Method for producing and screening multi-functional chimeric biologic libraries

Abstract

Disclosed herein are systems and methods for screening chimeric libraries for binding to a target of interest. Also disclosed herein are high throughput methods for identifying candidate chimeric biologics having improved biological activity against a target of interest.

Description

METHOD FOR PRODUCING AND SCREENING MULTI-FUNCTIONAL CHIMERIC BIOLOGIC LIBRARIESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 527,490, filed July 18, 2023, and U.S. Provisional Application No. 63 / 528,211, filed July 21, 2023, the entire contents of which are incorporated herein by reference.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Contract number 1R43HG012537-01A1 awarded by National Human Genome Research Institute. The government has certain rights in the invention.BRIEF SUMMARY

[0003] Provided herein are methods of identifying one or more member(s) of a library of fusion polypeptides, with functional activity against one or more target receptor(s) that is similar to or greater than a corresponding wildtype polypeptide, where each fusion polypeptide among the library of fusion polypeptides independently can comprise a first polypeptide directly or indirectly linked to a second polypeptide, and where the corresponding wildtype polypeptide comprises a wildtype of the first polypeptide or a wildtype of the second polypeptide, the method comprising: (a) transforming or transducing each host cell among the plurality of host cells individually with a unique vector that can comprise a barcode, and a polynucleotide sequence encoding a fusion polypeptide among the library of fusion polypeptides, thereby resulting in expression of the fusion polypeptide on a surface of the host cell that was transformed or transduced with the unique vector; (b) screening each fusion polypeptide expressed on the surface for each host cell for functional activity against the one or more target receptor(s), wherein the functional activity can comprise: (i) a level of binding of the fusion polypeptide to the one or more target receptor(s); or (ii) a level of signaling modulation of the one or more target receptor(s) facilitated by the fusion polypeptide; (c) identifying a subset of host cells that express one or more member(s) of the library of polypeptides that have similar or greater functional activity against the one or more target receptor(s) as compared to the corresponding wildtype polypeptide; and (d) sequencing the barcode of the unique vector present in the subset of host cells identified in (c), thereby identifying one or more member(s) of the library of fusion polypeptides that have similar or greater functional activity against the one or more target receptor(s) as compared to the corresponding wildtype polypeptide. In some embodiments, the first polypeptide is indirectly linked to the second polypeptide by indirectly linking the C-terminal amino acid of the first polypeptide to the N-terminal amino acid of the second polypeptide through a linker. In some embodiments, the linker can comprise from at least about: 1 to 5, 1 to 10, 1 to 20. or 1 to 50 amino acids. In some embodiments, the first polypeptide is directly linked to the second polypeptide by directly linking the C-terminal amino acid of the first polypeptide to the N-terminal amino acid of the second polypeptide through an amide bond. In some embodiments, the first polypeptide and / or the second polypeptide, prior to directly or indirectly being linked to each other, independently comprise an amino acid deletion, amino acid substitution, amino acid addition, or any combination of these, relative to a corresponding wildtype sequence. In some embodiments, the first polypeptide and / or the second polypeptide independently comprise at least about: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120. 130, 140, 150, 160, 170, 180, 190, 200. or 300 amino acids. In some embodiments, the method can further comprise introducing one or more post-translational modification (PTM) sites into at least one fusion polypeptide among the library of fusion polypeptides prior to transforming the unique vector into each host cell, wherein the one or more PTM sites are not present in a corresponding wildtype polypeptide. In some embodiments, the one or more PTM sites comprise a glycosylation site, a lipidation site, a phosphorylation site, a methylation site, an acetylation site, a sumoylation site, or a protease site, or any combination thereof. In some embodiments, the one or more PTM sites results in increased half-life, stability, solubility, excretion, serum half-life of the fusion polypeptide, as compared to the corresponding wildty pe polypeptide. In some embodiments, the one or more PTM sites results in reduced aggregation, and / or immunogenicity of the fusion polypeptide, as compared to the corresponding wildtype polypeptide.

[0004] Also provided herein are polypeptides comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the polypeptides can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the polypeptides can comprise the amino acid sequence of any one of SEQ ID NO: 24 to SEQ ID NO: 58.

[0005] Also provided herein are polypeptides comprising an amino acid sequence having at least about: 90%, 91%, 92 %. 93%. 94%, 95%, 96% 97%. 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 1. In some embodiments, the polypeptides can further comprise a modification selected from the group consisting of D2N, P4S, E5Q, DION, L18M, S22R, S27F, M31K, G37E, P52Q, A114E, S116F, K121R, R125Q, and any combination thereof, relative to SEQ ID NO: 1. In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %. 93%. 94%. 95%. 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 2, where thepolypeptide further comprises a modification selected from the group consisting of D4N, P6S, G12N, S13N, L20M, L28P. G39E, E54Q, T55A, P57S. I66T, K73E, D80E, E81Q, T82S, and any combination thereof, relative to SEQ ID NO: 2. In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 5, where the polypeptide further comprises a modification selected from the group consisting of H34Y, E40Q, E42V. H46N, V56A, L57F. E71K, D72N, E78D, Q79E, S80T, S86Y. T87I, Y90F, L93M, R121K, T132M, and any combination thereof, relative to SEQ ID NO: 5. In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 6, where the polypeptide further comprises a modification selected from the group consisting of D2N, P4S, S10G. SION, T14A, M16I. I17L. M18L. A19G, F152L, L156F, A158T, K159Q, G161R, E162K, R163G, E168D, and any combination thereof, relative to SEQ ID NO: 6. In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%. 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 8, where the polypeptide further comprises a modification selected from the group consisting of D95N, V106M, F116S, M132T, F152L, F154L, T156I, K159Q, G161R, R163K, R164S, D166E, and any combination thereof, relative to SEQ ID NO: 8. In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%. 96% 97%. 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 9, where the polypeptide further comprises a modification selected from the group consisting of R24G, G195S, A199T, I201M, L202I, L203M, R207G, R216G, E222G, D230G, K23IN, and any combination thereof, relative to SEQ ID NO: 9 In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %, 93%. 94%. 95%. 96% 97%, 98% or 99% sequence identity' and at least about: 90% sequence length to SEQ ID NO: 10, where the polypeptide further comprises a modification selected from the group consisting of G19A, R37G, I38L, R121K, I132M, E133G, R134K, and any combination thereof, relative to SEQ ID NO: 10. In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity' and at least about: 90% sequence length to SEQ ID NO: 11, where the polypeptide further comprises a modification selected from the group consisting of N2D, S4P, Q5E, N10D, S116F, K121R, M132T, L152F, Q159K, R161G, R161I, R163K, R164S, D166E, and any combination thereof, relative to SEQ ID NO: 11. In some embodiments, the polypeptides can comprise an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96%97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 15, where the polypeptide further comprises a K107M modification relative to SEQ ID NO: 15. In some embodiments, the polypeptides can bind to an interferon receptor or an interferon receptor subunit at a level greater than or equal to a corresponding wildtype IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by enzyme- linked immunosorbent assay (ELISA).

[0006] Provided herein are fusion polypeptides comprising: a first region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58; a second region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58; and a linker between the first region to the second region. In some embodiments, the amino acid sequence of the first region has at least 95% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid sequence of the first region can comprise any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid sequence of the second region has at least 95% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid sequence of the second region can comprise any one of SEQ ID NO: 24 to SEQ ID NO: 58. Also provided herein are fusion polypeptides comprising: a first region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23; a second region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23; and a linker between the first region to the second region. In some embodiments, the amino acid sequence of the first region has at least 95% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the first region can comprise any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the second region has at least 95% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the second region can comprise any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some embodiments, the linker comprises SEQ ID NO: 405. Also provided herein are fusion polypeptides comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 59 to SEQ ID NO: 404. In some embodiments, the fusion polypeptide can comprise an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 59 to SEQ ID NO: 404. In some embodiments, the fusion polypeptide can comprise an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 59 to SEQ ID NO: 404. In some embodiments, the fusion polypeptide can comprise the amino acid sequence of any one of SEQ ID NO: 59 to SEQ ID NO: 404. In some embodiments, the fusion polypeptide further can comprise: a third region comprising a protein antigen, a protease, a single domain antibody, a designed ankyrin repeat protein (dARPIN), an anti-calin, ora nanobody. In some embodiments, the fusion polypeptide can bind to an interferon receptor or an interferon receptor subunit at a level greater than or equal to a corresponding wildtype IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the fusion polypeptide is an agonist, a partial agonist, a selective agonist, an inverse agonist or an antagonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is an agonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is a partial agonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is an inverse agonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is an antagonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit at a level greater than a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by an assay comprising Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay or an ISRE luciferase reporter assay. In some embodiments, the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit resulting in a reduction of an extracellular level of a pro-inflammatory cytokine, as compared to a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured in an in vitro assay. In some embodiments, the pro-inflammatory cytokine is a chemokine, an interferon (IFN). an interleukin (IL), a lymphokine. a tumor necrosis factor (TNF), or any combination thereof. In some embodiments, the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit resulting in an increase of an extracellular level of an anti-inflammatory cytokine, as compared to a corresponding wildtype IFN protein having the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23, as measured in an in vitro assay. In some embodiments, the in vitro assay is an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming grow th factor (TGF), or any combination thereof. In some embodiments, the fusion polypeptide binds to an interferon receptor or an interferon receptor subunit at a level less than a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by an in vitro assay. In some embodiments, the fusion polypeptide binds and inhibits the interferon receptor or the interferon receptor subunit at a level greater than a corresponding wildtype IFN protein having the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23. as measured by an assay by an ISRE GFP reporter assay or an ISRE luciferase reporter assay.

[0007] Provided herein are nucleic acids encoding the polypeptide described previously, or the fusion polypeptide described previously. In some embodiments, the nucleic acid can further comprise a barcode sequence. Also provided herein are nucleic acid constructs that comprise comprises a polynucleotide sequence of any one of SEQ ID NO: 407 to SEQ ID NO: 763. In some embodiments, the nucleic acid construct can further comprise a reporter gene. In some embodiments, the reporter gene is a fluorescent reporter gene. Also provided herein are vectors comprising the nucleic acids described previously.

[0008] Also provided herein are pharmaceutical compositions comprising: the polypeptides described previously, the fusion polypeptides described previously, the nucleic acids or the nucleic constructs described previously, or the vectors described previously; and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is in unit dose form.

[0009] Also provided herein are vaccine compositions comprising: the polypeptides described previously, the fusion polypeptides described previously, the nucleic acids or the nucleic constructs described previously, or the vectors described previously; a microbial antigen, a tumor antigen, or a nucleic acid encoding the microbial antigen or the tumor antigen; and a pharmaceutically acceptable carrier diluent, or excipient.

[0010] Also provided herein are kits comprising: the polypeptides described previously, the fusion polypeptides described previously, the nucleic acids or the nucleic constructs described previously, or the vectors described previously; and a scaffold.

[0011] Also provided herein are genetically modified cell comprising the polypeptides described previously, the fusion polypeptides described previously, the nucleic acids or the nucleic constructs described previously, or the vectors described previously.

[0012] Provided herein are methods of treating a disease or condition in a subject in need thereof, the method comprising: administering a therapeutically effective amount of the polypeptides described previously, the fusion polypeptide described previously, or the pharmaceutical composition described previously to the subject in need thereof, thereby treating the disease or condition in the subject. In some embodiments, the disease or the condition can comprise a viral infection, a cancer, an autoimmune disease, multiple sclerosis, or a hemolytic disease. In some embodiments, the disease is an infection. In some embodiments, the infection is a viral infection. In some embodiments, the viral infection is a chronic hepatitis B (CHB), a chronic hepatitis C (CHC), a chronic granulomatous disease (CGD), a coronavirus disease (COVID), a genital wart or a human immunodeficiency virus (HIV). In some embodiments, the disease is a demyelinating disorder. In some embodiments, the demyelinating disorder is selected from the group consisting of Multiple Sclerosis (MS), Acute Disseminated Encephalomyelitis (ADEM), AcuteHemorrhagic Leucoencephalitis (AHLE), Balo’s disease (Concentric Sclerosis), Charcot-Marie- Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease). In some embodiments, the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms. In some embodiments, the disease is a cancer. In some embodiments, the cancer is a blood cancer, a bone cancer, a brain cancer, a breast cancer, a cervical cancer, a colon cancer, a heart cancer, a kidney cancer, a liver cancer, a lung cancer, a lymph node cancer, a lymphoma, a melanoma, a myeloma, a skin cancer, a stomach cancer, a pancreatic cancer, a testicular cancer, or a throat cancer. In some embodiments, the cancer is selected from the group consisting of astrocytoma, chronic myelogenous leukemia, follicular lymphoma, glioblastoma, hairy cell leukemia, Kaposi’s sarcoma, malignant melanoma, medulloblastoma, melanoma, multiple myeloma, non-Hodgkin lymphoma, and renal cell carcinoma. In some embodiments, the disease is an immune disorder. In some embodiments, the immune disorder is an autoimmune disorder. In some embodiments, the immune disorder is selected from the group consisting of lupus, systemic lupus erythematosus, asthma, atherosclerosis, atopic dermatitis, autoimmune hepatitis, Celiac disease, COPD, Crohn's disease, Graves' disease. Guillain-Barre syndrome, IgA nephropathy, inflammatory bowel disease, pelvic inflammatory disease, primary biliary cirrhosis, primary hemophagocytic lymphohistiocytosis, and ulcerative colitis. Also provided herein are methods of generating an immune response in a subject, the method comprising: administering to the subject the vaccine composition described previously, thereby generating an immune response in the subject. In some embodiments, the subject is a human. In some embodiments, the administering is intramuscular administration, intraocular administration, intranasal administration, subcutaneous administration, intravenous administration, intracranial administration, intrathecal administration, intertumoral administration, oral administration, sublingual administration, buccal administration, rectal administration, vaginal administration, or otic administration. In some embodiments, the administering is once every 24 hours, once every 48 hours, once every 72 hours, once every 96 hours, once every 120 hours, once every 144 hours, once every 168 hours, once every 280 hours, or once every 336 hours. In some embodiments, the administering occurs 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, or about once a year. In some embodiments, the method further comprises administering a second therapeutic. In some embodiments, the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof. In some embodiments, the method further comprises administering animmunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1, a PD-L1, CTLA-4 inhibitor.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0014] FIG. 1 depicts a flowchart for screening chimeric biologic libraries as described herein.

