L I L I L QI A L I AAttorney Docket No.217863-712601 55 G78N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL AAC|N80T- QQFQKEDAALTIYEMLQNIFAIFRQDSSSTNWTETIVENLLANVYHQI ACC NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L QI A L I A L I A L I L I A L QI A L I A L I A L I AAttorney Docket No.217863-712601 66 N80Q- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL CAG|E104N- QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWQETIVENLLANVYHQI AAC|L106T- NHLKTVLENKTEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHC L I L I L QI A L I A L I A L QI A L I A L I A L I A L IAttorney Docket No.217863-712601 77 N80Q- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL CAG|L106N- QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWQETIVENLLANVYHQI AAC|K108T- NHLKTVLEEKNETEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I L I A L I A L I A L I A L I A L I AAttorney Docket No.217863-712601 88 R71N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL AAC|D73T- QQFQKEDAALTIYEMLQNIFAIFNQTSSSTGWNETIVENLLANVYHQI ACC NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I A L I A L I A L I A L I L I A L I AAttorney Docket No.217863-712601 99 T77N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL AAC|W79T- QQFQKEDAALTIYEMLQNIFAIFRQDSSSNGTQETIVENLLANVYHQI ACC|N80Q- NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I A L I A L I L I A L I A L I A L I AAttorney Docket No.217863-712601 110 H131N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL AAC|L133T- QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI ACC NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILNYTKAKEYSHCA L I T L I A L I A L I A L I A L I L I A L I A L I A L I AAttorney Docket No.217863-712601 121 L160T-ACC MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I A L I A L I A L I L I A L I A L I AAttorney Docket No.217863-712601 132 V148N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL AAC|I150T- QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI ACC NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA L I A L I A L I A L I ASEQ ID Mutant Amino Acid Sequence NO: Q H TI Q H TI Q H TI Q H TI Q H TI Q H TI Q HAttorney Docket No.217863-712601 LKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTI VRVEILRNFYFINRLTGYLRN* Q H TI Q H TI Q H TI L N W Q H TI Q H TI Q H TI Q H TI Q H TI Q N W Q N W Q H TI Q H TIAttorney Docket No.217863-712601 157 F70N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQ AAC|Q72T- QFQKEDAALTIYEMLQNIFAINRTDSSSTGWNETIVENLLANVYHQINH ACC LKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTI Q H TI Q W Q W Q W Q W Q W Q W Q W Q H TI Q H TI Q H TI Q H TI Q H TIAttorney Docket No.217863-712601 171 M62N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQ AAC|Q64T- QFQKEDAALTIYENLTNIFAIFRQDSSSTGWNETIVENLLANVYHQINH ACC LKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTI Q H TI Q H TI Q W Q N Q H TI Q H TI Q H TI Q H TI Q H TI Q H TI Q W Q W Q H TIAttorney Docket No.217863-712601 185 V91N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQ AAC|H93T- QFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANNYTQINH ACC LKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTI Q H TI Q H TI Q H TI Q H TI Q H I Q H TI Q H TI Q H T Q H TI Q H TI Q H TI Q H TI Q H TIAttorney Docket No.217863-712601 199 S139N- MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQ AAC|C141T- QFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINH ACC LKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYNHTAWTI Q H TI Q H T Q H TI Q H TI Q H TI, g p g sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 136 can have an increase in biological activity as compared to wildtype IFN-beta having an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta proteins described having an amino acid sequence of any one of SEQ ID NO: 137 – SEQ ID NO: 204 can have a decrease in biological activity as compared to wildtype IFN-beta having an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta proteins can have more than biological activity of more than 100% of the biological activity of the wildtype IFN-beta having an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta proteins can have more than 90% of the biological activity of the wildtype IFN. In some embodiments, the engineered IFN- beta proteins can have more than 80% of the biological activity of the wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 70% of the biological activity of the wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 60% of the biological activity of the wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 50% of the biological activity of the wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 40% of the biological activity of the wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 30% of the biological activity of the wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 20% of the biological activity of theAttorney Docket No.217863-712601 wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 10% of the biological activity of the wildtype of which it is deduced (i.e., the wildtype IFN of which the coding sequence has been mutated to obtain the engineered IFN-beta).

[0081] In some embodiments, the engineered IFN-beta protein binds to an interferon-α / β receptor-1(IFNAR1) or interferon-α / β receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein. In some embodiments, the binding affinity is measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the engineered IFN-beta protein binds to an interferon-α / β receptor-1(IFNAR1) or interferon-α / β receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN- beta protein as measured by ELISA and / or a GigaAssay e.g., measuring signaling activity to an ISRE-GFP reporter in a live human cell line in vitro assay. In some embodiments, the engineered IFN-beta protein is an agonist of IFNAR1 or IFNAR2 subunits.

[0082] In some embodiments, the engineered IFN-beta protein binds and activates IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein is an agonist of IFNAR1 or IFNAR2 subunits In some embodiments, the engineered IFN- beta protein binds and inhibits IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein is an antagonist of IFNAR1 or IFNAR2 subunits. In some embodiments, the activity of IFNAR1 or IFNAR2 is measured by a reporter assay. In some embodiments, the reporter assay is an Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay or an ISRE luciferase reporter assay.

[0083] In some embodiments, the engineered IFN-beta protein reduces extracellular level of a pro-inflammatory cytokine as compared to wildtype IFN-beta protein. In some embodiments, the pro-inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof. In some embodiments, the pro-inflammatory cytokine is IL-1β, IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, TNF-β, or any combination thereof. In other embodiments, the engineered IFN-beta protein increases extracellular level of an anti-inflammatory cytokine as compared to wildtype IFN-beta protein. In some embodiments, the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof. In some embodiments, the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN-α, TGF-β, or any combination thereof.

[0084] In some embodiments, the engineered IFN-beta protein having amino acid sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 136 has reduced immunogenicity when administered toAttorney Docket No.217863-712601 a subject, as compared to administering the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the reduced immunogenicity comprises a reduced level of anti-drug antibody in the subject when the engineered IFN-beta protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject. In some embodiments, the engineered IFN-beta protein, when administered to a subject in need thereof, reduces a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering. In some embodiments, the reduced level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA. In some embodiments, the reduced level of inflammation in the subject results in reduced serum levels of C reactive protein as measured by high-sensitivity C-reactive protein (hs-CRP) test. In some embodiments, the reduced serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg / dL to about 2.9 mg / dL. In some embodiments, the administering of the engineered IFN-beta protein as disclosed herein to the subject results in reduced blood levels of a pro-inflammatory cytokine as compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA.

[0085] In some embodiments, the engineered IFN-beta protein has an amino acid substitution (e.g., one or more amino acid substitutions) recited in TABLE 1 or TABLE 2 below, relative to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1. An engineered IFN- beta protein variant having an amino acid substitution recited in TABLE 1 or TABLE 2 has similar biological activity (e.g., binding activity) to a wildtype IFN-beta protein.

[0086] In some embodiments, the engineered IFN-beta protein comprises an amino acid sequence having at least about: 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 137 – SEQ ID NO: 208.

[0087] In some embodiments, the non-naturally occurring protein having an amino acid substitution where the amino acid substitution results in the production of a post-translational modification (PTM) consensus sequence motif as recited in TABLE 5 below. In some embodiments, a “x” or “X” in the PTM consensus sequence motif as recited in TABLE 5 below can represent any amino acid.

[0088] In some embodiments, the non-naturally occurring protein having an amino acid substitution having an amino acid substitution as recited in TABLE 2 can be present as a salt, such as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable salt can include: acetate, acrylate, adipate, alginate, aspartate, benzoate,Attorney Docket No.217863-712601 benzenesulfonate, bisulfate, bisulfite, bitartrate, bromide, butyrate, butyn-1,4-dioate, camphorate, camphorsulfonate, caproate, caprylate, chlorobenzoate, chloride, citrate, cyclopentanepropionate, decanoate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hexyne-1,6-dioate, hydroxybenzoate, γ-hydroxybutyrate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isobutyrate, lactate, maleate, malonate, methanesulfonate, mandelate. metaphosphate, methanesulfonate, methoxybenzoate, methylbenzoate, monohydrogenphosphate, 1-napthalenesulfonate, 2-napthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, pyrosulfate, pyrophosphate, propiolate, phthalate, phenylacetate, phenylbutyrate, propanesulfonate, salicylate, succinate, sulfate, sulfite, succinate, suberate, sebacate, sulfonate, tartrate, thiocyanate, tosylate, undeconate and xylenesulfonate. TABLE 5. Exemplary IFN-beta Consensus Sequence Motif Amino Acid Motif AG KRAttorney Docket No.217863-712601 [IVL]Yxx[PF] [IVL]Yxxx[PF] ]Attorney Docket No.217863-712601 A[EA]EEY[FV]F[LFMIV]F AEV[IVLF]YAA[PF]FAttorney Docket No.217863-712601 ExxYxQ[GS] ExxYxQSAttorney Docket No.217863-712601 Rx[ST] Rx[ST]

[0089] In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 5 can be encoded by a vector (e.g., a DNA vector). In some cases, the vector can be a viral vector such as a lentiviral vector or an adeno-associated viral vector.

