Surrogate interferon agonists

Abstract

The present disclosure relates to compositions and methods relating to interferon agonists and their engineered polypeptides. The engineered polypeptides have specificity to receptors in immune systems mediated by Type I interferon (IFN). The present disclosure also relates to methods and system for identifying surrogate agonists for cell surface receptors including dimeric and trimeric receptors.

Description

SURROGATE INTERFERON AGONISTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 63 / 641,234, filed on May 1, 2024, the disclosure of which is incorporated by reference herein in its entirety, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which is hereby incorporated herein by reference in its entirety. The accompanying Sequence Listing XML file, named “078430- 542001WO_Sequence_Listing_ST26.XML,” was created on April 30, 2025 and is 253,952 bytes in size.FIELD

[0003] The present disclosure generally relates to, inter alia, the field of immunology. In particular, the disclosure provides methods and compositions for the identification of surrogate cytokine agonists for modulating transduction mediated by Type I interferon (IFN). The disclosure also provides compositions and methods useful for the treatment of health conditions associated with the inhibition of signal signaling mediated by Type I IFN.BACKGROUND

[0004] Cytokines, including interferons (IFNs), are gamering increasing interest as therapeutics given their powerful actions in the immune system, as well as other systems that regulate human biology. However, the process of therapeutic discovery for cytokines is generally limited to exploration of the intrinsic biological properties of the natural cytokine ligands, through modifications such as affinity maturation, half-life extension and / or tissue. More recently, cytokine engineering strategies have succeeded in demonstrating that cytokine pleiotropy can be mitigated by selective structure-based engineering and protein design. However, unlike multi-pass transmembrane proteins such as GPCRs and ion channels, cytokine systems that signal through Type I single-pass transmembrane receptors are not amenable to medicinal chemistry types of high-throughput approaches. This is due to two principal reasons.

[0005] First, cytokines are globular proteins that function to bind to receptor extracellular domains (ECDs) and dimerize them. The cytokine forms large protein-protein contact surfaces with the receptor ECDs to supply the binding energy needed to bridge two receptor subunits.In contrast, small molecules bind within pockets in G-protein-coupled receptors (GPCRs) and ion channel transmembrane helices. Thus, cytokine receptor systems are not amenable to small molecule library-based screening campaigns. Furthermore, cytokines themselves are single-domain four-helix bundle proteins that present structural limitations for ligand engineering, which is generally limited to interface mutagenesis.

[0006] Second, cytokine signaling has generally been assumed to be “on or off,” in contrast to tunable GPCR ( / .<?., biased) signaling. Thus, cytokine agonist therapeutics have largely been limited to variations of the natural cytokine. However, recent studies have shown that the orientation and proximity of dimeric receptor assemblies can profoundly influence signaling output and that cytokine receptor signaling is ‘tunable’. Furthermore, antibodies can, in some instances, act as cytokine agonists by dimerizing the cytokine receptors into appropriate signaling geometries.

[0007] Therefore, there is a need to bridge the gap between medicinal chemistry library approaches that can identify biased agonists, and traditional cytokine engineering approaches that do not access the full scope of cytokine receptor signaling plasticity. There is also a need for new cytokine agonists and methods for their discovery.SUMMARY

[0008] The present disclosure relates generally to the development of engineered polypeptides that are surrogate cytokine receptors comprising single-chain and two-chain bispecific ligands. The present disclosure also relates to recombinant nucleic acids and recombinant cells including nucleic acid sequences that encode the engineered polypeptides disclosed herein. Also provided are pharmaceutical compositions and methods useful for the treatment of health conditions associated with the inhibition of signal signaling mediated by Type I IFN.

[0009] In one aspect, provided herein are engineered polypeptides including a single-chain multispecific ligand that includes a first antigen-binding moiety specific for IFNAR1 and a second antigen-binding moiety, wherein the first antigen-binding moiety specific for IFNAR1 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10. In some embodiments, the second antigen-binding moiety is specific for IFNAR2, and wherein the second antigen-binding moiety comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

[0010] In one aspects, provided herein are engineered polypeptides including a single-chain multispecific ligand that includes a first antigen-binding moiety and a second antigen-binding moiety specific for INFAR2, wherein the second antigen-binding moiety specific for IFNAR2 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13. In some embodiments, the first antigen-binding moiety is specific for IFNAR1, and wherein the first antigen-binding moiety comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1- VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, 1FNAR1-VHH9, and IFNAR1-VHH10.