[0015] FIG. 2 depicts Phase A of the chimeric biologic library screening method described herein using an exemplary gene mutation library (GML) to test the influence of multi-receptor stimulation upon glucose-stimulated insulin secretion.

[0016] FIG. 3 depicts Phase B of the chimeric biologic library screening method described herein in which a library of missense substitutions, insertions, and deletions at the splice junction of the two peptides from the lead chimera / fusion polypeptide is screened.[0017| FIG. 4 depicts Phase C of the chimeric biologic library screening method described herein in which saturating mutagenesis is performed on the lead chimera / fusion polypeptide from Phase B.

[0018] FIG. 5 depicts Phase D of the chimeric biologic library screening method described herein in which one or more new N-linked glycosylation, lipidations, or other post-translational modification (PTM) sites are added into the lead chimera / fusion polypeptide from Phase C.

[0019] FIG. 6 depicts Phase E of the chimeric biologic library screening method described herein in which the receptor signaling of the receptors that are targeted by the peptide chimera / fusion polypeptide is analyzed.

[0020] FIG. 7 depicts Phase F of the chimeric biologic library screening method described herein in which inverse agonists are screened.

[0021] FIG. 8 depicts Phase G of the chimeric biologic library screening method described herein in which triple peptide or multi-peptide chimeras are prepared based on the results of Phase A-F.

[0022] FIG. 9 depicts a scatter plot of the activity scores of multiple replicates of the assay provided herein as measured using with an IFN-Sensitive Response Element (ISRE) reporter for each chimeric protein tested. Each dot indicates a separate fusion polypeptide as provided herein. X-axis: activity score for replicate 1. Y-axis: activity score for replicate 2. Pearson correlation coefficient: R2= 0.82.

[0023] FIG. 10 illustrates the biologic agent landscape using ISRE reporter signaling activity for different fusion polypeptides. The first position of the fusion polypeptide is on the Y-axis of the table and the second position of the fusion polypeptide is on the X-axis of the table.

[0024] FIG. 11 depicts the confidence levels polypeptides and fusion polypeptides. They key shows the approximate range of p values for each chimera. The first position of the fusion polypeptide is on the Y-axis of the table and the second position of the fusion polypeptide is on the X-axis of the table.

[0025] FIG. 12 depicts a scatter plot showing the relationship of signaling activity to IFNAR affinity . X-axis represents Interferon alpha receptor (IFNAR) 1 -interferon (IFN) Kd (nanomolar). Y-axis represents the assay reporter activity.DETAILED DESCRIPTION OF THE INVENTION

[0026] Disclosed herein are systems and methods comprise an assay that encompasses binding of the ligand, intracellular signaling and measures a transcriptional response. In some embodiments, the systems and methods for screening chimeric libraries for binding to a target of interest. Also disclosed herein are high throughput methods for identifying candidate chimeric biologies having improved biological activity against a target of interest.Definitions

[0027] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g, to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” "an" and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise.

[0029] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.

[0030] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain implementations include or do not include, certain features, elements, and / orsteps. Thus, such conditional language is not generally intended to imply that features, elements, and / or steps are in any way required for one or more implementations.[00311 Conjunctive language, such as the phrase "at least one of X. Y, and Z.” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain implementations require the presence of at least one of X, at least one of Y. and at least one of Z.

[0032] As used herein, the term, “about” or “approximately,” means within an acceptable error range for the particular value and includes a range of up to 10% of a given value. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%. more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. 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.

[0033] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0034] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0035] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0036] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0037] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.[0038| “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 can be 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.

[0039] “Sequence identity” as used herein, refers to the similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are the same at that position. The sequence identity between two sequences is a direct function of the number of matching positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are similar, the two sequences have 50% sequence identity, if 90% of the positions (e.g., 9 of 10), are matched, the two sequences have 90% sequence identity.

[0040] 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 undine.

[0041] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0042] The term “linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0043] The term “polynucleotide’' as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e.. the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0044] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0045] As used herein, the terms “chimera,” “tethered ligand chimera” or “hybrids” refers to a polypeptide or a fusion polypeptide as disclosed herein.

[0046] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g.. mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0047] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include nonplasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, butare not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.[0048| Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0049] Although this disclosure has been described in terms of certain implementations and uses, other implementations and other uses, including implementations and uses which do not provide all of the features and advantages set forth herein, are also within the scope of this disclosure. Components, elements, features, acts, or steps can be arranged or performed differently than described and components, elements, features, acts, or steps can be combined, merged, added, or left out in various implementations. All possible combinations and subcombinations of elements and components described herein are intended to be included in this disclosure. No single feature or group of features is necessary or indispensable.

[0050] Certain features that are described in this disclosure in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single implementation also can be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations, one or more features from a claimed combination can in some cases be excised from the combination, and the combination may be claimed as a subcombination or variation of a subcombination.

[0051] Any portion of any of the steps, processes, structures, and / or devices disclosed in one implementation or example in this disclosure can be combined or used with (or instead of) any other portion of any of the steps, processes, structures, and / or devices disclosed or illustrated in a different implementation, flowchart, or example. The implementations and examples described herein are not intended to be discrete and separate from each other. Combinations, variations, and some implementations of the disclosed features are within the scope of this disclosure.

[0052] While operations may be described in the specification in a particular order, such operations need not be performed in the particular order described or in sequential order, or that all operations be performed, to achieve desirable results. Other operations that are not depicted ordescribed can be incorporated in the example methods and processes. For example, one or more additional operations can be performed before, after, simultaneously, or between any of the described operations. Additionally, the operations may be rearranged or reordered in some implementations. Also, the separation of various components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products. Additionally, some implementations are within the scope of this disclosure.Overview

[0053] Disclosed herein are systems and methods comprise an assay that encompasses binding of the ligand, intracellular signaling and measures a transcriptional response. In some embodiments, a method for identifying bifunctional fusion polypeptides that are capable of binding one or more target receptors. Also disclosed herein are methods of functionally screening a library of fusion polypeptides in a plurality of host cells. As described herein, comprehensive profiles can be generated to model a particular gene under a particular condition (e.g., drug treatment), based on an assay in cell culture. A gene mutation library (GML) is analyzed using the workflow described herein to produce a comprehensive mutation effect on gene activity, as well as a mutation activity profile. Also provided herein are compositions, methods, assays, and kits for the development of biologic agents. Briefly, provided herein are (1) methods of producing, identifying, and screening biologic agents; (2) targeting biologic agents for a disease indication; (3) pharmaceutical compositions and delivery vehicles; (4) methods of treating a disease; and (5) kits. The present disclosure recognizes that these MEGA-Map™ produced from GigaAssays when combined into a pipeline can be used to screen for new Biologic fusion polypeptides (also referred to as chimera drugs). This application is uniquely suited to the GigaAssay technology and cannot be achieved with phage display, yeast display or any of the other existing technologies. This approach can be applied to all human ligand biologies (or a filtered FDA- approved set) to make comprehensive bifunctional, trifunctional, or multifunctional fusion polypeptides (or chimeric Biologic) libraries for all agonists and antagonists. This could be screened against any cell or molecular function assay in any cell type. The methods described herein is the first landscape ever produced and assayed for a large set of dual-functional chimera and thus the screen provides key insight into selection of the best performing drugs among this group as well as physiological properties of each gene or protein.Method of Screening

[0054] This workflow is exemplified using a soluble ligand autocrine (SLA) assay for discovery of new variant biologic drug leads. In the SLA assay, a clonal reporter cell line is established thatcontains a stably integrated reporter element. The reporter cell line is then transduced with a virus containing a single integrated copy of an encoded ligand chimera (fusion polypeptide) downstream of a Dox-inducible promoter. The tethered ligand chimera encodes an N-terminal fusion of a Type II Transmembrane Domain (Type II TMD), GGGGS linker, followed by the biologic ligand. In some embodiments, the fusion polypeptides can comprise a first polypeptide indirectly linked with a linker to a second polypeptide. 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 tethered ligand chimera. The tethered ligand chimera is translocated to the plasma membrane where it is tethered to the membrane with the Type II TMD. Upon routing to the cell surface, the tethered ligand interacts with its receptor to induce the reporter fluorescence. Cells are then sorted based on the GFP fluorescence as a marker for biological activity related to the ligands signaling pathway. This can be used to mutate a reference biologic to identify variant biosimilars and biobetters, or new molecular entities.

[0055] This workflow that includes the SLA- Assays can be combined into a pipeline that can be used to screen for new biologic chimera drugs in human cell lines. This combined capability is uniquely suited to the combined screening method described herein and cannot be achieved with phage display, yeast display or any of the other existing technologies. The pipeline can be broken down into seven phases of development (FIG. 1, phase A-G). FIGs. 2-8 depict the exemplary workflow applied to incretin biologies. Using the workflow outlined in FIG. 1 and described in detail in FIGs. 2-8, comprehensive bifunctional, trifunctional, or multifunction chimeric biologic libraries can be generated.

[0056] There are many possible types of chimera and hybrids of incretin-related hormones (of which we currently know less than 1 %), which represents a huge challenge for selecting the best diabetes and obesity biobetter therapeutics. Current methods directed at this problem fall far short as they mainly assess select variants and do so in the absence of the context of living human cells. In contrast, the present disclosure demonstrates a method to systematically assess a plurality of multi-agonist chimera and hybrids (exceeding tens or thousands of variants) for their impact en masse on glucose-stimulated insulin secretion in live human cells.

[0057] In phase A (FIG. 2), an exemplary gene mutation library (GML) is generated to test the influence of multi-receptor stimulation upon glucose-stimulated insulin secretion. The GML in this example is a comprehensive set of possible dual-peptide chimera with all pairs of up to 13 peptide ligands. This screen will identify one or more lead chimera that have the strongest effect on the glucose-stimulated insulin secretion assay. The screen also has the additional advantage of being against the desired pathological activity or drug target in human or mammalian cells.

[0058] In phase B (FIG. 3), a library of missense substitutions, insertions, and deletions at the splice junction of the two peptides from the lead chimera is screened in a second assay for the same insulin secretion function. This is used to select that best function for fusion of the peptides. In some embodiments, the linker can be functionalized to comprise a specific structural element.

[0059] In phase C (FIG. 4), the lead chimera with optimized fusion junctions is the basis for a saturating mutagenesis single variant GML library. This GML is analyzed in a third assay for the same insulin secretion function. This is used to select that best missense variants for one or more specific chimeras.

[0060] In phase D (FIG. 5), the new lead chimera is the basis for a GML library with all positions having one or more new N-linked glycosylation, lipidations, or other post-translational modification (PTM) sites. This GML is analyzed in a fourth assay for the same insulin secretion function. This is used to select that best chimera with PTM sites. These sites can be used to increase the half-life of the protein or improve other properties.

[0061] In phase E (FIG. 6), the receptor signaling of the receptors that are targeted by the peptide chimera is analyzed. This approach can be used to identify resistance mutations in the receptors that in diagnostic tests may be present in patients. This develops precision targeting of the chimera for specific patients based on a genetic test. If multiple chimeras are tested, this approach can be used to make a companion diagnostic to select among different chimera or fusion polypeptide based on mutants in a patient. For example. FIG. 6 shows a companion diagnostic for three drugs that target the IFN receptor.

[0062] In phase F (FIG. 7), the assay is used to screen a variant GML for antagonists, also called inverse agonists. As an example, saturating mutagenesis on GIP is performed, then the assay is performed in the presence of GIP stimulation. A GML for GIP mutants, or a chimera library can be screened to identify mutants that bind the GIP receptor but do not signal, thereby blocking the external GIP signal and resulting in the mutant being identified in the autocrine assay as a mutant that does not produce signal. This approach can be used to identify the best antagonists against a particular target. The approach can also be adapted for balanced hybrids, or unbalanced hybrid by designing new libraries and setting multiple orthogonal assays to test multiple activities.

[0063] These antagonists can be identified for any of the receptors shown using the autocrine assay. Once they are identified, they can be included in the Chimera GML library, allowing for a subsequent Phase A screening to be performed on chimera libraries that also contain these antagonists.

[0064] In phase G (FIG. 8), the same approach can be used to expand from double peptide chimera to triple peptide or multi-peptide chimera, with the resulting triple or multi-peptide chimera being available to screen in a subsequent Phase A screen.

[0065] This pipeline can also include orthogonal assays with one assay for each of the chimera peptide receptors. This can be used to tune the signal intensity of each peptide component. Alternatively, the same pipeline could be used to create balanced or unbalanced hybrids.

[0066] Also provided herein are methods of identifying one or more member(s) of a library of fusion polypeptides, with functional activity against one or more target receptor(s) that is similar to or greater than a corresponding wildtype polypeptide, where each fusion polypeptide among the library of fusion polypeptides independently comprise a first polypeptide directly or indirectly linked to a second polypeptide, and where the corresponding wildtype polypeptide comprises a wildtype of the first polypeptide or a wildtype of the second polypeptide. In some embodiments, the methods comprise (a) transforming or transducing each host cell among the plurality of host cells individually with a unique vector that comprises a barcode, and a polynucleotide sequence encoding a fusion polypeptide among the library of fusion polypeptides, thereby resulting in expression of the fusion polypeptide on a surface of the host cell that was transformed or transduced with the unique vector; (b) screening each fusion polypeptide expressed on the surface for each host cell for functional activity against the one or more target receptor(s), wherein the functional activity comprises: (i) a level of binding of the fusion polypeptide to the one or more target receptor(s); or (ii) a level of signaling modulation of the one or more target receptor(s) facilitated by the fusion polypeptide; (c) identifying a subset of host cells that express one or more member(s) of the library of polypeptides that have similar or greater functional activity against the one or more target receptor(s) as compared to the corresponding wildtype polypeptide; and (d) sequencing the barcode of the unique vector present in the subset of host cells identified in (c), thereby identify ing one or more member(s) of the library of fusion polypeptides that have similar or greater functional activity against the one or more target receptor(s) as compared to the corresponding wildty pe polypeptide.[0067| Further, the methods as provided herein include the generation of a gene mutation library of cDNAs that encodes for a library of fusion polypeptides. As used herein, the fusion polypeptide comprises a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide or the second polypeptide can comprise one or more amino acid substitution in a wildtype protein sequence. In some embodiments, the first polypeptide and / or the second polypeptide of the fusion polypeptidecan comprise a single amino acid substitution. In other cases, the first polypeptide and / or the second polypeptide of the fusion polypeptide can comprise at least two amino acid substitutions. In some embodiments, each fusion polypeptide is compared to the corresponding wildtype of the first polypeptide and / or the corresponding wildtype of the second polypeptide. In some cases, the comparison is based on a statistical model to test a specific hypothesis regarding the biological activity of each fusion polypeptide. In someembodiments, the specific hypothesis regarding the biological activity of each fusion polypeptide can include but is not limited to loss-of-function, gain-of-function, substantially similar activity to the wildtype target protein, a drug resistant, a drug susceptible, or a drug super-susceptible variant. In some embodiments, the specific hypothesis regarding the biological activity of each fusion polypeptide can comprise a loss-of-function variant, a gain-of-function variant, a fusion polypeptide with substantially similar activity to the wildtype target protein, a drug resistant variant, a drug susceptible variant, or a drug super-susceptible variant. In some embodiments, a fusion polypeptide is a loss-of-function variant, a gain-of-function variant, a variant with substantially similar activity to the wildtype protein, a drug resistant variant, or a drug susceptible variant. In some embodiments, each cDNA comprises a single fusion polypeptide. In some embodiments, a cDNA library is generated where each cDNA in the library encodes a fusion polypeptide comprising an amino acid substitution in a wildtype target protein. Moreover, the cDNA library can comprise cDNAs encoding fusion polypeptides each containing an amino acid substitution in a different amino acid in the wildtype protein or an INDEL, or systematically swapping each amino acid position, or adding or removing both N-terminal and C-terminal extensions. In some embodiments, the cDNA library can comprise mutations in each and every residue in the wildtype protein. In some instances, a cDNA library is generated comprising cDNAs each containing at least one amino acid substitution in a target protein. In some embodiments, the cDNAs results in the expression of the target protein comprising a mutation at one or more amino acid position(s).

