Engineered interferon-beta proteins

Abstract

Disclosed herein are engineered interferon-beta (IFN-beta) protein proteins having increased biological activity, relative to a wildtype IFN-beta protein. Also disclosed herein are vectors encoding the engineered IFN-beta protein variants described herein. Also disclosed herein are pharmaceutical compositions comprising the engineered IFN-beta protein variants described herein or a vector encoding the engineered IFN-beta protein variants described herein. Also disclosed herein are methods of treating a disease by administering to a subject the engineered IFN-beta protein variants described herein, a vector encoding the engineered IFN-beta protein variants described herein, or a pharmaceutical composition comprising the engineered IFN-beta protein variants described herein or a vector described herein.

Description

ENGINEERED INTERFERON-BETA PROTEINSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 537,905, filed September 12, 2023, and U.S. Provisional Application No. 63 / 537,946, filed September 12, 2023, the entire contents of which are incorporated herein by reference.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

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

[0003] Provided herein are engineered interferon beta (IFN-beta) proteins or a functional fragment thereof, comprising at least one amino acid substitution in a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution is selected from the amino acid substitutions in Table 1, and wherein the at least one amino acid substitution provides for an increase in a biological activity of the engineered IFN- beta protein as compared to the wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1. In some embodiments, the increase in the biological activity comprises an increase in an expression of one or more biological targets of the engineered IFN-beta protein as compared to wildtype IFN-beta protein, as measured by an in vitro assay. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises at least two amino acid substitutions, at least three amino acid substitutions or at least four amino acid substitutions from Table 1. In some embodiments, the at least two amino acid substitutions that are selected from the group consisting of E103F & L106K, E61Q & E104F, F67A & I83P, F67A & N86K, F67K & KI 081, F67Y & L98E, F70L & L87F, F70T & L87M, F67Y & L98E, G78M & Y92Q, I59K & Q72S, I59T & S75F, I66S & Y92H, I66W & E103G, K99I & DI 10R, L57M & L106V, L63M & Fl HR, L63M & L88R, N65A & L98Y, N86D & E107A, N86I & L98D, N86K & L102Y, N86W & L102P, Q18K & L57T, Q18K & T82W, Q64W & E103W, S75K & L106N, S75L & V101T, T58C & Fl 1 II, W79H & E104P, Y60M & E85V, R124L & I145V, Ml 17A & L164E, K115R & N153G, G114N & Y163S, T144V & R152R, N86W & L102P, T58I & D73G, Q64A & L87M, Y60N & I95T, E61C & DI 10Y, R71K & Y92F, N4K & L47L, L20E & I44M, R35V & K45N, S13Y & Y30V, N14A & D34I, S12F & K33K, L28C & E43N, W22I & E53M, Y30L & K45G, N80Q & F111E, S74N & I83K, S119H & A142S, Y126K & N158W, S119G & E137E, H121W & N153M, T112S & I157L, SI 19P & R152K, L21Y & W22S, S12I & E53Q,N4K & L47H, L6M & I44H, S13E & 144 V, Y3H & Q48Y, L6D & W22C, G114V & L164M, L9W & R35G, S13A & K52Q, L20R & Q49A, C17D & I44K, R27M & L47L, D34H & A55S, QI OS & W79L, L28F & MON, M62A & T100L, Y60K & A89V, Q18K & G78F, L57F & L87E, V91Q & E109N, Q64E & Y92V, A68I & D73N, R128M & T161P, M117Y & F156T, KI 15N & L164*, L120H & F156W, S13R & R35V, Q16A & K52P, W22R & E29N, R1 ID & Q51F, L47F & L120M, R11Y & F50G, G7A & I40F, S2F & G26F, K33N & N37A, L32Y & F50W, R35I & Q49H, R1 IF & Q46Y, G7A & Q18H, L6A & D34K, F8G & F50V, K19Y & R27K, F8F & S13E, S13P & R35T, K115I & H121P, H121N & H131K, Q51K & L160G, KI 15Y & G127T, T112L & Y132W, L122R & T161V, N158A & N166P, LI 16T & I15OG, and R113K & L160T. In some embodiments, the at least three amino acid substitutions from Table 1 that are L57Q & E81S & H97S, V84T & K108K & E109T, F8D & Q23P & K108N, R11Q & K52N & D54T, R11Y & E42D & E109D, R35M & E53P & V91V, G7M & R27M & G127W, L122V & V148A & L160R, and L120M & K134K & A135T. In some embodiments, the at least four amino acid substitutions from Table 1 that are N80Q & V84N & N86T & Fl 11 S. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises an amino acid sequence of any one of SEQ ID NO: 2- SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises a structural feature that is present in wildtype IFN-beta protein, as determined by modeling of the engineered IFN-beta structure. In some embodiments, the structural feature comprises an alpha-helix. In some embodiments, the structural feature comprises a helix-turn- helix structural motif. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof binds to an interferon-a / p receptor- l(IFNARl) or interferon-a / p receptor-2 (IFNAR2) heterodimer subunits at a level greater than the wildtype IFN-beta protein, as measured by an in vitro assay. In some embodiments, the in vitro assay comprises 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 IFN-beta protein or functional fragment thereof binds and activates IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein, as measured by an ISRE-GFP reporter assay or an ISRE luciferase reporter assay. In some embodiments, the engineered IFN-beta protein or functional fragment thereof is an agonist of IFNARl or IFNAR2 subunits. In some embodiments, the engineered IFN-beta protein or functional fragment thereof reduces an extracellular level of a pro-inflammatory cytokine, ascompared to the wildtype IFN-beta protein having 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-ip, IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN-y, TNF-a, TNF- , or any combination thereof. In some embodiments, the engineered IFN-beta protein or functional fragment thereof increases an extracellular level of an anti-inflammatory cytokine, as compared to the wildtype IFN-beta protein having 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-a, TGF-P, or any combination thereof. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof, when administered to a subject, reduces a level of inflammation in the 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 or a functional fragment thereof to the subject results in reduced blood levels of a pro- inflammatory cytokine, as compared to the blood levels of the pro-inflammatory cytokine before the administering, as measured by ELISA.

[0004] Also provided herein are methods of making an engineered interferon beta (IFN-beta) protein or a functional fragment thereof in a cell, comprising introducing into the cell a vector encoding the engineered IFN-beta protein as previously disclosed, wherein the introducing results in expression of the engineered IFN-beta protein in the cell. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof 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.

[0005] Also provided herein are polynucleotides encoding the engineered IFN-beta protein or a function fragment thereof as previously disclosed. In some embodiments, the polynucleotidecomprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 1609 - SEQ ID NO: 1711 or SEQ ID NO: 1713 - SEQ ID NO: 1939. In some embodiments, the polynucleotide comprises a nucleic acid sequence of any one of SEQ ID NO: 1609 - SEQ ID NO: 1711 or SEQ ID NO: 1713 - SEQ ID NO: 1939.

[0006] Provided herein are vectors encoding the polynucleotides disclosed herein.

[0007] Also provided herein are pharmaceutical compositions in unit dose form comprising (a) the engineered IFN-beta proteins as previously disclosed, the polynucleotides as previously disclosed, or the vector encoding the engineered IFN-beta protein of as previously disclosed, and (b) 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.

[0008] Provided herein are methods of treating a demyelinating disorder in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an engineered interferon beta (IFN-beta) protein or a functional fragment thereof that is sufficient to treat the demyelinating disorder, wherein the engineered IFN-beta protein comprises at least one amino acid substitution selected from the amino acid substitutions in Table 1 that provides for an increase in biological activity of the engineered IFN-beta protein, as compared to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1.

[0009] Also provided herein are methods of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of the engineered IFN-beta protein as previously disclosed, the polynucleotide as previously disclosed, the vector encoding the engineered IFN-beta protein as previously disclosed or the pharmaceutically composition of as previously disclosed, thereby treating the subject. In some embodiments, the disease is an infection. In some embodiments, the infection is a viral infection. In some embodiments, the viral infection is a 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-cistemal 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 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 the growth a tumor in the subject. In some embodiments, the method further comprises administering a second therapeutic. In some embodiments, the second therapeutic is an antibody, a peptide, an anti-body drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof. In some embodiments, the method further comprises further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1 or a PD-L1 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.

[0010] Also provided herein are kits comprising the engineered IFN-beta protein disclosed herein, the polynucleotide disclosed herein, the vector encoding the engineered IFN-beta protein disclosed herein or the pharmaceutically composition disclosed herein, and a container.INCORPORATION BY REFERENCE[OH] 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] FIGURE 1 illustrates the application of the high-throughput molecular function assay system to screen variants of IFN-beta for biological activity.

