WO2025194144A2 - Protéines bêta d'interféron modifiées glycosylées - Google Patents

Protéines bêta d'interféron modifiées glycosylées

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Publication number
WO2025194144A2
WO2025194144A2 PCT/US2025/020102 US2025020102W WO2025194144A2 WO 2025194144 A2 WO2025194144 A2 WO 2025194144A2 US 2025020102 W US2025020102 W US 2025020102W WO 2025194144 A2 WO2025194144 A2 WO 2025194144A2
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WO
WIPO (PCT)
Prior art keywords
protein
engineered
ifn
amino acid
beta
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/020102
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English (en)
Other versions
WO2025194144A3 (fr
Inventor
Martin Roy Schiller
Jerome I. Rotter
Christopher John GIACOLETTO
Lancer Brown
Elizabeth Joy VALENTE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Heligenics Inc
Original Assignee
Heligenics Inc
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Publication date
Application filed by Heligenics Inc filed Critical Heligenics Inc
Publication of WO2025194144A2 publication Critical patent/WO2025194144A2/fr
Publication of WO2025194144A3 publication Critical patent/WO2025194144A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • engineered interferon beta (IFN-beta) proteins or a functional fragments thereof comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1.
  • the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • the modification comprises an amino acid substitution, a deletion, or an insertion in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the modification is an amino acid substitution
  • the amino acid substitution comprises at least one amino acid substitution in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the modification is a deletion of one or more amino acids in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the modification comprises an insertion of one or more amino acids in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the at least one amino acid substitution results in an increase in a biological activity of the engineered IFN-beta protein as compared to a biological activity of the wildtype protein.
  • the at least one amino acid substitution results in a decrease in a biological activity of the engineered IFN-beta protein as compared to a biological activity of the wildtype protein.
  • the increase in the biological activity of biological activity comprises an increase in signaling, an increase in protein expression of one or more biological targets, an increase in mRNA expression, or any combination thereof, as measured by an in vitro assay.
  • the increase in signaling comprises an increase in IFNAR1-mediated signaling, as measured by an in vitro assay.
  • increase in signaling comprises an increase in IFNAR2-mediated signaling, as measured by an in vitro assay.
  • the modification comprises: (a) an amino acid substitution Attorney Docket No.217863-712601 that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a glycosylation motif into a glycosylation motif, thereby introducing a glycosylation motif into the engineered protein; (b) an amino acid substitution that results in a conversion of a glycosylation motif in the amino acid sequence of SEQ ID NO: 1 into a motif that is not a glycosylation motif, thereby eliminating a glycosylation motif from the engineered protein; or (c) a combination of (a) and (b).
  • the at least one amino acid substitution results in the conversion of 1, 2, 3, 4, or more motif(s) in the amino acid sequence of SEQ ID NO: 1 that are not glycosylation motifs into 1, 2, 3, 4, or more motif(s) that are glycosylation motif(s), thereby introducing 1, 2, 3, 4, or more glycosylation motif(s) into the engineered protein.
  • the at least one amino acid substitution results in the conversion of 1, 2, 3, 4, or more glycosylation motif(s) in the amino acid sequence of SEQ ID NO: 1 into 1, 2, 3, 4, or more motif(s) that are not glycosylation motifs, thereby eliminating 1, 2, 3, 4, or more glycosylation motif(s) into the engineered protein.
  • the glycosylation motif has a consensus sequence of N-X-S/T, wherein N is an Asparagine that is N-glycosylated at the amide bond in the side chain of the Asparagine, X is any amino acid except for proline, S is a serine, and T is a threonine.
  • the modification comprises an amino acid substitution that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a site for a post-translational modification into a post-translational modification consensus sequence motif, thereby introducing a site for a post translational modification into the engineered protein.
  • the post-translational modification consensus sequence motif is selected from the amino acid sequences in TABLE 5.
  • the amino acid substitution results in the addition of a post-translational modification on the engineered protein at the site of the post-translational modification consensus sequence motif that is not present in the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the post-translational modification is selected from the group consisting of: glycosylation, phosphorylation, lipidation, glycation, proline isomerization, methylation, acetylation, hydroxylation sites, and proteolysis.
  • the engineered protein is glycosylated.
  • the engineered protein is aglycosylated.
  • the engineered protein binds to an interferon- ⁇ / ⁇ receptor-1(IFNAR1) or an interferon- ⁇ / ⁇ receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by enzyme-linked immunosorbent assay (ELISA), or by measuring signaling activity of an Interferon Stimulated Response Element (ISRE)-Green fluorescent protein (GFP) reporter in a live human cell in vitro assay.
  • IFNAR1 interferon- ⁇ / ⁇ receptor-1
  • IFNAR2 interferon- ⁇ / ⁇ receptor-2
  • the engineered protein binds to and activates an IFNAR1 or an IFNAR2 heterodimer subunits at a level greater than or equal to the Attorney Docket No.217863-712601 wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA, or by measuring signaling activity of an ISRE-GFP reporter in a live human cell in vitro assay.
  • the engineered protein binds to and inhibits an activity of an IFNAR1 or an IFNAR2 heterodimer subunits at a level greater than or equal to the wildtype IFN- beta protein as measured by ELISA, or by measuring signaling activity of an ISRE-GFP reporter in a live human cell in vitro assay.
  • the engineered protein is an agonist of IFNAR1 or IFNAR2 subunits.
  • the engineered protein binds and activates IFNAR1 or IFNAR2 at a similar level than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE-GFP reporter assay or an ISRE luciferase reporter assay.
  • the engineered protein reduces an extracellular level of a pro-inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.
  • the pro- inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof.
  • the pro- inflammatory cytokine is IL-1 ⁇ , IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , or any combination thereof.
  • the engineered protein increases an extracellular level of an anti-inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.
  • the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof.
  • the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN- ⁇ , TGF- ⁇ , or any combination thereof.
  • the engineered protein binds to IFNAR1 or IFNAR2 subunits at a level less than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA. In some embodiments, the engineered protein is an antagonist of IFNAR1 or IFNAR2 subunits. In some embodiments, the engineered protein binds and inhibits an activity of the IFNAR1 or IFNAR2 at a level greater than wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE GFP reporter assay or an ISRE luciferase reporter assay.
  • the engineered protein is a recombinant IFN-beta or a portion thereof.
  • the engineered protein is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, conjugated to a polyethylene glycol (PEG) moiety, or any combination thereof.
  • PEG polyethylene glycol
  • a glycosylation site is introduced in the engineered protein that results in diminished binding of an antibody to the engineered protein, relative to an amount of binding of the antibody to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the modification results in a reduction of immunogenicity levels when administered to a subject, as compared to an Attorney Docket No.217863-712601 immunogenicity level when the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 is administered to a subject.
  • the reduction of immunogenicity levels comprises a reduced level of anti-drug antibody in the subject when the engineered protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN- beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject.
  • the engineered protein when administered to a subject in need thereof results in a reduction in a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering.
  • the reduction in the level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA.
  • the reduction in the level of inflammation in the subject results in a reduction in serum levels of C reactive protein as measured by high-sensitivity C-reactive protein (hs-CRP) test.
  • hs-CRP high-sensitivity C-reactive protein
  • the reduction in serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg/dL to about 2.9 mg/dL.
  • the administering to the subject results in a reduction in blood levels of a pro- inflammatory cytokine as compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA.
  • the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence length to any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the functional fragment thereof comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to any one of SEQ ID NO: 2 – SEQ ID NO: 208. In some embodiments, the engineered protein comprises an amino acid sequence that comprises a natural amino acid, or a non-natural amino acid.
  • Also provided herein are methods of making a engineered IFN-beta protein in a cell comprising introducing into the cell a vector encoding the engineered IFN-beta protein disclosed herein. In some embodiments, the introducing results in expression of the engineered IFN-beta protein in the cell. In some embodiments, the engineered protein is isolated from the cell. In some embodiments, the vector comprises a mammalian expression plasmid. In some embodiments, the engineered IFN-beta protein comprises a tag. In some embodiments, the tag is CBP, FLAG, GST, Myc, poly-His, or V5. In some embodiments, the tag comprises a cleavable tag or a non-cleavable tag.