[0011] In one aspect, provided herein are engineered polypeptides including a single-chain bispecific ligand that includes a first antigen-binding moiety specific for IFNAR1 and a second antigen-binding moiety specific for INFAR2, wherein: (a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and / or (b) the second antigenbinding moiety specific for 1FNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13. In some embodiments, (a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and / or (b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2- VHH4, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH9, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

[0012] In one aspect, provided herein are engineered polypeptides including a single-chain bispecific ligand that comprises a first antigen-binding moiety specific for IFNAR1 and a second antigen-binding moiety specific for INFAR2. In some embodiments, (a) the first antigen-binding moiety specific for IFNAR1 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1 -VHH2, IFNAR1 -VHH3, IFNAR1 -VHH4, IFNAR1 -VHH5, IFNAR1 -VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and (b) the second antigen-binding moiety specific for IFNAR2 comprises all three CDRs from an IFNAR2- VHH identified as such in Table 3B. In some embodiments, (a) the first antigen-binding moiety specific for IFNAR1 includes all three CDRs from an IFNAR2-VHH identified as such in FIG. 3A; and (b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2- VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2- VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13. In some embodiments, (a) the first antigen-binding moiety specific for IFNAR1 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1- VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and / or (b) the second antigen-binding moiety specific for IFNAR2 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH4, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH9, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

[0013] Non-limiting exemplary embodiments of the engineered polypeptides of the disclosure can include one or more of the following features. In some embodiments, the CDR sequences are indicated in FIGs. 3A-3B. In some embodiments, the CDR sequences of the IFNAR1 are selected from the group consisting of SEQ ID NO: 58-78 and 161-169. In some embodiments, the CDR sequences of the IFNAR2 are selected from the group consisting of SEQ ID NO: 79-99 and 170-187. In some embodiments, at least one of the CDRs includes one, two, three, four, or five substitutions. In some embodiments, the first antigen-binding moiety further includes a framework region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 100-127. In some embodiments, the first antigen-binding moiety includes anamino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7 and 15-23. In some embodiments, the first antigen-binding moiety includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7 and 15-23, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.

[0014] In some embodiments of the disclosure, the second antigen-binding moiety of the engineered polypeptides disclosed herein further includes a framework region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 128-155. In some embodiments, the second antigen-binding moiety comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-14 and 24-29. In some embodiments, the second antigen-binding moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-14 and 24-29, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.

[0015] In some embodiments of the disclosure, the single-chain bispecific ligand is a dimerizing-ligand for an IFNAR1 / IFNAR2 receptor heterodimer. In some embodiments, the engineered polypeptides of the disclosure are capable of inducing phosphorylation of STAT1, STAT2, and / or STAT3 in vitro. In some embodiments, the engineered polypeptides are capable of inducing phosphorylation of STAT1, STAT2, and / or STAT3 or a combination thereof in vivo. In some embodiments, the engineered polypeptides are capable of inhibiting viral replication. In some embodiments, the engineered polypeptides are capable of inhibiting viral replication in vitro. In some embodiments, the engineered polypeptides are capable of inhibiting viral replication in vivo. In some embodiments, the engineered polypeptides are capable of inhibiting viral replication in a cell without inducing the expression of pro- inflammatory cytokines. In some embodiments, the engineered polypeptides are capable of inhibiting viral replication in a cell without inducing the expression of anti-proliferative cytokines. In some embodiments, the engineered polypeptides are capable of inhibiting SARS- CoV-2 replication. In some embodiments, the engineered polypeptides are capable of inhibiting SARS-CoV-2 replication in vitro. In some embodiments, the engineered polypeptides are capable of inhibiting SARS-CoV-2 replication in vivo. In some embodiments, the engineered polypeptides are capable of inhibiting SARS-CoV-2 replication in a cellwithout inducing the expression of pro-inflammatory cytokines. In some embodiments, the engineered polypeptides are capable of inhibiting SARS-CoV-2 replication in a cell without inducing the expression of anti-proliferative cytokines. In some embodiments of the disclosure, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0016] In some embodiments of the disclosure, the engineered polypeptides disclosed herein further include a linker inserted between the first antigen-binding moiety and the second antigen-binding moiety. In some embodiments, the linker is a peptide linker. In some embodiments, the engineered polypeptide is an IFN agonist.

[0017] In one aspect, provided herein are recombinant nucleic acid molecules including a nucleic acid sequence that encodes an engineered polypeptide as disclosed herein. In some embodiments, the nucleic acid sequence is operably linked to a heterologous nucleic acid sequence. In some embodiments, the nucleic acid molecule is incorporated into an expression cassette or an expression vector.

[0018] In another aspect, provided herein are recombinant cells that include a recombinant nucleic acid molecule encoding an engineered polypeptide of the disclosure. In some embodiments, the recombinant cell is a prokaryotic cell. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In a related aspect, some embodiments disclosed herein relate to a cell culture that includes at least one recombinant cell of the disclosure and a culture medium.

[0019] In another aspect, some embodiments of the disclosure relate to pharmaceutical compositions which include one or more pharmaceutically acceptable excipients and one or more of the following: (a) an engineered polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; and (c) a recombinant cell as disclosed herein.