[0068] The fusion polypeptides as disclosed herein can comprise a wildtype of the first polypeptide comprising one or more amino acid substitutions directly or indirectly linked to a wildtype of the second polypeptide comprising one or more amino acid substitutions. In some embodiments, the wildtype of the first polypeptide and the wildtype of the second polypeptide can include, but is not limited to, a blood coagulation protein, an immunoglobulin, a cytokine, a chemokine, an angiopoietin, a grow th factor, a tissue growth factor, a fibroblast growth factor, a bone morphogenic protein, a neurotrophic factor, a stem cell factor (SCF), a tumor necrosis factor, a transforming growth factor, an interferon, an incretin, a colony stimulating factor, a soluble form of tumor necrosis factor receptors, an interleukin receptor and soluble forms of interleukin receptors, a hormone, a stimulating-hormone, albumin, a lipoprotein, a fetoprotein, an integrin, or any fusion proteins comprising any of the above mentioned proteins or fragments thereof.

[0069] Also provided herein are engineered human or mammalian cell lines that encode a receptor, optionally a surface-expressed receptor, operably linked to a reporter system. In some cases, the receptor and the reporter system can indicate if one of the fusion polypeptides binds toand modulates a biological activity of the receptor. In some embodiments, the receptor is a surface-expressed receptor. The term ‘‘surface-expressed receptor” refers to cell surface receptors (membrane receptors, transmembrane receptors) that are embedded in the plasma membrane of cells. For example, the surface-expressed receptor can include an immunoglobulin-binding receptor, a cytokine receptor, a chemokine receptor, an interferon receptor, an interleukin receptor, an activin receptor, an adrenomedullin receptor, an endothelin receptor, a G-protein coupled receptor, an angiopoietin receptor, a growth factor receptor, a tissue growth factor receptor, a fibroblast growth factor receptor, a bone morphogenic protein receptor, a neurotrophic factor receptor, a stem cell factor (SCF) receptor, a tumor necrosis factor receptor, a transforming growth factor receptor, a colony stimulating factor receptor, a hormone receptor, a stimulating- hormone receptor, albumin receptor, a lipoprotein receptor, a fetoprotein receptor, an integrin receptor, or any fusion proteins comprising any of the above mentioned proteins or fragments thereof.

[0070] As used herein, the term “library,” refers to a pool of clones with at least two polynucleotides, cell clones, molecules, or proteins. In certain embodiments, a library is used to screen for cDNAs encoding fusion polypeptides. In some embodiments, the library is a plasmid cDNA library. In other embodiments, the library is a viral library. The viral library, for example, can comprise a lentiviral library, an adenoviral library, or a retroviral library. In other embodiments, a plasmid cDNA library of cDNA is converted into a viral library. In other embodiments, a library of protein drugs is mixed with a single cell clone, and then a biological assay is used to discern which drugs cause a response in the cell clone. A plasmid cDNA library can comprise 100, 1,000, 10,000, 100,000, or 1 million different cDNA molecules. In other embodiments, a plasmid cDNA library comprises 100, 1,000, 10,000, 100,000, 1 million, 10 million, or 100 million cDNAs further comprising a unique molecular identifier (UMI) or a barcode group, or cells engineered to express the cDNA with UMIs or barcodes as RNA.

[0071] In some embodiments, each cDNA of the cDNA library is incorporated into one plasmid, such that each cDNA of the cDNA library is individually and independently incorporated into its own plasmid, forming a plasmid cDNA library. In some embodiments, each cDNA of the cDNA library expresses a fusion polypeptide. In some embodiments, each plasmid of the plasmid cDNA library has only one unique cDNA of the plurality of cDNAs, and wherein each plasmid in the plasmid cDNA library further comprises a unique molecular identifier (UMI) or a barcode group. In some embodiments, each plasmid of the plasmid cDNA library comprises two unique cDNA of the plurality of cDNAs, that expresses a fusion polypeptide and wherein each plasmid in the plasmid cDNA library further comprises a unique molecular identifier (UMI) or a barcode group. In some embodiments, the cDNA library is packaged into a viral vector, thereby generating aviral library. In some embodiments, the viral library comprises virions. In some embodiments, each virion comprises only one viral plasmid of the cDNA library. In some embodiments, the viral library is a retroviral library, an adenoviral library, or a lentiviral library. In some embodiments, the viral library is a lentiviral library. In some embodiments, the lentiviral vector includes an expression cassette. In some embodiments, the expression cassette can include a promoter operably linked to the polynucleotide sequence encoding a fusion polypeptide. In some embodiments, the promoter operably linked to the polynucleotide encoding the fusion polypeptide is inducible. In some embodiments, the target cells are transduced with the cDNA library lentiviral pool at a low multiplicity of infection (MOI) such that >95% of the target cells express a single fusion polypeptide. In other embodiments, the target cells are transduced with the lentiviral library lentiviral pool at a high multiplicity of infection so that target cells express fusion polypeptides. The terms “multiplicity of infection” or “MOI” are used according to its plain ordinary meaning in Virology and refers to the ratio of infectious agent (e.g., a virus) to the target (e.g., a cell) in a given area or volume. In embodiments, the area or volume is assumed to be homogenous. In some embodiments, the MOI is at least 0.001. at least 0.01, at least 0.01, at least 0.1. at least 0.2, at least 0.3, at least 0.4. at least 0.5, at least 0.6, at least 0.7. at least 0.8, at least 0.9, or at least 1.

[0072] In some embodiments presented herein is a method of preparing a plurality of a plasmid cDNA library from a plurality of cDNAs, the method comprising incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNA sample encodes a fusion polypeptide. In one embodiment, the tag comprises a cell-specific identifier sequence and a unique molecular identifier (UMI) sequence. In some embodiments, the tag comprises a cell-specific identifier sequence without the UMI. In some embodiments, the tag comprises a barcode. The method further comprises pooling the tagged cDNA samples; optionally amplifying the pooled cDNA samples to generate a cDNA library comprising doublestranded cDNA, thereby generating a plurality of tagged cDNA. In some embodiments, the cDNA of the plurality of cDNAs further comprises a UMI or a barcode group. In some embodiments, the UMI or the barcode groups can be immobilized to a solid support. For example, the solid support can be one or more beads. Thus, in certain embodiments, a plurality of beads can be presented, wherein each bead in the plurality bears a unique sample barcode and / or UMI sequence. In some embodiments, each cDNA of the cDNA library is contacted with one or more beads having a unique set of sample barcodes and / or UMI sequences in order to identify the cDNA of the cDNA library. In some embodiments, purified nucleic acid from a transduced cell are contacted with one or more beads having a unique set of sample barcodes and / or UMI sequences in order to identify the purified cDNA from the transduced cell.

[0073] “Constitutive activity” is defined as the elevated basal activity of a receptor which is independent of the presence of an agonist. Constitutive activity of a receptor may be measured using a number of different methods, including cellular (e.g., membrane) preparations, purified reconstituted receptors with or without the associated G-protein in phospholipid vesicles, and functional cellular assays.

[0074] “Agonist” is defined as a compound that increases the activity of a receptor when it contacts the receptor.

[0075] An “antagonist” is defined as a compound that competes with an agonist for binding to a receptor, thereby blocking the action of an agonist on the receptor. However, an antagonist (also known as a “neutral” antagonist) binds, but has no effect on constitutive receptor activity .

[0076] An “inverse agonist” is defined as a compound that decreases the basal activity of a receptor (i.e., signaling mediated by the receptor). Such compounds are also known as negative antagonists. An inverse agonist is a ligand for a receptor that causes the receptor to adopt an inactive state relative to a basal state occurring in the absence of any ligand. Thus, while an antagonist can inhibit the activity of an agonist, an inverse agonist is a ligand that can alter the conformation of the receptor in the absence of an agonist. Agonists are proposed to stabilize the receptor in an active conformation. Conversely, inverse agonists are believed to stabilize an inactive receptor conformation. Thus, while an antagonist manifests its activity by virtue of inhibiting an agonist, an inverse agonist can additionally manifest its activity in the absence of an agonist by inhibiting the spontaneous conversion of an unliganded receptor to an active conformation.Method of identifying IFN variant polypeptides, and fusion polypeptides

[0077] Disclosed herein are polypeptides and fusion polypeptides comprising a first polypeptide, and a second polypeptide, where the first polypeptide, the second polypeptide or both comprise an interferon (IFN) polypeptide. In some embodiments, the polypeptide comprises an IFN polypeptide. In some embodiments, the polypeptide comprises an IFN variant polypeptide. In some embodiments, the fusion polypeptide comprises at least two fusion polypeptides. Disclosed herein are fusion polypeptides comprising an amino acid substitution, relative to a wildtype IFN protein, where the fusion polypeptides comprising the first polypeptide and second polypeptide has substantially similar or greater biological activity as compared to a corresponding wildtype of the first polypeptide or wildtype of the second polypeptide biological activity. Such fusion polypeptides can be used as a treatment for diseases or conditions such as inflammatory conditions, multiple sclerosis and cancer. In some embodiments, the fusion polypeptide comprises two IFN polypeptides. In some embodiments, the IFN polypeptides can comprise apolypeptide from a Type I IFN, a Type II IFN, or a Type III IFN. In some cases, the Type I IFN protein can include, but is not limited to, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6. IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNω (IFNW1), IFNε (IFNE), IFNκ (IFNK), IFNβ (IFNB1), any psuedoproteins or functional fragments thereof. In some embodiments, the Type II IFN protein includes IFNγ (IFNG). In some embodiments, the Type III IFN protein can comprise IFN-λ1 (IFNL1 or IL29), IFN-λ2 (IFNL2 or IL28A), IFN-λ3 (IFNL3 or IL28B), and IFN-λ4 (IFNL4).

[0078] In some embodiments, the fusion polypeptide can have increased functional activity as compared to a corresponding wildtype IFN protein. In some embodiments, the corresponding wildty pe IFN protein can comprise IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA14, IFNA16. IFNA17, IFNA21, IFNB1, IFNE1, IFNK, IFNL1. IFNL2, IFNL3, IFNL4. IFNW1_21 or IFNW1_23. In some embodiments, the corresponding wildtype IFN protein can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence similarity to any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some embodiments, the corresponding wildtype sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence length to any one of SEQ ID NO: 1 to SEQ ID NO: 23.

[0079] TABLE 1. Exemplary IFN amino acid sequencesIFN variant polypeptides

[0080] In some embodiments, the wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23 can comprise an amino acid substitution. In some embodiments, the wildtype IFN protein comprising the amino acid substitution is a variant IFN polypeptide. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identify and at least about: 90% sequence length to SEQ ID NO: 1. In some cases, the variant polypeptide further comprises a modification selected from the group consisting of D2N, P4S, E5Q, DION, L18M, S22R, S27F, M31K, G37E. P52Q, A114E. S116F. K121R, R125Q and any combination thereof, relative to SEQ ID NO: 1. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identify to any one of SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, the variant polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %. 93%, 94%, 95%, 96% 97%. 98% or 99% sequence identify and at least about: 90% sequence length to SEQ ID NO: 2. In some cases, the variant polypeptide further comprises a modification selected from the group consisting of D4N, P6S, G12N, S13N, L20M, L28P, G39E, E54Q, T55A, P57S, I66T, K73E, D80E, E81Q, T82S, and any combination thereof, relative to SEQ ID NO: 2. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identify to SEQ ID NO: 26. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 5. In some cases, the variant IFN polypeptide further comprises a modification selected from the group consisting of H34Y, E40Q, E42V, H46N, V56A, L57F, E71 K, D72N, E78D, Q79E, S80T, S86Y, T87I, Y90F, L93M, R121K, T132M, and any combination thereof, relative to SEQ ID NO: 5. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%. 98% or 99% sequence identify to SEQ ID NO: 27 or SEQ ID NO: 28. In some embodiments, the variant IFNpolypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 6. In some cases, the variant IFN polypeptide further comprises a modification selected from the group consisting of D2N, P4S, S10G, SION, T14A. M16I, I17L, M18L, A19G, F152L, L156F, A158T, K159Q, G161R, E162K, R163G, E168D, and any combination thereof, relative to SEQ ID NO: 6. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 29 - SEQ ID NO: 33. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 8. In some cases, the variant IFN polypeptide further comprises a modification selected from the group consisting of D95N, VI06M, F116S, M132T, F152L, F154L, T156I, K159Q, G161R, R163K, R164S, D166E, and any combination thereof, relative to SEQ ID NO: 8. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%. 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 37 - SEQ ID NO: 39. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 9. In some cases, the variant IFN polypeptide further comprises a modification selected from the group consisting of R24G, G195S, A199T. I201M, L202I, L203M, R207G. R216G, E222G, D230G, K231N, and any combination thereof, relative to SEQ ID NO: 9. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 42. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%. 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 10. In some cases, the variant IFN polypeptide further comprises a modification selected from the group consisting of G19A, R37G, I38L, R121K, I132M, E133G, R134K, and any combination thereof, relative to SEQ ID NO: 10. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 43 - SEQ ID NO: 45. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 11. In some cases, the variant IFN polypeptide further comprises a modification selected from the group consisting of N2D,S4P, Q5E, N10D, S116F, K121R, M132T, L152F, Q159K, R161G, R161I, R163K, R164S, D166E, and any combination thereof, relative to SEQ ID NO: 11. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%. 91%. 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 46 - SEQ ID NO: 50. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%. 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 15. In some cases, the variant IFN polypeptide further comprises a K107M modification relative to SEQ ID NO: 15. In some embodiments, the variant IFN polypeptide comprises an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity to SEQ ID NO: 58.