  • an engineered interferon beta (IFN-beta) protein or a functional fragment thereof comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1.
  • the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • the polynucleotide comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 209 – SEQ ID NO: 342. In some embodiments, the polynucleotide comprises any one of SEQ ID NO: 209 – SEQ ID NO: 342. In some embodiments, the polynucleotide comprises at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence length to any one of SEQ ID NO: 209 – SEQ ID NO: 342.
  • each codon in the polynucleotide can independently be replaced by a degenerate codon.
  • the polynucleotide comprise one or more degenerate codons.
  • the polynucleotide comprises a nucleic acid mimic.
  • the nucleic acid mimic comprises a phosphorothioate nucleic acid, a phosphoramidate nucleic acid, a morpholino nucleic acid, a hexitol nucleic acid (HNA), a peptide nucleic acid (PNA), or a locked nucleic acid (LNA).
  • one or more codons in the polynucleotide is modified.
  • one or more residues of an amino acid in the engineered IFN-beta protein is substituted for a non-natural amino acid.
  • vectors comprising the polynucleotides disclosed herein.
  • pharmaceutical compositions in unit dose form comprising: (a) the engineered proteins or the polynucleotides disclosed herein; and a pharmaceutically acceptable excipient, carrier, or diluent.
  • the pharmaceutical composition is encapsulated.
  • the pharmaceutical composition is in the form of a liquid.
  • the pharmaceutical composition is formulated for local, systemic, or topical administration.
  • the pharmaceutical composition is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parental, intravenous, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for controlled-release. In some embodiments, the pharmaceutical composition is formulated for single-dosage administration. [0007] Also provided herein are methods of treating a disease in a subject, the methods comprising: administering to the subject a therapeutically effective amount of a composition comprising (a) an engineered interferon beta (IFN-beta) protein or a functional fragment thereof; and (b) a pharmaceutically acceptable excipient, carrier, or diluent.
  • IFN-beta engineered interferon beta
  • the engineered IFN-beta protein comprises a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1.
  • the engineered IFN-beta protein Attorney Docket No.217863-712601 comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • the disease is an infection.
  • the infection is a viral infection.
  • the viral infection is chronic Hepatitis B infection, or a chronic Hepatitis C infection.
  • the disease is a demyelinating disorder.
  • 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).
  • the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.
  • the subject is a human subject.
  • 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.
  • 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.
  • the administering is at a dose selected from the group consisting of about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, or about 2000 mg.
  • 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.
  • the administering reduces a volume of a tumor in the subject.
  • the administering inhibits or diminishes the growth a tumor in the subject.
  • the methods further comprise administering a second therapeutic.
  • the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.
  • the methods further comprise administering an immunotherapy.
  • the immunotherapy comprises a checkpoint inhibitor.
  • the checkpoint inhibitor is a PD-1, a PD-L1, CTLA-4 inhibitor.
  • the administering induces activation of an IFN-stimulated response element (ISRE) or a Gamma interferon activation site (GAS) element, wherein the activation of the ISRE or GAS induces an interferon-like activity as measured by a luciferase reporter assay.
  • ISRE IFN-stimulated response element
  • GAS Gamma interferon activation site
  • methods of treating a disease or a condition in a subject comprising: administering to the subject a therapeutically effective amount of Attorney Docket No.217863-712601 engineered proteins disclosed herein, the polynucleotides disclosed herein, or the pharmaceutical composition disclosed herein, thereby treating the subject.
  • a demyelinating disorder comprising: administering to the subject a therapeutically effective amount of a composition comprising: (a) an engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208; and (b) a pharmaceutically acceptable excipient, carrier or diluent.
  • IFN-beta engineered interferon beta
  • 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).
  • the demyelinating disorder is a MS, wherein the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.
  • kits comprising the engineered proteins disclosed herein, the polynucleotides disclosed herein, or the pharmaceutical composition disclosed herein,, and a container.
  • FIG.1 depicts the design and application of the high-throughput molecular function assay system for assessing mutational effects on gene activity (MEGA) and to produce MEGA- maps.
  • FIG.2 illustrates the application of the high-throughput molecular function assay system to screen variants of IFN-beta for biosimilar activity.
  • FIG.3 depicts an overview of the high-throughput molecular function assay system to assess tethered IFN-beta autocrine signaling.
  • FIG.4 depicts a graph flow-cytometry profile of ISRE reporter activity in IFN-beta treated cells vs. untreated. Untreated HEK-293 interferon-responsive reporter cells is used as control (grey; untreated) to compare with HEK-293 cells treated with purified wildtype IFN ⁇ ligand (dark grey; treated). The x-axis depicts relative level fluorescence of ZsGreen and the y- axis depicts the number events (cell counts).
  • FIG.5 depicts a schematic of the purification of HIS-tagged engineered protein as described herein.
  • FIGs.6A-6B depicts a schematic for determination of EC50 of IFN-beta signaling in engineered protein as described herein.
  • FIG 6A depicts a 96-well plate assay for measuring EC50 of IFN signaling for comparing IFN chimera.
  • FIG 6B Dose response curve output from the 96-well ISRE reporter assay.
  • FIG.7 depicts an assay to determine immunogenicity of IFN leads using Peripheral Blood Mononuclear Cells (PBMCs).
  • PBMCs Peripheral Blood Mononuclear Cells
  • FIG.8A-8B depicts a schematic of the purification of un-tagged engineered protein as described herein.
  • FIG.8A depicts a schematic of the purification.
  • FIG.8B depicts amino acid sequence of 6xHIS and the Enterokinase Light Chain Cleavage Site (EKCS) protease site upstream of the engineered protein described herein, with the cleavage position of enterokinase indicated by a dashed vertical line.
  • FIG.9 depicts the dose response of the engineered proteins described herein to determine the EC50 values in an antiviral Cytopathic Effect (CPE) assay.
  • CPE Cytopathic Effect
  • FIG.10 illustrates a schematic to determine biomarker expression in human peripheral blood mononuclear cells (PBMCs) after treatment with the engineered proteins described herein.
  • FIG.11 depicts the prophylactic assessment of engineered proteins in Experimental Autoimmune Encephalomyelitis (EAE) mouse model for Multiple Sclerosis (MS).
  • EAE Experimental Autoimmune Encephalomyelitis
  • MS Multiple Sclerosis
  • FIG.12 depicts the liver toxicity assessment of engineered proteins described herein in mice.
  • FIG.13 depicts the pharmacokinetic study of the engineered proteins described herein in humans.
  • FIG.14 depicts the immunogenicity study of the engineered proteins described herein in humans.
  • FIGs.15A-15N depict the verification of the engineered proteins described herein on SDS-PAGE.
  • FIGs.16A-16L depict the plate reader output after cells were treated with exemplary engineered HIS-tagged engineered IFN-beta proteins or HIS-tagged wildtype IFN-beta protein.
  • the x-axis depicts the ligand concentration, and the y-axis depicts the ISRE reporter activity.
  • FIGs.17A-17G depict the flow cytometry measurements in cells treated with exemplary HIS-tagged engineered IFN-beta proteins increased activity (GOF variant) as compared to wildtype at different concentrations.
  • the x-axis depicts the ISRE reporter activity, and the y-axis depicts the cell counts.
  • FIGs.18A-18F depict quantitative RT-PCR determination of OAS1 mRNA expression in cells in expressing exemplary HIS-tagged engineered IFN-beta proteins as compared to HIS- tagged wildtype IFN beta protein.
  • DETAILED DESCRIPTION OF THE INVENTION [0031] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains.
  • Detecting the presence of includes determining the amount of something present, as well as determining whether it may be present or absent.
  • 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.