[0090] In some embodiments, the non-naturally occurring protein as disclosed herein is a recombinant IFN-beta protein. In some embodiments, the non-naturally occurring protein binds to an interferon-α / β receptor-1(IFNAR1) or interferon-α / β receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein. In some embodiments, the binding affinity is measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the non-naturally occurring protein binds to an interferon-α / β receptor-1(IFNAR1) or interferon-α / β receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to theAttorney Docket No.217863-712601 wildtype IFN-beta protein as measured by ELISA and / or a GigaAssay e.g., measuring signaling activity to an ISRE-GFP reporter in a live human cell line in vitro assay. In some embodiments, the non-naturally occurring protein is an agonist of IFNAR1 or IFNAR2 subunits. In other embodiments, the non-naturally occurring protein binds and activates IFNAR1 or IFNAR2 at a level substantially similar to the wildtype IFN-beta protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the non-naturally occurring protein binds and activates IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the non-naturally occurring protein is an agonist of IFNAR1 or IFNAR2 subunits In some embodiments, the non-naturally occurring protein binds and inhibits IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the wildtype IFN- beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the non- naturally occurring protein is an antagonist of IFNAR1 or IFNAR2 subunits. In some embodiments, the activity of IFNAR1 or IFNAR2 is measured by a reporter assay. In some embodiments, the reporter assay is an Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay or an ISRE luciferase reporter assay.

[0091] In some embodiments, the non-naturally occurring protein reduces extracellular level of a pro-inflammatory cytokine as compared to wildtype IFN-beta protein. In some embodiments, the pro-inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof. In some embodiments, the pro-inflammatory cytokine is IL-1β, IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, TNF-β, or any combination thereof. In other embodiments, the non-naturally occurring protein increases extracellular level of an anti-inflammatory cytokine as compared to wildtype IFN-beta protein. In some embodiments, the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof. In some embodiments, the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN-α, TGF-β, or any combination thereof.

[0092] In some embodiments, a glycosylation site is introduced in the non-naturally occurring protein that results in diminished binding of an antibody to the non-naturally occurring protein relative to an amount of binding of the antibody to the wildtype IFN-beta protein. In some embodiments, the non-naturally occurring beta protein has reduced immunogenicity when administered to a subject, as compared to administering the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the reduced immunogenicity comprises a reduced level of anti-drug antibody in the subject when the non-naturally occurring protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN-Attorney Docket No.217863-712601 beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject. In some embodiments, the non-naturally occurring beta protein, when administered to a subject in need thereof, reduces a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering. In some embodiments, the reduced level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA. In some embodiments, the reduced level of inflammation in the subject results in reduced serum levels of C reactive protein as measured by high-sensitivity C-reactive protein (hs-CRP) test. In some embodiments, the reduced serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg / dL to about 2.9 mg / dL. In some embodiments, the administering of the non-naturally occurring protein as disclosed herein to the subject results in reduced blood levels of a pro-inflammatory cytokine as compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA. Polynucleotides encoding engineered IFN-beta proteins

[0093] Provided herein are polynucleotides encoding the engineered IFN-beta proteins disclosed herein. In some embodiments, the polynucleotide disclosed herein encodes an engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. Also provided herein are nucleic acids encoding the engineered IFN-beta proteins disclosed herein. In some embodiments, the nucleic acid sequence comprise a sequence at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar to any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some cases, the nucleic acid is a nucleic acid construct. In some embodiments, a nucleic acid construct comprises a polynucleotide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. The polynucleotide of claim 55, wherein polynucleotide sequence at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 209 – SEQ ID NO: 342. In some embodiments, the polynucleotide sequence comprises a nucleotide sequence derived from a full length engineered protein, or a fragment thereof.

[0094] In some embodiments, each codon in the polynucleotide can independently be replaced by a degenerate codon. In some embodiments, the polynucleotide sequence comprise one or more degenerate codons. In some embodiments, the polynucleotide comprises a nucleic acid mimic. In some embodiments, the nucleic acid mimic comprises a phosphorothioate nucleic acid,Attorney Docket No.217863-712601 a phosphoramidate nucleic acid, a morpholino nucleic acid, a hexitol nucleic acid (HNA), a peptide nucleic acid (PNA), or a locked nucleic acid (LNA). In some embodiments, each codon independently codes for one or more amino acids. In some embodiments, the one or more amino acids comprise alanine, arginine, aspartic acid, glutamine, glutamic acid, glycine, praline, serine, leucine, cysteine, valine, lysine, methionine, tryptophan, phenylalanine, arginine, tyrosine, threonine, isoleucine, histidine, lysine and asparagine. In some embodiments, one or more codons in the polynucleotide is modified.

[0095] In some embodiments, one or more residues of an amino acid in the engineered protein is substituted for a non-natural amino acid. Non-limiting examples of non-natural amino acids include azidohomoalanine, homoproparglyglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine and ethynylephenylalanine. In some embodiments, the polynucleotide encodes a polypeptide comprising a non-natural amino acid. In some embodiments, a chemical moiety is added to the non-natural amino acid. In some embodiments, the chemical moiety comprises cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, poly(ethylene) glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides. In some cases, the non-natural amino acid residue is fluorinated, electroactive or unsaturated. In some embodiments, the polypeptide further comprises a sequence encoding a linker, or a tag.

[0096] In some embodiments, the engineered IFN-beta protein as disclosed herein is encoded by a vector (e.g., a DNA vector). In some cases, a vector comprise the nucleic acids or nucleic acid constructs disclosed herein. In some cases, the vector is a viral vector such as a lentiviral vector or an adeno-associated viral vector. In some embodiments, the engineered IFN-beta protein as disclosed herein is encoded by a nucleic acid sequence. TABLE 6. Polynucleotide sequences encoding exemplary engineered proteins SEQ Mutant Sequence ID NOAttorney Docket No.217863-712601 209 K105N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|E10 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC 7T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G G A G C A A A G A G C A A A G G A G C A A A G AAttorney Docket No.217863-712601 214 L6T-ACC ATGAGCTACAACTTGACCGGATTCCTACAAAGAAGCAGCAATTTTCAG TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A C C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 219 S13N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAACAATACCCAG AAC|F15 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A C C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 224 Q23N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|N25 TGTCAGAAGCTCCTGTGGAACCTTACCGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G A A A G A G C A A A G AAttorney Docket No.217863-712601 229 Y3N- ATGAGCAACAACACCCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|L5T- TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 234 R35N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|N37 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAACATGACCTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A A G A G C A A A A G A G C A A A A G A G C A A A G AAttorney Docket No.217863-712601 239 I44N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|Q46 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGAACAAGACCCTGCA A A A G A G C A A A G A G C A A A G A G C A A A A G A G C A A C G GAttorney Docket No.217863-712601 244 Q49N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|Q51 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A C G G G C A A A G A G C A A A G AAttorney Docket No.217863-712601 249 E53N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|A55 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A T C A C G G G C A A A G A G C A A A G AAttorney Docket No.217863-712601 254 L9N- ATGAGCTACAACTTGCTTGGATTCAACCAAACCAGCAGCAATTTTCAG AAC|R11 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 259 E109N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|F111 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 264 I83N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|E85 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 269 L57N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|I59T TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC -ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A C G G G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 274 N80Q- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG CAG|E10 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC 7N- AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 279 N80Q- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG CAG|I83N TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC - AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 284 N80Q- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG CAG|L10 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC 6N- AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A C A G A G C A A A G AAttorney Docket No.217863-712601 289 N80Q- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG CAG|T82 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC N- AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 294 Q94N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|N96 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A G A G C A A A G A G C A A A G A G C A A A A G A G C A A A A G AAttorney Docket No.217863-712601 299 S75N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A A G A G C A A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 304 T58N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|Y60 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A C G G G C A A A A G A G C A A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 309 V84N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|N86 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 314 G114N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|L11 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC 6T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G G A G C A A A G A G C A A A G A G C A A A G G C A A A G AAttorney Docket No.217863-712601 319 I145N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|R14 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC 7T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G A G C A A A G G AAttorney Docket No.217863-712601 324 L122N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|R12 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC 4T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 329 L164N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|N16 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC 6T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 334 R159N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G C G C A A A G G A G C A A A G A G C A A A G A G C A A A G AAttorney Docket No.217863-712601 339 V148N- ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAG AAC|I150 TGTCAGAAGCTCCTGTGGCAACTTAATGGGAGGCTTGAATACTGCCTC T-ACC AAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA A A A G A G C A A A G A G C A A A G A G C A A A G ACompositions

[0097] Provided herein are compositions comprising an engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, and a pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments, the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, anAttorney Docket No.217863-712601 engineered, non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or a vector encoding the engineered protein having an amino acid substitution recited in TABLE 1, can be present in a composition. In some cases, the composition can be a pharmaceutical composition with a pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments, an engineered, non-naturally occurring protein having an amino acid substitution recited in TABLE 2, or a vector encoding the engineered protein having an amino acid substitution recited in TABLE 2, can be present in a composition. In some cases, the composition can be a pharmaceutical composition with a pharmaceutically acceptable excipient, diluent, or carrier.