[0014] FIGURE 2 depicts the activation of the Interferon Stimulation Response Element (ISRE) pathway in response to expression of the engineered IFN-beta protein disclosed herein. The x- axis depicts the IFN signaling score, and the y-axis depicts the number of non-naturally occurring IFN-beta proteins.

[0015] FIGURE 3 depicts the variant classification matrix. The rows represent the TOST p- value, and the column represents the score p-value.

[0016] FIGURES 4A-4C depicts the activation of the ISRE pathway in response to expression of the engineered IFN-beta protein as compared to wildtype IFN-beta.

[0017] FIGURE 4A depicts the activity measurements of engineered IFN-beta proteins with activity less that wildtype IFN beta.

[0018] FIGURE 4B depicts the activity measurements of engineered IFN-beta proteins with activity equal to wildtype IFN beta.

[0019] FIGURE 4C depicts the activity measurements of engineered IFN-beta proteins with activity greater than the wildtype IFN beta. The x-axis depicts the IFN signaling score, and the y- axis depicts the number of non-naturally occurring IFN-beta proteins.

[0020] FIGURES 5A-5D depict the verification of an exemplary loss-of-function (LOF) engineered IFN-beta protein.

[0021] FIGURE 5A is histogram depicting the score distribution for 145 independent barcode measurements for the LOF engineered IFN-beta protein (left, dark gray) compared to -37,000 independent barcode measurements for wildtype IFN-beta protein (right, gray). The x-axis depicts the barcode score, and the y-axis depicts the barcode count.

[0022] FIGURE 5B is a histogram depicting the flow cytometry measurements in cells treated with the exemplary loss-of-function (LOF) engineered IFN-beta protein (left), or wildtype IFN- beta protein (right). The x-axis depicts the ISRE reporter activity, and the y-axis depicts the cell counts.

[0023] FIGURES 5C-5D is a graph and table depicting the plate reader output after cells were treated with the engineered IFN-beta protein (gray), or wildtype IFN-beta protein (black). The x- axis depicts the ligand concentration, and the y-axis depicts the ISRE reporter activity.

[0024] FIGURES 6A-6J depict the verification of an exemplary engineered IFN-beta protein with similar signaling activity (WT-like variant) to wildtype IFN-beta.

[0025] FIGURE 6A is histogram depicting the score distribution for 106 independent barcode measurements for the WT-like variant (dark gray) compared to -37,000 independent barcode measurements for wildtype IFN-beta protein (gray). The x-axis depicts the barcode score, and the y-axis depicts the barcode count.

[0026] FIGURES 6B-6C is a graph and table depicting the plate reader output after cells were treated with the engineered IFN-beta protein (gray), or wildtype IFN-beta protein (black). The x- axis depicts the ligand concentration, and the y-axis depicts the ISRE reporter activity.

[0027] FIGURES 6D-6G are histograms depicting the flow cytometry measurements in cells treated with the engineered IFN-beta protein with similar signaling activity (WT-like variant) to wildtype IFN-beta, and the wildtype IFN-beta protein at varying concentrations (0.313 ng / mL (FIGURE 6D), 0.625 ng / mL (FIGURE 6E), 2.5 ng / mL (FIGURE 6F), 20 ng / mL (FIGURE 6G)). The x-axis depicts the ISRE reporter activity, and the y-axis depicts the cell counts.

[0028] FIGURE 6H is a western blot, first lane represents protein ladder, and second lane demonstrates protein variant expression in cells.

[0029] FIGURES 61-6 J show quantitative RT-PCR determination of OAS1 mRNA expression in cells in expressing the tagged non-naturally occurring WT-like variant, and tagged wildtype IFN beta protein (FIGURE 61), and OAS1 expression normalized to house-keeping gene, HPRT (FIGURE 6 J)

[0030] FIGURES 7A-7K depict the verification of an exemplary engineered IFN-beta protein with signaling activity greater than wildtype IFN-beta (GOF variant).

[0031] FIGURE 7A is histogram depicting the score distribution for 138 independent barcode measurements for the WT-like variant (dark gray) compared to -37,000 independent barcode measurements for wildtype IFN-beta protein (gray).

[0032] FIGURES 7B-7C is a graph and table depicting the plate reader output after cells were treated with the engineered IFN-beta protein (gray), or wildtype IFN-beta protein (black). The x- axis depicts the ligand concentration, and the y-axis depicts the ISRE reporter activity.

[0033] FIGURES 7D-7G are histograms depicting the flow cytometry measurements in cells treated with the engineered IFN-beta protein with similar signaling activity (WT-like variant) to wildtype IFN-beta, and the wildtype IFN-beta protein at varying concentrations (0.313 ng / mL (FIGURE 7D), 0.625 ng / mL (FIGURE 7E), 2.5 ng / mL (FIGURE 7F), 20 ng / mL (FIGURE 7G)). The x-axis depicts the ISRE reporter activity, and the y-axis depicts the cell counts.

[0034] FIGURE 7H-7I are graphs depicting the flow cytometry measurements in cells treated with the engineered IFN-beta protein increased activity (GOF variant) to wildtype at differentconcentrations. The x-axis depicts the ISRE reporter activity, and the y-axis depicts the cell counts.

[0035] FIGURES 7J-7K show quantitative RT-PCR determination of OAS1 mRNA expression in cells in expressing the tagged engineered IFN-beta protein, and tagged wildtype IFN beta protein (FIGURE 7 J), and OAS1 expression normalized to house-keeping gene, HPRT (FIGURE 7K)DETAILED DESCRIPTION OF THE INVENTIONOverview

[0036] Disclosed herein are engineered interferon beta (IFN-beta) proteins having increased signaling activity through an IFNAR receptor, which is comprised of a heterodimer of IFNAR1 and IFNAR2, as compared to wildtype IFN-beta proteins. IFN-beta is a biologic used to reduce inflammation and treat inflammatory conditions, such as Multiple Sclerosis and cancers. Also disclosed herein are compositions comprising the engineered IFN beta protein, methods of making the engineered IFN beta protein, and methods for treating a disease. In some embodiments, the engineered IFN beta protein as described herein can act as a signaling agent. In some embodiments, the biological activity of the engineered IFN beta proteins disclosed herein have greater activity than wildtype IFN-beta. In some embodiments, the biological activity of the engineered IFN beta proteins disclosed herein have similar or substantially similar to wildtype IFN-beta.Definitions

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The term “operably coupled” refers to functional linkage between a regulatory sequence and a nucleic acid sequence resulting in expression of the latter

[0041] Unless otherwise indicated, open terms for example “contain,” “containing,” “include,” “including,” and the like mean comprising.

[0042] 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.

[0043] 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).

[0044] 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 suspected 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 subjectin 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0001] “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 ofother 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.

[0002] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0003] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0004] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0005] “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.

[0006] 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 nonplasmid 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.

[0007] “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 identitybetween 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.

[0008] 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.

[0009] 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).

[0010] 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.

[0011] 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 in 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.

[0012] 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 alsocommonly 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.

[0013] As used herein, the terms “interferon beta,” “IFN-beta,” “IFN-P” and “IFNP” are used interchangeably, and refer to a polypeptide or protein with at least 90% sequence identity to SEQ ID NO: 1.

[0014] 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 a variant interferon-beta polypeptide or protein.

[0015] Ranges: throughout this disclosure, various aspects of the invention is 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.

[0016] 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.

[0017] 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 in 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 aclaimed 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.

[0018] 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.Engineered IFN-beta proteins

[0049] Disclosed herein are engineered, engineered IFN-beta protein s comprising an amino acid substitution, relative to a wild-type IFN-beta protein, that results in an increase in signaling activity of the IFN-protein variant relative to the wild-type IFN-beta protein. Such variant proteins are used as a treatment for inflammatory diseases or conditions such as multiple sclerosis.

[0050] 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 below, relative to a wild-type IFN- beta protein having an amino acid sequence of SEQ ID NO: 1 (MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALT IYEMLQNIFAIFRQDS S STGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMS SLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN). A engineered IFN-beta protein having an amino acid substitution recited in Table 1 has an increase in activity (e g., signaling activity), relative to a wild-type IFN-beta protein.TABLE 1. Exemplary IFN-beta amino acid substitutionsrecited in Table 1 is present as a salt, such as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable salt includes, but is not limited to: acetate, acrylate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, bisulfite, bitartrate, bromide, butyrate, butyn-l,4-dioate, camphorate, camphorsulfonate, caproate, caprylate, chlorobenzoate, chloride, citrate, cyclopentanepropionate, decanoate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hexyne-1,6- dioate, hydroxybenzoate, y-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.