  • the term “operably coupled” refers to functional linkage between a regulatory sequence and a nucleic acid sequence resulting in expression of the latter.
  • open terms for example “contain,” “containing,” “include,” “including,” and the like mean comprising.
  • 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.
  • 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.
  • “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).
  • the term “homology” can refer to the relationship among sequences whereby there is some extent of likeness, typically due to descent from a common ancestral sequence. Homologous sequences can share homology based on genic, structural, functional and/or behavioral properties.
  • Similarity of a polypeptide to a reference polypeptide is generally determined based on conservation of certain properties of amino acids (such as conservative substitutions), and the degree of similarity can be determined using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis.53711).
  • the term “native” or “wildtype,” in reference to interferon beta (IFN-beta) refers to biologically active, naturally-occurring IFN-beta, including biologically active, naturally- occurring IFN-beta variants.
  • interferon beta As used herein, the terms “interferon beta,” “IFN-beta,” “IFN- ⁇ ” and “IFN ⁇ ” are used interchangeably, and refer to a polypeptide or protein with at least 95% sequence identity to SEQ ID NO: 1. [0042] The term includes the 166 amino acid human IFN-beta mature sequence (SEQ ID NO: 1). [0043] The term “glycosylation” refers to post-translational protein modification in which carbohydrates, particularly glycans, are attached to functional groups of amino acids.
  • Glycosylation is the process by which carbohydrates are converted to nitrogen on the side chain of asparagine or arginine (N-glycosylation); hydroxyl oxygens of serine, threonine, tyrosine, Attorney Docket No.217863-712601 hydroxylysine or hydroxyproline side chains (O-glycosylation); phosphate of phosphoserine (phosphoglycosylation); or attached to a carbon of the tryptophan side chain (C-glycosylation).
  • N-glycosylation N-glycosylation
  • hydroxyl oxygens of serine, threonine, tyrosine Attorney Docket No.217863-712601 hydroxylysine or hydroxyproline side chains (O-glycosylation); phosphate of phosphoserine (phosphoglycosylation); or attached to a carbon of the tryptophan side chain (C-glycosylation).
  • glycosylation motif refers to an amino-acid residue sequence that tends to be post-translationally modified by glycosylation (i.e., enzyme-catalyzed covalent linkage of one or more carbohydrate moieties) during protein biosynthesis.
  • glycosylation i.e., enzyme-catalyzed covalent linkage of one or more carbohydrate moieties
  • glycosylation motif refers to a consensus sequence motif that contains a site for glycosylation.
  • the site for glycosylation comprises an amino acid residue that is capable of forming a covalent linkage, wherein the covalent linkage is to a carbohydrate moiety.
  • the covalent linkage occurs via a nitrogen atom.
  • the nitrogen atom is in a sidechain amide group of an asparagine [Asn] residue).
  • subject refers 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.
  • suitable mammal can be administered a composition as described herein or be treated by a method as described herein.
  • 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.
  • 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.
  • 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.
  • a subject has or is suspected Attorney Docket No.217863-712601 of having a disease or condition, such as a cancer.
  • the subject is a patient, such as a patient being treated for a condition or a disease, such as a cancer.
  • a subject is a responder to cancer therapy. In some cases, a subject is a non-responder to a cancer therapy. A subject is predisposed to a risk of developing a condition or a disease. A subject is in remission from a condition or a disease. In some instances, a subject is healthy. A subject may be a subject in need thereof. A subject may have received a positive diagnosis of the cancer. A subject may have received a cancer therapy that failed to treat a cancer. [0048]
  • a “therapeutically effective amount” refers to an amount of a composition as disclosed herein with or without additional agents that is effective to achieve its intended purpose, for example to treat a disease. Individual patient needs may vary.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • 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.
  • endogenous refers to any material from or produced inside an organism, cell, tissue, or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue, or system.
  • 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.
  • 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.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non- plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • Sequence identity 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.
  • sequence identity between two sequences is a direct function of the number of matching positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are similar, the two sequences have 50% sequence identity, if 90% of the positions (e.g., 9 of 10), are matched, the two sequences have 90% sequence identity.
  • sequence identity means the percentage of identical subunits at corresponding positions in two sequences (e.g., nucleic acid sequences, amino acid sequences) when the two sequences are aligned to maximize subunit matching, therefore, taking into account gaps and insertions resulting from indels.
  • nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. [0060] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • 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).
  • linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • 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.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • polynucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which is hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides is hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in Attorney Docket No.217863-712601 the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • 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.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • the engineered proteins as described herein have binding affinity to an interferon- ⁇ / ⁇ receptor-1 (IFNAR1) or interferon- ⁇ / ⁇ receptor-2 (IFNAR2) heterodimer subunits, which is substantially similar or greater than the binding activity of the wildtype IFN-beta protein.
  • IFN- beta is a biologic used to reduce inflammation and treat inflammatory conditions, such as Multiple Sclerosis.
  • These novel engineered proteins are designed to have increased serum half- life, increased solubility, increased signaling, and reduced immunogenicity.
  • compositions comprising non-naturally occurring protein, methods of making the engineered protein, and methods for treating disease.
  • the engineered protein as described herein is a recombinant IFN-beta protein.
  • the engineered protein can act as a signaling agent.
  • the biological activity of the engineered IFN-beta protein is identical to wildtype IFN-beta.
  • Glycosylated engineered proteins Attorney Docket No.217863-712601 [0071] Disclosed herein are engineered interferon beta (IFN-beta) proteins or functional fragments thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • engineered, non-naturally occurring interferon (IFN)-beta protein variants comprising an amino acid substitution that results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a glycosylation motif into a glycosylation motif, thereby introducing a glycosylation motif into the engineered protein.
  • the amino acid substitution results in the conversion of a glycosylation motif in the amino acid sequence of SEQ ID NO: 1 into a motif that is not a glycosylation motif, thereby eliminating a glycosylation motif from the engineered protein.
  • the amino acid substitution introduces, eliminates or both introduces and eliminates one or more glycosylation sites in the amino acid sequence of SEQ ID NO: 1.
  • the engineered proteins has substantially wildtype IFN-beta like biological activity.
  • the engineered proteins described herein comprise a polypeptide sequence having a sequence with at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • Such variants can be used as a treatment for inflammatory diseases or conditions such as multiple sclerosis and cancer.
  • the engineered protein has an amino acid substitution (e.g., one or more amino acid substitutions) recited in TABLE 1 below, relative to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1 (MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALT IYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMS SLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN).
  • amino acid substitution e.g., one or more amino acid substitutions
  • the conversion results in the introduction of a glycosylation motif into the non-naturally occurring protein.
  • the amino acid substitution results in the conversion of 1, 2, 3, 4, or more glycosylation motif(s) in the amino acid sequence of SEQ ID NO: 1 into 1, 2, 3, 4, or more motif(s) that are not glycosylation motifs.
  • the conversion results in the elimination of a glycosylation motif into the non-naturally occurring protein.
  • a single glycosylation site is introduced.
  • a double-glycosylation site is introduced.
  • the double-glycosylation site contains a native glycosylation site of wildtype IFN-beta (Asn 80) protein.
  • the native glycosylation site of wildtype IFN- beta protein is eliminated in the non-naturally occurring protein.
  • the non-naturally occurring protein is aglycosylated.
  • the non-naturally occurring protein as disclosed herein having an amino acid substitution as recited in TABLE 1 can be present as a salt, such as a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt can Attorney Docket No.217863-712601 include: acetate, acrylate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, bisulfite, bitartrate, bromide, butyrate, butyn-1,4-dioate, camphorate, camphorsulfonate, caproate, caprylate, chlorobenzoate, chloride, citrate, cyclopentanepropionate, decanoate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hexyne-1,6-dioate, hydroxybenzoate, ⁇ -hydroxybutyrate,
  • 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.
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1 can be encoded by a vector (e.g., a DNA vector).
  • the vector can be a viral vector such as a lentiviral vector or an adeno-associated viral vector.