[0020] In yet another aspect, provided herein are methods for modulating IFN-mediated signaling, the method include administering to the subject a composition including one or more of the following: (a) an engineered polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; (c) a recombinant cell as disclosed herein; and (d) a pharmaceutical composition as disclosed herein.

[0021] In another aspect, provided herein are methods for the treatment of a health condition in a subject in need thereof, the methods include administering to the subject a composition including one or more of the following: (a) an engineered polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; (c) a recombinant cell as disclosed herein; and (d)a pharmaceutical composition as disclosed herein.

[0022] Non-limiting exemplary embodiments of the embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the administered composition results in an induced downstream signaling activity. In some embodiments, the downstream signaling activity includes STAT1, STAT2, STAT3, STAT5, STAT6, Akt, S6, or ERK activity, or a combination of any thereof. In some embodiments, the downstream signaling activity includes activation of innate and / or adaptive immune responses. In some embodiments, the downstream signaling activity includes differential induction of an interferon stimulated gene (ISG) as a metric for surrogate IFN activity. In some embodiments, the ISG is selected from the group consisting of MX1, OAS1, IFIT1, IFITM1, TRAIL, CXCL10, ISG15, CH25CH, cGAS, BST2, and NCOA7ISG. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject has or is suspected of having a health disease associated with IFN-mediated signaling. In some embodiments, the health condition is a cancer, an immune disease, or an infection. In some embodiments, the infection is a viral infection.

[0023] In another aspect, provided herein are kits for modulating IFN-mediated signaling and / or for treating a health condition, the kit including one or more of the following: (a) an engineered polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; (c) a recombinant cell as disclosed herein; and (d) a pharmaceutical composition as disclosed herein.

[0024] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIGs. 1A-1O illustrate that Type I interferon surrogate agonists exhibit biased signaling and inhibit viral replication. (A) Schematic representation of exemplary bispecific type I IFN surrogate ligands which heterodimerize IFNAR1 and IFNAR2 left). A collection of 11 IFNAR1 binders (1 scFv, 10 VHH) were paired with 6 IFNAR2 binders (VHH), resulting in 66 combinations of IFNAR1-IFNAR2 fusion molecules connected via a 5-AA linker right . Twelve of these molecules induced pSTATl activity on YT-1 cells (pink shading). The IFNAR2-specific scFv “3F11” was identified from the patent US7662381B2. Seven of the hits, “HIS 1-7,” were selected for further analysis. (B-D) Dose-responserelationship of STAT1 phosphorylation evoked by IFNto or surrogate agonists. YT-1 cells (B), A549 cells (C), or PBMCs (D) were stimulated with saturating ligand concentration for 20 min., fixed and permeabilized, then stained with a-STATl(pY701)-AlexaFluor647 and analyzed via flow cytometry. (E) Heatmap representation of STAT1-STAT6 phosphorylation evoked by surrogate agonists in YT-1 cells at different time points and normalized to the activation induced by IFNto (F) Heatmap representation of STAT1 and STAT2 phosphorylation evoked by surrogate agonists in A549 cells at varying time points, normalized to activation induced by IFNto (G) qRT-PCR analysis of SeV RNA in A549 cells pre-treated with lOnM surrogate ligands or IFNto for 24 hours, followed by SeV infection (MOI=0.1) for 24 hours. (H) SARS-CoV-2 nLUC A549-hACE2 Antiviral Assay. A549-hACE2 cells were treated with varying concentration of surrogate ligands, IFNto or negative control (monomer VHH “Al”) for 24 hours, prior to infection with SARS-CoV-2 nLUC. SARS-CoV-2 nLUC replication (relative light units) for triplicate wells per VHH dilution is shown. (I- J) Heatmap representation of selected ISGs induced by surrogate ligands in A549 cells (I) or human primary bronchial / tracheal epithelial cells (J). Gene expression is normalized to the level induced by IFNto (K) qRT-PCR analysis of SeV RNA in PBMCs pre-treated with lOnM surrogate ligands or IFNto for 24 hours, followed by SeV infection (MOI=0.5) for 24 hours. (L) Heatmap representation of selected ISGs induced by surrogate ligands in PBMCs. (M) CellTiter-GLO assay of human primary bronchial / tracheal epithelial cells treated with lOnM surrogate ligands or IFNto for 72 hours. (N) Identification of four mouse IFN surrogate agonists with pSTATl activity on J774.2 cells left) and on mouse embryonic fibroblasts (right). (O) Mouse IFN surrogates exert antiviral activity against SeV.

[0026] FIGs. 2A-2D depict signaling kinetics and gene expression driven by Type I Interferon surrogate ligands. (A) SPR sensorgrams displaying dose-dependent binding of IFNAR2 VHHs to immobilized human IFNAR2 ECD (extracellular domain). Binding constants were determined from kinetic fitting and summarized in (B). (C) qRT-PCR analysis of mRNA level of indicated genes in human primary bronchial / tracheal epithelial cells treated with 10 nM surrogate ligands or IFNto for 8 hours. (D) qRT-PCR analysis of mRNA level of indicated genes in PBMCs treated with lOnM surrogate ligands or IFNto for 8 hours.