[0081] In some embodiments, the variant IFN polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence similarity to any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the variant IFN polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence length to any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid modifications is any mutation disclosed in Table 2.

[0082] TABLE 2. IFN variant polypeptides

[0083] In some embodiments, the polypeptides described having an amino acid sequence of any one of SEQ ID NO: 24 - SEQ ID NO: 58 can have a biological activity of less than 70% of the biological activity of the wildtype IFN having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the polypeptides can have less than 60% of the biological activity of the wildtype IFN. In some embodiments, the polypeptides can have less than 50% of the biological activity of the wildtype IFN. In some embodiments, the polypeptides can have less than 40% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have less than 30% of the biological activity of the wildtype IFN. In some embodiments, the polypeptides can have less than 20% of the biological activity of the wildtype IFN. In some embodiments, the polypeptides can have less than 10% of the biological activity of the wildtype of which it is deduced (i.e., the wildty pe IFN of which the coding sequence has been mutated to obtain the mutant IFN).

[0084] In some embodiments, the polypeptides described having an amino acid sequence of any one of SEQ ID NO: 24 - SEQ ID NO: 58 can have a biological activity of more than 70% of the biological activity of the wildtype IFN having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the polypeptides can have more than 60% of the biological activity of the wildtype IFN. In some embodiments, the polypeptides can have more than 50% of the biological activity of the wildtype IFN. In some embodiments, the polypeptides can have more than 40% of the biological activity of the wildty pe IFN. In some embodiments, the polypeptides can have more than 30% of the biological activity of the wildty pe IFN. In some embodiments, the polypeptides can have more than 20% of the biological activity of the wildtype IFN. In some embodiments, the polypeptides can have more than 10% of the biological activityof the wildtype of which it is deduced (i.e., the wildtype IFN of which the coding sequence has been mutated to obtain the mutant IFN).IFN Fusion Polypeptides

[0085] Disclosed herein are fusion polypeptides comprising a first polypeptide directly or indirectly linked to a second polypeptide. In some cases, the fusion polypeptides disclosed herein can comprise a first polypeptide directly linked to a second polypeptide For example, the first polypeptide, can be directly linked to the second polypeptide by directly linking the C-terminal amino acid of the first polypeptide to the N-terminal amino acid of the second polypeptide through an amide bond. In some embodiments, the first polypeptide and / or the second polypeptide, prior to directly or indirectly being linked to each other, independently comprise an amino acid deletion, amino acid substitution, amino acid addition, or any combination of these, relative to a corresponding wildtype sequence. In some embodiments, the first polypeptide and / or the second polypeptide is a variant IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 24 - SEQ ID NO: 58. In some embodiments, the first polypeptide is a variant IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 24 - SEQ ID NO: 58. In some embodiments, the second polypeptide is a variant IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 24 - SEQ ID NO: 58. In some embodiments, the fusion polypeptide can comprise a first polypeptide and a second polypeptide are indirectly linked using a linker. In some embodiments, the linker comprises from at least about: 1 to 5, 1 to 10, 1 to 20, or 1 to 50 amino acids. In some cases, the linker is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15. 16. 17. 18. 19. 20. 25. 30. 35. 40. 45 or 50 amino acids in length. In some embodiments, the linker can comprise any amino acids. In some embodiments, the linker can comprise GGGGS, GGGGSGGGGS (SEQ ID NO: 406), or GGGGSGGGGSGGGGS (SEQ ID NO: 405). In some embodiments, the fusion polypeptide can comprise any one of SEQ ID NO: 59 - SEQ ID NO: 404. In some embodiments, the linker can be functionalized with a specific structural element. For example, the specific structural element can comprise removing digestive protease sites, and other such structural elements. In some embodiments, the fusion polypeptides can be expressed in a genetically modified cell.

[0086] TABLE 3: IFN fusion polypeptides

[0087] In some embodiments, the fusion polypeptides described having an amino acid sequence of any one of SEQ ID NO: 59 - SEQ ID NO: 404 can have a biological activity of less than 70% of the biological activity of the wildtype IFN having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the fusion polypeptides can have less than 60% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have less than 50% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have less than 40% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have less than 30% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have less than 20% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have less 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 mutant IFN).

[0088] In some embodiments, the fusion polypeptides described having an amino acid sequence of any one of SEQ ID NO: 59 - SEQ ID NO: 404 can have a biological activity of more than 70% of the biological activity of the wildtype IFN having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the fusion polypeptides can have more than 60% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have more than 50% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have more than 40% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have more than 30% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides can have more than 20% of the biological activity of the wildtype IFN. In some embodiments, the fusion polypeptides 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 mutant IFN).

[0089] In some embodiments, the fusion polypeptide as disclosed herein comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence similarity to any one of SEQ ID NO: 59 - SEQ ID NO: 404. In some embodiments, the fusion polypeptide binds to one or more target receptors. In some embodiments, the one or more target receptors comprise an interferon receptor or an interferon receptor subunit. In some embodiments, the fusion polypeptide binds to an interferon receptor or an interferon receptor subunit. In some embodiments, the interferon receptor is interferon-α / β receptor- 1 (IFN AR1), interferon-α / β receptor-2 (IFNAR2), interferon gamma receptor 1 (IFNGR1), interferon gamma receptor 2(IFNGR2), or interferon lambda receptor 1 (IFNLR1). In some embodiments, the fusion polypeptide can bind to an interleukin receptor. In some embodiments, the interleukin receptor is interleukin 10 receptor subunit beta (IL-10R2). In some embodiments, the fusion polypeptides bind to IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL-10R2 heterodimer subunits at a level greater than or equal to the corresponding wildtype IFN protein. In some embodiments, the binding affinity is measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the fusion polypeptide bind to an IFNAR1, IFNAR2, IFNGR1, IFNGR2. IFNLR1 or IL-10R2 heterodimer subunits heterodimer subunits at a level greater than or equal to the corresponding wildtype IFN protein as measured by ELISA and / or a GigaAssay e.g., measuring signaling activity to an ISRE-GFP reporter in a live human cell line in vitro assay. In some embodiments, the fusion polypeptide is an agonist of IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL-10R2 heterodimer subunits. In some embodiments, the fusion polypeptide is a partial agonist of IFN ARI, IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL-10R2 heterodimer subunits. In some embodiments, the fusion polypeptide is an inverse agonist of IFN ARI, IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL-10R2 heterodimer subunits. In some embodiments, the fusion polypeptide is an antagonist of IFNAR1. IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL- I0R2 heterodimer subunits. In some embodiments, the fusion polypeptide is a neutralizing antagonist of IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL-10R2 heterodimer subunits. In other embodiments, the fusion polypeptide binds and activates IFNAR1. IFNAR2, IFNGR1, IFNGR2. IFNLR1 or IL-10R2 heterodimer subunits at a level substantially similar to the wildtype IFN protein with the wildtype IFN-beta protein with the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the fusion polypeptide binds and activates IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL-10R2 at a level greater than the wildtype IFN protein with the wildtype IFN-beta protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the fusion polypeptide binds and inhibits IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1 or IL-10R2 at a level greater than the wildtype IFN protein with the wildtype IFN protein with the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the activity of IFNAR1, IFNAR2, IFNGR1, IFNGR2. IFNLR1 or IL-10R2 is measured by a reporter assay. In some embodiments, the reporter assay is an Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay, an ISRE luciferase reporter assay or a gamma-Activated Sequence (GAS) reporter assay.

[0090] In some embodiments, the fusion polypeptide reduces extracellular level of a pro- inflammatory cytokine as compared to wildtype IFN-beta protein. In some embodiments, the pro- inflammatory cytokine is a chemokine, an interferon (IFN), an interleukin (IL), a lymphokine, atumor necrosis factor (TNF), or any combination thereof. In some embodiments, the pro- inflammatory cytokine is IL-ip, IL-6, IL-8, IL-9. IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, TNF-β. or any combination thereof. In other embodiments, the fusion polypeptide increases extracellular level of an anti-inflammatory cytokine as compared to wildtype IFN-beta protein. In some embodiments, the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming grow th factor (TGF), or any combination thereof. In some embodiments, the anti-inflammatory cytokine is IL-4, IL-10, IL-11, IL-13, IFN-α, TGF-β. or any combination thereof.

[0091] In some embodiments, a glycosylation site is introduced in the fusion polypeptide that results in diminished binding of an antibody to the fusion polypeptide relative to an amount of binding of the antibody to the wildtype IFN-beta protein. In some embodiments, the non- naturally occurring beta protein has reduced immunogenicity when administered to a subject, as compared to administering the wildtype IFN-beta with the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23. In some embodiments, the reduced immunogenicity comprises a reduced level of anti-drug antibody in the subject when the fusion polypeptide is administered to the subject, as compared a level of anti-drug antibody when the wildtype IFN-β (IFN-beta) with the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23 is administered, as measured by ELISA on a sample obtained from the subject. In some embodiments, the non-naturally occurring beta protein, when administered to a subject in need thereof, reduces a level of inflammation in a subject, relative to a level of inflammation in the subject prior to the administering. In some embodiments, the reduced level of inflammation is determined by analyzing levels of an autoantibody, a C reactive protein (CRP), a proteolytic enzyme, an inflammatory mediator, a marker of ongoing inflammation, or any combination thereof, as measured by ELISA. In some embodiments, the reduced level of inflammation in the subject results in reduced serum levels of C reactive protein as measured by high-sensitivity C-reactive protein (hs-CRP) test. In some embodiments, the reduced serum levels of C reactive protein comprises a plasma concentration in the subject of from about 0.1 mg / dL to about 2.9 mg / dL. In some embodiments, the administering of the fusion polypeptide 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.

[0092] In some embodiments, a fusion polypeptide can comprise a first region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58, a second region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58. and a linker between the first region to the second region. In some cases, the amino acid sequence of the first region has at least 95% identity to any one of SEQ IDNO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid sequence of the first region comprises any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid sequence of the first region has at least 95% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some cases, the amino acid sequence of the first region comprises any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the second region has at least 95% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid sequence of the second region comprises any one of SEQ ID NO: 24 to SEQ ID NO: 58. In some embodiments, the amino acid sequence of the second region has at least 95% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the second region comprises any one of SEQ ID NO: 1 to SEQ ID NO: 23. In some cases, the linker comprises SEQ ID NO: 405. In some embodiments, the fusion polypeptide further comprises: a third region comprising a protein antigen, a protease, a single domain antibody, a designed ankyrin repeat protein (dARPIN), an anti-calin, or a nanobody. In some embodiments, the fusion polypeptide binds to an interferon receptor or an interferon receptor subunit at a level greater than or equal to a corresponding wildtype IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the fusion polypeptide is an agonist, a partial agonist, a selective agonist, an inverse agonist or an antagonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is an agonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is a partial agonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is an inverse agonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide is an antagonist of the interferon receptor or the interferon receptor subunit. In some embodiments, the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit at a level greater than a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by an assay comprising Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay or an ISRE luciferase reporter assay. In some embodiments, the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit resulting in a reduction of an extracellular level of a pro-inflammatory cytokine, as compared to a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured in an in vitro assay. In some embodiments, the pro-inflammatory cytokine is a chemokine. an interferon (IFN), an interleukin (IL), a lymphokine. a tumor necrosis factor (TNF), or any combination thereof. In some embodiments, the polypeptide or the fusion polypeptide binds and activates the interferonreceptor or the interferon receptor subunit resulting in an increase of an extracellular level of an anti-inflammatory cytokine, as compared to a corresponding wildtype IFN protein having the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23, as measured in an in vitro assay. In some embodiments, the in vitro assay is an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the anti-inflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming grow th factor (TGF), or any combination thereof. In some embodiments, the fusion polypeptide binds to an interferon receptor or an interferon receptor subunit at a level less than a corresponding wildtype IFN protein having an ammo acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by an in vitro assay. In some embodiments, the fusion polypeptide binds and inhibits the interferon receptor or the interferon receptor subunit at a level greater than a corresponding wildtype IFN protein having the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23. as measured by an assay by an ISRE GFP reporter assay or an ISRE luciferase reporter assay. In some embodiments, the fusion polypeptides can be expressed in a genetically modified cell.Nucleic Acid Constructs encoding variant library

[0093] A plasmid can be made by introducing compatible restriction enzyme sites (e.g., EcoRI, Sall, and AsiSI) and inserting a desired clone of the IFN variant library. An IFN variant encoding can be PCR amplified from a template with a Polymerase and cloned into the digested plasmid. In some embodiments, the plasmid is a retrovirus. In some embodiments, the plasmid is a lentiviral plasmid. In some embodiments, the plasmid is an AAV plasmid. In some embodiments, the viral vector corresponds to a virus of a specific serotype. In some examples, the serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV11 serotype, an AAV12 serotype, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.

[0094] In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some examples, the plasmid comprises circular double-stranded DNA. In some examples, the plasmid may be linear. Various selectable markers can be used to select for plasmid transduction such as puromycin or blasticidin S resistance genes. For example, the selectable marker GLP1 or IFN variant amplicons can be fused by inverse PCR using a polymerase. The fused amplicons are then cloned into the plasmid digested with a compatible restriction enzyme.

[0095] In some embodiments, a double stranded (ds) DNA library containing nucleic acid sequences that encode possible fusion polypeptides. In some cases, the nucleic acid sequences comprise all the possible single amino acid variants that are synthesized. The dsDNA from eachwell can be pooled and a single round of overlap PCR extension appended random-mers oligonucleotides to the 3’ untranslated region. The synthesized dsDNA library has a 3’-overhang sequence after the stop codon that overlaps with the 5’ overhang sequence upstream of the random-mers oligonucleotide sequence. The pooled ds DNA library and the random oligomer can be mixed in 1:2, 1:5, 1: 10, 1 :20, and 1 :50 molar ratio, denatured, and annealed. Hybridized DNA can be extended with a DNA Polymerase for one cycle of PCR. The PCR reaction mix can then be treated with an exonuclease and purified. The purified DNA can be digested with compatible restriction enzymes and ligated into the digested plasmid with a ligase. Ligation reactions can be pooled, purified, and dialyzed. The purified ligation reaction mixture can be electroporated into electrocompetent cells, plated, and incubated. Transformants can be scrapped from the plates and the plasmid library from the pooled cell suspension can be isolated.