  • the non-naturally occurring protein variant has an amino acid substitution (e.g., one or more amino acid substitutions) recited in TABLE 2 below, relative to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1.
  • a non-naturally occurring protein variant having an amino acid substitution where the amino acid substitution introduces a N-linked glycosylation site recited in TABLE 2 has similar biological activity (e.g., binding activity) to a wildtype IFN-beta protein. TABLE 2.
  • Exemplary IFN-beta amino acid substitutions Amino Acid Substitution – Mutated Codon
  • Attorney Docket No.217863-712601 S13N-AAC & F15T-ACC S2N-AAC & N4T-ACC ally occurring protein having an amino acid substitution as recited in TABLE 2 introduces a glycosylation motif.
  • the non-naturally occurring IFN-beta is modified to comprise 1, 2, 3, 4, or more glycosylation sites than wildtype IFN-beta protein.
  • introduction of glycosylation site is accomplished by insertion of a consensus sequence motif.
  • the consensus sequence motif is a post-translational modification (PTM) consensus sequence motif as recited in TABLE 5 below.
  • the glycosylation motif has a consensus sequence motif.
  • the consensus motif is (i) N-X-S/T, where X is independently selected from any amino acid except proline; (ii) D/E-X-N-Z-S/T, where X and Z are independently selected from any amino acid except proline; or (iii) N-X-C, where X is independently selected from any amino acid except proline.
  • the engineered, non-naturally occurring protein having an amino acid substitution as recited in TABLE 2 introduces an amino acid substitution and can be further post-translationally modified.
  • the modifications can include: glycosylation, phosphorylation, lipidation, glycation, proline isomerization, methylation, acetylation, hydroxylation sites, and proteolysis.
  • the non-naturally occurring protein is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, conjugated to a polyethylene glycol (PEG) moiety, or any combination thereof.
  • PEG polyethylene glycol
  • the engineered IFN-beta protein or the functional fragment thereof is glycosylated. In some embodiments, the engineered IFN-beta protein or the functional fragment thereof is double glycosylated. In some embodiments, the engineered IFN-beta protein or the functional fragment thereof is multi-glycosylated. In some embodiments, the engineered IFN- beta protein or the functional fragment thereof is aglycosylated. [0079] In some embodiments, the engineered IFN-beta protein or a functional fragment thereof comprises at least one amino acid substitutions. In some embodiments, the at least one amino acid substitution is selected from TABLE 1 or TABLE 2.
  • the engineered Attorney Docket No.217863-712601 IFN-beta protein comprises an amino acid sequence having at least about: 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 2 – SEQ ID NO: 136. TABLE 3.
  • Exemplary engineered IFN-beta protein sequences SEQ ID Mutant Amino Acid Sequence NO: L I A L I A L I L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A L I A Attorney Docket No.217863-712601 11 S12N- MSYNLLGFLQRNSTFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL AAC
  • the engineered IFN-beta proteins described having an amino acid sequence of any one of SEQ ID NO: 137 – SEQ ID NO: 204 can have a decrease in biological activity as compared to wildtype IFN-beta having an amino acid sequence of SEQ ID NO: 1.
  • 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.
  • the engineered IFN-beta proteins can have more than 90% of the biological activity of the wildtype IFN.
  • the engineered IFN- beta proteins can have more than 80% of the biological activity of the wildtype IFN.
  • 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.
  • the engineered IFN-beta proteins can have more than 20% of the biological activity of the Attorney Docket No.217863-712601 wildtype IFN. In some embodiments, the engineered IFN-beta proteins can have more than 10% of the biological activity of the wildtype of which it is deduced (i.e., the wildtype IFN of which the coding sequence has been mutated to obtain the engineered IFN-beta). [0081] In some embodiments, the engineered IFN-beta protein binds to an interferon- ⁇ / ⁇ receptor-1(IFNAR1) or interferon- ⁇ / ⁇ receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein.
  • IFNAR1 interferon- ⁇ / ⁇ receptor-1
  • IFNAR2 interferon- ⁇ / ⁇ receptor-2
  • the binding affinity is measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the engineered IFN-beta protein binds to an interferon- ⁇ / ⁇ receptor-1(IFNAR1) or interferon- ⁇ / ⁇ receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN- beta protein as measured by ELISA and/or a GigaAssay e.g., measuring signaling activity to an ISRE-GFP reporter in a live human cell line in vitro assay.
  • the engineered IFN-beta protein is an agonist of IFNAR1 or IFNAR2 subunits.
  • the engineered IFN-beta protein binds and activates IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the engineered IFN-beta protein is an agonist of IFNAR1 or IFNAR2 subunits
  • 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.
  • the engineered IFN-beta protein is an antagonist of IFNAR1 or IFNAR2 subunits.
  • the activity of IFNAR1 or IFNAR2 is measured by a reporter assay.
  • the reporter assay is an Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay or an ISRE luciferase reporter assay.
  • ISRE Interferon Stimulated Response Element
  • GFP Green fluorescent protein
  • the engineered IFN-beta protein reduces extracellular level of a pro-inflammatory cytokine as compared to wildtype IFN-beta protein.
  • the pro-inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof.
  • the pro-inflammatory cytokine is IL-1 ⁇ , IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , or any combination thereof.
  • the engineered IFN-beta protein increases extracellular level of an anti-inflammatory cytokine as compared to wildtype IFN-beta protein.
  • the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof.
  • the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN- ⁇ , TGF- ⁇ , or any combination thereof.
  • the engineered IFN-beta protein having amino acid sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 136 has reduced immunogenicity when administered to Attorney Docket No.217863-712601 a subject, as compared to administering the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • hs-CRP high-sensitivity C-reactive protein
  • 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.
  • 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.
  • the engineered IFN-beta protein has an amino acid substitution (e.g., one or more amino acid substitutions) recited in TABLE 1 or TABLE 2 below, relative to a wildtype IFN-beta protein having an amino acid sequence of SEQ ID NO: 1.
  • an engineered IFN- beta protein variant having an amino acid substitution recited in TABLE 1 or TABLE 2 has similar biological activity (e.g., binding activity) to a wildtype IFN-beta protein.
  • the engineered IFN-beta protein comprises an amino acid sequence having at least about: 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 137 – SEQ ID NO: 208.
  • PTM post-translational modification
  • a “x” or “X” in the PTM consensus sequence motif as recited in TABLE 5 below can represent any amino acid.
  • the non-naturally occurring protein having an amino acid substitution having an amino acid substitution as recited in TABLE 2 can be present as a salt, such as a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt can include: acetate, acrylate, adipate, alginate, aspartate, benzoate, Attorney Docket No.217863-712601 benzenesulfonate, bisulfate, bisulfite, bitartrate, bromide, butyrate, butyn-1,4-dioate, camphorate, camphorsulfonate, caproate, caprylate, chlorobenzoate, chloride, citrate, cyclopentanepropionate, decanoate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hexyne-1,6-dioate, hydroxybenzoate, ⁇ -hydroxybutyrate,
  • 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.
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 5 can be encoded by a vector (e.g., a DNA vector).
  • the vector can be a viral vector such as a lentiviral vector or an adeno-associated viral vector.
  • the non-naturally occurring protein as disclosed herein is a recombinant IFN-beta protein.
  • the non-naturally occurring protein binds to an interferon- ⁇ / ⁇ receptor-1(IFNAR1) or interferon- ⁇ / ⁇ receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein.
  • the binding affinity is measured by enzyme-linked immunosorbent assay (ELISA).
  • the non-naturally occurring protein binds to an interferon- ⁇ / ⁇ receptor-1(IFNAR1) or interferon- ⁇ / ⁇ receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the Attorney Docket No.217863-712601 wildtype IFN-beta protein as measured by ELISA and/or a GigaAssay e.g., measuring signaling activity to an ISRE-GFP reporter in a live human cell line in vitro assay.
  • the non-naturally occurring protein is an agonist of IFNAR1 or IFNAR2 subunits.