[0098] In some embodiments, a pharmaceutical composition can comprise an excipient, such as a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, a coloring agent. In some instances, a pharmaceutical composition can comprise a diluent such as water, glycerol, methanol, ethanol, and other similar biocompatible diluents. In some embodiments, the pharmaceutical composition can be in unit dose form.

[0099] In some embodiments, the pharmaceutical composition can be in the form of a tablet, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a suppository, and any combination thereof. Methods of treatment

[0100] Also provided herein are methods for treating a disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective dose of engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, an engineered, non-naturally occurring protein having an amino acid substitution recited in TABLE 1 or TABLE 2, a vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or a pharmaceutical composition comprising the non-naturally occurring protein or vector, can be administered to a subject to treat a disease or condition. For example, the non- naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non-naturally occurring protein or vector, can be used to treat an inflammatory condition. In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding theAttorney Docket No.217863-712601 non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non-naturally occurring protein or vector, can be used to treat a cancer (e.g., a melanoma, a bladder cancer, a head or neck cancer, a kidney cancer, a liver cancer, a non-small cell lung cancer, a viral infection (e.g., a hepatitis infection such as chronic Hepatitis B infection or a chronic Hepatitis C infection), a systemic lupus (e.g., erythematosus), or multiple sclerosis (e.g., relapse remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms).

[0101] In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector, can be administered to a subject by a route of administration selected from: inhalation, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebroventricular, intracisternal, intracorneal, intracoronal, intracoronary, intracorpous cavernaosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, retrobulbar, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, vaginal, infraorbital, intraparenchymal, intrathecal, intraventricular, stereotactic, or any combination thereof.

[0102] In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector, can be co-administered along with a second therapeutic. In some embodiments, the second therapeutic can be an antibody, a peptide, an antibody drug conjugate, or any combination thereof. In some cases, the second therapeutic can be an immunotherapy agent such as a checkpoint inhibitor. In some embodiments, the checkpointAttorney Docket No.217863-712601 inhibitor can be a PD-1 inhibitor such as nivolumab or pembrolizumab, a CTLA-4 inhibitor such as ipilimumab, or a PD-L1 inhibitor such as atezolizumab, avelumab, or durvalumab.

[0103] In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector, can be administered 1, 2, 3, or 4 times a day; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 times a week; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 times a month. In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non-naturally occurring protein or vector, can be administered every day, every other day, once a week, or once a month.

[0104] In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector, can independently be administered at a dose selected from the group consisting of about: 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, and 10 mg / kg; each with respect to a body weight of the subject.

[0105] In some embodiments, the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector, can independently be administered at a dose of about: 10 ng, 11 ng, 12 ng, 13 ng, 14 ng, 15 ng, 16 ng, 17 ng, 18 ng, 19 ng, 20 ng, 21 ng, 22 ng, 23 ng, 24 ng, 25 ng, 26 ng, 27 ng, 28 ng, 29 ng, 30 ng, 31 ng, 32 ng, 33 ng, 34 ng, 35 ng, 36 ng, 37 ng, 38 ng, 39 ng, 40 ng, 41 ng, 42 ng, 43 ng, 44 ng, 45 ng, 46 ng, 47 ng, 48 ng, 49 ng, 50 ng, 51 ng, 52 ng, 53 ng, 54 ng, 55 ng, 56 ng, 57 ng, 58 ng, 59 ng, 60 ng, 61 ng, 62 ng, 63 ng, 64 ng, 65 ng, 66 ng, 67 ng, 68 ng, 69 ng, 70 ng, 71 ng, 72 ng, 73 ng, 74 ng, 75 ng, 76 ng, 77 ng, 78 ng, 79 ng, 80 ng, 81 ng, 82 ng, 83 ng, 84 ng, 85 ng, 86 ng, 87 ng, 88 ng, 89 ng, 90 ng, 91 ng, 92 ng, 93 ng, 94 ng, 95 ng, 96 ng, 97 ng, 98 ng, 99 ng, 100 ng, 110 ng, 120 ng, 130 ng, 140 ng, 150 ng, 160 ng, 170 ng, 180 ng, 190 ng, 200 ng, 210 ng, 220 ng, 230 ng, 240 ng, 250 ng, 260 ng, 270 ng, 280 ng,Attorney Docket No.217863-712601 290 ng, 300 ng, 310 ng, 320 ng, 330 ng, 340 ng, 350 ng, 360 ng, 370 ng, 380 ng, 390 ng, 400 ng, 410 ng, 420 ng, 430 ng, 440 ng, 450 ng, 460 ng, 470 ng, 480 ng, 490 ng, 500 ng, 510 ng, 520 ng, 530 ng, 540 ng, 550 ng, 560 ng, 570 ng, 580 ng, 590 ng, 600 ng, 610 ng, 620 ng, 630 ng, 640 ng, 650 ng, 660 ng, 670 ng, 680 ng, 690 ng, 700 ng, 710 ng, 720 ng, 730 ng, 740 ng, 750 ng, 760 ng, 770 ng, 780 ng, 790 ng, 800 ng, 810 ng, 820 ng, 830 ng, 840 ng, 850 ng, 860 ng, 870 ng, 880 ng, 890 ng, 900 ng, 910 ng, 920 ng, 930 ng, 940 ng, 950 ng, 960 ng, 970 ng, 980 ng, 990 ng, 1 µg, 1.1 µg, 1.2 µg, 1.3 µg, 1.4 µg, 1.5 µg, 1.6 µg, 1.7 µg, 1.8 µg, 1.9 µg, 2 µg, 2.1 µg, 2.2 µg, 2.3 µg, 2.4 µg, 2.5 µg, 2.6 µg, 2.7 µg, 2.8 µg, 2.9 µg, 3 µg, 3.1 µg, 3.2 µg, 3.3 µg, 3.4 µg, 3.5 µg, 3.6 µg, 3.7 µg, 3.8 µg, 3.9 µg, 4 µg, 4.1 µg, 4.2 µg, 4.3 µg, 4.4 µg, 4.5 µg, 4.6 µg, 4.7 µg, 4.8 µg, 4.9 µg, 5 µg, 5.1 µg, 5.2 µg, 5.3 µg, 5.4 µg, 5.5 µg, 5.6 µg, 5.7 µg, 5.8 µg, 5.9 µg, 6 µg, 6.1 µg, 6.2 µg, 6.3 µg, 6.4 µg, 6.5 µg, 6.6 µg, 6.7 µg, 6.8 µg, 6.9 µg, 7 µg, 7.1 µg, 7.2 µg, 7.3 µg, 7.4 µg, 7.5 µg, 7.6 µg, 7.7 µg, 7.8 µg, 7.9 µg, 8 µg, 8.1 µg, 8.2 µg, 8.3 µg, 8.4 µg, 8.5 µg, 8.6 µg, 8.7 µg, 8.8 µg, 8.9 µg, 9 µg, 9.1 µg, 9.2 µg, 9.3 µg, 9.4 µg, 9.5 µg, 9.6 µg, 9.7 µg, 9.8 µg, 9.9 µg, 10 µg, 11 µg, 12 µg, 13 µg, 14 µg, 15 µg, 16 µg, 17 µg, 18 µg, 19 µg, 20 µg, 21 µg, 22 µg, 23 µg, 24 µg, 25 µg, 26 µg, 27 µg, 28 µg, 29 µg, 30 µg, 31 µg, 32 µg, 33 µg, 34 µg, 35 µg, 36 µg, 37 µg, 38 µg, 39 µg, 40 µg, 41 µg, 42 µg, 43 µg, 44 µg, 45 µg, 46 µg, 47 µg, 48 µg, 49 µg, 50 µg, 51 µg, 52 µg, 53 µg, 54 µg, 55 µg, 56 µg, 57 µg, 58 µg, 59 µg, 60 µg, 61 µg, 62 µg, 63 µg, 64 µg, 65 µg, 66 µg, 67 µg, 68 µg, 69 µg, 70 µg, 71 µg, 72 µg, 73 µg, 74 µg, 75 µg, 76 µg, 77 µg, 78 µg, 79 µg, 80 µg, 81 µg, 82 µg, 83 µg, 84 µg, 85 µg, 86 µg, 87 µg, 88 µg, 89 µg, 90 µg, 91 µg, 92 µg, 93 µg, 94 µg, 95 µg, 96 µg, 97 µg, 98 µg, 99 µg, 100 µg, 110 µg, 120 µg, 130 µg, 140 µg, 150 µg, 160 µg, 170 µg, 180 µg, 190 µg, 200 µg, 210 µg, 220 µg, 230 µg, 240 µg, 250 µg, 260 µg, 270 µg, 280 µg, 290 µg, 300 µg, 310 µg, 320 µg, 330 µg, 340 µg, 350 µg, 360 µg, 370 µg, 380 µg, 390 µg, 400 µg, 410 µg, 420 µg, 430 µg, 440 µg, 450 µg, 460 µg, 470 µg, 480 µg, 490 µg, 500 µg, 510 µg, 520 µg, 530 µg, 540 µg, 550 µg, 560 µg, 570 µg, 580 µg, 590 µg, 600 µg, 610 µg, 620 µg, 630 µg, 640 µg, 650 µg, 660 µg, 670 µg, 680 µg, 690 µg, 700 µg, 710 µg, 720 µg, 730 µg, 740 µg, 750 µg, 760 µg, 770 µg, 780 µg, 790 µg, 800 µg, 810 µg, 820 µg, 830 µg, 840 µg, 850 µg, 860 µg, 870 µg, 880 µg, 890 µg, 900 µg, 910 µg, 920 µg, 930 µg, 940 µg, 950 µg, 960 µg, 970 µg, 980 µg, 990 µg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg,Attorney Docket No.217863-712601 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, or 100 mg. EMBODIMENTS

[0106] Embodiment 1. A non-naturally occurring protein, comprising an amino acid sequence having an amino acid substitution with respect to a wildtype interferon beta (IFN-beta) protein with the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution is selected from the amino acid substitutions in TABLE 1.