[0052] In some instances, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 is encoded by a vector (e.g., a DNA vector). 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 or a functional fragment thereof comprises an amino acid sequence having at least about: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein or functional fragment thereof comprises at least one amino acid substitution in a region comprising residues 2 to 166 of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises at least one amino acid substitution selected from the group consisting of: S2, Y3, N4, L5, L6, G7, F8, L9, Q10, RI 1, S12, S13, N14, F15, Q16, C17, Q18, K19, L20, L21, W22, Q23, L24, N25, G26, R27, L28, E29, Y30, L32, D34, R35, M36, F38, D39, 140, P41, E42, 144, K45, Q46, L47, Q49, Q51, D54, L57, T58, 159, Y60, E61, M62, L63, Q64, N65, 166, F67, A68, F70, Q72,574, S75, S76, T77, G78, W79, N80, E81, T82, 183, V84, E85, N86, L87, L88, N90, Y92, H97, L98, K99, V101, L102, E103, E104, L106, E107, K108, E109, DUO, Fi l l, G114, L116, M117, L120, L122, K123, R124, G127, Y126, R128, 1129, L130, A135, Y155, R147, V148, L151, N158, F156, G162, and R165. In some embodiments, the engineered IFN-beta protein or functional fragment thereof comprises at least one amino acid substitution in a region comprising residues 2 to 166 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from Table 1. In some embodiments, the engineered IFN-beta protein or functional fragment thereof comprises at least two amino acid substitutions in a region comprising residues 2 to 166 of SEQ ID NO: 1. In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises at least two amino acid substitutions selected from the group consisting of: Q16, Q18, K52, L57, T58, 159, Y60, E61, L63, Q64, N65, 166, F67, Q72,575, G78, W79, T82, 183, E85, N86, L87, L88, Y92, L98, K99, V101, L102, E103, E104, L106, E107, K108, DUO, Fi l l, G114, L116, M117, L120, L122, K123, R124, G127, Y126, R128, 1129, L130, A135, Y155, R147, V148, L151, N158, F156, G162, and R165. In some embodiments, the at least two amino acid substitutions are selected from Table 1. In some cases, the at least two amino acid substitutions selected from Table 1 are E103F & L106K, E61Q & E104F, F67A & I83P, F67A & N86K, F67K & K108I, F67Y & L98E, F70L & L87F, F70T & L87M, F67Y & L98E, G78M & Y92Q, I59K & Q72S, I59T & S75F, I66S & Y92H, I66W &E103G, K99I & DI 10R, L57M & L106V, L63M & Fl 11R, L63M & L88R, N65A & L98Y, N86D & E107A, N86I & L98D, N86K & L102Y, N86W & L102P, Q18K & L57T, Q18K & T82W, Q64W & E103W, S75K & L106N, S75L & V1O1T, T58C & Fl 1 II, W79H & E104P, Y60M & E85V, R124L & I145V, Ml 17A & L164E, KI 15R & N153G, G114N & Y163S, T144V & R152R, N86W & L102P, T58I & D73G, Q64A & L87M, Y60N & I95T, E61C & DI 1OY, R71K & Y92F, N4K & L47L, L20E & I44M, R35V & K45N, S13Y & Y30V, N14A & D34I, S12F & K33K, L28C & E43N, W22I & E53M, Y30L & K45G, N80Q & Fl HE, S74N & I83K, S119H & A142S, Y126K & N158W, S119G & E137E, H121W & N153M, T112S & I157L, S119P & R152K, L21Y & W22S, S12I & E53Q, N4K & L47H, L6M & I44H, S13E & I44V, Y3H & Q48Y, L6D & W22C, G114V & L164M, L9W & R35G, S13A & K52Q, L20R & Q49A, C17D & I44K, R27M & L47L, D34H & A55S, Q1OS & W79L, L28F & HON, M62A & T1OOL, Y60K & A89V, Q18K & G78F, L57F & L87E, V91Q & E109N, Q64E & Y92V, A68I & D73N, R128M & T161P, M117Y & F156T, KI 15N & L164*, L120H & F156W, S13R & R35V, Q16A & K52P, W22R & E29N, R1 ID & Q51F, L47F & L120M, R11Y & F5OG, G7A & HOF, S2F & G26F, K33N & N37A, L32Y & F50W, R35I & Q49H, R1 IF & Q46Y, G7A & Q18H, L6A & D34K, F8G & F5OV, K19Y & R27K, F8F & S13E, S13P & R35T, KI 151 & H121P, H121N & H131K, Q51K & L160G, K115Y & G127T, T112L & Y132W, L122R & T161V, N158A & N166P, LI 16T & I15OG, and R113K & L160T. In some embodiments, the at least three amino acid substitutions are selected from Table 1. In some cases, the at least three amino acid substitutions selected from Table 1 are L57Q & E81S & H97S, V84T & K108K & E109T, F8D & Q23P & K108N, R1 IQ & K52N & D54T, R11Y & E42D & E109D, R35M & E53P & V91V, G7M & R27M & G127W, L122V & V148A & L160R, and L120M & K134K & A135T. In some embodiments, the at least four amino acid substitutions are selected from Table 1. In some cases, the at least four amino acid substitutions selected from Table 1 are N80Q & V84N & N86T & Fl 1 IS. In some embodiments, the engineered IFN-beta protein comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332.TABLE 2: Engineered IFN-beta proteins with increased activity as compared to wildtype IFN-beta.

[0053] In some embodiments, the engineered IFN-beta proteins described having an amino acid sequence of any one of SEQ ID NO: 2 - SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332 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 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 the 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).

[0054] In some embodiments, the engineered IFN-beta protein binds to an interferon-a / p receptor- 1 (IFN ARI) or interferon-a / p 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-a / p receptor- l(IFNARl) or interferon-a / p receptor-2 (IFNAR2) heterodimer subunits and signals through the IFNARl or IFNAR2 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 IFNARl or IFNAR2 subunits.

[0055] In some embodiments, the engineered IFN-beta protein binds and activates IFNARl 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, theengineered 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.

[0056] 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-ip, IL-6, IL-8, IL-9, IL-12, IL-15, IL- 17, IL- 18, IFN-y, TNF-a, TNF-P, 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-a, TGF-P, or any combination thereof.

[0057] In some embodiments, the engineered IFN-beta protein having amino acid sequence of any one of SEQ ID NO: 2 - SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332 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 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.

[0058] In some embodiments, the engineered IFN-beta protein has an amino acid substitution (e.g., one or more amino acid substitutions) recited in Table 3 below, relative to a wild-type 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 3 has similar biological activity (e.g., signaling activity) to a wild-type IFN-beta protein.TABLE 3. Exemplary IFN-beta amino acid substitutions

[0059] In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises an amino acid sequence having at least about: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 1. In some embodiments, the engineered IFN- beta protein or functional fragment thereof comprises at least one amino acid substitution in a region comprising residues 2 to 166 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from Table 3. In some embodiments, the engineered IFN-beta protein comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 335 - SEQ ID NO: 1606 or SEQ ID NO: . In other embodiments, the engineered IFN-beta 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.TABLE 4: Engineered IFN-beta proteins with substantially similar activity to wildtype

[0060] Provided herein are nucleic acids encoding the engineered IFN-beta proteins disclosed herein. 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: 1380 - SEQ ID NO: 1483. 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: 1380 to SEQ ID NO: 1482. 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 5: Nucleic acid sequences encoding the engineered IFN-beta proteins disclosed herein with increased biological activity.

[0061] Provided herein are nucleic acids encoding the engineered IFN-beta proteins disclosed herein. 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: 1484 - SEQ ID NO: 2755. 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: 1484 to SEQ ID NO: 2755. 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: Nucleic acid sequences encoding the engineered IFN-beta proteins disclosed herein.Compositions

[0062] Disclosed herein are pharmaceutical compositions comprising the engineered IFN-beta proteins disclosed herein, the nucleic acids disclosed herein, the nucleic acid constructs disclosed herein or the vectors disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, an engineered IFN-beta protein having an amino acid substitution recited in Table 1, or have an amino acid sequence of any one of SEQ ID NO: 2 - SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332, is present in a composition. In some embodiments, a vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1, or have an amino acid sequence of any one of SEQ ID NO: 2 - SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332 is present in a composition. In some embodiments, the engineered IFN-beta protein disclosed herein, the polynucleotides disclosed herein, or the vector encoding the engineered IFN-beta protein as disclosed herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable: excipient, carrier, or diluent. In some cases, the composition is a pharmaceutical composition with a pharmaceutically acceptable excipient, diluent, or carrier.