  • the non-naturally occurring protein binds and activates IFNAR1 or IFNAR2 at a level substantially similar to the wildtype IFN-beta protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the non-naturally occurring protein binds and activates IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1.
  • the non-naturally occurring protein is an agonist of IFNAR1 or IFNAR2 subunits In some embodiments, the non-naturally occurring protein binds and inhibits IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the wildtype IFN- beta protein with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the non- naturally occurring protein is an antagonist of IFNAR1 or IFNAR2 subunits. In some embodiments, the activity of IFNAR1 or IFNAR2 is measured by a reporter assay.
  • the reporter assay is an Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay or an ISRE luciferase reporter assay.
  • ISRE Interferon Stimulated Response Element
  • GFP Green fluorescent protein
  • the non-naturally occurring protein reduces extracellular level of a pro-inflammatory cytokine as compared to wildtype IFN-beta protein.
  • the pro-inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof.
  • the pro-inflammatory cytokine is IL-1 ⁇ , IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , or any combination thereof.
  • the non-naturally occurring protein increases extracellular level of an anti-inflammatory cytokine as compared to wildtype IFN-beta protein.
  • the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming growth factor (TGF), or any combination thereof.
  • the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN- ⁇ , TGF- ⁇ , or any combination thereof.
  • a glycosylation site is introduced in the non-naturally occurring protein that results in diminished binding of an antibody to the non-naturally occurring protein relative to an amount of binding of the antibody to the wildtype IFN-beta protein.
  • the non-naturally occurring beta protein has reduced immunogenicity when administered to a subject, as compared to administering the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1.
  • the reduced immunogenicity comprises a reduced level of anti-drug antibody in the subject when the non-naturally occurring protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN- Attorney Docket No.217863-712601 beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject.
  • the non-naturally occurring beta protein when administered to a subject in need thereof, reduces a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering.
  • 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.
  • CRP C reactive protein
  • 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.
  • 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.
  • the administering of the non-naturally occurring protein as disclosed herein to the subject results in reduced blood levels of a pro-inflammatory cytokine as compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA.
  • Polynucleotides encoding engineered IFN-beta proteins [0093] Provided herein are polynucleotides encoding the engineered IFN-beta proteins disclosed herein.
  • the polynucleotide disclosed herein encodes an engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1, wherein the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • IFN-beta interferon beta
  • the nucleic acid sequence comprise a sequence at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar to any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • the nucleic acid is a nucleic acid construct.
  • a nucleic acid construct comprises a polynucleotide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • polynucleotide of claim 55 wherein polynucleotide sequence at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 209 – SEQ ID NO: 342.
  • the polynucleotide sequence comprises a nucleotide sequence derived from a full length engineered protein, or a fragment thereof.
  • each codon in the polynucleotide can independently be replaced by a degenerate codon.
  • the polynucleotide sequence comprise one or more degenerate codons.
  • the polynucleotide comprises a nucleic acid mimic.
  • the nucleic acid mimic comprises a phosphorothioate nucleic acid, Attorney Docket No.217863-712601 a phosphoramidate nucleic acid, a morpholino nucleic acid, a hexitol nucleic acid (HNA), a peptide nucleic acid (PNA), or a locked nucleic acid (LNA).
  • each codon independently codes for one or more amino acids.
  • the one or more amino acids comprise alanine, arginine, aspartic acid, glutamine, glutamic acid, glycine, praline, serine, leucine, cysteine, valine, lysine, methionine, tryptophan, phenylalanine, arginine, tyrosine, threonine, isoleucine, histidine, lysine and asparagine.
  • one or more codons in the polynucleotide is modified.
  • one or more residues of an amino acid in the engineered protein is substituted for a non-natural amino acid.
  • Non-limiting examples of non-natural amino acids include azidohomoalanine, homoproparglyglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine and ethynylephenylalanine.
  • the polynucleotide encodes a polypeptide comprising a non-natural amino acid.
  • a chemical moiety is added to the non-natural amino acid.
  • the chemical moiety comprises cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, poly(ethylene) glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.
  • the non-natural amino acid residue is fluorinated, electroactive or unsaturated.
  • the polypeptide further comprises a sequence encoding a linker, or a tag.
  • the engineered IFN-beta protein as disclosed herein is encoded by a vector (e.g., a DNA vector).
  • a vector comprise the nucleic acids or nucleic acid constructs disclosed herein.
  • the vector is a viral vector such as a lentiviral vector or an adeno-associated viral vector.
  • the engineered IFN-beta protein as disclosed herein is encoded by a nucleic acid sequence. TABLE 6.
  • the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • an Attorney Docket No.217863-712601 engineered, non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or a vector encoding the engineered protein having an amino acid substitution recited in TABLE 1, can be present in a composition.
  • the composition can be a pharmaceutical composition with a pharmaceutically acceptable excipient, diluent, or carrier.
  • an engineered, non-naturally occurring protein having an amino acid substitution recited in TABLE 2, or a vector encoding the engineered protein having an amino acid substitution recited in TABLE 2 can be present in a composition.
  • the composition can be a pharmaceutical composition with a pharmaceutically acceptable excipient, diluent, or carrier.
  • a pharmaceutical composition can comprise an excipient, such as a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, a coloring agent.
  • a pharmaceutical composition can comprise a diluent such as water, glycerol, methanol, ethanol, and other similar biocompatible diluents.
  • the pharmaceutical composition can be in unit dose form.
  • the pharmaceutical composition can be in the form of a tablet, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a suppository, and any combination thereof.
  • Methods of treatment comprising administering to a subject in need thereof a therapeutically effective dose of engineered interferon beta (IFN-beta) protein or a functional fragment thereof, comprising a modification in a wildtype IFN-beta protein with an amino acid sequence of SEQ ID NO: 1.
  • the engineered IFN-beta protein comprises a polypeptide sequence of any one of SEQ ID NO: 2 – SEQ ID NO: 208.
  • an engineered, non-naturally occurring protein having an amino acid substitution recited in TABLE 1 or TABLE 2 a vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or a pharmaceutical composition comprising the non-naturally occurring protein or vector, can be administered to a subject to treat a disease or condition.
  • the non- naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non-naturally occurring protein or vector can be used to treat an inflammatory condition.
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the Attorney Docket No.217863-712601 non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non-naturally occurring protein or vector can be used to treat a cancer (e.g., a melanoma, a bladder cancer, a head or neck cancer, a kidney cancer, a liver cancer, a non-small cell lung cancer, a viral infection (e.g., a hepatitis infection such as chronic Hepatitis B infection or a chronic Hepatitis C infection), a systemic lupus (e.g., erythematosus), or multiple sclerosis (e.g., relapse remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms).
  • a cancer e.g., a melanoma, a bladder cancer, a head or neck cancer, a kidney cancer
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector can be administered to a subject by a route of administration selected from: inhalation, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebroventricular, intracisternal, intracorneal, intracoronal, intracoronary, intracorpous cavernaosum, intradermal,
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector can be co-administered along with a second therapeutic.
  • the second therapeutic can be an antibody, a peptide, an antibody drug conjugate, or any combination thereof.
  • the second therapeutic can be an immunotherapy agent such as a checkpoint inhibitor.
  • the checkpoint Attorney Docket No.217863-712601 inhibitor can be a PD-1 inhibitor such as nivolumab or pembrolizumab, a CTLA-4 inhibitor such as ipilimumab, or a PD-L1 inhibitor such as atezolizumab, avelumab, or durvalumab.
  • a PD-1 inhibitor such as nivolumab or pembrolizumab
  • a CTLA-4 inhibitor such as ipilimumab
  • a PD-L1 inhibitor such as atezolizumab, avelumab, or durvalumab.
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1 can be administered 1, 2, 3, or 4 times a day; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 times a week; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1 can be administered every day, every other day, once a week, or once a month.