[0107] Embodiment 2. The non-naturally occurring protein of embodiment 1, wherein: a. the amino acid substitution results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a glycosylation motif into a glycosylation motif, thereby introducing a glycosylation motif into the non-naturally occurring protein; b. the amino acid substitution results in a conversion of a glycosylation motif in the amino acid sequence of SEQ ID NO: 1 into a motif that is not a glycosylation motif, thereby eliminating a glycosylation motif from the non- naturally occurring protein; or c. a combination of (a) and (b).

[0108] Embodiment 3. The non-naturally occurring protein of embodiment 2, wherein the amino acid substitution results in the conversion of 1, 2, 3, 4, or more motif(s) in the amino acid sequence of SEQ ID NO: 1 that are not glycosylation motifs into 1, 2, 3, 4, or more motif(s) that are glycosylation motif(s), thereby introducing 1, 2, 3, 4, or more glycosylation motif(s) into the non-naturally occurring protein.

[0109] Embodiment 4. The non-naturally occurring protein of embodiment 2, wherein the amino acid substitution results in the conversion of 1, 2, 3, 4, or more glycosylation motif(s) in the amino acid sequence of SEQ ID NO: 1 into 1, 2, 3, 4, or more motif(s) that are not glycosylation motifs, thereby eliminating 1, 2, 3, 4, or more glycosylation motif(s) into the non- naturally occurring protein.

[0110] Embodiment 5. The non-naturally occurring protein of any one of embodiments 1-4, wherein the glycosylation motif has a consensus sequence of N-X-S / T, wherein N is an Asparagine that is N-glycosylated at the amide bond in the side chain of the Asparagine, X is any amino acid except for proline, S is a serine, and T is a threonine.Attorney Docket No.217863-712601

[0111] Embodiment 6. The non-naturally occurring protein of embodiment 5, wherein the amino acid substitution in the non-naturally occurring protein is selected from the amino acid substitutions in TABLE 2.

[0112] Embodiment 7. The non-naturally occurring protein of embodiment 5, wherein an asparagine, a serine, a threonine, or any combination thereof, is substituted.

[0113] Embodiment 8. The non-naturally occurring protein of embodiment 1, wherein the amino acid substitution results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a site for a post-translational modification into a post-translational modification consensus sequence motif, thereby introducing a site for a post translational modification into the non-naturally occurring protein.

[0114] Embodiment 9. The non-naturally occurring protein of embodiment 8, wherein the post-translational modification consensus sequence motif is selected from the amino acid sequences in TABLE 5.

[0115] Embodiment 10. The non-naturally occurring protein of embodiment 9, wherein the amino acid substitution results in the addition of a post-translational modification on the non- naturally occurring protein at the site of the post-translational modification consensus sequence motif that is not present in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.

[0116] Embodiment 11. The non-naturally occurring protein of embodiment 10, wherein the post-translational modification is selected from the group consisting of: glycosylation, phosphorylation, lipidation, glycation, proline isomerization, methylation, acetylation, hydroxylation sites, and proteolysis.

[0117] Embodiment 12. The non-naturally occurring protein of any one of embodiments 1-11, wherein the non-naturally occurring protein is glycosylated.

[0118] Embodiment 13. The non-naturally occurring protein of any one of embodiments 1-11, wherein the non-naturally occurring protein is aglycosylated.

[0119] Embodiment 14. The non-naturally occurring protein of any one of embodiments 1-13, wherein the non-naturally occurring protein binds to an interferon-α / β receptor-1(IFNAR1) or interferon-α / β receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein as measured by enzyme-linked immunosorbent assay (ELISA) or by measuring signaling activity of an Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter in a live human cell in vitro assay.

[0120] Embodiment 15. The non-naturally occurring protein of any one of embodiments 1-14, wherein the non-naturally occurring protein is an agonist of IFNAR1 or IFNAR2 subunits.Attorney Docket No.217863-712601

[0121] Embodiment 16. The non-naturally occurring protein of embodiment 15, wherein the non-naturally occurring protein binds and activates IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE-GFP reporter assay or an ISRE luciferase reporter assay.

[0122] Embodiment 17. The non-naturally occurring protein of any one of embodiments 1-13, wherein the non-naturally occurring protein reduces an extracellular level of a pro-inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

[0123] Embodiment 18. The non-naturally occurring protein of embodiment 17, wherein the pro-inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof.

[0124] Embodiment 19. The non-naturally occurring protein of embodiment 18, wherein the pro-inflammatory cytokine is IL-1β, IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, TNF-β, or any combination thereof.

[0125] Embodiment 20. The non-naturally occurring protein of any one of embodiments 1-13, wherein the non-naturally occurring protein increases an extracellular level of an anti- inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

[0126] Embodiment 21. The non-naturally occurring protein of embodiment 20, wherein the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof.

[0127] Embodiment 22. The non-naturally occurring protein of embodiment 21, wherein the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN-α, TGF-β, or any combination thereof.

[0128] Embodiment 23. The non-naturally occurring protein of any one of embodiments 1-13, wherein the non-naturally occurring protein binds to IFNAR1 or IFNAR2 subunits at a level less than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

[0129] Embodiment 24. The non-naturally occurring protein of any one of embodiments 1-14, wherein the non-naturally occurring protein is an antagonist of IFNAR1 or IFNAR2 subunits.

[0130] Embodiment 25. The non-naturally occurring protein of embodiment 24, wherein the non-naturally occurring protein binds and inhibits the activity of the IFNAR1 or IFNAR2 at a level greater than wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE GFP reporter assay or an ISRE luciferase reporter assay.Attorney Docket No.217863-712601

[0131] Embodiment 26. The non-naturally occurring protein of any one of embodiments 1-25, wherein the non-naturally occurring protein is a recombinant IFN-beta or a portion thereof.

[0132] Embodiment 27. The non-naturally occurring protein of any one of embodiments 1-26, wherein the non-naturally occurring protein is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, conjugated to a polyethylene glycol (PEG) moiety, or any combination thereof.

[0133] Embodiment 28. The non-naturally occurring protein of embodiment 1-27, wherein a glycosylation site is introduced in the non-naturally occurring protein that results in diminished binding of an antibody to the non-naturally occurring protein relative to an amount of binding of the antibody to the wildtype IFN-beta protein.

[0134] Embodiment 29. The non-naturally occurring protein of any one of embodiments 1-28, wherein the non-naturally occurring beta protein has reduced immunogenicity when administered to a subject, as compared to administering the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1.

[0135] Embodiment 30. The non-naturally occurring protein of embodiment 28, wherein the reduced immunogenicity comprises a reduced level of anti-drug antibody in the subject when the non-naturally occurring protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject.

[0136] Embodiment 31. The non-naturally occurring protein of any one of embodiments 1-30, wherein the non-naturally occurring beta protein, when administered to a subject in need thereof, reduces a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering.

[0137] Embodiment 32. The non-naturally occurring protein of embodiment 31, wherein the reduced level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA.

[0138] Embodiment 33. The non-naturally occurring protein of embodiment 31, wherein the reduced level of inflammation in the subject results in reduced serum levels of C reactive protein as measured by high-sensitivity C-reactive protein (hs-CRP) test.

[0139] Embodiment 34. The non-naturally occurring protein of embodiment 33, wherein the reduced serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg / dL to about 2.9 mg / dL.

[0140] Embodiment 35. The non-naturally occurring protein of embodiment 34, wherein the administering to the subject results in reduced blood levels of a pro-inflammatory cytokine asAttorney Docket No.217863-712601 compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA.

[0141] Embodiment 36. A method of making a non-naturally occurring protein in a cell, comprising introducing into the cell a vector encoding the non-naturally occurring protein of any one of embodiments 1-35, wherein the introducing results in expression of the non-naturally occurring protein in the cell.