[0063] In some embodiments, a pharmaceutical composition 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 comprise a diluent such as water, glycerol, methanol, ethanol, and other similar biocompatible diluents. In some embodiments, the pharmaceutical composition is in unit dose form.

[0064] In some embodiments, the pharmaceutical composition is 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. 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.Methods of treatment

[0065] Provided herein are methods of treating a demyelinating disorder in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an engineered interferon beta (IFN-beta) protein or a functional fragment thereof that is sufficient to treat the demyelinating disorder, wherein the engineered IFN-beta protein comprises at least one amino acid substitution selected from the amino acid substitutions in Table 1 that provides for an increase in biological activity of the engineered IFN-beta protein, as compared to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1. Also provided herein are methods of treating a demyelinating disorder in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an engineered interferon beta (IFN-beta) protein or a functional fragment thereof that is sufficient to treat the demyelinating disorder, wherein the engineered IFN-beta protein comprises at least one amino acid substitution selected from the amino acid substitutions in Table 3 that provides for a biological activity substantially similar a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1. 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).

[0019] Further provided herein are methods of treating a disease or a condition in a subject, the method comprising: administering to the subject a therapeutically effective amount of the engineered IFN-beta protein as disclosed herein, the polynucleotide as disclosed herein, the vector encoding the engineered IFN-beta protein as disclosed herein or the pharmaceutically composition as disclosed herein, thereby treating the subject. In some embodiments, the disease is an infection. In some embodiments, the infection is a viral infection. In some embodiments, the viral infection is a 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 disease or condition is 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 HepatitisB 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).

[0020] In some embodiments, the disease or the condition comprises a viral infection, a cancer, an autoimmune disease, multiple sclerosis, or a hemolytic disease. In some embodiments, the disease is an infection. In some cases, the infection is a viral infection. In some embodiments, the viral infection is a chronic hepatitis B (CHB), a chronic hepatitis C (CHC), a chronic granulomatous disease (CGD), a coronavirus disease (COVID), a genital wart or a human immunodeficiency virus (HIV). In some cases, the disease is a demyelinating disorder. In some embodiments, the demyelinating disorder is 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 cases, the disease is a cancer. In some embodiments, the cancer is a blood cancer, a bone cancer, a brain cancer, a breast cancer, a cervical cancer, a colon cancer, a heart cancer, a kidney cancer, a liver cancer, a lung cancer, a lymph node cancer, a lymphoma, a melanoma, a myeloma, a skin cancer, a stomach cancer, a pancreatic cancer, a testicular cancer, or a throat cancer. In some embodiments, the cancer is selected from the group consisting of astrocytoma, chronic myelogenous leukemia, follicular lymphoma, glioblastoma, hairy cell leukemia, Kaposi’s sarcoma, malignant melanoma, medulloblastoma, melanoma, multiple myeloma, non-Hodgkin lymphoma, and renal cell carcinoma. In some cases, the disease is an immune disorder. In some embodiments, the immune disorder is selected from the group consisting of lupus, systemic lupus erythematosus, asthma, atherosclerosis, atopic dermatitis, autoimmune hepatitis, Celiac disease, COPD, Crohn's disease, Graves' disease, Guillain-Barre syndrome, IgA nephropathy, inflammatory bowel disease, pelvic inflammatory disease, primary biliary cirrhosis, primary hemophagocytic lymphohistiocytosis, ulcerative colitis, and other autoimmune disorders.

[0066] 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 adose 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 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 the growth a tumor in the subject. In some embodiments, the method further comprises administering a second therapeutic. In some embodiments, the second therapeutic is an antibody, a peptide, an anti-body drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof. In some embodiments, the method further comprises further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1 or a PD-L1 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.

[0067] In some embodiments, an engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, a vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or a pharmaceutical composition comprising the engineered IFN-beta protein or vector, is administered to a subject to treat a disease or condition. For example, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN- beta protein or vector, is used to treat an inflammatory condition. In some instances, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN-beta protein or vector, is 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).

[0068] In some embodiments, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN-beta protein or vector, is 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, intraci sternal, 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.

[0069] In some embodiments, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN-beta protein or vector, is co-administered along with a second therapeutic. In some embodiments, the second therapeutic is an antibody, a peptide, an anti-body drug conjugate, or any combination thereof. In some cases, the second therapeutic is an immunotherapy agent such as a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is 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.

[0070] In some embodiments, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, vector encoding the engineered IFN-beta protein having an aminoacid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN-beta protein or vector, is 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 engineered IFN- beta protein having an amino acid substitution recited in Table 1 or Table 3, vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN-beta protein or vector, is administered every day, every other day, once a week, or once a month.

[0071] In some embodiments, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN-beta protein or vector, is 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.

[0072] In some embodiments, the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, an engineered IFN-beta protein having an amino acid sequence recited in Table 2 or Table 4, vector encoding the engineered IFN-beta protein having an amino acid substitution recited in Table 1 or Table 3, or pharmaceutical composition comprising the engineered IFN-beta protein or vector, is 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,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 gg,1.1 gg, 1.2 gg, 1.3 gg, 1.4 gg, 1.5 gg, 1.6 gg, 1.7 gg, 1.8 gg, 1.9 gg, 2 gg, 2.1 gg, 2.2 gg, 2.3 gg, 2.4 gg, 2.5 gg, 2.6 gg, 2.7 gg, 2.8 gg, 2.9 gg, 3 gg, 3.1 gg, 3.2 gg, 3.3 gg, 3.4 gg, 3.5 gg,3.6 gg, 3.7 gg, 3.8 gg, 3.9 gg, 4 gg, 4.1 gg, 4.2 gg, 4.3 gg, 4.4 gg, 4.5 gg, 4.6 gg, 4.7 gg, 4.8 gg, 4.9 gg, 5 gg, 5.1 gg, 5.2 gg, 5.3 gg, 5.4 gg, 5.5 gg, 5.6 gg, 5.7 gg, 5.8 gg, 5.9 gg, 6 gg, 6.1 gg, 6.2 gg, 6.3 gg, 6.4 gg, 6.5 gg, 6.6 gg, 6.7 gg, 6.8 gg, 6.9 gg, 7 gg, 7.1 gg, 7.2 gg, 7.3 gg,7.4 gg, 7.5 gg, 7.6 gg, 7.7 gg, 7.8 gg, 7.9 gg, 8 gg, 8.1 gg, 8.2 gg, 8.3 gg, 8.4 gg, 8.5 gg, 8.6 gg, 8.7 gg, 8.8 gg, 8.9 gg, 9 gg, 9.1 gg, 9.2 gg, 9.3 gg, 9.4 gg, 9.5 gg, 9.6 gg, 9.7 gg, 9.8 gg,9.9 gg, 10 gg, 11 gg, 12 gg, 13 gg, 14 gg, 15 gg, 16 gg, 17 gg, 18 gg, 19 gg, 20 gg, 21 gg, 22 gg, 23 gg, 24 gg, 25 gg, 26 gg, 27 gg, 28 gg, 29 gg, 30 gg, 31 gg, 32 gg, 33 gg, 34 gg, 35 gg, 36 gg, 37 gg, 38 gg, 39 gg, 40 gg, 41 gg, 42 gg, 43 gg, 44 gg, 45 gg, 46 gg, 47 gg, 48 gg, 49 gg, 50 gg, 51 gg, 52 gg, 53 gg, 54 gg, 55 gg, 56 gg, 57 gg, 58 gg, 59 gg, 60 gg, 61 gg, 62 gg, 63 gg, 64 gg, 65 gg, 66 gg, 67 gg, 68 gg, 69 gg, 70 gg, 71 gg, 72 gg, 73 gg, 74 gg, 75 gg, 76 gg, 77 gg, 78 gg, 79 gg, 80 gg, 81 gg, 82 gg, 83 gg, 84 gg, 85 gg, 86 gg, 87 gg, 88 gg, 89 gg, 90 gg, 91 gg, 92 gg, 93 gg, 94 gg, 95 gg, 96 gg, 97 gg, 98 gg, 99 gg, 100 gg, 110 gg, 120 gg, 130 gg, 140 gg, 150 gg, 160 gg, 170 gg, 180 gg, 190 gg, 200 gg, 210 gg, 220 gg, 230 gg, 240 gg, 250 gg, 260 gg, 270 gg, 280 gg, 290 gg, 300 gg, 310 gg, 320 gg, 330 gg, 340 gg, 350 gg,360 gg, 370 gg, 380 gg, 390 gg, 400 gg, 410 gg, 420 gg, 430 gg, 440 gg, 450 gg, 460 gg, 470 gg, 480 gg, 490 gg, 500 gg, 510 gg, 520 gg, 530 gg, 540 gg, 550 gg, 560 gg, 570 gg, 580 gg,590 gg, 600 gg, 610 gg, 620 gg, 630 gg, 640 gg, 650 gg, 660 gg, 670 gg, 680 gg, 690 gg, 700 gg, 710 gg, 720 gg, 730 gg, 740 gg, 750 gg, 760 gg, 770 gg, 780 gg, 790 gg, 800 gg, 810 gg,820 gg, 830 gg, 840 gg, 850 gg, 860 gg, 870 gg, 880 gg, 890 gg, 900 gg, 910 gg, 920 gg, 930 gg, 940 gg, 950 gg, 960 gg, 970 gg, 980 gg, 990 gg, 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, 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, 34mg, 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

[0073] 1. A non-naturally occurring interferon beta (IFN-beta) protein, comprising an amino acid sequence having an amino acid substitution with respect to the IFN-beta protein amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution is selected from the amino acid substitutions in Table 1.