[0027] FIGs. 3A-3B show sequences of individual VHH binding modules of Human Type I IFN surrogate agonists. Each VHH includes four framework regions (FR1-4) and three hypervariable regions (CDRs) with the following structure: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4). The sequences of CDR1, CDR2, and CDR3 are shown in underlined bold letters.

[0028] FIG. 4 show sequences of individual VHH binding modules of Mouse Type I IFN surrogate agonists.

[0029] FIGs. 5A-5B show sequences of active molecules from the initial screen of Human Type I IFN surrogate agonists.

[0030] FIG. 6 shows sequences of active molecules from Mouse Type I IFN surrogate agonists.

[0031] FIG. 7 shows sequences of molecules selected for functional studies of Human Type I IFN surrogate agonists (see Figs. 1A and 1B-1M).DETAILED DESCRIPTION OF THE DISCLOSURE

[0032] The present disclosure generally relates to compositions and methods pertaining to engineered polypeptides that are cytokine agonists. The present disclosure also relates to platforms for the generation and screening of agonists for naturally- and non-naturally occurring combinations of receptors. The engineered polypeptides of the present disclosure include ligands that have the capacity to dimerize cell surface receptors in ways that are structurally inaccessible to natural or engineered cytokines. The ligands are single chain bispecific ligands that can include one or more antibody domains. The domains (binders) can include one or more nanobodies (VHH) and / or scFvs that can be mixed and matched in modular fashion to create libraries of dimerizing ligands (see, e.g., FIGs. 1 A and IB). The engineered polypeptides can dimerize Type I IFN systems.

[0033] The present disclosure also relates to methods of and systems for identifying such cytokine agonists. These methods and systems can be used for any multimeric cell surface receptors including dimeric receptors (e.g. cytokine, Receptor Tyrosine Kinase (RTK) and IgSF family), trimeric receptors {e.g., death receptors), and other systems. The methods and systems can also be used for systems with limited or nonexistent structural knowledge, or for creating surrogate ligands when the cognate ligands present biochemical challenge. The methods and systems can be used in both natural and non-natural receptor combinations and can be used to explore new receptor combinations for drug discovery.

[0034] Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. Disclosed herein are strategies and methods for the discovery of surrogate cytokine agonists using modular ligands with the capability of exploring receptor dimer geometry as a pharmacological variable. The strategies and methods are amenable to high-throughputscreening. As described in greater detail herein, combinatorial matrices of single chain bispecific ligands that exhibited a diverse spectrum of agonist strengths, signaling biases and functional activities that have been inaccessible through traditional cytokine engineering have been generated using VHH and scFv to Type I Interferon receptors. As described in greater detail below, this modular approach can enable the engineering of a ligand that compels the formation of heterodimers on T and NK cells, generating a non-canonical activation signal.

[0035] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. GENERAL TECHNIQUES

[0036] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005).Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.II. DEFINITIONS

[0037] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those ofskill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0038] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B.”

[0039] The terms “cell,” “cell culture,” and “cell line” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.

[0040] The term “linker,” as used herein, refers to an amino acid or sequence of amino acids that that is optionally located between two amino acid sequences in a fusion polypeptide of the invention.

[0041] The binding activity of the single chain bispecific ligands of the disclosure can be assayed by any suitable method known in the art. A ligand that "preferentially binds" or "specifically binds" to a target protein or target epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also known in the art. An antibody or polypeptide is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular protein or epitope than it does with alternative proteins or epitopes. A ligand "specifically binds" is “specific to” or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. Also, a ligand "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration to that target in asample than it binds to other substances present in the sample. It is also understood by reading this definition, for example, that a ligand which specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Preferably, “specific,” in reference to binding, means that to the extent that a molecule forms complexes with other molecules or complexes, it forms at least fifty percent of the complexes with the molecule or complex for which it has specificity. Generally, the molecules or complexes have areas on their surfaces or in cavities giving rise to specific recognition between the two binding moieties. Exemplary of specific binding are antibodyantigen interactions, enzyme-substrate interactions, polynucleotide hybridizations and / or formation of duplexes, cellular receptor-ligand interactions, and so forth.

[0042] A variety of assay formats may be used to select a single chain bispecific ligand that specifically binds a molecule of interest. For example, solid-phase ELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence- activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with an antigen or a receptor, or ligand binding portion thereof, that specifically binds with a cognate ligand or binding partner. Typically, a specific or selective reaction will be at least twice the background signal or noise, more typically more than 10 times background, even more typically, more than 50 times background, more typically, more than 100 times background, yet more typically, more than 500 times background, even more typically, more than 1000 times background, and even more typically, more than 10,000 times background. Also, an antibody is said to "specifically bind" an antigen when the equilibrium dissociation constant (KD) is < 7 nM.