[0096] Viral libraries (e.g., lenti viral libraries) can be produced in transformant compatible cells (e.g., LentiX 293T cells). In some embodiments, tens of thousands of cells to billions of cells are seeded into petri dishes. In some embodiments, approximately 3 million LentiX293T cells can be seeded in a petri dish and grown in complete media (e.g., DMEM+10% Fetal Calf serum). In some embodiments, the plasmid includes one or more regulatory elements. In some embodiments, the plasmid comprises packaging elements used in transformation of the viral plasmid. For example, the plasmid library; a vector encoding packaging elements Gag and Pol; a vector encoding a Rev packaging element; and a vector encoding an envelope protein are combined. In some embodiments, CaCl2 can be added to plasmid mixture. In some embodiments, 2x HEPES buffered saline can be added to the above transfection mix with stirring. The transfection mix can be incubated and added to the cells in a petri dish. The cells can be incubated in a CO2 incubator at 37°C with a 5% CO2 atmosphere. Post-transfection, the calcium phosphate-containing medium can be replaced with complete media (DMEM+10%FBS) and incubated in CO2 incubator at 37°C with a 5% CO2 atmosphere. In some cases, spent media from confluent transfected cells (e g., LentiX 293T) is filtered. Aliquots of the filtered spent media with the lentivirus can be frozen and stored.

[0097] Viral vectors for specific clones can be produced in cells (e.g., LentiX 293T). For example, the cells are seeded in a well of a well plate. After 24 hours, cells are co-transfected with the plasmid clone; one or more packaging elements or envelope proteins; and transfecting with a transfection reagent (e.g., Lipofectamine LTX (Invitrogen)). After incubation, media is replaced, and cells are cultured in complete media. Cell supernatants are collected, filtered, frozen, and stored.

[0098] Viruses can be titered by seeding cells in a well of a well plate and culturing in complete media (e.g., DMEM+10% FBS). In some cases, serial dilutions of virus can be added afterremoving majority of the spent media from the wells and incubated. Complete media can be added and incubated. In some embodiments, spent media is removed, replaced with complete media containing a selection compound (e.g.. puromycin), and incubated. In some embodiments, cells are inspected for viability under the microscope and colonies are counted to calculate the infectious unit / ml.

[0099] In some embodiments, the host cells can comprise a reporter. In some embodiments, the reporter can be GFP. In some embodiments, a plurality cells can be seeded in the well of a well plate and grown in complete media (e.g., DMEM) and then transfected with a reporter plasmid. For example, a GFP reporter plasmid carrying LTR-GFP and a resistance marker (e.g., blasticidin S-resistance (BSR) gene) is transfected in each host cell (e.g., LentiX 293T) and incubated. Transfected cells can be selected for marker resistance (e.g., blasticidin S). In some cases, the media (e.g.. DMEM ) with the marker can be exchanged every 3 days. In some cases, cells can be trypsinized and then serially diluted in well plates. In some embodiments, this process generates single colonies expressing the plasmids described herein that can- be expanded and screened.

[0100] In some embodiments. gDNA can be isolated to confirm viral integration. Tat amplicons can be subcloned and sequenced. In some cases, each clone in the host cell line can be assessed for Tat transcriptional activity' in a subculture. For example, cells cultured in well plates can be transfected with wildtype Tat expression vector, and transactivation-induced GFP expression can be evaluated by epifluorescence microscopy. In some cases, the host cells expressing the plasmids can generate a clonal cell line. In some cases, the clonal reporter cell line can be propagated, frozen, and stored.Nucleic Acids and Vectors encoding the polypeptides and fusion polypeptides

[0101] Provided herein are nucleic acids encoding the fusion polypeptides disclosed herein. Also provided herein are nucleic acids encoding the polypeptides disclosed herein. In some embodiments, the nucleic acid comprises a barcode sequence. In some embodiments, the nucleic acid sequence can 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: 407 - SEQ ID NO: 763. In some cases, the nucleic acid is a nucleic acid construct. In some embodiments, a nucleic acid construct comprises a polynucleotide sequence of any one of SEQ ID NO: 407 to SEQ ID NO: 763. In some embodiments, the nucleic acid construct further comprises a reporter gene. In some cases, the reporter gene is a fluorescent reporter gene. In some embodiments, the fusion polypeptide as disclosed herein can be encoded by a vector (e.g., a DNA vector). In some cases, a vector can comprise the nucleic acids or nucleic acid constructs disclosed herein. In some cases, the vector can be a viral vector such as a lentiviral vector or an adeno-associated viral vector. Insome embodiments, the fusion polypeptide as disclosed herein can be encoded by a nucleic acid sequence.

[0102] TABLE 4: Nucleic acid sequences encoding polypeptides and fusion polypeptides disclosed herein.-Ill-Compositions and Kits

[0103] Disclosed herein are pharmaceutical compositions comprising the polypeptides disclosed herein, the fusion polypeptides disclosed herein, the nucleic acids disclosed herein, the nucleic acid constructs disclosed herein or the vectors disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, a fusion polypeptide, or a vector encoding the fusion polypeptide disclosed herein, can be present in a composition. In some cases, the composition can be a pharmaceutical composition with a pharmaceutically acceptable excipient, diluent, or carrier. In some cases, the pharmaceutical composition or the composition is in unit dose form. In some embodiments, the pharmaceutical composition or the 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.

[0104] In some embodiments, a pharmaceutical composition can comprise an excipient, such as a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, a coloring agent. In some instances, a pharmaceutical composition can comprise a diluent such as water, glycerol, methanol, ethanol, and other similar biocompatible diluents. In some embodiments, the pharmaceutical composition can be in unit dose form.

[0105] Also disclosed herein are vaccine compositions comprising the polypeptides disclosed herein, the fusion polypeptides disclosed herein, the nucleic acids disclosed herein, the nucleic acid constructs disclosed herein or the vectors disclosed herein, a microbial antigen, a tumor antigen, or a nucleic acid encoding the microbial antigen or the tumor antigen; and a pharmaceutically acceptable carrier diluent, or excipient.

[0106] Also disclosed herein are kits comprising the polypeptides disclosed herein, the fusion polypeptides disclosed herein, the nucleic acids disclosed herein, the nucleic acid constructs disclosed herein or the vectors disclosed herein, and a scaffold.Methods of treatment

[0107] Also provided herein are methods of treating a disease or condition in a subject in need thereof comprising administering a fusion polypeptide, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the vector to a subject to treat a disease or condition. For example, the fusion polypeptide as disclosed herein, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the vector, can be used to treat an inflammatory condition. In some embodiments, the fusion polypeptide as disclosed herein, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the vector, can be used to treat a disease or condition. In some embodiments, the disease or condition is a cancer (e g., a melanoma, a bladder cancer, a head or neck cancer, a kidney cancer, a liver cancer, a non-small cell lung cancer), a viral infection (e.g., a hepatitis infection such as chronic 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).

[0108] In some embodiments, the disease or the condition comprises a viral infection, a cancer, an autoimmune disease, multiple sclerosis, or a hemolytic disease. In some embodiments, the disease is an infection. In some cases, the infection is a viral infection. In some embodiments, the viral infection is a chronic hepatitis B (CHB), a chronic hepatitis C (CHC), a chronic granulomatous disease (CGD), a coronavirus disease (COVID), a genital wart or a humanimmunodeficiency virus (HIV). In some cases, the disease is a demyelinating disorder. In some embodiments, the demyelinating disorder is Multiple Sclerosis (MS). Acute Disseminated Encephalomyelitis (ADEM), Acute Hemorrhagic Leucoencephalitis (AHLE), Balo’s disease (Concentric Sclerosis), Charcot-Marie-Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease). In some embodiments, the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms. In some cases, the disease is a cancer. In some embodiments, the cancer is a blood cancer, a bone cancer, a brain cancer, a breast cancer, a cervical cancer, a colon cancer, a heart cancer, a kidney cancer, a liver cancer, a lung cancer, a lymph node cancer, a lymphoma, a melanoma, a myeloma, a skin cancer, a stomach cancer, a pancreatic cancer, a testicular cancer, or a throat cancer. In some embodiments, the cancer is selected from the group consisting of astrocytoma, chronic myelogenous leukemia, follicular lymphoma, glioblastoma, hairy cell leukemia, Kaposi’s sarcoma, malignant melanoma, medulloblastoma, melanoma, multiple myeloma, non-Hodgkin lymphoma, and renal cell carcinoma. In some cases, the disease is an immune disorder. In some embodiments, the immune disorder is selected from the group consisting of lupus, systemic lupus erythematosus, asthma, atherosclerosis, atopic dermatitis, autoimmune hepatitis, Celiac disease, COPD, Crohn's disease, Graves' disease, Guillain-Barre syndrome, IgA nephropathy, inflammatory bowel disease, pelvic inflammatory disease, primary biliary cirrhosis, primary hemophagocytic lymphohistiocytosis, ulcerative colitis, and other autoimmune disorders.

[0109] Also provided herein are methods of generating an immune response in a subject, the method comprising administering to the subject the vaccine composition, thereby generating an immune response in the subject. In some cases, the subject is a human.

[0110] In some embodiments, a fusion polypeptide as disclosed herein, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the 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, intracavemous, intracavitary, intracerebroventricular, intracistemal, intracorneal, intracoronal, intracoronary, intracorpous cavemaosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial,intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic. intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, retrobulbar, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic. ureteral, urethral, vaginal, infraorbital, intraparenchymal, intrathecal, intraventricular, stereotactic, or any combination thereof. In some embodiments, the administering is intramuscular administration, intraocular administration, intranasal administration, subcutaneous administration, intravenous administration, intracranial administration, intrathecal administration, intertumoral administration, oral administration, sublingual administration, buccal administration, rectal administration, vaginal administration, or otic administration. In some embodiments, the administering is once every 24 hours, once every 48 hours, once every 72 hours, once every 96 hours, once every 120 hours, once every 144 hours, once every 168 hours, once every 280 hours, or once every 336 hours. In some embodiments, the administering occurs from about once a day, about twice a day, about once a week, about twice a week, about once every two weeks, about once a month, about once every 3 months, about once every 6 months, about once every 9 months, to about once a year.

[0111] In some embodiments, the method further comprises administering a second therapeutic. In some embodiments, the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof. In some embodiments, the method further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1, a PD-L1, CTLA-4 inhibitor. In some embodiments, the fusion polypeptide as disclosed herein, a vector encoding the fusion peptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the vector, can be co-administered along with a second therapeutic. In some embodiments, the second therapeutic can be an antibody, a peptide, an antibody drug conjugate, or any combination thereof. In some cases, the second therapeutic can be an immunotherapy agent such as a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor can be a PD- 1 inhibitor such as nivolumab or pembrolizumab, a CTLA-4 inhibitor such as ipihmumab, or a PD-L1 inhibitor such as atezolizumab, avelumab, or durvalumab.

[0112] In some embodiments, the fusion polypeptide as disclosed herein, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the vector, can be administered 1, 2, 3. or 4 times a day; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12. 14, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 times a week; or 1, 2. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74. 75. 76. 77, 78, 79, 80, 81, 82, 83, 84. 85. 86, 87, 88, 89. or 90 times a month. In some embodiments, fusion polypeptide as disclosed herein, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the vector, can be administered every day, every other day, once a week, or once a month.

[0113] In some embodiments, the fusion polypeptide as disclosed herein, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the 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.