[0074] 2. The non-naturally occurring IFN-beta protein of embodiment 1, wherein the non- naturally occurring IFN-beta has a structural feature that is present in naturally occurring IFN- beta protein, as determined by modeling of the non-naturally occurring IFN-beta structure.

[0075] 3. The non-naturally occurring IFN-beta protein of embodiment 2, wherein the structural feature comprises an alpha-helix.

[0076] 4 The non-naturally occurring IFN-beta protein of embodiment 3, wherein the structural feature is a helix-tum-helix structural motif.

[0077] 5. The non-naturally occurring IFN-beta protein of embodiment 1, wherein the non- naturally occurring IFN-beta protein binds to the interferon-o / p receptor- 1 / interferon-a / p receptor-2 heterodimer at a level greater than or equal to the naturally occurring IFN-beta.

[0078] 6. The non-naturally occurring IFN-beta protein of embodiment 1, wherein the non- naturally occurring IFN-beta protein, when administered to a subject, reduces a level of inflammation in the subject, relative to a level of inflammation in the subject prior to the administering.

[0079] 7 The non-naturally occurring IFN-beta protein of any one of embodiments 1-6, wherein the protein is a recombinant IFN-beta or a portion thereof.

[0080] 8. The non-naturally occurring IFN-beta protein of any one of embodiments 1-7, wherein the non-naturally occurring IFN-beta protein comprises a post-translational modification.

[0081] 9. A method of making a non-naturally occurring interferon beta (IFN-beta) protein in a cell, comprising: (a) transforming the cell with a vector encoding the non-naturally occurring IFN-beta protein of any one of embodiments 1-8; (b) expressing the IFN-beta protein in the cell; and (c) isolating the non-naturally occurring IFN-beta protein.

[0082] 10. A pharmaceutical composition in unit dose form comprising: (a) the non-naturally occurring interferon beta (IFN-beta) protein of any one of embodiments 1-8 or a vector encoding the non-naturally occurring IFN-beta protein of any one of embodiments 1-8; and (b) a pharmaceutically acceptable: excipient, carrier, or diluent.

[0083] 11. The pharmaceutical composition of embodiment 10, wherein the pharmaceutical composition is encapsulated.

[0084] 12. The pharmaceutical composition of embodiment 10, wherein the pharmaceutical composition is in the form of a liquid.

[0085] 13. 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 IFN-beta protein of any one of embodiments 1-8, or the pharmaceutically composition of any one of embodiments 10-12, thereby treating the subject.

[0086] 14. The method of embodiment 13, wherein the disease is an infection.

[0087] 15. The method of embodiment 14, wherein the infection is a viral infection.

[0088] 16. The method of embodiment 15, wherein the viral infection is a chronic Hepatitis B infection, or a chronic Hepatitis C infection.

[0089] 17. The method of embodiment 13, wherein the disease is multiple sclerosis (MS).

[0090] 18. The method of embodiment 17, wherein the MS is relapse remitting MS, nonrelapsing MS, clinically isolated syndrome, and secondary progressive forms.

[0091] 19. The method of any one of embodiments 13-18, wherein the subject is a human subject.

[0092] 20. The method of any one of embodiments 13-19, wherein the administering is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra-ci sternal injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, a surgical route, or any combination thereof.

[0093] 21. The method of embodiment 13, 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.

[0094] 22. The method of embodiment 13, 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 2000 mg.

[0095] 23. The method of embodiment 13, 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.

[0096] 24. The method of embodiment 13, wherein the administering reduces a volume of a tumor in the subject.

[0097] 25. The method of embodiment 13, wherein the administering inhibits the growth a tumor in the subject.

[0098] 26. The method of embodiment 13, wherein the method further comprises administering a second therapeutic.

[0099] 27. The method of embodiment 26, wherein the second therapeutic is an antibody, a peptide, an anti-body drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.

[0100] 28. The method of embodiment 13, wherein the method further comprises further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor.

[0101] 29. The method of embodiment 28, wherein the checkpoint inhibitor is a PD-1 or a PD- L1 inhibitor.

[0102] 30. The method of embodiment 13, wherein the administering induces activation of an IFN-stimulated response element (ISRE) or an Gamma interferon activation site (GAS) element, wherein the activation of the ISRE or GAS induces an interferon-like activity.

[0103] 31. A kit comprising the non-naturally occurring interferon beta protein of any one of embodiments 1-8, or the pharmaceutically composition of any one of embodiments 10-12, and a container.

[0104] 32. A non-naturally occurring interferon beta (IFN-beta) protein, comprising an amino acid sequence having an amino acid substitution with respect to the IFN-beta protein amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution is selected from the amino acid substitutions in Table 3.

[0105] 33. The non-naturally occurring IFN-beta protein of embodiment 32, wherein the non- naturally occurring IFN-beta has a structural feature that is present in naturally occurring IFN- beta protein, as determined by modeling of the non-naturally occurring IFN-beta structure.

[0106] 34. The non-naturally occurring IFN-beta protein of embodiment 33, wherein the structural feature comprises an alpha-helix.

[0107] 35. The non-naturally occurring IFN-beta protein of embodiment 34, wherein the structural feature is a helix-turn-helix structural motif.

[0108] 36. The non-naturally occurring IFN-beta protein of embodiment 32, wherein the non- naturally occurring IFN-beta protein binds to the interferon-a / p receptor- 1 / interferon-a / p receptor-2 heterodimer at a level substantially equal to the naturally occurring IFN-beta.

[0109] 37. The non-naturally occurring IFN-beta protein of embodiment 32, wherein the non- naturally occurring IFN-beta protein, when administered to a subject, reduces a level of inflammation in the subject, relative to a level of inflammation in the subject prior to the administering.

[0110] 38. The non-naturally occurring IFN-beta protein of any one of embodiments 32-37, wherein the protein is a recombinant IFN-beta or a portion thereof.[OlH] 39. The non-naturally occurring IFN-beta protein of any one of embodiments 32-38, wherein the non-naturally occurring IFN-beta protein comprises a post-translational modification.

[0112] 40. A method of making a non-naturally occurring interferon beta (IFN-beta) protein in a cell, comprising: a) transforming the cell with a vector encoding the non-naturally occurring IFN-beta protein of any one of embodiments 32-39; b) expressing the IFN-beta protein in the cell; and c) isolating the non-naturally occurring IFN-beta protein.

[0113] 41. A pharmaceutical composition in unit dose form comprising: (a) the non-naturally occurring interferon beta (IFN-beta) protein of any one of embodiments 32-39 or a vector encoding the non-naturally occurring IFN-beta protein of any one of embodiments 32-39; and (b) a pharmaceutically acceptable: excipient, carrier, or diluent.

[0114] 42. The pharmaceutical composition of embodiment 41, wherein the pharmaceutical composition is encapsulated.

[0115] 43. The pharmaceutical composition of embodiment 41, wherein the pharmaceutical composition is in the form of a liquid.

[0116] 44. 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 IFN-beta protein of any one of embodiments 32-39, or the pharmaceutically composition of any one of embodiments 41-43, thereby treating the subject.

[0117] 45. The method of embodiment 44, wherein the disease is an infection.

[0118] 46. The method of embodiment 45, wherein the infection is a viral infection.

[0119] 47. The method of embodiment 46, wherein the viral infection is a chronic Hepatitis B infection, or a chronic Hepatitis C infection.