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, vector encoding the non-naturally occurring protein having an amino acid substitution recited in TABLE 1, or pharmaceutical composition comprising the non- naturally occurring protein or vector can independently be administered at a dose selected from the group consisting of about: 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, and 10 mg/kg; each with respect to a body weight of the subject.
  • 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
  • the non-naturally occurring protein having an amino acid substitution recited in TABLE 1 can independently be administered at a dose of about: 10 ng, 11 ng, 12 ng, 13 ng, 14 ng, 15 ng, 16 ng, 17 ng, 18 ng, 19 ng, 20 ng, 21 ng, 22 ng, 23 ng, 24 ng, 25 ng, 26 ng, 27 ng, 28 ng, 29 ng, 30 ng, 31 ng, 32 ng, 33 ng, 34 ng, 35 ng, 36 ng, 37 ng, 38 ng, 39 ng, 40 ng, 41 ng, 42 ng, 43 ng, 44 ng, 45 ng, 46 ng, 47 ng, 48 ng, 49 ng, 50 ng, 51 ng, 52 ng, 53 ng, 54 ng
  • Embodiment 1 A non-naturally occurring protein, comprising an amino acid sequence having an amino acid substitution with respect to a wildtype interferon beta (IFN-beta) protein with the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution is selected from the amino acid substitutions in TABLE 1.
  • Embodiment 2. The non-naturally occurring protein of embodiment 1, wherein: a. the amino acid substitution results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a glycosylation motif into a glycosylation motif, thereby introducing a glycosylation motif into the non-naturally occurring protein; b.
  • Embodiment 3 The non-naturally occurring protein of embodiment 2, wherein the amino acid substitution results in the conversion of 1, 2, 3, 4, or more motif(s) in the amino acid sequence of SEQ ID NO: 1 that are not glycosylation motifs into 1, 2, 3, 4, or more motif(s) that are glycosylation motif(s), thereby introducing 1, 2, 3, 4, or more glycosylation motif(s) into the non-naturally occurring protein.
  • Embodiment 4 The non-naturally occurring protein of embodiment 2, wherein the amino acid substitution results in the conversion of 1, 2, 3, 4, or more glycosylation motif(s) in the amino acid sequence of SEQ ID NO: 1 into 1, 2, 3, 4, or more motif(s) that are not glycosylation motifs, thereby eliminating 1, 2, 3, 4, or more glycosylation motif(s) into the non- naturally occurring protein.
  • N is an Asparagine that is N-glycosylated at the amide bond in the side chain of the Asparagine
  • X is any amino acid except for proline
  • S is a serine
  • T is a threonine.
  • Embodiment 8 The non-naturally occurring protein of embodiment 1, wherein the amino acid substitution results in a conversion of a motif in the amino acid sequence of SEQ ID NO: 1 that is not a site for a post-translational modification into a post-translational modification consensus sequence motif, thereby introducing a site for a post translational modification into the non-naturally occurring protein.
  • Embodiment 9 The non-naturally occurring protein of embodiment 8, wherein the post-translational modification consensus sequence motif is selected from the amino acid sequences in TABLE 5.
  • Embodiment 11 The non-naturally occurring protein of embodiment 10, wherein the post-translational modification is selected from the group consisting of: glycosylation, phosphorylation, lipidation, glycation, proline isomerization, methylation, acetylation, hydroxylation sites, and proteolysis.
  • the non-naturally occurring protein of any one of embodiments 1-13 wherein the non-naturally occurring protein binds to an interferon- ⁇ / ⁇ receptor-1(IFNAR1) or interferon- ⁇ / ⁇ receptor-2 (IFNAR2) heterodimer subunits at a level greater than or equal to the wildtype IFN-beta protein as measured by enzyme-linked immunosorbent assay (ELISA) or by measuring signaling activity of an Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter in a live human cell in vitro assay.
  • ELISA enzyme-linked immunosorbent assay
  • ISRE Interferon Stimulated Response Element
  • GFP Green fluorescent protein
  • Attorney Docket No.217863-712601 [00121]
  • Embodiment 16 The non-naturally occurring protein of embodiment 15, wherein the non-naturally occurring protein binds and activates IFNAR1 or IFNAR2 at a level greater than the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by an ISRE-GFP reporter assay or an ISRE luciferase reporter assay.
  • Embodiment 18 The non-naturally occurring protein of embodiment 17, wherein the pro-inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, a tumor necrosis factor (TNF), or any combination thereof.
  • IFN interferon
  • IL interleukin
  • TNF tumor necrosis factor
  • Embodiment 20 The non-naturally occurring protein of any one of embodiments 1-13, wherein the non-naturally occurring protein increases an extracellular level of an anti- inflammatory cytokine, as compared to the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1, as measured by ELISA.
  • Embodiment 21 Embodiment 21.
  • Embodiment 22 The non-naturally occurring protein of embodiment 21, wherein the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN- ⁇ , TGF- ⁇ , or any combination thereof.
  • Embodiment 23 Embodiment 23.
  • Embodiment 24 The non-naturally occurring protein of any one of embodiments 1-14, wherein the non-naturally occurring protein is an antagonist of IFNAR1 or IFNAR2 subunits.
  • Embodiment 25 Embodiment 25.
  • Attorney Docket No.217863-712601 [00131]
  • Embodiment 26 The non-naturally occurring protein of any one of embodiments 1-25, wherein the non-naturally occurring protein is a recombinant IFN-beta or a portion thereof.
  • Embodiment 28 The non-naturally occurring protein of embodiment 1-27, wherein a glycosylation site is introduced in the non-naturally occurring protein that results in diminished binding of an antibody to the non-naturally occurring protein relative to an amount of binding of the antibody to the wildtype IFN-beta protein.
  • Embodiment 29 Embodiment 29.
  • Embodiment 30 The non-naturally occurring protein of embodiment 28, wherein the reduced immunogenicity comprises a reduced level of anti-drug antibody in the subject when the non-naturally occurring protein is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 is administered, as measured by ELISA on a sample obtained from the subject.
  • Embodiment 31 Embodiment 31.
  • Embodiment 32 The non-naturally occurring protein of any one of embodiments 1-30, wherein the non-naturally occurring beta protein, when administered to a subject in need thereof, reduces a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering.
  • Embodiment 32 The non-naturally occurring protein of embodiment 31, wherein the reduced level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA.
  • CRP C reactive protein
  • Embodiment 33 Embodiment 33.
  • Embodiment 34 The non-naturally occurring protein of embodiment 33, wherein the reduced serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg/dL to about 2.9 mg/dL.
  • Embodiment 35 The non-naturally occurring protein of embodiment 34, wherein the administering to the subject results in reduced blood levels of a pro-inflammatory cytokine as Attorney Docket No.217863-712601 compared to the blood levels of one or more pro-inflammatory cytokines before the administering, as measured by ELISA.
  • Embodiment 36 A method of making a non-naturally occurring protein in a cell, comprising introducing into the cell a vector encoding the non-naturally occurring protein of any one of embodiments 1-35, wherein the introducing results in expression of the non-naturally occurring protein in the cell.
  • Embodiment 37 The method of embodiment 36, wherein the non-naturally occurring protein is isolated from the cell.
  • Embodiment 38 The method of embodiment 36, wherein the vector comprises a mammalian expression plasmid.
  • Embodiment 39 The method of embodiment 36, wherein the non-naturally occurring protein comprises a tag.
  • Embodiment 40 A method of making a non-naturally occurring protein in a cell, comprising introducing into the cell a vector encoding the non-naturally occurring protein of any one of embodiments 1-35, wherein the introducing results in expression of the non-naturally occurring protein in the cell.
  • Embodiment 41 The method of embodiment 39, wherein the tag is CBP, FLAG, GST, Myc, poly-His, or V5.
  • Embodiment 41 The method of embodiment 39, wherein the tag comprises a cleavable tag or a non-cleavable tag.
  • Embodiment 42 A polynucleotide encoding the non-naturally occurring protein of any one of embodiments 1-35.