[0043] The term "binding affinity" is herein used as a measure of the strength of a non- co valent interaction between two molecules, e.g., an antibody or portion thereof and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules may be quantified by determination of the dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka(or kon) and dissociation rate constant kd (or koff), respectively. KD is related to kaand kd through theequation KD = kd / ka. The value of the dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the KD may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432). Other standard assays to evaluate the binding ability of antibodies or polypeptides of the present disclosure towards target antigens are known in the art, including for example, ELIS As, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA.

[0044] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and / or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants thereof.

[0045] The term “percent identity,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and / or additions, as well as those that havesubstitutions. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al. , ] Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0046] The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions.

[0047] The term “engineered” or “recombinant” polypeptide as used herein, refers to a polypeptide that has been altered through human intervention. As non-limiting examples, an engineered polypeptide can be one which: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques; 2) includes conjoined polypeptide sequences that are not conjoined in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more amino acids with respect to the naturally occurring polypeptide sequence; and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring polypeptide.

[0048] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those includedlimits are also included in the disclosure.

[0049] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantia] equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0050] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0051] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub combination wasindividually and explicitly disclosed herein.

[0052] Some embodiments of the disclosure relate to new cytokine ligands with properties of partial or complete agonisms of the downstream signal transduction mediated through Type I Interferons (IFN).

[0053] Type I interferons (IFNs) are a network of homologous cytokines that bind to a shared, heterodimeric cell surface receptor interferon-alpha / beta receptor (which has two transmembrane subunits - alpha chain IFN ARI and beta chain - IFNAR2), and engage signaling pathways that activate innate and adaptive immune responses. The IFNs have a wide range of immunomodulatory, anti-viral, and anti-proliferative actions which are mediated by 16 different sub-types of IFN cytokines that dimerize IFNAR1 / IFNAR2 to activate several STATs, principally STAT1 (Ng. et al., 2016).III. COMPOSITIONS

[0054] The compositions disclosed herein include surrogate cytokine agonists that comprise bispecific single-chain ligands. The single-chain ligands are made up of antibody domains (binders) that can be mixed and matched to create libraries of dimerizing ligands described throughout the disclosure, figures and Examples presented below. For instance, various combinations of antibody domains are shown in FIGS. 1A, 5A-5B, and 6-7 and in the Informal Sequence Listing. A person of skill in the art would appreciate that while several embodiments have the scFvs or VHHs fused together, other formats, (e.g., zippers and Fc heterodimers) may also be used.

[0055] The single-chain bispecific ligands can be assembled pursuant to methods known to those skilled in the art. In some embodiments, the ligands are heterodimers. In some embodiments, the heterodimers can be expressed as Fc fusions, which can then self-dimerize via their Fc domains to generate bispecific homodimers. Single-chain bispecific ligands can be fused to the Fc domain of IgG to extend its half-life, e.g. by pegylation, glycosylation, and the like as known in the art. Fc-fusion can also endow alternative Fc receptor mediated properties in vivo. The “Fc region” can be a naturally occurring or synthetic polypeptide that is homologous to an IgG C-terminal domain produced by digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The single-chain bispecific ligands can include the entire Fc region, or a smaller portion that retains the ability to extend the circulating half-life of a chimeric polypeptide of which it is a part. In addition, full-length or fragmented Fc regions can be variants of the wild-type molecule. That is, they can containmutations that may or may not affect the function of the polypeptides. In some embodiments, the domains can be separately fused to acidic or basic zippers, which when co-expressed, selfassemble to generate bispecific heterodimers. In some embodiments, the ligands are hetero trimers. In other embodiments, the ligands can be homodimers. In some embodiments, the ligands are two-chain ligands wherein the ligand is encoded by a single polypeptide and wherein two molecules of the polypeptide self-assemble to make a homodimer.

[0056] In some embodiments, the ligands are multi-chain agonists that include fusions to oligomeric zippers. Oligomeric zippers are known in the art, such as described by Harbuy et al. 1993.

[0057] The ligands of the present disclosure are surrogate cytokine agonists. Measuring agonism is within the standard knowledge of a skilled artisan. For example, in some embodiments, measuring agonism may be done by stimulating cells expressing the receptor(s)-of-interest with the candidate ligand, then assaying a biological output such as proximal signaling. In some embodiments of the present disclosure, measuring agonism can be performed by assaying phosphorylation of STATs. In some embodiments, measuring agonism can be performed by assaying downstream function(s) (for example, assaying proliferation, differentiation, antiviral activity, etc. using known methods).