[0142] Embodiment 37. The method of embodiment 36, wherein the non-naturally occurring protein is isolated from the cell.

[0143] Embodiment 38. The method of embodiment 36, wherein the vector comprises a mammalian expression plasmid.

[0144] Embodiment 39. The method of embodiment 36, wherein the non-naturally occurring protein comprises a tag.

[0145] Embodiment 40. The method of embodiment 39, wherein the tag is CBP, FLAG, GST, Myc, poly-His, or V5.

[0146] Embodiment 41. The method of embodiment 39, wherein the tag comprises a cleavable tag or a non-cleavable tag.

[0147] Embodiment 42. A polynucleotide encoding the non-naturally occurring protein of any one of embodiments 1-35.

[0148] Embodiment 43. A pharmaceutical composition in unit dose form comprising: a. the non-naturally occurring protein of any one of embodiments 1-35, a vector encoding the non- naturally occurring protein of any one of embodiments 1-35, or the polynucleotide of embodiment 42; and b. a pharmaceutically acceptable excipient, carrier, or diluent.

[0149] Embodiment 44. The pharmaceutical composition of embodiment 43, wherein the pharmaceutical composition is encapsulated.

[0150] Embodiment 45. The pharmaceutical composition of embodiment 43, wherein the pharmaceutical composition is in the form of a liquid.

[0151] Embodiment 46. The pharmaceutical composition of any one of embodiments 43-45, wherein the pharmaceutical composition is formulated for local, systemic, or topical administration.

[0152] Embodiment 47. The pharmaceutical composition of any one of embodiments 43-46, wherein the pharmaceutical composition is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parental, intravenous, or intramuscular administration.

[0153] Embodiment 48. The pharmaceutical composition of any one of embodiments 43-47, wherein the pharmaceutical composition is formulated for controlled-release.Attorney Docket No.217863-712601

[0154] Embodiment 49. The pharmaceutical composition of any one of embodiments 43-48, wherein the pharmaceutical composition is formulated for single-dosage administration.

[0155] Embodiment 50. A method of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of the non-naturally occurring protein of any one of embodiments 1-35, the polynucleotide of embodiment 42, or the pharmaceutical composition of any one of embodiments 43-49, thereby treating the subject.

[0156] Embodiment 51. The method of embodiment 50, wherein the disease is an infection.

[0157] Embodiment 52. The method of embodiment 51, wherein the infection is a viral infection.

[0158] Embodiment 53. The method of embodiment 52, wherein the viral infection is chronic Hepatitis B infection, or a chronic Hepatitis C infection.

[0159] Embodiment 54. The method of embodiment 50, wherein the disease is a demyelinating disorder.

[0160] Embodiment 55. The method of embodiment 54, wherein the demyelinating disorder is selected from the group consisting of Multiple Sclerosis (MS), Acute Disseminated Encephalomyelitis (ADEM), Acute Hemorrhagic Leucoencephalitis (AHLE), Balo’s disease (Concentric Sclerosis), Charcot-Marie-Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease).

[0161] Embodiment 56. The method of embodiment 55, wherein the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.

[0162] Embodiment 57. The method of any one of embodiments 50-56, wherein the subject is a human subject.

[0163] Embodiment 58. The method of any one of embodiments 50-57, wherein the administering is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra- cisternal injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, a surgical route, or any combination thereof.

[0164] Embodiment 59. The method of embodiment 50, wherein the administering occurs from about once a day, about twice a day, about once a week, about twice a week, about once every two weeks, about once a month, about once every 3 months, about once every 6 months, about once every 9 months, to about once a year.

[0165] Embodiment 60. The method of embodiment 50, wherein the administering is at a dose selected from the group consisting of about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, or about 2000 mg.Attorney Docket No.217863-712601

[0166] Embodiment 61. The method of embodiment 50, wherein the administering is at a dose selected from the group consisting of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, and 10 mg / kg.

[0167] Embodiment 62. The method of embodiment 50, wherein the administering reduces a volume of a tumor in the subject.

[0168] Embodiment 63. The method of embodiment 50, wherein the administering inhibits or diminishes the growth a tumor in the subject.

[0169] Embodiment 64. The method of embodiment 50, wherein the method further comprises administering a second therapeutic.

[0170] Embodiment 65. The method of embodiment 64, wherein the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.

[0171] Embodiment 66. The method of embodiment 50, wherein the method further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor.

[0172] Embodiment 67. The method of embodiment 66, wherein the checkpoint inhibitor is a PD-1, a PD-L1, CTLA-4 inhibitor.

[0173] Embodiment 68. The method of embodiment 50, wherein the administering induces activation of an IFN-stimulated response element (ISRE) or a Gamma interferon activation site (GAS) element, wherein the activation of the ISRE or GAS induces an interferon-like activity as measured by a luciferase reporter assay.

[0174] Embodiment 69. A kit comprising the non-naturally occurring protein of any one of embodiments 1-35, the polynucleotide of embodiment 42, or the pharmaceutical composition of any one of embodiments 43-49, and a container. EXAMPLES Example 1 – Generation of a plasmid-based Gene Mutation Library

[0175] A library of non-naturally occurring Interferon beta variants was generated. The library was a plasmid library designed for the generation of a lentiviral library and contains the cDNA for human Interferon beta having the sequence of SEQ ID NO: 1 with variants as described in TABLE 1, and TABLE 2. (FIG.1). A clonal reporter cell line was established that contains a stably integrated ZsGreen1-DR fluorescent protein downstream of an interferon-responsive promoter containing 3xISREs (Interferon Stimulated Response Elements). (FIG.2) The reporter cell line was then transduced so that it contains a single integrated copy of a non-naturallyAttorney Docket No.217863-712601 occurring protein as described herein downstream of a Dox-inducible promoter. The non- naturally occurring protein as described herein consists of an N-terminal fusion of a Type II Transmembrane Domain (Type II TMD) and GGGGS linker. Upon addition of doxycycline, the Tet Transactivator binds doxycycline and changes conformation to allow for binding to the Tet response elements in the Dox-inducible promoter leading to the induced expression of the non- naturally occurring protein as described herein. The non-naturally occurring protein as described herein is then translocated to the plasma membrane where it is tethered to the membrane with the Type II TMD. On the cell surface, non-naturally occurring protein as described herein interacts with the Type I IFN receptor (consisting of IFNAR1 / 2 receptor subunits) to induce the interferon-stimulated signaling pathway. Phosphorylated STAT1 / 2 translocates to the nucleus, binds to the ISRE promoter upstream of GFP leading to induction of GFP expression. Cells can be sorted based on fluorescence as a marker for biological activity related to the interferon signaling pathway. (FIG.2). The non-naturally occurring protein as described herein was saturation mutagenized in order to produce amino acid substitutions at each position thereby introducing or eliminating adding mutations in a consensus sequence as disclosed herein for N- linked glycosylation at the asparagine (N), serine (S), or threonine (T) residues of the consensus sequence motif of Nx[S / T]. IFN-beta variants in the assay are designed so that N-linked glycosylation sites are added at each amino acid position within the protein coding sequence. Each clone generated contained a Unique Molecular Identifier (UMI) region which was used to quantify and associate the UMI in each sorted population with the corresponding gene mutation. This information is used to create MEGA-Maps associating gene mutations with biological activity. Example 2 – High-throughput screening of non-naturally occurring protein

[0176] The resulting IFN-beta variant lentiviral library produced in Example 1 was transduced into a cell population of about 30 million cells, stimulated with Doxycycline, and plated at low density. Plating densities were titrated to minimize paracrine (intercellular) signaling. Cells stably expressing IFN-β plus a red fluorescent protein (mScarlet) were co-cultured with cells expressing endogenous IFNAR1 / 2 and an ectopic ISRE reporter driving expression of a green fluorescent protein (ZsGreen1-DR). In this assay, only cells that undergo paracrine signaling become positive for ZsGreen1. This density was used for autocrine assays. Cells were harvested, fixed, and partitioned into bins by Flow Sorting based on zsGreen fluorescence (as a marker of biological activity) into four bins: Bin 1: Low fluorescence = Low biological activity Bin 2: Mid-Low fluorescence = Mid-Low biological activityAttorney Docket No.217863-712601 Bin 3: Mid-High fluorescence = Mid-High biological activity Bin 4: High fluorescence = High biological activity

[0177] This population of barcodes was compared to the set of barcodes for wildtype IFN-beta (control) and used to identify variants that have binding activity similar to that of WT, which were separated from variants that have significantly lower activity than wildtype IFN-beta (FIG. 3, TABLE 7). A portion of the variants are aglycosylated, single-glycosylated, or double- glycosylated, where the double-glycosylated variants include a de novo glycosylation site in combination with the native glycosylation site left intact. A wide dynamic range of fluorescence in HEK-293 ISRE reporter cells was observed when comparing control (left) to cells stimulated by the non-naturally occurring proteins described herein (right) by flow cytometry (FIG.4). TABLE 7. Exemplary Engineered, Non-Naturally Occurring protein classification Non-Naturally Occurring protein class Number Reduced activity compared to wildtype IFN-beta protein 241