[0120] 48. The method of embodiment 44, wherein the disease is multiple sclerosis (MS).

[0121] 49. The method of embodiment 48, wherein the MS is relapse remitting MS, nonrelapsing MS, clinically isolated syndrome, and secondary progressive forms.

[0122] 50. The method of any one of embodiments 44-49, wherein the subject is a human subject.

[0123] 51. The method of any one of embodiments 44-50, wherein the administering is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra-ci sternal injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, a surgical route, or any combination thereof.

[0124] 52. The method of embodiment 44, 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.

[0125] 53. The method of embodiment 44, 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 2000 mg.

[0126] 54. The method of embodiment 44, 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.

[0127] 55. The method of embodiment 44, wherein the administering reduces a volume of a tumor in the subject.

[0128] 56. The method of embodiment 44, wherein the administering inhibits the growth a tumor in the subject.

[0129] 57. The method of embodiment 44, wherein the method further comprises administering a second therapeutic.

[0130] 58. The method of embodiment 57, wherein the second therapeutic is an antibody, a peptide, an anti-body drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.

[0131] 59. The method of embodiment 44, wherein the method further comprises further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor.

[0132] 60. The method of embodiment 59, wherein the checkpoint inhibitor is a PD-1 or a PD- L1 inhibitor.

[0133] 61. The method of embodiment 44, 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.

[0134] 62. A kit comprising the non-naturally occurring interferon beta protein of any one of embodiments 32-39, or the pharmaceutically composition of any one of embodiments 41-43, and a container.EXAMPLESEXAMPLE 1 - Generation of Engineered Interferon Variants

[0135] A library of potential improved engineered interferon Beta (IFN-beta) variants was generated. The library was a plasmid library designed for generation of a lentiviral library and contains the cDNA for human Interferon Beta library with variants. The cDNAs were expressed under a Doxycycline inducible protomer operably-linked to a polynucleotide encoding a Type II transmembrane domain of human Transferrin receptor fused to the coding region of mature IFNP, separated by a short linker sequence (GGGGS) and a Myc affinity tag, such that the C- terminus of IFNP extends into the extracellular space. This arrangement of the transmembrane domain of the transferrin receptor and the piece of the intracellular domain allows the fusion protein to be shuttled to the plasma membrane, resulting in subsequent expression of IFN-beta on the cell surface. When induced with Doxycycline, the IFN-beta region of the expressed fusion protein binds the endogenous or ectopically expressed IFNAR1 and IFNAR2 receptor heterodimer and, in the same cell (autocrine) signals through the Jak / Stat pathway to the Interferon Stimulation Response Element (ISRE) upstream of a zsGreen cDNA to drive expression of this transcriptional reporter (FIG. 1). The IFN-beta polypeptide was saturation mutagenized in order to produce amino acid substitutions at each position. Each unique clone in the plasmid library was barcoded with a unique molecular identifier and sequenced with long reads by NGS. Each unique molecule encoding a variant is associated with a unique barcode.EXAMPLE 2 - High-throughput screening of IFN variants

[0136] 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-P plus a red fluorescent protein (mScarlet) were co-cultured with cells expressing endogenous IFNAR and an ectopic ISRE reporter driving expression of a green fluorescent protein (ZsGreenl-DR). In this assay, only cells that undergo paracrine signaling become positive for ZsGreenl. At a cell density of 2,500 cells / cm2, <2.5% of the reporter cells became green. This density was used for autocrine assays.

[0137] In cell lines transduced with both IFN -mScarlet and ISRE- ZsGreenl, >80% of cells become green when IFNP expression is stimulated with doxycycline, validating robust autocrinesignaling. No IFNp ligand shedding was detected in conditioned media from tethered-IFNP- expressing cells. Cells were harvested, fixed, and partitioned into bins by Flow Sorting. Libraries for NGS were prepared for each bin and the number of times each barcode occurs in each pool is quantified by NGS. These data were used to create an activity score and each variant will generally have 100s of barcodes (FIG. 2). This population of barcodes for a single variant was compared to the set of barcodes for wild type IFN-beta (reference) and used to identify variants that have signaling activity significantly higher than WT (Gain of function), which were separated from variants that are significantly lower than WT (Loss of function).

[0138] From this screen, novel engineered IFN-beta variants having more signaling activity significantly higher than WT IFN-beta were discovered, with these variants recited in Table 1 and Table 3. The reporter activity was a weighted mean derived from the distribution of the reads in each of the cell sorted bins, ranked by fluorescence intensity (1-4 with 4 being the highest, labeled as “score” in the following tables) of the frequency of each barcode in each bin. The reporter activities for all barcodes for each of the engineered IFN-beta proteins was compared to that of wildtype IFN beta (Tables 7 - 8). Several of the engineered IFN-beta proteins identified had activity similar to wildtype and were classified as “WT,” while other engineered IFN-beta proteins with increased activity and were classified as gain of function (“GOF”) or gain of function-Like (“GOF-like”).TABLE 7: Engineered IFN-beta with increased activity as compared to corresponding wildtype IFN-beta.TABLE 8: Engineered IFN-beta with substantially similar activity as compared to corresponding wildtype IFN-beta.

[0139] The coding sequences of the exemplary were synthesized and fused to a 6x HIS affinity tag on the N-terminus and cloned into the mammalian expression vector pCDNA3.4 (containing a CMV promoter, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE),and HSV-TK poly(A) sequence). Proteins expressed during transient transfection in TurboCHO cells were purified from the media by the TurboCHO High-Throughput protein purification service. Purity was assessed via SDS-PAGE, capillary electrophoresis, and HPLC, and the protein concentration measured via Bradford Assay. Proteins were further purified by gel filtration or hydrophobic interaction columns, taking advantage of the small size and high hydrophobicity of IFNp. The purified protein was lyophilized for storage and reconstituted in pharmaceutically acceptable excipients.

[0140] Signaling activity of purified engineered IFN-beta proteins was measured by FACS, and cells were scored from 1.0 to 3.0, with WT IFNP having an average score of 2.15 (FIG. 3). Sequence analysis showed that most of the engineered IFN-beta proteins had near WT activity (FIGs. 4A-4D). However, 250,000 additional variants were created from errors in oligo synthesis. These clones included 1,000s of Loss of Function (LOF) or Gain of Function (GOF) variants (FIGs. 4A-4D). These random variants have less barcode coverage, but they encode a rich subset of GOF engineered IFN-beta proteins that can be examined as an alternative approach if PTMs do not vastly improve IFN activity. Assay designed to identify biosimilar variants by separating LOF activity from WT. GOF variants were expected to receive WT classification.

[0141] The depth of data achieved in a GigaAssay is illustrated in a bin plot for an exemplary engineered IFN-beta protein of the -250,000 engineered IFN-beta proteins (FIGs. 5A). This engineered IFN-beta protein had an average score of 2.15 in the GigaAssay and was classified as an IFNP LOF variant. The activity scores for 145 independently barcoded cells ranged predominantly from 1.6 to 2.1, whereas 37,000 such measurements for WT IFNP were centered at 2.0 to 2.6 (FIG. 5A). The loss of function was also verified by flow cytometry in cells stimulated with purified engineered IFN-beta proteins or WT IFN-beta protein (FIG. 5B).

[0142] A bin plot for an exemplary engineered IFN-beta protein of the -250,000 engineered IFN-beta proteins (FIG. 6A). This engineered IFN-beta protein had an average score of 1.72 in the GigaAssay and was classified as an IFN-P biosimilar. The activity scores for 106 independently barcoded cells ranged predominantly from 2.0 to 2.6, which is comparable with the 37,000 such measurements for WT IFNP were centered at 2.0 to 2.6 (FIG. 6A). The engineered IFN-beta protein with biosimilar activity was also verified by flow cytometry in cells stimulated with purified engineered IFN-beta protein (FIG. 6H) or WT proteins (FIGs. 6B - 6F). EC50 for cell-based reporter assay was measured in ng / m, and p-values were False Discovery Rate (FDR) adjusted (FIG. 6C). IFNP-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. TotalRNA was extracted and subjected to qPCR targeting the p2-micoglobulin and 2’-5’ oligoadenylate synthetase 1 genes (OAS1) (FIGs. 61 - 6J). OAS1 expression was normalized to house-keeping gene, HPRT. Error bars represent mean + / - SD for n=3 biological replicate, ns = p = >0.05, * p = <0.05. Untreated cells were included as controls.