[0058] As used herein, the term “VHH” or “nanobody” are used herein interchangeably to refer to variable domain of a heavy-chain antibody. A nanohody is the smallest antigenbinding fragment or single variable domain derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids (Hamers- Casterman el al. 1993; Desmyter el al. 1996). In the family of “camelids,” immunoglobulins devoid of light polypeptide chains are found.“Camelids” comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Lama paccos, Lama glama, Lama guanicoe, and Lama vicugna). The single variable domain heavy chain antibody is herein designated as a nanobody or a VHH antibody. Nanobodies can also be derived from sharks. In some embodiments, the antibody domain is an antigen-binding fragment of a VHH (nanobody) or a single-chain variable fragment (scFv). In some embodiments, the single chain bispecific ligand includes two nanobodies, a first nanobody that is specific to IFNAR1 and a second nanobody that is specific to IFNAR2.

[0059] In one aspect, provided herein are engineered polypeptides including a single-chainmultispecific ligand that includes a first antigen-binding moiety specific for IFNAR1 and a second antigen-binding moiety, wherein the first antigen-binding moiety specific for IFNAR1 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10. In some embodiments, the second antigen-binding moiety is specific for IFNAR2, and wherein the second antigen-binding moiety comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2- VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

[0060] In one aspects, provided herein are engineered polypeptides including a single-chain multispecific ligand that includes a first antigen-binding moiety and a second antigen-binding moiety specific for INFAR2, wherein the second antigen-binding moiety specific for IFNAR2 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13. In some embodiments, the first antigen-binding moiety is specific for IFNAR1, and wherein the first antigen-binding moiety comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1- VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10.

[0061] In one aspect, provided herein are engineered polypeptides including a single-chain bispecific ligand that includes a first antigen-binding moiety specific for IFNAR1 and a second antigen-binding moiety specific for INFAR2, wherein: (a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and / or (b) the second antigenbinding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7,IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13. In some embodiments, (a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and / or (b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2- VHH4, TFNAR2-VHH6, TFNAR2-VHH7, TFNAR2-VHH9, IFNAR2-VHH11 , IFNAR2- VHH12, and IFNAR2-VHH13.

[0062] In one aspect, provided herein are engineered polypeptides including a single-chain bispecific ligand that comprises a first antigen-binding moiety specific for IFNAR1 and a second antigen -binding moiety specific for INFAR2. In some embodiments, (a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, 1FNAR1-VHH2, 1FNAR1-VHH3, 1FNAR1-VHH4, 1FNAR1-VHH5, 1FNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and (b) the second antigen-binding moiety specific for IFNAR2 comprises all three CDRs from an IFNAR2- VHH identified as such in Table 3B. In some embodiments, (a) the first antigen-binding moiety specific for IFNAR1 comprises all three CDRs from an IFNAR2-VHH identified as such in Table 3A; and (b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2- VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2- VHH10, 1FNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13. In some embodiments of the disclosure, (a) the first antigen-binding moiety specific for IFNAR1 includes all three CDRs from a VHH selected from the group consisting of IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and / or (b) the second antigen-binding moiety specific for IFNAR2 includes all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH4, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH9, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

[0063] Non-limiting exemplary embodiments of the engineered polypeptides of the disclosure can include one or more of the following features. In some embodiments, theantigen-binding moiety is or comprises an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single-domain antibody (dAb), a VNAR domain, or a VHH domain. In some embodiments, the antigen -binding moiety includes a VH domain and a VL domain. In some embodiments, the antigen-binding moiety is or comprises a single-chain antigen-binding (scFab) fragment. In some embodiments, the antigen-binding moiety is or comprises a singlechain variable fragment (scFv).

[0064] In some embodiments, the CDR sequences are indicated in FIGs. 3A-3B. In some embodiments, at least one of the CDRs includes one, two, three, four, or five substitutions. In some embodiments, the first antigen-binding moiety further includes a framework region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 100-127.

[0065] In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ IDNO: 6. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0066] In some embodiments, the first antigen-binding moiety includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 1, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 2, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 3, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 4, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigenbinding moiety includes the amino acid sequence of SEQ ID NO: 5, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 6, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 7, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.

[0067] In some embodiments of the disclosure, the second antigen-binding moiety of the engineered polypeptides disclosed herein further includes a framework region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 128-155.

[0068] In some embodiments, the second antigen-binding moiety comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-14. In some embodiments, the first antigen-binding moietyincludes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first antigenbinding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first antigen-binding moiety includes an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the first antigen-binding moiety includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0069] In some embodiments, the second antigen-binding moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-14, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 8, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 9, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 10, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 11, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 12, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 13, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the first antigen-binding moiety includes the amino acid sequence of SEQ ID NO: 14, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.

[0070] Provided herein are compositions including an engineered polypeptide with a single chain bispecific ligand wherein a first specificity of the ligand is to IFNAR1 and a second specificity of the ligand is to IFNAR2 and wherein the engineered polypeptide is a cytokine agonist.