  • Embodiment 43 A pharmaceutical composition in unit dose form comprising: a. the non-naturally occurring protein of any one of embodiments 1-35, a vector encoding the non- naturally occurring protein of any one of embodiments 1-35, or the polynucleotide of embodiment 42; and b.
  • Embodiment 44 The pharmaceutical composition of embodiment 43, wherein the pharmaceutical composition is encapsulated.
  • Embodiment 45 The pharmaceutical composition of embodiment 43, wherein the pharmaceutical composition is in the form of a liquid.
  • Embodiment 46 The pharmaceutical composition of any one of embodiments 43-45, wherein the pharmaceutical composition is formulated for local, systemic, or topical administration.
  • Embodiment 47 The pharmaceutical composition of any one of embodiments 43-46, wherein the pharmaceutical composition is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parental, intravenous, or intramuscular administration.
  • Embodiment 48 The pharmaceutical composition of any one of embodiments 43-47, wherein the pharmaceutical composition is formulated for controlled-release. Attorney Docket No.217863-712601 [00154] Embodiment 49. The pharmaceutical composition of any one of embodiments 43-48, wherein the pharmaceutical composition is formulated for single-dosage administration. [00155] Embodiment 50. A method of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of the non-naturally occurring protein of any one of embodiments 1-35, the polynucleotide of embodiment 42, or the pharmaceutical composition of any one of embodiments 43-49, thereby treating the subject. [00156] Embodiment 51.
  • Embodiment 50 The method of embodiment 50, wherein the disease is an infection.
  • Embodiment 52 The method of embodiment 51, wherein the infection is a viral infection.
  • Embodiment 53 The method of embodiment 52, wherein the viral infection is chronic Hepatitis B infection, or a chronic Hepatitis C infection.
  • Embodiment 54 The method of embodiment 50, wherein the disease is a demyelinating disorder.
  • Embodiment 55 Embodiment 55.
  • 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).
  • MS Multiple Sclerosis
  • AHLE Acute Hemorrhagic Leucoencephalitis
  • Balo’s disease Concentric Sclerosis
  • Charcot-Marie-Tooth disease CMT
  • GFS Guillain-Barre Syndrome
  • HAM HTLV-I Associated Myelopathy
  • HAM Neuromyelitis Optica
  • Embodiment 58 The method of any one of embodiments 50-57, wherein the administering is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra- cisternal injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, a surgical route, or any combination thereof.
  • Embodiment 59 The method of embodiment 50, wherein the administering occurs from about once a day, about twice a day, about once a week, about twice a week, about once every two weeks, about once a month, about once every 3 months, about once every 6 months, about once every 9 months, to about once a year.
  • Embodiment 60 The method of embodiment 50, wherein the administering is at a dose selected from the group consisting of about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, or about 2000 mg.
  • Attorney Docket No.217863-712601 [00166] Embodiment 61.
  • Embodiment 50 wherein the administering is at a dose selected from the group consisting of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, and 10 mg/kg.
  • Embodiment 62 The method of embodiment 50, wherein the administering reduces a volume of a tumor in the subject.
  • Embodiment 63 The method of embodiment 50, wherein the administering inhibits or diminishes the growth a tumor in the subject.
  • Embodiment 64 The method of embodiment 50, wherein the method further comprises administering a second therapeutic.
  • Embodiment 65 Embodiment 65.
  • Embodiment 64 wherein the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.
  • Embodiment 66 The method of embodiment 50, wherein the method further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor.
  • Embodiment 67 The method of embodiment 66, wherein the checkpoint inhibitor is a PD-1, a PD-L1, CTLA-4 inhibitor.
  • Embodiment 68 Embodiment 68.
  • Embodiment 69 A kit comprising the non-naturally occurring protein of any one of embodiments 1-35, the polynucleotide of embodiment 42, or the pharmaceutical composition of any one of embodiments 43-49, and a container.
  • EXAMPLES Example 1 – Generation of a plasmid-based Gene Mutation Library [00175] A library of non-naturally occurring Interferon beta variants was generated.
  • the library was a plasmid library designed for the generation of a lentiviral library and contains the cDNA for human Interferon beta having the sequence of SEQ ID NO: 1 with variants as described in TABLE 1, and TABLE 2.
  • FIG.1 A clonal reporter cell line was established that contains a stably integrated ZsGreen1-DR fluorescent protein downstream of an interferon-responsive promoter containing 3xISREs (Interferon Stimulated Response Elements).
  • the reporter cell line was then transduced so that it contains a single integrated copy of a non-naturally Attorney Docket No.217863-712601 occurring protein as described herein downstream of a Dox-inducible promoter.
  • the non- naturally occurring protein as described herein consists of an N-terminal fusion of a Type II Transmembrane Domain (Type II TMD) and GGGGS linker.
  • Type II TMD Type II Transmembrane Domain
  • GGGGS linker Upon addition of doxycycline, the Tet Transactivator binds doxycycline and changes conformation to allow for binding to the Tet response elements in the Dox-inducible promoter leading to the induced expression of the non- naturally occurring protein as described herein.
  • the non-naturally occurring protein as described herein is then translocated to the plasma membrane where it is tethered to the membrane with the Type II TMD.
  • non-naturally occurring protein as described herein interacts with the Type I IFN receptor (consisting of IFNAR1/2 receptor subunits) to induce the interferon-stimulated signaling pathway.
  • Phosphorylated STAT1/2 translocates to the nucleus, binds to the ISRE promoter upstream of GFP leading to induction of GFP expression.
  • Cells can be sorted based on fluorescence as a marker for biological activity related to the interferon signaling pathway. (FIG.2).
  • the non-naturally occurring protein as described herein was saturation mutagenized in order to produce amino acid substitutions at each position thereby introducing or eliminating adding mutations in a consensus sequence as disclosed herein for N- linked glycosylation at the asparagine (N), serine (S), or threonine (T) residues of the consensus sequence motif of Nx[S/T].
  • IFN-beta variants in the assay are designed so that N-linked glycosylation sites are added at each amino acid position within the protein coding sequence.
  • Each clone generated contained a Unique Molecular Identifier (UMI) region which was used to quantify and associate the UMI in each sorted population with the corresponding gene mutation. This information is used to create MEGA-Maps associating gene mutations with biological activity.
  • UMI Unique Molecular Identifier
  • Example 2 High-throughput screening of non-naturally occurring protein
  • 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- ⁇ plus a red fluorescent protein (mScarlet) were co-cultured with cells expressing endogenous IFNAR1/2 and an ectopic ISRE reporter driving expression of a green fluorescent protein (ZsGreen1-DR). In this assay, only cells that undergo paracrine signaling become positive for ZsGreen1. This density was used for autocrine assays.
  • a portion of the variants are aglycosylated, single-glycosylated, or double- glycosylated, where the double-glycosylated variants include a de novo glycosylation site in combination with the native glycosylation site left intact.
  • a wide dynamic range of fluorescence in HEK-293 ISRE reporter cells was observed when comparing control (left) to cells stimulated by the non-naturally occurring proteins described herein (right) by flow cytometry (FIG.4). TABLE 7.
  • Exemplary non-naturally occurring proteins comprised a glycosylation site in an immune epitope without any overlap to amino acid residues associated with binding to the IFNAR1/2 receptor subunits.
  • the major immuno epitopes that patients develop during treatment with wildtype IFN-beta were amino acids 1-12, 151-162, and 121-132, with the first two being favored for neutralizing antibodies and amino acids 1-12 being the preferred epitope.
  • epitopes 1-12 were directly juxtaposed to epitopes 151-162 and 121-132, with 121-132 on the opposing face of the protein.
  • Attorney Docket No.217863-712601 None of the epitopes were near the native N-linked glycosylation site of wildtype IFN- BETA at amino acid N80.
  • a model of the structure of a human IFN-beta paralog, IFN alpha 2 complexed with IFNAR receptor (PDB: 3SE3) was used to assess the contacts of the non-naturally occurring protein as described herein with the IFNAR receptor.