[0143] A bin plot for an exemplary engineered IFN-beta protein with increased activity of the -250,000 engineered IFN-beta proteins (FIG. 7A). This engineered IFN-beta protein had an average score of 2.25 in the GigaAssay and was classified as an IFN-P Biobetter, or an engineered IFN-beta protein with increased biological activity. The activity scores for 138 independently barcoded cells ranged predominantly from 2.20 to 3, whereas 37,000 such measurements for WT IFNP were centered at 2.0 to 2.6 (FIG. 7A). EC50 for cell-based reporter assay was measured in ng / m, and p-values were False Discovery Rate (FDR) adjusted (FIG. 7B- 7C). The engineered 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. 7D - 71). IFNP-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 p2-micoglobulin and 2’-5’ oligoadenylate synthetase 1 genes (OAS1) (FIGs. 7J - 7K). OAS1 expression was normalized to house-keeping gene, HPRT. Error bars represent mean + / - SD for n=3 biological replicate, ns = p = >0.05, * p = <0.05. Untreated cells were included as controls.EXAMPLE 3 - Methods of treating a disease or a condition using an engineered IFN-beta protein

[0021] We note that any of the engineered IFN-beta proteins could be modified with glycosylation sites or pegylated, modifications used in many known drugs. The engineered IFN- beta proteins as disclosed herein are relevant to diseases involving viral infectious diseases, melanoma, genital warts, lupus, and psoriasis. Examples for each category are discussed.Antiviral Therapies with engineered IFN-beta proteins

[0022] Engineered IFN-beta proteins disclosed herein can act as potent antiviral treatment.

[0023] A human subject diagnosed with human immunodeficiency virus (HIV) is administered a therapeutically effective amount of the engineered IFN-beta proteins disclosed herein, and the engineered IFN-beta proteins alleviate the symptoms associate with HIV. Several engineered IFN-beta proteins described in EXAMPLE 1 and 2 have significant increased ISRE signaling activity as compared to wildtype IFN-beta and could be used as agonists for HIV and Acquired immunodeficiency syndrome (AIDS) suppression therapeutics.

[0024] A human subject diagnosed with coronavirus disease 2019 (COVID-19) is administered a therapeutically effective amount of the engineered IFN-beta proteins disclosed herein, and the engineered IFN-beta proteins alleviate the symptoms associate with COVID-19. Several engineered IFN-beta proteins described in EXAMPLE 1 and 2 have significant increased ISRE signaling activity and could be to prevent COVID-19 hospitalization and progression.

[0025] A human subject diagnosed with Chronic hepatitis B (CHB) is administered a therapeutically effective amount of the engineered IFN-beta proteins disclosed herein, and the engineered IFN-beta proteins alleviate the symptoms associate with CHB. Several engineered IFN-beta proteins described in EXAMPLES 1 and 2 have significant increased ISRE signaling activity and are proposed efficacious therapeutic method of alleviating CHB symptoms.Oncological Therapies With Engineered IFN-beta proteins

[0026] Engineered IFN-beta proteins disclosed herein can act as potent anti-cancer treatment.

[0027] A human subject diagnosed with melanoma is administered a therapeutically effective amount of the engineered IFN-beta proteins disclosed herein, and the engineered IFN-beta proteins alleviate the symptoms associate with Melanoma. Melanoma is skin neoplasm derived from melanocytes. Several engineered IFN-beta proteins described in EXAMPLE 1 and 2 have significant increased ISRE signaling activity and could be to treat melanoma.

[0028] A human subject diagnosed with glioblastoma multiforme (GBM) is administered a therapeutically effective amount of the engineered IFN-beta proteins disclosed herein, and the engineered IFN-beta proteins alleviate the symptoms associate with GBM. GBM is a brain neoplasm. Several engineered IFN-beta proteins described in EXAMPLE 1 and 2 have significant increased ISRE signaling activity and could be to treat GBM.Autoimmune Therapies With Engineered IFN-beta proteins

[0029] Engineered IFN-beta proteins disclosed herein can act as potent autoimmune therapies.

[0030] A human subject diagnosed with Multiple Sclerosis (MS) is administered a therapeutically effective amount of the engineered IFN-beta proteins disclosed herein, and the engineered IFN-beta proteins alleviate the symptoms associate with MS. Single IFNP and modified IFNP polypeptides are used to treat MS. Several engineered IFN-beta proteins described in EXAMPLE 1 and 2 have significant increased ISRE signaling activity as compared to wildtype and some modified IFNP polypeptides. Thus, the engineered IFN-beta proteins disclosed herein are better therapeutics for treating and alleviating the symptoms of MS. These include any of the engineered IFN-beta proteins screened and identified in EXAMPLE 1 and 2.Other indications

[0031] The engineered IFN-beta proteins is used to target specific indication. For any desired indication, purified His-tagged engineered IFN-beta proteins or purified engineered IFN-beta proteins are tested for signaling potency in a reporter cell plate readers assay to determined EC50 and compared to other drugs including FDA-approved reference interferon-beta drug. Similar experiments are repeated to determine a cytopathic effect assay which is a standard test for IFN therapies. Then cells related to the indication is stimulated with the engineered IFN-beta proteins and transcripts induced are compared to control cells to determine which engineered IFN-beta proteins are most appropriate and potent for the indication. The engineered IFN-beta proteins are then further tested in pre-clinical animal models followed by human studies.

[0032] 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

CLAIMSWhat is claimed is:

1. An engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising at least one amino acid substitution in a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution is selected from the amino acid substitutions in Table 1, and wherein the at least one amino acid substitution provides for an increase in a biological activity of the engineered IFN-beta protein as compared to the wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1.

2. The method of claim 1, wherein the increase in the biological activity comprises an increase in an expression of one or more biological targets of the engineered IFN-beta protein as compared to wildtype IFN-beta protein, as measured by an in vitro assay.

3. The engineered IFN-beta protein of claim 1, wherein the engineered IFN-beta protein or a functional fragment thereof comprises at least two amino acid substitutions, at least three amino acid substitutions or at least four amino acid substitutions from Table 1.

4. The engineered IFN-beta protein of claim 3, comprising the at least two amino acid substitutions that are selected from the group consisting of E103F & L106K, E61Q & E104F, F67A & I83P, F67A & N86K, F67K & KI 081, F67Y & L98E, F70L & L87F, F70T & L87M, F67Y & L98E, G78M & Y92Q, I59K & Q72S, I59T & S75F, I66S & Y92H, I66W & E103G, K99I & DI 10R, L57M & L106V, L63M & Fl 11R, L63M & L88R, N65A & L98Y, N86D & E107A, N86I & L98D, N86K & L102Y, N86W & L102P, Q18K & L57T, Q18K & T82W, Q64W & E103W, S75K & L106N, S75L & V101T, T58C & Fl 1 II, W79H & E104P, Y60M & E85V, R124L & I145V, Ml 17A & L164E, KI 15R & N153G, G114N & Y163S, T144V & R152R, N86W & L102P, T58I & D73G, Q64A & L87M, Y60N & I95T, E61C & DI 10Y, R71K & Y92F, N4K & L47L, L20E & I44M, R35V & K45N, S13Y & Y30V, N14A & D34I, S12F & K33K, L28C & E43N, W22I & E53M, Y30L & K45G, N80Q & F111E, S74N & I83K, SI 19H & A142S, Y126K & N158W, S119G & E137E, H121W & N153M, T112S & I157L, S119P & R152K, L21Y & W22S, S12I & E53Q, N4K & L47H, L6M & I44H, S13E & I44V, Y3H & Q48Y, L6D & W22C, G114V & L164M, L9W & R35G, S13A & K52Q, L20R & Q49A, C17D & I44K, R27M & L47L, D34H & A55S, Q10S & W79L, L28F & MON, M62A & T100L, Y60K & A89V, Q18K & G78F, L57F & L87E, V91Q & E109N, Q64E & Y92V, A68I & D73N, R128M & T161P, Ml 17Y & F156T, K115N & L164*, L120H & F156W, S13R & R35V, Q16A & K52P, W22R & E29N, R1 ID & Q51F, L47F & L120M, R11Y & F50G, G7A & I40F, S2F & G26F, K33N & N37A, L32Y & F50W, R35I & Q49H, R1 IF & Q46Y, G7A & Q18H, L6A & D34K, F8G &F50V, K19Y & R27K, F8F & S13E, S13P & R35T, KI 151 & H121P, H121N & H131K, Q51K & L160G, KI 15Y & G127T, T112L & Y132W, L122R & T161V, N158A & N166P, L116T & I150G, and R113K & L160T.