[0071] In some embodiments, the single chain bispecific ligand comprises a first nanobody (i.e., VHH or scFv) specific to IFNAR1 and a second nanobody (z.<?., VHH or scFv) specific to IFNAR2. In some embodiments, the first nanobody comprises an amino acid sequence that is the amino acid sequence set forth in any one of SEQ. ID. NOS.: 1-7 and 15-23 (FIGs. 3A- 3B). In some embodiments, the first nanobody comprises an amino acid sequence that has at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , or any values in between, sequence identity to the amino acid sequence set forth in any one of SEQ. ID. NOS.: 1-7 and 15-23. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the first nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0072] In some embodiments, the second the second nanobody includes an amino acid sequence set forth in any one of SEQ. ID. NOS.: 8-14 and 24-29. In some embodiments, the second nanobody comprises an amino acid sequence that has at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any values in between, sequence identity to the amino acid sequence set forth in any one of SEQ. ID. NOS.: 8-14 and 24-29. In some embodiments, the second nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the second nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the second nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the second nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the second nanobody includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second nanobodyincludes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0073] In some embodiments, the single chain bispecific ligand comprises the amino acid sequence set forth in any one of SEQ. ID. NOS.: 30-48 (FIGs. 5A-5B, FIG. 6). In some embodiments, the single chain bispecific ligand comprises an amino acid sequence that has at least 80%, or 85%, or 87%, or 90%, or 91 %, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or any values in between, sequence identity to the amino acid sequence set forth in any one of SEQ. ID. NOS.: 30-48. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37. In some embodiments,the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the single chain bispecific ligand includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48.

[0074] In some embodiments, the single chain bispecific ligand is a dimerizing-ligand for an IFNAR1 / IFNAR2 receptor heterodimer. In some embodiments, the engineered polypeptide with a single chain bispecific ligand having a first specificity to IFNAR1 and a second specificity to IFNAR2 is capable of inducing phosphorylation of STAT1, or STAT2 or STAT3 or a combination thereof in vitro and / or in vivo. Methods of measuring phosphorylation of STAT1, or STAT2 or STAT3 is known to the skilled in the art and are described above. In some embodiments the single-chain bispecific ligand is capable of inducing STAT 5 phosphorylation in vitro and / or in vivo. Measuring phosphorylation of STAT5 is known to the skilled in the art and includes, for example, western blot and phospho flow cytometry. In some embodiments, assaying the phosphorylation of STAT1, or STAT2 or STAT3 is performed as described in the Examples below.

[0075] Deficiency of IFN signaling is one of the most important reasons for the immune dysfunction and even the resistance or failure of common cancer therapeutic strategies. Studies have suggested that the expression of interferon- stimulated genes (ISGs) may be impaired in lymphocytes from patients with breast cancer, melanoma, and gastrointestinal cancer (Zhang X et al. Oncoimmunology. 2021 ; 10(1): 1929005). In some embodiments, the single-chain bispecific ligands promote cytolytic ability against tumors in vitro and / or in vivo. In some embodiments, the cytolytic ability of the single-chain bispecific ligands described herein can be measured using a cytolytic assay, e.g., an assay examining the ability of lymphocytes to detect and kill cancer cells.

[0076] The surrogate IFNs of the disclosure exhibit anti-viral activity. Type I IFNs, for example, can exhibit antiviral ability by inducing interferon stimulated genes (ISGs). As described in the Examples, surrogate IFN ligands of the disclosure showed biased induction of ISGs (as compared with IFN co; FIGS. II and 2D). In human primary airway epithelial cells, “Human Interferon Surrogates” (HIS) ligands induced high levels of the antiviral genes MX1 and OAS1 with minimal induction of pro-inflammatory genes CXCL9 and CXCL10 (FIG. 1 J). Moreover, HIS agonists effectively inhibited SeV replication in PBMCs while barely inducing pro-inflammatory cytokine expression (FIGs. 1K-1L). In addition to anti-viral activity against SeV, anti-viral activity against other viruses is also contemplated. Examples of viruses against which the surrogate IFNS of the present disclosure can exhibit anti-viral activity include, but are not limited to, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Varicella-zoster virus (can cause chickenpox and shingles, VZV), Herpes Simplex Virus (can cause herpes and encephalitis, HSV), Dengue Virus (can cause dengue fever, DENV), Vesicular StomatitisVirus (VSV), Influenza A virus (IAV), HIV-1, Human Cytomegalovirus (HCMV), Ebola Virus Disease (EVD), and Human Papilloma Virus (HPV).