[0114] In some embodiments, the fusion polypeptide as disclosed herein, a vector encoding the fusion polypeptide as disclosed herein, or a pharmaceutical composition comprising the fusion polypeptide or the vector, can independently be administered at a dose of about: 10 ng, 11 ng, 12 ng, 13 ng, 14 ng. 15 ng, 16 ng, 17 ng. 18 ng, 19 ng, 20 ng, 21 ng, 22 ng, 23 ng, 24 ng, 25 ng. 26 ng, 27 ng, 28 ng, 29 ng, 30 ng, 31 ng, 32 ng, 33 ng, 34 ng, 35 ng, 36 ng, 37 ng, 38 ng, 39 ng, 40 ng, 41 ng, 42 ng, 43 ng, 44 ng, 45 ng, 46 ng, 47 ng, 48 ng, 49 ng, 50 ng, 51 ng, 52 ng, 53 ng, 54 ng, 55 ng, 56 ng, 57 ng, 58 ng, 59 ng, 60 ng, 61 ng, 62 ng, 63 ng, 64 ng, 65 ng, 66 ng, 67 ng, 68 ng, 69 ng, 70 ng. 71 ng, 72 ng, 73 ng. 74 ng, 75 ng, 76 ng, 77 ng, 78 ng, 79 ng, 80 ng, 81 ng. 82 ng. 83 ng, 84 ng. 85 ng. 86 ng, 87 ng. 88 ng, 89 ng, 90 ng. 91 ng, 92 ng, 93 ng. 94 ng, 95 ng. 96 ng, 97 ng, 98 ng, 99 ng, 100 ng, 110 ng, 120 ng, 130 ng, 140 ng, 150 ng, 160 ng, 170 ng, 180 ng, 190 ng, 200 ng, 210 ng, 220 ng, 230 ng, 240 ng, 250 ng, 260 ng, 270 ng, 280 ng, 290 ng, 300 ng,310 ng, 320 ng, 330 ng, 340 ng, 350 ng. 360 ng, 370 ng, 380 ng, 390 ng, 400 ng, 410 ng, 420 ng,430 ng, 440 ng, 450 ng, 460 ng. 470 ng. 480 ng, 490 ng, 500 ng, 510 ng. 520 ng, 530 ng, 540 ng,550 ng, 560 ng, 570 ng, 580 ng, 590 ng, 600 ng, 610 ng, 620 ng, 630 ng, 640 ng, 650 ng, 660 ng,670 ng, 680 ng, 690 ng, 700 ng, 710 ng, 720 ng, 730 ng, 740 ng, 750 ng, 760 ng, 770 ng, 780 ng,790 ng. 800 ng, 810 ng, 820 ng, 830 ng, 840 ng, 850 ng, 860 ng, 870 ng, 880 ng, 890 ng, 900 ng,910 ng. 920 ng, 930 ng, 940 ng, 950 ng. 960 ng, 970 ng, 980 ng, 990 ng, 1 μg, 1.1 μg, 1.2 μg, 1.3 μg, 1.4 μg, 1.5 μg, 1.6 μg, 1.7 μg, 1.8 μg, 1.9 μg, 2 μg, 2.1 μg, 2.2 μg, 2.3 μg, 2.4 μg, 2.5 μg, 2.6 μg, 2.7 μg, 2.8 μg, 2.9 μg, 3 μg, 3.1 μg, 3.2 μg, 3.3 μg, 3.4 μg, 3.5 μg, 3.6 μg, 3.7 μg, 3.8 μg, 3.9 μg, 4 μg, 4.1 μg, 4.2 μg, 4.3 μg, 4.4 μg, 4.5 μg, 4.6 μg, 4.7 μg, 4.8 μg, 4.9 μg, 5 μg, 5.1 μg, 5.2 μg, 5.3 μg, 5.4 μg, 5.5 μg, 5.6 μg, 5.7 μg, 5.8 μg, 5.9 μg, 6 μg, 6.1 μg, 6.2 μg, 6.3 μg, 6.4 μg, 6.5 μg. 6.6 μg. 6.7 μg. 6.8 μg. 6.9 μg. 7 μg. 7.1 μg. 7.2 μg. 7.3 μg. 7.4 μg. 7.5 μg. 7.6 μg, 7.7 μg, 7.8 μg, 7.9 μg, 8 μg, 8.1 μg, 8.2 μg, 8.3 μg, 8.4 μg, 8.5 μg, 8.6 μg, 8.7 μg, 8.8 μg, 8.9 μg, 9 μg, 9.1μg, 9.2 μg, 9.3 μg, 9.4 μg, 9.5 μg, 9.6 μg, 9.7 μg, 9.8 μg, 9.9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, 25 μg, 26 μg, 27 μg,28 μg, 29 μg, 30 μg, 31 μg. 32 μg, 33 μg, 34 μg, 35 μg, 36 μg. 37 μg, 38 μg, 39 μg, 40 μg. 41 μg, 42 μg, 43 μg, 44 μg, 45 μg, 46 μg, 47 μg, 48 μg, 49 μg, 50 μg, 51 μg, 52 μg, 53 μg, 54 μg,55 μg, 56 μg, 57 μg, 58 μg, 59 μg, 60 μg, 61 μg, 62 μg, 63 μg, 64 μg, 65 μg, 66 μg, 67 μg, 68 μg, 69 μg, 70 μg, 71 μg, 72 μg, 73 μg, 74 μg, 75 μg, 76 μg, 77 μg, 78 μg, 79 μg, 80 μg, 81 μg,82 μg, 83 μg, 84 μg, 85 μg. 86 μg, 87 μg, 88 μg, 89 μg, 90 μg, 91 μg, 92 μg, 93 μg, 94 μg. 95 μg, 96 μg, 97 μg, 98 μg, 99 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg,290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg. 460 μg, 470 μg, 480 μg, 490 μg, 500 μg, 510 μg,520 μg, 530 μg, 540 μg. 550 μg, 560 μg. 570 μg, 580 μg, 590 μg. 600 μg, 610 μg. 620 μg, 630 μg, 640 μg, 650 μg, 660 μg, 670 μg, 680 μg, 690 μg, 700 μg, 710 μg, 720 μg, 730 μg, 740 μg,750 μg, 760 μg, 770 μg, 780 μg, 790 μg, 800 μg, 810 μg, 820 μg, 830 μg, 840 μg, 850 μg, 860 μg, 870 μg, 880 μg, 890 μg, 900 μg, 910 μg. 920 μg, 930 μg, 940 μg, 950 μg, 960 μg, 970 μg,980 μg, 990 μg, 1 mg. 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg. 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg. 6.6 mg, 6.7 mg, 6.8 mg, 6.9 mg, 7 mg. 7.1 mg, 7.2 mg. 7.3 mg, 7.4 mg, 7.5 mg. 7.6 mg, 7.7 mg. 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg. 28 mg. 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg. 38 mg. 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg. 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg. 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, or 100 mg.EXAMPLESEXAMPLE 1: METHOD OF FUNCTIONALLY SCREENING A LIBRARY OF FUSION POLYPEPTIDESGeneration of a library of fusion polypeptides

[0115] An IFN dual agonist chimera library that was assayed. 21 IFN genes were synthesized (IDT) and cloned into the tethered soluble ligand autocrine GigaAssay vector system, under control of the Dox-inducible 1xTRE promoter and chicken HS4 insulator. This system can accommodate either single ligands or dual-ligand fusion polypeptides, that is fusion polypeptides as disclosed herein capable of binding one target receptor or at least two target receptors. A Type II transmembrane domain at the N-terminus is fused in-frame with a GGGGS (x1) flexible linker, FLAG affinity tag, and the fusion polypeptide coding sequence. To create a library of fusion polypeptides, each of the 21 first polypeptide sequences (SEQ ID NO: 24 - SEQ ID NO: 58) was linked to a GGGGS (x3) linker (GGGGSGGGGSGGGGS (SEQ ID NO: 405), followed in turn by one of the random 21 second polypeptide sequences (SEQ ID NO: 24 - SEQ ID NO: 58) in the second position (creating a total of 441 fusion polypeptides). The length of the linker can vary anywhere from 5 amino acids in length to 30 amino acids in length. After the genes were cloned, each cDNA molecule in the plasmid library was barcoded with a Unique Molecular Identifier (UMI) in the 3’UTR. Software was engineered to identify the first and second polypeptides in the fusion polypeptide, as well as any fragments that might be exchanged with other IFN genes by homologous recombination.

[0116] The resulting library of fusion polypeptides containing polypeptides with single receptor activity and fusion polypeptides with dual-receptor activity was sequenced by long CCS-read PacBio sequencing to associate the various chimeras to their UMI. In addition to the 21 single genes, all possible 441 dual wildtype IFN chimera combinations were identified, nearly all represented with 100-275 UMls (Table 5). This library was designed to contain both a first polypeptide or a second polypeptide that correlated with a Type I and Type III IFNs for versatility. In the cell lines not expressing receptor subunits for Type III signaling, the polypeptides containing the Type III IFNs could serve as negative controls. Due to the high degree of sequence similarity between the first and second polypeptides, significant recombination occurred in about 50% of the library clones during the cloning process. These fusion polypeptides were hybrids of one IFN protein with at least one other IFN protein. The library of fusion polypeptides contained 53,299 fusion polypeptides.

[0117] Table 5: Fusion polypeptide sequencing resultsIdentification of fusion polypeptides using a high-throughput screening assay

[0118] After the screening, the library of fusion polypeptides was transduced into reporter cells. There was no drop out for any of the 441 designed polypeptides with single receptor activity and fusion polypeptides with dual-receptor activity with each having an average measurement of 275 unique barcodes. The reporter activity was a weighted mean derived from the distribution of the reads in each of the cell sorted bins, ranked by fluorescence intensity (1-4 with 4 being the highest, labeled as “score” in the following tables) of the frequency of each barcode in each bin. The reporter activities for all barcodes for each of the fusion polypeptides was compared to that of wildtype IFN beta, resulting in p values ranged to as low as 10-296(Tables 6 - Table 9). Such confident p values, reflect the well-designed architecture of the high throughput screening assay which eliminated most false positive results. The q-value represented the False discovery rate adjust p-value of a t-test comparing the fusion polypeptide against its reference. Several of the fusion polypeptides identified had activity similar to wildtype and were classified as “REF,” while other fusion polypeptides with increased activity and were classified as gain of function("GOF") or gain of function-Like (“GOF-like”).

[0119] Table 6: Fusion polypeptide activity as compared to corresponding wildtype IFNA2

[0120] TABLE 7: Polypeptide and Fusion polypeptide activity from the high throughput assay as compared to IFNA2 activity

[0121] This experiment was completed for two separate technical replicates that produced highly similar results with a high Pearson correlation coefficient among activity measurement for each fusion polypeptide (R2 of 0.82; FIG. 9; TABLE 8).

[0122] TABLE 8: Fusion polypeptide activity from high throughput assay as compared to the corresponding wildtype protein.*Where X is any amino acid.

[0123] The results for the IFN chimera and single IFN activity scores and p values are shown in FIGs 10 and 11. As shown in FIG. 10, the color of each cell is the level of ISRE signaling activity of a fusion polypeptide with single receptor activity, whereas other cells refer to the dual activity fusion polypeptide with dual receptor activity as compared to corresponding wildtype IFN protein. Activities are shaded by activity scores with the maximum score being 3.0 for the fusion polypeptide with single receptor activity and the minimum IFN score being 1.8 for the IFNε -IFNK fusion polypeptide. The lowest score was 1.23 for an IFN fusion polypeptide containing a stop codon (p < 9.9 x 10-11) indicating that all fusion polypeptides have some ISRE signaling activity, but some fusion polypeptides were much lower.

[0124] This experiment describes the first landscape ever produced and assayed for a large set of dual-functional fusion polypeptides and thus the screen provides key insight into selection of the best performing fusion polypeptides for therapeutic purposes among this group as well as physiological properties of each IFN gene.

[0125] Fusion polypeptides can contain two identical or different polypeptides (for example, an IFN polypeptide having amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 405) connected by a short linker. Given that the goal of identifying therapeutic agents is high efficacy, molecular or cellular activity of the target receptor (or drug target) is a more reliable measure than affinity to the receptor. Here, we demonstrate that the method disclosed herein directly measured molecular and / or cell activity, and it only has a weak relationship with affinity ( FIG. 12).

[0126] Thus, disclosed herein is a method of measuring activity of Type I and III Single IFN Signaling, which is a novel way for identifying drug leads and superior to the prior approach of first screening by affinity. There is a weak trend of the ISRE signaling activity derived from the GigaAssay to match the IFN subtype rank order derived from published affinities of different Class 1 IFNs for the IFNAR1 or IFNAR1 / IFNAR2 receptor complex (R2 of 0.15; FIG. 10). In both cases, IFNβ has the strongest affinity and signaling with IFNω , the second highest for both. However, in most cases, either the signaling activity is stronger (e.g., IFNα1O) or weaker (e.g., IFNε) than the affinity relative to the dotted trendline.

[0127] We identify for the first time that IFNε and IFNλs have poor ISRE signaling activity, which is very relevant when combined in a fusion polypeptide. IFNε is a type I IFN that has detectable, but very low signaling activity when compared to other Type I IFNs. IFNλs are Type III IFNs and signal through a different receptor heterodimer. IFNλs activate transcription of genes with either a ISRE or GAS transcriptional response element. The ISRE reporter cells have the Type III IFNλ receptors. However, all four IFNλ show weak, but detectable activation of the ISRE signaling reporter (TABLE 6 - TABLE 9).

[0128] We have learned that the polypeptide variants (SEQ ID NO: 24 - SEQ ID NO: 58) have signaling activity that is equal to, or greater than that of the corresponding wildtype protein. Thus, making fusion polypeptides with the same polypeptide sequence for the first polypeptide and the second polypeptide did not increase efficacy for Type I and III IFNs receptors,

[0129] We observed that fusion polypeptides, depending on the combinations of different Type I and III IFNs, can have a range of activities that is greater than either of the single component interferons (e.g., IFNα2-IFNλ2), equal to the component IFNs (e.g., IFNα1-IFNα14), or is reduced from one or both component IFNs (e.g., IFNα1 -IFNK). We also observed that the order of the component IFNs in the chimera can markedly affect the activity of the fusion polypepitde (e.g.. I IFNλ2-IFNα16 does not equal I IFNα16-IFNλ2). A number of fusion polypeptides generated herein were determined as agonist drug leads, even in previously poorly characterized IFN family members (e.g., IFNα5-IFNα14).

[0130] TABLE 8: Type I and Type III IFN containing Fusion polypeptide activity from high throughput assay as compared to IFNA2 activity

[0131] Similarly, fusion polypeptide were also demonstrated to be antagonists drug leads. A number of fusion polypeptides were identified as antagonists that inhibit the endogenous physiological actions of IFNs (e.g., IFNε -IFNK or IFNK-IFNε blocks the activity of single component IFNK). We observed a number of fusion polypeptides antagonist drug leads, even in previously poorly characterized IFN family members such as IFNε. Approximately half of IFNαs. when combined with a IFNλ in a chimera had increased activity over the IFNα despite the inhibitor nature of the IFNλs.EXAMPLE 2: METHOD OF TREATING A DISEASE OR DISORDER USING AFUSION POLYPEPTIDE

[0132] In this invention, we disclose the relative ISRE signaling activity for all possible pairs of type 1 and type 3 IFN fusion polypeptides. Since that are measured in the same cells, under thesame conditions, and in the same experiment, all measurements are standardized against each other. As potential therapeutics, the chimera can be broken down into 3 distinct classifications some with established specificity for indications, although it is likely relevant to evaluate each class for each indication given the lack of general knowledge about the interactions of stimulation with more than IFN. The 3 classes are those fusion polypeptides that have strong agonism, strong antagonism, and modulators, in which different IFN agonist and antagonists are mixed to modulate the immune system. Specific examples are provided in this section.

[0133] We note that any of the proposed fusion polypeptides could be modified with glycosylation sites or pegylated, modifications used in many known drugs. Since the type 3 IFNλ is specific to epithelial cells, fusion polypeptides with these IFNs may be relevant to diseases involving cells in these tissues such as viral infectious diseases, melanoma, genital warts, lupus, and psoriasis. Examples for each category are discussed.Antiviral Therapies with fusion polypeptides

[0134] Fusion polypeptides disclosed herein, for example those containing IFNλ1-IFNλ4, can act as potent antiviral treatment.

[0135] A human subject diagnosed with human immunodeficiency virus (HIV) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with HIV. Different IFNs can be used to control HIV infection. For example, combining IFNα and IFNβ reduces the number of latent cells, IFN P and IFNα14 together suppress HIV replication, and IFNα4, IFNα8, and IFNα14 activate natural killer cells. The combined actions of different agonist suggest that fusion polypeptides may be good therapeutics. We observed several fusion polypeptides described in EXAMPLE 1 that contain these IFN components that have significant ISRE signaling activity and could be used as agonists for HIV and Acquired immunodeficiency syndrome (AIDS) suppression therapeutics (IFNα2-IFNβ, IFNα4-IFNβ, IFNα8-IFNβ, IFNα14-IFNβ, IFNα2-IFNα14, IFNβ-IFNα2, IFNβ-IFNα4, IFNβ-IFNα8, IFNβ-IFNα14, IFNα14-IFNα2, and the trimeric IFNα2-IFNα14- IFNβ is a natural extension of these fusion polypeptide. These include any of the fusion polypeptides screened and identified in EXAMPLE 1 that contain IFNλ1-IFNλ4.