5. The engineered IFN-beta protein of claim 3, comprising the at least three amino acid substitutions from Table 1 that are L57Q & E81S & H97S, V84T & K108K & E109T, F8D & Q23P & K108N, R1 IQ & K52N & D54T, R11 Y & E42D & E109D, R35M & E53P & V91V, G7M & R27M & G127W, L122V & V148A & L160R, and L120M & K134K & A135T.

6. The engineered IFN-beta protein of claim 3, comprising the at least four amino acid substitutions from Table 1 that are N80Q & V84N & N86T & F 111 S.

7. The engineered IFN-beta protein of any one of claims 1 to 6, wherein the engineered IFN- beta protein or a functional fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332.

8. The engineered IFN-beta protein of claim 6, wherein the engineered IFN-beta protein or a functional fragment thereof comprises an amino acid sequence of any one of SEQ ID NO: 2- SEQ ID NO: 104, or SEQ ID NO: 106 - SEQ ID NO: 332.

9. The engineered IFN-beta protein of any one of claims 1 to 8, wherein the engineered IFN- beta protein or a functional fragment thereof comprises a structural feature that is present in wildtype IFN-beta protein, as determined by modeling of the engineered IFN-beta structure.

10. The engineered IFN-beta protein of claim 9, wherein the structural feature comprises an alpha-helix.

11. The engineered IFN-beta protein of claim 9, wherein the structural feature comprises a helixturn-helix structural motif.

12. The engineered IFN-beta protein of any one of claims 1 to 11, wherein the engineered IFN- beta protein or a functional fragment thereof binds to an interferon-a / p receptor- l(IFNARl) or interferon-a / p receptor-2 (IFNAR2) heterodimer subunits at a level greater than the wildtype IFN-beta protein, as measured by an in vitro assay.

13. The engineered IFN-beta protein of claim 12, wherein the in vitro assay comprises 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.

14. The engineered IFN-beta protein of any one of claims 1 to 13, wherein the engineered IFN- beta protein or functional fragment thereof binds and activates IFNAR1 or IFNAR2 at a levelgreater than the wildtype IFN-beta protein, as measured by an ISRE-GFP reporter assay or an ISRE luciferase reporter assay.

15. The engineered IFN-beta protein of claim 14, wherein the engineered IFN-beta protein or functional fragment thereof is an agonist of IFNAR1 or IFNAR2 subunits.

16. The engineered IFN-beta protein of any one of claims 1 to 15, wherein the engineered IFN- beta protein or functional fragment thereof reduces an extracellular level of a pro- inflammatory cytokine, as compared to the wildtype IFN-beta protein having the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

17. The engineered IFN-beta protein of claim 16, wherein the pro-inflammatory cytokine is a chemokine, an interferon (TFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof.

18. The engineered IFN-beta protein of claim 16, wherein the pro-inflammatory cytokine is IL- 10, IL-6, IL-8, IL-9, IL- 12, IL- 15, IL- 17, IL- 18, IFN-y, TNF-a, TNF-0, or any combination thereof.

19. The engineered IFN-beta protein of any one of claims 1 to 15, wherein the engineered IFN- beta protein or functional fragment thereof increases an extracellular level of an antiinflammatory cytokine, as compared to the wildtype IFN-beta protein having the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.

20. The engineered IFN-beta protein of claim 19, 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.

21. The engineered IFN-beta protein of claim 19, wherein the anti-inflammatory cytokine is IL- 4, IL-10, IL-11, IL-13, IFN-a, TGF-0, or any combination thereof.

22. The engineered IFN-beta protein of any one of claims 1 to 21, wherein the engineered IFN- beta protein or a functional fragment thereof, when administered to a subject, reduces a level of inflammation in the subject, relative to a level of inflammation in the subject prior to the administering.

23. The engineered IFN-beta protein of claim 22, 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.

24. The engineered IFN-beta protein of claim 22, 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.

25. The engineered IFN-beta protein of claim 24, 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.

26. The engineered IFN-beta protein of claim 22, wherein the administering of the engineered IFN-beta protein or a functional fragment thereof to the subject results in reduced blood levels of a pro-inflammatory cytokine, as compared to the blood levels of the pro- inflammatory cytokine before the administering, as measured by ELISA.

27. A method of making an engineered interferon beta (IFN-beta) protein or a functional fragment thereof in a cell, comprising introducing into the cell a vector encoding the engineered IFN-beta protein of any one of claims 1 to 26, wherein the introducing results in expression of the engineered IFN-beta protein in the cell.

28. The method of claim 27, wherein the engineered IFN-beta protein or a functional fragment thereof is isolated from the cell.

29. The method of claim 27, wherein the vector comprises a mammalian expression plasmid.

30. The method of claim 27, wherein the engineered IFN-beta protein comprises a tag.

31. The method of claim 30, wherein the tag is CBP, FLAG, GST, Myc, poly-His, or V5.

32. The method of claim 30, wherein the tag comprises a cleavable tag or a non-cleavable tag.

33. A polynucleotide encoding the engineered IFN-beta protein or a functional fragment thereof of any one of claims 1 to 26.

34. The polynucleotide of claim 33, wherein the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 1609 - SEQ ID NO: 1711 or SEQ ID NO: 1713 - SEQ ID NO: 1939.

35. The polynucleotide of claim 33, wherein the polynucleotide comprises a nucleic acid sequence of any one of SEQ ID NO: 1609 - SEQ ID NO: 1711 or SEQ ID NO: 1713 - SEQ ID NO: 1939.

36. A vector encoding the polynucleotide of any one of claims 33 to 35.

37. A pharmaceutical composition in unit dose form comprising: a) the engineered IFN-beta protein of any one of claims 1 to 26, the polynucleotide of any one of claims 33 to 35, or the vector encoding the engineered IFN-beta protein of claim 36; and (b) a pharmaceutically acceptable: excipient, carrier, or diluent38. The pharmaceutical composition of claim 37, wherein the pharmaceutical composition is encapsulated.

39. The pharmaceutical composition of claim 37, wherein the pharmaceutical composition is in the form of a liquid.

40. The pharmaceutical composition of any one of claims 37 to 39, wherein the pharmaceutical composition is formulated for local, systemic, or topical administration.

41. The pharmaceutical composition of any one of claims 37 to 40, wherein the pharmaceutical composition is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parental, intravenous, or intramuscular administration.

42. The pharmaceutical composition of any one of claims 37 to 41, wherein the pharmaceutical composition is formulated for controlled-release.

43. The pharmaceutical composition of any one of claims 37 to 42, wherein the pharmaceutical composition is formulated for single-dosage administration.

44. A method of treating a demyelinating disorder in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an engineered interferon beta (IFN-beta) protein or a functional fragment thereof that is sufficient to treat the demyelinating disorder, wherein the engineered IFN-beta protein comprises at least one amino acid substitution selected from the amino acid substitutions in Table 1 that provides for an increase in biological activity of the engineered IFN-beta protein, as compared to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1.

45. A method of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of the engineered IFN-beta protein of any one of claims 1 to 26, the polynucleotide of any one of claims 33 to 35, the vector encoding the engineered IFN-beta protein of claim 36 or the pharmaceutically composition of any one of claims 37 to 43, thereby treating the subject.

46. The method of claim 45, wherein the disease is an infection.

47. The method of claim 46, wherein the infection is a viral infection.

48. The method of claim 47, wherein the viral infection is a chronic Hepatitis B infection, or a chronic Hepatitis C infection.

49. The method of claim 45, wherein the disease is a demyelinating disorder.

50. The method of claim 49, 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).

51. The method of claim 50, wherein the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.

52. The method of any one of claims 45 to 50, wherein the subject is a human subject.

53. The method of claim any one of claims 45 to 52, wherein the administering is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra-ci sternal injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, a surgical route, or any combination thereof.

54. The method of claim 45, 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.

55. The method of claim 45, 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 2000 mg.

56. The method of claim 45, 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.

57. The method of claim 45, wherein the administering reduces a volume of a tumor in the subject.

58. The method of claim 45, wherein the administering inhibits the growth a tumor in the subj ect.

59. The method of claim 45, wherein the method further comprises administering a second therapeutic.

60. The method of claim 59, wherein the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.

61. The method of claim 45, wherein the method further comprises further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor.

62. The method of claim 61, wherein the checkpoint inhibitor is a PD-1 or a PD-L1 inhibitor.

63. The method of claim 45, 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.

64. A kit comprising the engineered IFN-beta protein of any one of claims 1 to 26, the polynucleotide of any one of claims 33 to 35, the vector encoding the engineered IFN-beta protein of claim 36 or the pharmaceutically composition of any one of claims 37 to 43, and a container.

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