[0178] T the wildtype IFN-beta pg q p d approximately 887 mutants, including glycosylation mutants that had signaling activity equivalent to the wildtype IFN-beta protein (TABLE 7). Exemplary non-naturally occurring proteins comprised a glycosylation site in an immune epitope without any overlap to amino acid residues associated with binding to the IFNAR1 / 2 receptor subunits. From this screen, novel non-naturally interferon beta variants containing de novo glycosylation site(s) or aglycosylation site(s) as described herein were discovered to have substantially similar, decreased signaling activity, or increased signaling activity to wildtype IFN-beta. Example 3 – Comparison between variants functional elements and structure

[0179] Based on the foregoing examples, several non-naturally occurring proteins were engineered to have receptor stimulation efficacy similar to that of wildtype IFN-beta. These novel non-naturally occurring proteins were designed to have increased serum half-life, increased solubility and reduced immunogenicity. The major immuno epitopes that patients develop during treatment with wildtype IFN-beta were amino acids 1-12, 151-162, and 121-132, with the first two being favored for neutralizing antibodies and amino acids 1-12 being the preferred epitope. When plotted on the 3D structure of IFNAR2 (PDB: 3SE3), epitopes 1-12 were directly juxtaposed to epitopes 151-162 and 121-132, with 121-132 on the opposing face of the protein.Attorney Docket No.217863-712601 However, none of the epitopes were near the native N-linked glycosylation site of wildtype IFN- BETA at amino acid N80.

[0180] A model of the structure of a human IFN-beta paralog, IFN alpha 2 complexed with IFNAR receptor (PDB: 3SE3) was used to assess the contacts of the non-naturally occurring protein as described herein with the IFNAR receptor. The model was consistent with the contacts observed in the structure of the mouse IFNAR1 / IFN-beta complex (PDB: 3WCY). IFNAR2 binds to IFN-beta making contacts with amino acids 19, 22, 26, 26, 30, 33, 151, 155, 156, and 159. Then, IFNAR2 was recruited and contacts IFN-beta at amino acids 65, 67, 68, 88, 92, 119, 123, 126, and 127. IFNAR1 makes no contacts with IFNAR2, rather both directly bind IFN-beta.

[0181] The non-naturally occurring proteins containing the amino acid substitutions in TABLE 2 were mapped on the X-ray structure and a new IFNAR1 binding contact was discovered to be essential for signaling. The crystal structure of IFN-beta shows an asymmetric dimer with a patch of residues forming intermolecular contacts. IFN-beta was a hydrophobic protein and was prone to aggregation and may form dimers, although the relevance of these dimers to signaling was not well understood. The mutations in the non-naturally occurring proteins having increased biological activity were found to be juxtaposed to the dimerization site. Moreover, two clusters of non-naturally occurring variants were immediately juxtaposed to the dimerization interface, thus these mutations can affect the dimerization or oligomerization. Notably these patches were not in immuno epitopes or IFNAR 1 / 2 receptor binding sites. Additionally, protein aggregation can generate an immune response and induction of antidrug antibodies, however, introduction of de novo glycosylation sites as described herein over the surface of the non-naturally occurring protein can block hydrophobic surfaces resulting in reduced protein aggregation, increased solubility and reduced immunogenicity when administered to a human subject. Example 4 – Purification of HIS-tagged non-naturally occurring proteins

[0182] TurboCHO cells is transfected with a mammalian expression plasmid to express HIS- tagged non-naturally occurring proteins (FIG.5). A secretory signal is fused on the N-terminus of the 6xHIS tag to facilitate secretion of the protein into supernatant during cell culture. The secretory signal gets cleaved from the HIS-tagged IFN protein during protein trafficking. The supernatant from transfected cells is loaded onto a Nickle-NTA (Ni-NTA) affinity column, which binds the HIS residue of the HIS-tagged non-naturally occurring protein. Wash buffers is loaded into the column to wash away unbound material, and elution buffer is used to release the purified HIS-tagged IFN protein from the column. The purified non-naturally occurring protein as described is checked for quality control using SDS-PAGE gels (to confirm purity and size;Attorney Docket No.217863-712601 FIGs.15A-15N) and Bradford assay (to measure concentration). The yield and purity of each protein is listed in TABLE 8. TABLE 8. Purification results. Protein SEQ Class Purification Yield Purity ID (mg) from 30 mL (%) N

[0183] HEK-293 cells stably expressing ZsGreen1-DR fluorescent protein downstream of an interferon-responsive promoter containing 3xISREs (Interferon-Stimulated Response Elements) were plated in a 96-well plate and allowed to attach overnight (FIG.6A). Once attached, cells were treated with serially-diluted purified engineered, non-naturally occurring proteins as described herein (EXAMPLE 4) (in triplicate; n=3) at varying concentrations covering the range of the dose curve. Fluorescence of the treated cells was measured on a plate reader 24 hours post- treatment and EC50 values for ISRE reporter activity was determined for each IFN lead. TABLE 9 provides the amino acid sequence for the two control sequences used in this example. TABLE 9. Control IFN-beta sequences SEQ ID MUTANT Amino Acid Sequence E N RIAttorney Docket No.217863-712601 344 R11N, M117N MSYNLLGFLQNSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPE EIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN IEC50 of 0.1161 ng / mL for wildtype IFN-beta protein (FIG.6B; FIGs.16A-16L). EC50 for cell- based reporter assay was measured in ng / m, and p-values were False Discovery Rate (FDR) adjusted (FIGs.16A-16L). The HIS-tagged IFN-beta protein with biosimilar activity was also verified by flow cytometry in cells stimulated with purified engineered IFN-beta proteins or WT proteins (FIGs.17A-17G). IFNβ-stimulated gene (ISG) expression is a hallmark of IFN activity. To assess this transcriptional response, HEK-293 cells were treated for 24 hours with the optimal dose of the engineered IFN-beta or controls to saturate ISRE signaling. Total RNA was extracted and subjected to qPCR targeting the β2-micoglobulin and 2’-5’ oligoadenylate synthetase 1 genes (OAS1). OAS1 expression was normalized to house-keeping gene, HPRT (FIGs.18A- 18F). Error bars represent mean + / - SD for n=3 biological replicate, ns = p = >0.05, * p = <0.05. Untreated cells were included as controls. These results demonstrate that some of the engineered proteins disclosed herein exhibited increased biological activity (“Biobetter”), than wildtype IFN-beta (SEQ ID NO: 1), while other engineered proteins exhibited similar biological activity (“Biosimilar”) to wildtype IFN-beta (SEQ ID NO: 1). Notably, exemplary engineered proteins disclosed herein demonstrated reduced function or activity. Representative engineered proteins for each category was tabulated in TABLE 10 below. TABLE 10. Properties of HIS-tagged engineered, non-naturally occurring proteins. Flow- r rAttorney Docket No.217863-712601 Y92N_Q94T 137ReducedReduced Reduced Reduced FunctionFunction Function Function Rd dr Mononuclear Cells (PBMCs)

[0185] Cryopreserved PBMCs are harvested from healthy donors that have never been exposed to interferon therapy (FIG.7). The non-naturally occurring proteins as described herein are taken up by antigen presenting cells (APCs) and presented to T cells in culture. If the non-naturally occurring protein is found to be immunogenic, which can lead to T cell activation and concurrent cytokine secretion. Immunogenicity assessment can then be performed by phenotyping the T cells using flow cytometry following stimulation with the non-naturally occurring proteins. Example 7 – Purification of untagged non-naturally occurring proteins

[0186] CHO cells are transfected with a mammalian expression plasmid to express HIS-tagged the non-naturally occurring proteins (FIG 8A-8B). A secretory signal is fused on the N-terminus of the 6xHIS tag to facilitate secretion of the protein into supernatant during cell culture. The secretory signal gets cleaved from the HIS-tagged non-naturally occurring protein during protein trafficking. Supernatant from transfected cells is loaded onto a Nickle-NTA (Ni-NTA) affinity column which binds the HIS residue of the HIS-tagged non-naturally occurring protein. Wash buffers are loaded into the column to wash away unbound material, and elution buffer is used to release the purified HIS-tagged non-naturally occurring protein from the column. Treatment with Enterokinase Light Chain proteinase is used to cleave the Enterokinase Light Chain Cleavage Site (EKCS) peptide sequence, separating the HIS-EKCS tag from the mature non-naturally occurring protein. The reaction is loaded into a purification column to remove the cleaved tag, resulting in purified untagged non-naturally occurring protein with no extra residues from the tags. Purified non-naturally occurring protein as described herein is checked for quality control using SDS-PAGE gels (to confirm purity and size), analytical size-exclusion (to assess the tag removal), Bradford assay (to measure concentration), and mass spectrometry (to confirm N-term.Attorney Docket No.217863-712601 and C-term peptides) (FIG.8A). Samples are lyophilized and resuspended in a pharmaceutically acceptable solvent. Amino acid sequence of 6xHIS and the EKCS protease site upstream of mature non-naturally occurring protein, with the cleavage position of enterokinase indicated by a dashed vertical line (FIG.8B). Example 8 – Dose response of non-naturally occurring protein to compare EC50 values with an antiviral Cytopathic Effect (CPE) assay