[0136] A human subject diagnosed with coronavirus disease 2019 (COVID-19) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with COVID- 19. IFNI is an antiviral cytokine that is a type 3 IFN. A PEGylated version of IFNλ was previously used in a randomized controlled clinical study of preventing COVID hospitalization or emergency showed a significance 51% reduction, suggesting that this cytokine may be useful for early treatment of COVID- 19. IFNλ is an agonist that binds the IFNLR1 / IL10R2 receptors in epithelial cells signaling through the ISREpathway. We observed several chimeras with IFNλ components that have significant, but modulated ISRE signaling activity and could be to prevent COVID- 19 hospitalization and progression. These include any of the fusion polypeptides screened and identified in EXAMPLE 1 that contain IFNλ1 -IFNλ4.

[0137] A human subject diagnosed with Chronic hepatitis B (CHB) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with CHB. Therefore, several of our IFNα2 or IFNα14 fusion polypeptides with other IFN subtypes may be more efficacious than IFNα2. IFNλ is a type 3 IFN agonist that binds the IFNLR1 / IL10R2 receptors in epithelial cells signaling through the ISRE pathway. Several fusion polypeptides that comprise IFNλ with IFNα2 or IFNα14 have significant activity, some of which modulated ISRE signaling activity and are proposed efficacious therapeutic method of alleviating CHB symptoms. These include any of the fusion polypeptides screened and identified in EXAMPLE 1.

[0138] A human subject diagnosed with Chronic hepatitis C (CHC) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with CHC. CHC is the leading cause of chronic liver disease and treated with FDA-approved pegylated IFNα2. Several of our fusion polypeptides containing IFNα2 is demonstrated more efficacious than IFNα2 used to treat CHC. IFNλs are type 3 IFN agonist that binds the IFNLR1 / IL10R2 receptors in epithelial cells signaling through the ISRE pathway. There is a genetic association between HPC and IFNλ3, in which IFNλ3 is protective: IFNλ4 is also implicated. Several fusion polypeptides with IFNλ and IFNα2 components (including fusion polypeptides with IFNλ1-IFNλ4) have significant, but modulated ISRE signaling activity, provide for an efficacious method of treating CHC in a subject. These include any of the fusion polypeptides screened and identified in EXAMPLE 1 that contain IFNλ1-IFNλ4.

[0139] A human subject diagnosed with Chronic hepatitis D (CHD) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with CHD. CHD is the most severe form of hepatitis viral infection and can lead to hepatocellular carcinoma. Several of our IFNα2 chimera may be more efficacious than known pegylated IFNα2. IFNλ is a ty pe 3 IFN agonist that binds the IFNLR1 / IL10R2 receptors in epithelial cells signaling through the ISRE pathway. Fusion polypeptides with IFNλ components that have significant, but modulated ISRE signaling activity and are better therapies for alleviating the symptoms associated with CHD. These include any of the fusion polypeptides screened and identified in EXAMPLE 1 that contain IFNλ1-IFNλ4.Oncological Therapies With Fusion Polypeptides

[0140] Fusion polypeptides disclosed herein can act as potent anti-cancer treatment.

[0141] A human subject diagnosed with melanoma is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with Melanoma. Melanoma is skin neoplasm derived from melanocytes. Fusion polypeptides comprising IFNβ and IFNα2 are considered treatments. We observed fusion polypeptides that had agonist activity with different IFNα genes and could be used as agonists for treating melanoma. For example, fusion polypeptides comprising IFNα2-IFNβ. IFNα4-IFNβ, IFNα8-IFNβ, IFNα14-IFNβ, IFNα2-IFNα14, IFNβ-IFNα2, IFNβ-IFNα4, IFNβ-IFNα8, IFNβ- IFNα14, and IFNα14-IFNα2 are used to treat a subject afflicted with melanoma. Several IFNα- IFNλ fusion polypeptide that have significant, but modulated ISRE signaling activity and could be to treat melanoma. For example, IFNλ2-IFNα2, IFNλ3-IFNα2. IFNα2-IFNλ2 and IFNα2- IFNλ3 had substantial ISRE signaling and as such are efficacious in treating melanoma. These include any of the fusion polypeptides screened and identified in EXAMPLE 1.

[0142] A human subject diagnosed with glioblastoma multiforme (GBM) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with GBM. GBM is a brain neoplasm. IFNβ and IFNα2 are considered as potential treatments. We observed fusion polypeptides (IFNα2-IFNβ, IFNα4-IFNβ, IFNα8-IFNβ, IFNα14-IFNβ, IFNα2-IFNα14, IFNβ-IFNα2, IFNβ-IFNα4. IFNβ- IFNα8, IFNβ-IFNα14, IFNα14-IFNα2) that had agonist activity with different IFNα genes and could be used as agonists for GBM suppression therapeutics. We observed several IFNα-IFNλ fusion polypeptides that have significant, but modulated ISRE signaling activity and could be to treat GBM. For example, IFNλ2-IFNα2, IFNλ3-IFNα2, -FNα2-IFNλ2 and IFNα2-IFNλ3 had substantial ISRE signaling. These include any of the fusion polypeptides screened and identified in EXAMPLE 1

[0143] A human subject diagnosed with polycythemia vera (PV) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with PV. PV is a JAK2-mutated neoplasm characterized by eiythrocytosis. While, treating subjects with PEG-IFNα2 reduced driver mutation burden in a Phase III trial, and PEG-IFNα2 is the first line therapy for younger patients with disease, the fusion polypeptides disclosed herein is more efficacious in treating subjects with PV. We observed fusion polypeptides (IFNα2-IFNβ, IFNα4-IFNβ, IFNα8-IFNβ, IFNα14- IFNβ, IFNα2-IFNα14, IFNβ-IFNα2, IFNβ-IFNα4, IFNβ-IFNα8, IFNβ-IFNα14, IFNα14- IFNα2) that had agonist activity with different IFNα genes and could be used as agonists for PV suppression therapeutics. We observed several IFNα-IFNλ fusion polypeptides that have significant, but modulated ISRE signaling activity and could be to treat PV. For example, IFNλ2-IFNα2, IFNλ3-IFNα2, IFNα2-IFNλ2 and IFNα2-IFNλ3 had substantial ISRE signaling. These include any of the fusion polypeptides screened and identified in EXAMPLE 1.Autoimmune Therapies With Fusion Polypeptides

[0144] Fusion polypeptides disclosed herein can act as potent autoimmune therapies.

[0145] A human subject diagnosed with Multiple Sclerosis (MS) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with MS. Single IFNβ and modified IFNβ polypeptides are used to treat MS. However, IFNβ-IFNλ and IFNλ-IFNβ fusion polypeptides have modest ISRE activity reduction when compared to IFNβ, but increased efficacy with these fusion polypeptides stem from IFNλs variant polypeptides that activate the GAS response element signaling pathway which synergizes with the ISRE pathway. Thus, the fusion polypeptides disclosed herein are better therapeutics for treating and alleviating the symptoms of MS. These include any of the fusion polypeptides screened and identified in EXAMPLE 1.

[0146] A human subject diagnosed with Systemic Lupus Ery thematosus (SLE) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with SLE. SLE is a rheumatologic disorder thought to be B-cell mediated and IFNα hyperactivity is central to the disease. A current strategy is to block the actions of IFNα with a neutralizing antibody to its receptor. One alternative would be to use inverse antagonist to either block or module IFN signaling. Our screen identified several likely inverse agonists fusion polypeptides (e.g., IFNε -IFNκ or IFNκ-IFNε blocks the activity of single component IFNκ) that inhibit signaling and the endogenous physiological actions of IFNs. IFNλ is a type 3 IFN agonist that binds the IFNLR1 / IL10R2 receptors in epithelial cells signaling through the ISRE pathway. We observed several fusion polypeptides with IFNλ components that have significant, but modulated ISRE signaling activity' and are better therapies. These include any of the fusion polypeptides screened and identified in EXAMPLE 1

[0147] A human subject diagnosed with Genital warts is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with Genital warts. Genital warts are the most commonly transmitted sexual disease. Locally -injected IFNα2 is FDA approved for treating genital warts. In this role, IFNs are thought to modulate the immune response. We observed several fusion polypeptides that had agonist activity with different IFNα genes (IFNα2-IFNβ, IFNα4-IFNβ, IFNα8-IFNβ, IFNα14- IFNβ, IFNα2-IFNα14, IFNβ-IFNα2, IFNβ-IFNα4, IFNβ-IFNα8, IFNβ-IFNα14, IFNα14- IFNα2) and could be used as agonists for Genital Wart treatment. We also observed several IFNα-IFNλ fusion polypeptides that have significant, but modulated ISRE signaling activity andcould be to teat genital warts. For example, IFNλ2-IFNα2, IFNλ3-IFNα2. -FNα2-IFNλ2 and IFNα2-IFNλ3 had substantial ISRE signaling. Nevertheless, we suspect that any of the fusion polypeptides tested may be an effective therapy. These include any of the fusion polypeptides screened and identified in EXAMPLE 1.

[0148] A human subject diagnosed with Psoriasis is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with Psoriasis. Currently IFNs are not approved by the FDA for treatment of Psoriasis. However, Psoriasis is induced and exacerbated by Type I IFNs, for example, IFNα and IFNβ in skin lesions. IFNκ also induces psoriasis in mice and is upregulated in blood of afflicted subjects. Blocking IFN signaling blunts cell-mediated skin inflammation and psoriasis-like inflammatory diseases. Thus, several of our fusion polypeptides (IFNε -IFNκ or IFNκ-IFNε or most fusion polypeptides that contain IFNλ1 -IFNλ4) that have antagonism are therapeutically useful for inhibiting Type I IFN signaling and treating psoriasis. However, we suspect that any of the fusion polypeptides tested may be an effective therapy. These include any of the fusion polypeptides screened and identified in EXAMPLE 1.

[0149] Type 1 IFNs play a role in the pathogenesis for rheumatoid arthritis (RA), diabetes mellitus, Sjogren’s syndrome, dermatomyositis (DM), and systemic sclerosis (SS), thus these diseases may be treated with one or more of our IFN fusion polypeptides. For that matter, these fusion polypeptides may be generally useful for many indications that would benefit from treatment with antivirals, antiproliferative factors, and immunomodulatory agents. A human subject diagnosed with rheumatoid arthritis (RA) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with RA. A human subject diagnosed with diabetes mellitus (DM) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with DM. A human subject diagnosed with diabetes mellitus is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with diabetes mellitus. A human subject diagnosed with Sjogren’s syndrome is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with Sjogren’s syndrome. A human subject diagnosed with dermatomy ositis (DM) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with DM. A human subject diagnosed with systemic sclerosis (SS) is administered a therapeutically effective amount of the fusion polypeptides disclosed herein, and the fusion polypeptides alleviate the symptoms associate with SS.Other indications

[0150] The fusion polypeptides can be used to target specific indication. For any desired indication, purified His-tagged fusion polypeptides or purified fusion polypeptides are tested for signaling potency in a reporter cell plate readers assay to determined EC50 and compared to other drugs including FDA-approved reference IFNs. Similar experiments are repeated to determine a cytopathic effect assay which is a standard test for IFN therapies. Then cells related to the indication can be stimulated with the fusion polypeptides and transcripts induced are compared to control cells to determine which fusion polypeptides are most appropriate and potent for the indication. The fusion polypeptides are then further tested in pre-clinical animal models followed by human studies.

[0151] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of identifying one or more member(s) of a library of fusion polypeptides, with functional activity against one or more target receptor(s) that is similar to or greater than a corresponding wildtype polypeptide, wherein each fusion polypeptide among the library of fusion polypeptides independently comprise a first polypeptide directly or indirectly linked to a second polypeptide, and wherein the corresponding wildtype polypeptide comprises a wildtype of the first polypeptide or a wildtype of the second polypeptide, the method comprising:(a) transforming or transducing each host cell among the plurality of host cells individually with a unique vector that comprises a barcode, and a polynucleotide sequence encoding a fusion polypeptide among the library of fusion polypeptides, thereby resulting in expression of the fusion polypeptide on a surface of the host cell that was transformed or transduced with the unique vector;(b) screening each fusion polypeptide expressed on the surface for each host cell for functional activity against the one or more target receptor(s), wherein the functional activity comprises: (i) a level of binding of the fusion polypeptide to the one or more target receptor(s); or (ii) a level of signaling modulation of the one or more target receptor(s) facilitated by the fusion polypeptide;(c) identifying a subset of host cells that express one or more member(s) of the library of polypeptides that have similar or greater functional activity against the one or more target receptor(s) as compared to the corresponding wildtype polypeptide; and(d) sequencing the barcode of the unique vector present in the subset of host cells identified in (c), thereby identifying one or more member(s) of the library of fusion polypeptides that have similar or greater functional activity against the one or more target receptor(s) as compared to the corresponding wildtype polypeptide.

2. The method of claim 1. wherein the first polypeptide is indirectly linked to the second polypeptide by indirectly linking the C-terminal amino acid of the first polypeptide to the N-terminal amino acid of the second polypeptide through a linker.

3. The method of claim 2, wherein the linker comprises from at least about: 1 to 5, 1 to 10, 1 to 20, or 1 to 50 amino acids.

4. The method of claim 1. wherein the first polypeptide is directly linked to the second polypeptide by directly linking the C-terminal amino acid of the first polypeptide to the N-terminal amino acid of the second polypeptide through an amide bond.

5. The method of claim 1, wherein the first polypeptide and / or the second polypeptide, prior to directly or indirectly being linked to each other, independently comprise an amino acid deletion, amino acid substitution, amino acid addition, or any combination of these, relative to a corresponding wildtype sequence.

6. The method of any one of claims 1 to 5, wherein the first polypeptide and / or the second polypeptide independently comprise at least about: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130. 140, 150, 160, 170, 180, 190, 200. or 300 amino acids.

7. The method of any one of claims 1 to 6, wherein the method further comprises introducing one or more post-translational modification (PTM) sites into at least one fusion polypeptide among the library of fusion polypeptides prior to transforming the unique vector into each host cell, wherein the one or more PTM sites are not present in a corresponding wildtype polypeptide.