  • the model was consistent with the contacts observed in the structure of the mouse IFNAR1/IFN-beta complex (PDB: 3WCY).
  • IFNAR2 binds to IFN-beta making contacts with amino acids 19, 22, 26, 26, 30, 33, 151, 155, 156, and 159. Then, IFNAR2 was recruited and contacts IFN-beta at amino acids 65, 67, 68, 88, 92, 119, 123, 126, and 127.
  • IFNAR1 makes no contacts with IFNAR2, rather both directly bind IFN-beta.
  • the non-naturally occurring proteins containing the amino acid substitutions in TABLE 2 were mapped on the X-ray structure and a new IFNAR1 binding contact was discovered to be essential for signaling.
  • the crystal structure of IFN-beta shows an asymmetric dimer with a patch of residues forming intermolecular contacts.
  • IFN-beta was a hydrophobic protein and was prone to aggregation and may form dimers, although the relevance of these dimers to signaling was not well understood.
  • the mutations in the non-naturally occurring proteins having increased biological activity were found to be juxtaposed to the dimerization site.
  • Example 4 Purification of HIS-tagged non-naturally occurring proteins
  • TurboCHO cells is transfected with a mammalian expression plasmid to express HIS- tagged non-naturally occurring proteins (FIG.5).
  • a secretory signal is fused on the N-terminus of the 6xHIS tag to facilitate secretion of the protein into supernatant during cell culture.
  • the secretory signal gets cleaved from the HIS-tagged IFN protein during protein trafficking.
  • the supernatant from transfected cells is loaded onto a Nickle-NTA (Ni-NTA) affinity column, which binds the HIS residue of the HIS-tagged non-naturally occurring protein.
  • Ni-NTA Nickle-NTA
  • Wash buffers is loaded into the column to wash away unbound material, and elution buffer is used to release the purified HIS-tagged IFN protein from the column.
  • the purified non-naturally occurring protein as described is checked for quality control using SDS-PAGE gels (to confirm purity and size; Attorney Docket No.217863-712601 FIGs.15A-15N) and Bradford assay (to measure concentration). The yield and purity of each protein is listed in TABLE 8. TABLE 8. Purification results.
  • EXAMPLE 4 serially-diluted purified engineered, non-naturally occurring proteins as described herein
  • Control IFN-beta sequences SEQ ID MUTANT Amino Acid Sequence E N RI Attorney Docket No.217863-712601 344 R11N, M117N MSYNLLGFLQNSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPE EIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN I EC50 of 0.1161 ng/mL for wildtype IFN-beta protein (FIG.6B; FIGs.16A-16L).
  • EC50 for cell- based reporter assay was measured in ng/m, and p-values were False Discovery Rate (FDR) adjusted (FIGs.16A-16L).
  • FDR False Discovery Rate
  • the HIS-tagged IFN-beta protein with biosimilar activity was also verified by flow cytometry in cells stimulated with purified engineered IFN-beta proteins or WT proteins (FIGs.17A-17G).
  • IFN ⁇ -stimulated gene (ISG) expression is a hallmark of IFN activity.
  • HEK-293 cells were treated for 24 hours with the optimal dose of the engineered IFN-beta or controls to saturate ISRE signaling.
  • exemplary engineered proteins disclosed herein demonstrated reduced function or activity.
  • Representative engineered proteins for each category was tabulated in TABLE 10 below. TABLE 10.
  • Properties of HIS-tagged engineered, non-naturally occurring proteins Flow- r r Attorney Docket No.217863-712601 Y 92N_Q94T 137 Reduced Reduced Reduced Reduced F unction Function Function Function Function R d d r M ononuclear Cells (PBMCs) [00185]
  • Cryopreserved PBMCs are harvested from healthy donors that have never been exposed to interferon therapy (FIG.7).
  • the non-naturally occurring proteins as described herein are taken up by antigen presenting cells (APCs) and presented to T cells in culture.
  • APCs antigen presenting cells
  • Example 7 Purification of untagged non-naturally occurring proteins
  • CHO cells are transfected with a mammalian expression plasmid to express HIS-tagged the non-naturally occurring proteins (FIG 8A-8B).
  • a secretory signal is fused on the N-terminus of the 6xHIS tag to facilitate secretion of the protein into supernatant during cell culture. The secretory signal gets cleaved from the HIS-tagged non-naturally occurring protein during protein trafficking.
  • Supernatant from transfected cells is loaded onto a Nickle-NTA (Ni-NTA) affinity column which binds the HIS residue of the HIS-tagged non-naturally occurring protein.
  • Wash buffers are loaded into the column to wash away unbound material, and elution buffer is used to release the purified HIS-tagged non-naturally occurring protein from the column.
  • Treatment with Enterokinase Light Chain proteinase is used to cleave the Enterokinase Light Chain Cleavage Site (EKCS) peptide sequence, separating the HIS-EKCS tag from the mature non-naturally occurring protein.
  • EKCS Enterokinase Light Chain Cleavage Site
  • the reaction is loaded into a purification column to remove the cleaved tag, resulting in purified untagged non-naturally occurring protein with no extra residues from the tags.
  • Purified non-naturally occurring protein as described herein is checked for quality control using SDS-PAGE gels (to confirm purity and size), analytical size-exclusion (to assess the tag removal), Bradford assay (to measure concentration), and mass spectrometry (to confirm N-term. Attorney Docket No.217863-712601 and C-term peptides) (FIG.8A). Samples are lyophilized and resuspended in a pharmaceutically acceptable solvent.
  • CPE Cytopathic Effect
  • cytopathic encephalomyocarditis EMC virus at a concentration that causes 100% cell death in untreated cell cultures after 24 hours.
  • EMC cytopathic encephalomyocarditis
  • ViralTox Glo Promega reagent is added 24 hours post-infection to measure ATP levels as an indicator of cytopathic effect, luminescence is quantified on a plate reader, and EC50 values for antiviral activity are determined for the non-naturally occurring proteins described herein (FIG.9).
  • PBMCs Peripheral Blood Mononuclear Cells
  • RNA is extracted and used for either RNAseq to sequence the transcriptome, or RT-PCR (for key biomarkers: B2M, 2',5'- OAS1, MxA) to assess the level of induction of interferon-stimulated genes between IFN leads compared to control wildtype IFN-beta.
  • EAE Experimental Autoimmune Encephalomyelitis
  • MS Multiple Sclerosis
  • Example 12 Pharmacokinetic study of non-naturally occurring proteins [00191] Healthy subjects are injected with a single subcutaneous dose of 60 mcg of non- naturally occurring protein (FIG.13).

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Abstract

Sont divulgués dans la présente invention des variants de protéine d'origine non naturelle modifiés présentant des sites de glycosylation de novo ayant une activité biologique de type sauvage d'interféron bêta de type sauvage. Sont également divulgués dans la présente invention des vecteurs codant pour les variants de protéine d'origine non naturelle selon l'invention. Sont de même divulguées dans la présente invention des compositions pharmaceutiques comprenant les variants de protéine d'origine non naturelle selon l'invention ou un vecteur codant pour les variants de protéine d'origine non naturelle selon l'invention. Sont en outre divulguées dans la présente invention des méthodes de traitement d'une maladie par administration à un sujet des variants de protéine d'origine non naturelle selon l'invention, d'un vecteur codant pour les variants de protéine d'origine non naturelle selon l'invention, ou d'une composition pharmaceutique comprenant les variants de protéine d'origine non naturelle selon l'invention ou un vecteur selon l'invention.
PCT/US2025/020102 2024-03-15 2025-03-14 Protéines bêta d'interféron modifiées glycosylées Pending WO2025194144A2 (fr)

Applications Claiming Priority (2)

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US202463566081P 2024-03-15 2024-03-15
US63/566,081 2024-03-15

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WO2025194144A2 true WO2025194144A2 (fr) 2025-09-18
WO2025194144A3 WO2025194144A3 (fr) 2026-02-05

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