[0187] A549 cells are plated in a 96-well plate and allowed to attach overnight. Once attached, cells are treated with serially-diluted purified non-naturally occurring proteins in parallel to a control wildtype IFN-beta, in triplicate (n=3) at varying concentrations covering the range of the dose curve. After 24 hours, media is replaced with media containing cytopathic encephalomyocarditis (EMC) virus at a concentration that causes 100% cell death in untreated cell cultures after 24 hours. ViralTox Glo (Promega) reagent is added 24 hours post-infection to measure ATP levels as an indicator of cytopathic effect, luminescence is quantified on a plate reader, and EC50 values for antiviral activity are determined for the non-naturally occurring proteins described herein (FIG.9). Example 9 – Interferon-induced biomarker expression in human PBMCs

[0188] PBMCs (Peripheral Blood Mononuclear Cells), either fresh or cryopreserved, are treated with exemplary non-naturally occurring proteins as described herein in parallel to a control wildtype IFN-beta, in triplicate (n=3) (FIG.10). After treatment, RNA is extracted and used for either RNAseq to sequence the transcriptome, or RT-PCR (for key biomarkers: B2M, 2',5'- OAS1, MxA) to assess the level of induction of interferon-stimulated genes between IFN leads compared to control wildtype IFN-beta. Example 10 – Prophylactic assessment of non-naturally occurring protein in Experimental Autoimmune Encephalomyelitis (EAE) mouse model for Multiple Sclerosis (MS)

[0189] Mice are immunized with MOG35-55-peptide at Day 0 to induce EAE. Also at Day 0, mice receive 30 mg / kg of purified, untagged IFN-β1 lead candidate proteins in parallel to a control wildtype IFN-beta (10 mice per test subject; n=10) from Day 0-7 post-EAE induction (FIG.11). Development of EAE symptoms are monitored through Day 36, covering the peak and chronic phases of EAE development. Splenocytes and spinal cord are harvested at Day 36, histopathology and FACS analysis of T cells and subsets of T cells is performed. Example 11 – Liver toxicity assessment of non-naturally occurring protein in miceAttorney Docket No.217863-712601

[0190] Mice are treated with purified, untagged IFN-β1 lead candidate proteins in parallel to a control wildtype IFN-beta (3 mice per test subject; n=3) and monitored for up to 2 days post- treatment (FIG.12). Blood samples are collected for liver function tests, and liver slides are prepared for pathology. Example 12 – Pharmacokinetic study of non-naturally occurring proteins

[0191] Healthy subjects are injected with a single subcutaneous dose of 60 mcg of non- naturally occurring protein (FIG.13). Peak serum concentration (Cmax) with median time of peak serum concentration (Tmax), serum elimination half-life, and area under the serum concentration versus time curve from 0 to 96 hours are assessed. Example 13 – Immunogenicity study of non-naturally occurring proteins

[0192] The presence of neutralizing antibodies (NAb) to the non-naturally occurring protein is determined by collecting and analyzing serum pre-study and at 6-month time intervals during 2 years of the clinical trial (FIG.14). Data reflects percentage of patients whose test results were considered positive for antibodies to the non-naturally occurring protein using antiviral cytopathic effect assay. Observed incidence of NAb positive in assay can be influence by several factors including sample handling, timing of sample collection, concomitant medications, and underlying disease.

[0193] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No.217863-712601 CLAIMS WHAT IS CLAIMED IS:

1. An engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO:

208.

2. The engineered protein of claim 1, wherein the modification comprises an amino acid substitution, a deletion, or an insertion in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO:

1.

3. The engineered protein of claim 2, wherein the modification is an amino acid substitution, and the amino acid substitution comprises at least one amino acid substitution in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO:

1.

4. The engineered protein of claim 2, wherein the modification is a deletion of one or more amino acids in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO:

1.

5. The engineered protein of claim 2, wherein the modification comprises an insertion of one or more amino acids in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO:

1.

6. The engineered protein of claim 3, wherein the at least one amino acid substitution results in an increase in a biological activity of the engineered IFN-beta protein as compared to a biological activity of the wildtype protein.

7. The engineered protein of claim 3, wherein the at least one amino acid substitution results in a decrease in a biological activity of the engineered IFN-beta protein as compared to a biological activity of the wildtype protein.

8. The engineered protein of claim 7, wherein the increase in the biological activity of biological activity comprises an increase in signaling, an increase in protein expression of one or more biological targets, an increase in mRNA expression, or any combination thereof, as measured by an in vitro assay.

9. The engineered protein of claim 8, wherein the increase in signaling comprises an increase in IFNAR1-mediated signaling, as measured by an in vitro assay.

10. The engineered protein of claim 8, wherein the increase in signaling comprises an increase in IFNAR2-mediated signaling, as measured by an in vitro assay.

11. The engineered protein of claim 1, wherein the modification comprises:Attorney Docket No.217863-712601 a. an amino acid substitution that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a glycosylation motif into a glycosylation motif, thereby introducing a glycosylation motif into the engineered protein; b. an amino acid substitution that results in a conversion of a glycosylation motif in the amino acid sequence of SEQ ID NO: 1 into a motif that is not a glycosylation motif, thereby eliminating a glycosylation motif from the engineered protein; or c. a combination of (a) and (b).

12. The engineered protein of claim 3, wherein the at least one amino acid substitution results in the conversion of 1, 2, 3, 4, or more motif(s) in the amino acid sequence of SEQ ID NO: 1 that are not glycosylation motifs into 1, 2, 3, 4, or more motif(s) that are glycosylation motif(s), thereby introducing 1, 2, 3, 4, or more glycosylation motif(s) into the engineered protein.

13. The engineered protein of claim 3, wherein the at least one amino acid substitution results in the conversion of 1, 2, 3, 4, or more glycosylation motif(s) in the amino acid sequence of SEQ ID NO: 1 into 1, 2, 3, 4, or more motif(s) that are not glycosylation motifs, thereby eliminating 1, 2, 3, 4, or more glycosylation motif(s) into the engineered protein.

14. The engineered protein of claim 11, wherein the glycosylation motif has a consensus sequence of N-X-S / T, wherein N is an Asparagine that is N-glycosylated at the amide bond in the side chain of the Asparagine, X is any amino acid except for proline, S is a serine, and T is a threonine.

15. The engineered protein of claim 1, wherein the modification comprises an amino acid substitution that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a site for a post-translational modification into a post-translational modification consensus sequence motif, thereby introducing a site for a post translational modification into the engineered protein.

16. The engineered protein of claim 15, wherein the post-translational modification consensus sequence motif is selected from the amino acid sequences in TABLE 5.

17. The engineered protein of claim 15, wherein the amino acid substitution results in the addition of a post-translational modification on the engineered protein at the site of the post-translational modification consensus sequence motif that is not present in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO:

1.

18. The engineered protein of claim 17, wherein the post-translational modification is selected from the group consisting of: glycosylation, phosphorylation, lipidation, glycation, proline isomerization, methylation, acetylation, hydroxylation sites, and proteolysis.Attorney Docket No.217863-712601 19. The engineered protein of any one of claims 1-18, wherein the engineered protein is glycosylated.

20. The engineered protein of any one of claims 1-18, wherein the engineered protein is aglycosylated.

21. The engineered protein of claim 1, wherein the engineered protein binds to an interferon- α / β receptor-1(IFNAR1) or an interferon-α / β receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by enzyme-linked immunosorbent assay (ELISA), or by measuring signaling activity of an Interferon Stimulated Response Element (ISRE)-Green fluorescent protein (GFP) reporter in a live human cell in vitro assay.

22. The engineered protein of claim 1, wherein the engineered protein binds to and activates an IFNAR1 or an IFNAR2 heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA, or by measuring signaling activity of an ISRE-GFP reporter in a live human cell in vitro assay.

23. The engineered protein of claim 1, wherein the engineered protein binds to and inhibits an activity of an IFNAR1 or an IFNAR2 heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein as measured by ELISA, or by measuring signaling activity of an ISRE-GFP reporter in a live human cell in vitro assay.

24. The engineered protein of claim 1, wherein the engineered protein is an agonist of IFNAR1 or IFNAR2 subunits.

25. The engineered protein of claim 1, wherein the engineered protein binds and activates IFNAR1 or IFNAR2 at a similar level than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE-GFP reporter assay or an ISRE luciferase reporter assay.

26. The engineered protein of claim 1, wherein the engineered protein reduces an extracellular level of a pro-inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

27. The engineered protein of claim 26, wherein the pro-inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof.

28. The engineered protein of claim 27, wherein the pro-inflammatory cytokine is IL-1β, IL- 6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, TNF-β, or any combination thereof.Attorney Docket No.217863-712601 29. The engineered protein of claim 1, wherein the engineered protein increases an extracellular level of an anti-inflammatory cytokine, as compared to the wildtype IFN- beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

30. The engineered protein of claim 29, wherein the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof.

31. The engineered protein of claim 30, wherein the anti-inflammatory cytokine is IL-4, IL- 10, IL-11, IL-13, IFN-α, TGF-β, or any combination thereof.