8. The method of claim 7, wherein the one or more PTM sites comprise a glycosylation site, a lipidation site, a phosphory lation site, a methylation site, an acetylation site, a sumoylation site, or a protease site, or any combination thereof.

9. The method of claims 7 or 8. wherein the one or more PTM sites results in increased half-life, stability, solubility, excretion, serum half-life of the fusion polypeptide, as compared to the corresponding wildtype polypeptide.

10. The method of claims 7 or 8, wherein the one or more PTM sites results in reduced aggregation, and / or immunogenicity of the fusion polypeptide, as compared to the corresponding wildty pe polypeptide.

11. A polypeptide comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58.

12. The polypeptide of claim 11, wherein the polypeptide comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58.

13. The polypeptide of claim 11, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 24 to SEQ ID NO: 58.

14. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%. 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 1, wherein the polypeptide further comprises a modification selected from the group consisting of D2N, P4S, E5Q, DION, L18M, S22R, S27F, M3 IK, G37E, P52Q, Al 14E, SI 16F, K121R, R125Q, and any combination thereof, relative to SEQ ID NO: 1.

15. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%. 92 %, 93%, 94%, 95%, 96% 97%. 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 2, wherein the polypeptide further comprises a modificationselected from the group consisting of D4N, P6S, G12N, S13N, L20M, L28P, G39E, E54Q, T55A, P57S, I66T. K73E, D80E, E81Q. T82S, and any combination thereof, relative to SEQ ID NO: 2.

16. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 5, wherein the polypeptide further comprises a modification selected from the group consisting of H34Y, E40Q, E42V, H46N, V56A, L57F, E71K, D72N. E78D, Q79E, S80T, S86Y, T87I, Y90F, L93M, R121K, T132M, and any combination thereof, relative to SEQ ID NO: 5.

17. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO:

6. wherein the polypeptide further comprises a modification selected from the group consisting of D2N, P4S, S10G, SION, T14A, M16I, I17L, M18L, A19G, F152L, L156F, A158T, K159Q, G161R, E162K, R163G, E168D, and any combination thereof, relative to SEQ ID NO: 6.

18. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 8, wherein the polypeptide further comprises a modification selected from the group consisting of D95N, V106M, F116S, M132T, F152L, F154L, T156I, K159Q, G161R, R163K. R164S, D166E, and any combination thereof, relative to SEQ ID NO: 8.

19. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity' and at least about: 90% sequence length to SEQ ID NO: 9, wherein the polypeptide further comprises a modification selected from the group consisting of R24G, G195S, A199T, I201M, L202I, L203M, R207G, R216G, E222G, D230G, K23IN, and any combination thereof, relative to SEQ ID NO: 9.

20. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 10, wherein the polypeptide further comprises a modification selected from the group consisting of G19A, R37G, I38L, R121K, I132M, E133G, R134K, and any combination thereof, relative to SEQ ID NO: 10.

21. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%. 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO:

11. wherein the polypeptide further comprises a modification selected from the group consisting of N2D, S4P, Q5E, N10D, SI 16F, K121R, M132T, L152F,Q159K, R161G, R161I, R163K, R164S, D166E, and any combination thereof, relative to SEQID NO: 11.

22. A polypeptide comprising an amino acid sequence having at least about: 90%, 91%, 92 %, 93%, 94%, 95%, 96% 97%, 98% or 99% sequence identity and at least about: 90% sequence length to SEQ ID NO: 15, wherein the polypeptide further comprises a K107M modification relative to SEQ ID NO: 15.

23. The polypeptide of any one of claims 11 to 22, wherein the polypeptide binds to an interferon receptor or an interferon receptor subunit at a level greater than or equal to a corresponding wildtype IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by enzyme-linked immunosorbent assay (ELISA).

24. A fusion polypeptide comprising:(a) a first region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58;(b) a second region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58; and(c) a linker between the first region to the second region.

25. The fusion polypeptide of claim 24, wherein the amino acid sequence of the first region has at least 95% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58.

26. The fusion polypeptide of claim 24, wherein the amino acid sequence of the first region comprises any one of SEQ ID NO: 24 to SEQ ID NO: 58.

27. The fusion polypeptide of claim 24, wherein the amino acid sequence of the second region has at least 95% identity to any one of SEQ ID NO: 24 to SEQ ID NO: 58.

28. The fusion polypeptide of claim 24, wherein the amino acid sequence of the second region comprises any one of SEQ ID NO: 24 to SEQ ID NO: 58.

29. A fusion polypeptide comprising:(a) a first region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23;(b) a second region comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23; and(c) a linker between the first region to the second region.

30. The fusion polypeptide of claim 29, wherein the amino acid sequence of the first region has at least 95% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23.

31. The fusion polypeptide of claim 29, wherein the amino acid sequence of the first region comprises any one of SEQ ID NO: 1 to SEQ ID NO: 23.

32. The fusion polypeptide of claim 29, wherein the amino acid sequence of the second region has at least 95% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 23.

33. The fusion polypeptide of claim 29, wherein the amino acid sequence of the second region comprises any one of SEQ ID NO: 1 to SEQ ID NO: 23.

34. The fusion polypeptide of any one of claims 24 to 33, wherein the linker comprises SEQ ID NO: 405.

35. A fusion polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of 40SEQ ID NO: 59 to SEQ ID NO: 404.

36. The fusion polypeptide of claim 35, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 59 to SEQ ID NO: 404.

37. The fusion polypeptide of claim 35, wherein the fusion polypeptide comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 59 to SEQ ID NO: 404.

38. The fusion polypeptide of claim 35, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 59 to SEQ ID NO: 404.

39. The fusion polypeptide of claim any one of claims 24 to 34, wherein the fusion polypeptide further comprises: a third region comprising a protein antigen, a protease, a single domain antibody, a designed ankyrin repeat protein (dARPIN), an anti-calin, or a nanobody.

40. The fusion polypeptide of any one of claims 24 to 35, wherein the fusion polypeptide binds to an interferon receptor or an interferon receptor subunit at a level greater than or equal to a corresponding wildtype IFN polypeptide having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by enzyme-linked immunosorbent assay (ELISA).

41. The fusion polypeptide of claim 40, wherein the fusion polypeptide is an agonist, a partial agonist, a selective agonist, an inverse agonist or an antagonist of the interferon receptor or the interferon receptor subunit.

42. The fusion polypeptide of claim 40, wherein the fusion polypeptide is an agonist of the interferon receptor or the interferon receptor subunit.

43. The fusion polypeptide of claim 40, wherein the fusion polypeptide is a partial agonist of the interferon receptor or the interferon receptor subunit.

44. The fusion polypeptide of claim 40, wherein the fusion polypeptide is an inverse agonist of the interferon receptor or the interferon receptor subunit.

45. The fusion polypeptide of claim 40, wherein the fusion polypeptide is an antagonist of the interferon receptor or the interferon receptor subunit.

46. The fusion polypeptide of claim 41, wherein the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit at a level greater than a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1- SEQ ID NO: 23, as measured by an assay comprising Interferon Stimulated Response Element (ISRE) Green fluorescent protein (GFP) reporter assay or an ISRE luciferase reporter assay.

47. The fusion polypeptide of claim 46, wherein the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit resulting in a reduction of an extracellular level of a pro-inflammatory cytokine, as compared to a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 23, as measured in an in vitro assay.

48. The fusion polypeptide of claim 47, 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.

49. The polypeptide or the fusion polypeptide of claim 47, wherein the fusion polypeptide binds and activates the interferon receptor or the interferon receptor subunit resulting in an increase of an extracellular level of an anti-inflammatory cytokine, as compared to a corresponding wildtype IFN protein having the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23, as measured in an in vitro assay.

50. The polypeptide or the fusion polypeptide of claims 47 or 49, wherein the in vitro assay is an enzyme-linked immunosorbent assay (ELISA).

51. The polypeptide or the fusion polypeptide of claim 49, wherein the antiinflammatory cytokine is a chemokine, an IFN, an IL, a lymphokine, a TNF, a Transforming grow th factor (TGF), or any combination thereof.

52. The fusion polypeptide of any one of claims 24 to 3835, wherein the fusion polypeptide binds to an interferon receptor or an interferon receptor subunit at a level less than a corresponding wildtype IFN protein having an amino acid sequence of any one of SEQ ID NO: 1- SEQ ID NO: 23, as measured by an in vitro assay.

53. The fusion polypeptide of claim 52, wherein the fusion polypeptide binds and inhibits the interferon receptor or the interferon receptor subunit at a level greater than a corresponding wildlype IFN protein having the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 23, as measured by an assay by an ISRE GFP reporter assay or an ISRE luciferase reporter assay.

54. A nucleic acid encoding the polypeptide of any one of claims 11 to 23, or the fusion polypeptide of any one of claims 24 to 38.

55. The nucleic acid of claim 54, further comprising a barcode sequence.

56. A nucleic acid construct that comprises a polynucleotide sequence of any one of SEQ ID NO: 407 to SEQ ID NO: 763.

57. The nucleic acid of any one of claims 54 to 56, further comprising a reporter gene.

58. The nucleic acid of claim 57, wherein the reporter gene is a fluorescent reporter gene.

59. A vector comprising the nucleic acid or the nucleic acid construct of any one of claims 54 to 58.

60. A pharmaceutical composition comprising: the polypeptide of any one of claims 11 to 23, the fusion polypeptide of any one of claims 24 to 35, the nucleic acid of any one of claims 54 to 58, or the vector of claim 59; and a pharmaceutically acceptable carrier, diluent, or excipient.

61. The pharmaceutical composition of claim 60, that is in unit dose form.

62. A vaccine composition comprising: the polypeptide of any one of claims 11 to 23, the fusion polypeptide of any one of claims 24 to 35, the nucleic acid of any one of claims 54 to 58, or the vector of claim 59; a microbial antigen, a tumor antigen, or a nucleic acid encoding the microbial antigen or the tumor antigen; and a pharmaceutically acceptable carrier diluent, or excipient.

63. A kit comprising : the polypeptide of any one of claims 11 to 23, the fusion polypeptide of any one of claims 24 to 35. the nucleic acid of any one of claims 54 to 58. or the vector of claim 59; and a scaffold.

64. A genetically modified cell comprising the polypeptide of any one of claims 11 to 23, the fusion polypeptide of any one of claims 24 to 38, the nucleic acid of any one of claims 54 to 58, or the vector of claim 59.

65. A method of treating a disease or condition in a subject in need thereof, the method comprising: administering a therapeutically effective amount of the polypeptide of any one of claims 11 to 23, the fusion polypeptide of any one of claims 24 to 38, or the pharmaceutical composition of claim 60 or 61 to the subject in need thereof, thereby treating the disease or condition in the subject.

66. The method of claim 65, wherein the disease or the condition comprises a viral infection, a cancer, an autoimmune disease, multiple sclerosis, or a hemolytic disease.

67. The method of claim 65, wherein the disease is an infection.

68. The method of claim 67, wherein the infection is a viral infection.

69. The method of claim 68, wherein the viral infection is a chronic hepatitis B (CHB). a chronic hepatitis C (CHC), a chronic granulomatous disease (CGD), a coronavirus disease (COVID), a genital wart or a human immunodeficiency virus (HIV).

70. The method of claim 65, wherein the disease is a demyelinating disorder.

71. The method of claim 70, wherein the demyelinating disorder is selected from the group consisting of Multiple Sclerosis (MS), Acute Disseminated Encephalomyelitis (ADEM), Acute Hemorrhagic Leucoencephalitis (AHLE). Balo’s disease (Concentric Sclerosis). Charcot- Marie-Tooth disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), and Neuromyelitis Optica (Devic’s Disease).

72. The method of claim 71, wherein the MS is relapsing remitting MS, non-relapsing MS, clinically isolated syndrome, and secondary progressive forms.

73. The method of claim 65, wherein the disease is a cancer.

74. The method of claim 73, wherein the cancer is a blood cancer, a bone cancer, a brain cancer, a breast cancer, a cervical cancer, a colon cancer, a heart cancer, a kidney cancer, a liver cancer, a lung cancer, a lymph node cancer, a lymphoma, a melanoma, a myeloma, a skin cancer, a stomach cancer, a pancreatic cancer, a testicular cancer, or a throat cancer.

75. The method of claim 73, wherein the cancer is selected from the group consisting of astrocytoma, chronic myelogenous leukemia, follicular lymphoma, glioblastoma, hairy cell leukemia, Kaposi’s sarcoma, malignant melanoma, medulloblastoma, melanoma, multiple myeloma, non-Hodgkin lymphoma, and renal cell carcinoma.

76. The method of claim 65, wherein the disease is an immune disorder.

77. The method of claim 76, wherein the immune disorder is an autoimmune disorder.

78. The method of claim 76, wherein the immune disorder is selected from the group consisting of lupus, systemic lupus erythematosus, asthma, atherosclerosis, atopic dermatitis, autoimmune hepatitis, Celiac disease, COPD, Crohn's disease, Graves' disease. Guillain-Barre syndrome, IgA nephropathy, inflammatory bowel disease, pelvic inflammatory disease, primary biliary cirrhosis, primary- hemophagocytic lymphohistiocytosis, and ulcerative colitis.

79. A method of generating an immune response in a subject, the method comprising: administering to the subject the vaccine composition of claim 62, thereby generating an immune response in the subject.

80. The method of any one of claims 65 to 79, wherein the subject is a human.

81. The method of any one of claims 65 to 80, wherein the administering is intramuscular administration, intraocular administration, intranasal administration, subcutaneous administration, intravenous administration, intracranial administration, intrathecal administration,intertumoral administration, oral administration, sublingual administration, buccal administration, rectal administration, vaginal administration, or otic administration.

82. The method of any one of claims 65 to 81. wherein the administering is once every 24 hours, once every 48 hours, once every 72 hours, once every 96 hours, once every 120 hours, once every 144 hours, once every 168 hours, once every 280 hours, or once every 336 hours.

83. The method of any one of claims 65 to 81, wherein the administering occurs 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, or about once a year.

84. The method of any one of claims 65 to 83, wherein the method further comprises administering a second therapeutic.

85. The method of claim 84, wherein the second therapeutic is an antibody, a peptide, an antibody drug conjugate, a CAR-T, a cancer vaccine, or any combination thereof.

86. The method of any one of claims 65 to 85, wherein the method further comprises administering an immunotherapy, and wherein the immunotherapy comprises a checkpoint inhibitor.

87. The method of claim 86, wherein the checkpoint inhibitor is a PD-1, a PD-L1,CTLA-4 inhibitor.

Images