32. The engineered protein of claim 1, wherein the engineered protein binds to IFNAR1 or IFNAR2 subunits at a level less than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

33. The engineered protein of claim 1, wherein the engineered protein is an antagonist of IFNAR1 or IFNAR2 subunits.

34. The engineered protein of claim 33, wherein the engineered protein binds and inhibits an activity of the IFNAR1 or IFNAR2 at a level greater than wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE GFP reporter assay or an ISRE luciferase reporter assay.

35. The engineered protein of any one of claims 1-34, wherein the engineered protein is a recombinant IFN-beta or a portion thereof.

36. The engineered protein of any one of claims 1-35, wherein the engineered protein is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, conjugated to a polyethylene glycol (PEG) moiety, or any combination thereof.

37. The engineered protein of claim 1, wherein a glycosylation site is introduced in the engineered protein that results in diminished binding of an antibody to the engineered protein, relative to an amount of binding of the antibody to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO:

1.

38. The engineered protein of claim 1, wherein the modification results in a reduction of immunogenicity levels when administered to a subject, as compared to an immunogenicity level when the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 is administered to a subject.

39. The engineered protein of claim 38, wherein the reduction of immunogenicity levels comprises a reduced level of anti-drug antibody in the subject when the engineered protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject.Attorney Docket No.217863-712601 40. The engineered protein of claim 1, wherein the engineered protein, when administered to a subject in need thereof results in a reduction in a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering.

41. The engineered protein of claim 40, wherein the reduction in the level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA.

42. The engineered protein of claim 40, wherein the reduction in the level of inflammation in the subject results in a reduction in serum levels of C reactive protein as measured by high-sensitivity C-reactive protein (hs-CRP) test.

43. The engineered protein of claim 42, wherein the reduction in serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg / dL to about 2.9 mg / dL.

44. The engineered protein of claim 43, wherein the administering to the subject results in a reduction in blood levels of a pro-inflammatory cytokine as compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA.

45. The engineered protein of claim 1, wherein the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 2 – SEQ ID NO:

208.

46. The engineered protein of claim 1, wherein the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence length to any one of SEQ ID NO: 2 – SEQ ID NO:

208.

47. The engineered protein of claim 1, wherein the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to any one of SEQ ID NO: 2 – SEQ ID NO:

208.

48. The engineered protein of claim 1, wherein the engineered protein comprises an amino acid sequence that comprises a natural amino acid, or a non-natural amino acid.

49. A method of making a engineered IFN-beta protein in a cell, comprising introducing into the cell a vector encoding the engineered IFN-beta protein of any one of claims 1-4744, wherein the introducing results in expression of the engineered IFN-beta protein in the cell.

50. The method of claim 49, wherein the engineered IFN-beta protein is isolated from the cell.

51. The method of claim 49, wherein the vector comprises a mammalian expression plasmid.Attorney Docket No.217863-712601 52. The method of claim 49, wherein the engineered IFN-beta protein comprises a tag.

53. The method of claim 52, wherein the tag is CBP, FLAG, GST, Myc, poly-His, or V5.

54. The method of claim 52, wherein the tag comprises a cleavable tag or a non-cleavable tag.

55. A polynucleotide encoding an engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO:

208.

56. The polynucleotide of claim 55, wherein the polynucleotide comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 209 – SEQ ID NO:

342.

57. The polynucleotide of claim 55, wherein the polynucleotide comprises any one of SEQ ID NO: 209 – SEQ ID NO:

342.

58. The polynucleotide of claim 55, wherein the polynucleotide comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence length to any one of SEQ ID NO: 209 – SEQ ID NO:

342.

59. The polynucleotide of claim 55, wherein each codon in the polynucleotide can independently be replaced by a degenerate codon.

60. The polynucleotide of claim 55, wherein the polynucleotide comprise one or more degenerate codons.

61. The polynucleotide of claim 55, wherein the polynucleotide comprises a nucleic acid mimic.

62. The polynucleotide of claim 61, wherein the nucleic acid mimic comprises a phosphorothioate nucleic acid, a phosphoramidate nucleic acid, a morpholino nucleic acid, a hexitol nucleic acid (HNA), a peptide nucleic acid (PNA), or a locked nucleic acid (LNA).

63. The polynucleotide of claim 55, wherein one or more codons in the polynucleotide is modified.

64. The polynucleotide of claim 55, wherein one or more residues of an amino acid in the engineered IFN-beta protein is substituted for a non-natural amino acid.

65. A vector comprising the polynucleotide of any one of claims 55-64.

66. A pharmaceutical composition in unit dose form comprising: a. the engineered protein of any one of claims 1-48, or the polynucleotide of any one of claims 55-64; and b. a pharmaceutically acceptable excipient, carrier, or diluent.Attorney Docket No.217863-712601 67. The pharmaceutical composition of claim 66, wherein the pharmaceutical composition is encapsulated.

68. The pharmaceutical composition of claim 66, wherein the pharmaceutical composition is in the form of a liquid.

69. The pharmaceutical composition of any one of claims 66-68, wherein the pharmaceutical composition is formulated for local, systemic, or topical administration.

70. The pharmaceutical composition of any one of claims 66-69, wherein the pharmaceutical composition is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parental, intravenous, or intramuscular administration.

71. The pharmaceutical composition of any one of claims 66-70, wherein the pharmaceutical composition is formulated for controlled-release.

72. The pharmaceutical composition of any one of claims 66-71, wherein the pharmaceutical composition is formulated for single-dosage administration.

73. A method of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising (a) an engineered interferon beta (IFN-beta) protein or a functional fragment thereof; and (b) a pharmaceutically acceptable excipient, carrier, or diluent, wherein the engineered IFN- beta protein comprises a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, and wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208,.

74. The method of claim 73, wherein the disease is an infection.

75. The method of claim 74, wherein the infection is a viral infection.

76. The method of claim 75, wherein the viral infection is chronic Hepatitis B infection, or a chronic Hepatitis C infection.

77. The method of claim 73, wherein the disease is a demyelinating disorder.

78. The method of claim 77, wherein the demyelinating disorder is selected from the group consisting of Multiple Sclerosis (MS), Acute Disseminated Encephalomyelitis (ADEM), Acute Hemorrhagic Leucoencephalitis (AHLE), Balo’s disease (Concentric Sclerosis), Charcot-Marie-Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease).

79. The method of claim 78, wherein the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.

80. The method of any one of claims 73-79, wherein the subject is a human subject.

81. The method of any one of claims 73-80, wherein the administering is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra-cisternal injection,Attorney Docket No.217863-712601 intratumoral injection, subcutaneous injection, intraperitoneal injection, a surgical route, or any combination thereof.

82. The method of claim 73, wherein the administering occurs from about once a day, about twice a day, about once a week, about twice a week, about once every two weeks, about once a month, about once every 3 months, about once every 6 months, about once every 9 months, to about once a year.

83. The method of claim 73, wherein the administering is at a dose selected from the group consisting of about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, or about 2000 mg.

84. The method of claim 73, wherein the administering is at a dose selected from the group consisting of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, and 10 mg / kg.

85. The method of claim 73, wherein the administering reduces a volume of a tumor in the subject.

86. The method of claim 73, wherein the administering inhibits or diminishes the growth a tumor in the subject.

87. The method of claim 73, wherein the method further comprises administering a second therapeutic.

88. The method of claim 87, wherein the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.

89. The method of claim 73, wherein the method further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor.

90. The method of claim 89, wherein the checkpoint inhibitor is a PD-1, a PD-L1, CTLA-4 inhibitor.

91. The method of claim 73, wherein the administering induces activation of an IFN- stimulated response element (ISRE) or a Gamma interferon activation site (GAS) element, wherein the activation of the ISRE or GAS induces an interferon-like activity as measured by a luciferase reporter assay.

92. A method of treating a disease or a condition in a subject, the method comprising: administering to the subject a therapeutically effective amount of engineered protein of any one of claims 1-48, the polynucleotide of any one of claims 55-64, or the pharmaceutical composition of any one of claims 66-72, thereby treating the subject.

93. A method of treating a demyelinating disorder, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising: (a) anAttorney Docket No.217863-712601 engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208; and (b) a pharmaceutically acceptable excipient, carrier or diluent.

94. The method of claim 93, wherein the demyelinating disorder is selected from the group consisting of Multiple Sclerosis (MS), Acute Disseminated Encephalomyelitis (ADEM), Acute Hemorrhagic Leucoencephalitis (AHLE), Balo’s disease (Concentric Sclerosis), Charcot-Marie-Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease).

95. The method of claim 93, wherein the demyelinating disorder is a MS, wherein the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.

96. A kit comprising the engineered protein of any one of claims 1-48, or the polynucleotide of any one of claims 55-64 or the pharmaceutical composition of any one of claims 66- 72, and a container.

Images