[0077] The surrogate IFNs of the disclosure can be components of pharmaceutical compositions. Pharmaceutical compositions can be anti-viral compositions that can be used, for example in, but not limited to, the treatment of viral infections. Any viral infection, such as infection with e.g. retrovirus, lentivirus, hepadnavirus, herpes viruses, pox viruses, human papilloma viruses, etc., is within the scope of the disclosure including hepatitis B and hepatitis C infections (Li et. al, 2018.)

[0078] In some embodiments, the engineered polypeptide with a single chain bispecific ligand having a first specificity to IFNAR1 and a second specificity to IFNAR2 is capable of inhibiting SARS-CoV-2 replication in vitro or in vivo. Assaying the inhibition of SARS-CoV- 2 replication is known to the skilled in the art (for example, Hou et al. 2020). In some embodiments, assaying the inhibition of SARS-CoV-2 replication is done as follows: plates are seeded with A549-hACE2 cells. A549 is a human lung epithelial cell line stably expressing the SARS-CoV-2 receptor, hACE2, to facilitate efficient infection for antiviral assays (Hou et al., 2020). Cells are infected with recombinant SARS-CoV-2 engineered to express nanoluciferase at a multiplicity of infection of 0.25. After incubation, input virus is removed, cells are washed and infection medium is added. After 48 hours of infection, levels of virus replication can be measured by Promega NanoGio assay measured on a Promega GloMax Luminometer. Similarly treated uninfected sister plates can be generated in order to gauge potential cytotoxicity by Promega CellTiter Gio assay read on a Promega GloMax Luminometer. In some embodiments, assaying the inhibition of SARS-CoV-2 replication is done as described in Example 8 below.

[0079] In some embodiments, antiviral ability can be assayed by observing the induction of expression of interferon stimulated genes (ISGs). Such genes include, but are not limited to, for example, MX1, OAS1, IFIT1, 1FITM1, TRAIL, CXCL10, 1SG15, CH25CH, cGAS, BST2, and NCOA7. In some embodiments, the differential ISG induction can be used as a metric for screening surrogate IFNs for activity. In some embodiments, the analysis for differential ISG induction involves comparing the levels of specific ISGs and determining whether certain functional categories are preferentially reduced or weakened by surrogate ligands compared to endogenous interferons.

[0080] In some embodiments, the engineered polypeptides with a single chain bispecific ligand having a first specificity to IFNAR1 and a second specificity to IFNAR2 inhibit SARS-CoV-2 replication in a cell without inducing pro-inflammatory cytokine expression. Techniques for measuring the expression of pro-inflammatory cytokines are known to the skilled in the art. In some embodiments, pro-inflammatory cytokines include CCL2, CCL3, CXCL9, CXCL10 and others. In some embodiments, mRNA expression of pro-inflammatory cytokines is assayed. In some embodiments, polypeptide levels of pro-inflammatory cytokines are assayed as known by the skilled in the art (Metzemaekers 2018).

[0081] In some embodiments, inhibiting SARS-CoV-2 replication in a cell occurs without inducing anti-proliferative cytokine expression, including BAX, BAK1, FAS, and others.

[0082] In some embodiments, inhibiting SARS-CoV-2 replication in a cell occurs without inducing pro-apoptotic gene expression, such as TRAIL, ISG12, TNFSF10, and IFIT2.Linkers

[0083] In some embodiments, the antibody domains (e.g., antigen-binding moieties or binders) of the engineered polypeptides of the disclosure are operably joined to one another by an intervening linker. There is no particular limitation in regard to the linkers that can be used in the chimeric polypeptides described herein. In some embodiments, the linker is a synthetic compound linker such as, for example, a chemical cross-linking agent. Non-limiting examples of suitable cross-linking agents that are available on the market include N- hydroxy succinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis [2- (sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0084] In some embodiments, the linker is a peptide linker. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 aminoacid residues.

[0085] In some embodiments, VHH and scFv binders to human IFNAR1 and IFNAR2, are fused via 2-amino acid linkers or 5-amino acid linkers (FIG. 1 A). A person of skill in the art readily appreciates testing different linker lengths to modulate activity.

[0086] In some embodiments, the linkers are flexible Gly-Ser linkers. Examples of such polypeptide linkers include but are not limited to: GS, GGS, GGGS (SEQ ID NO: 156). In some embodiments, the linker can include other amino acids such as A or T. Examples include but are not limited to GTSAS (SEQ ID NO: 157), GGGGTSAS (SEQ ID NO: 158), GGGSGGGGTSAS (SEQ ID NO: 159), and GGGSGGGS GGGGTSAS (SEQ ID NO: 160).

[0087] The antibody domains or binders of the disclosure can be linked in a Forward or Reverse orientation (as illustrated, for example in FIG. 1 A and Table 1 below).

[0088] In some embodiments, the antibody domains or binders of the engineered polypeptides of the disclosure are joined directly to one another, e.g., they are not joined via a linker.TABLE 1: Summary of exemplary binders and antibody domains described in the present disclosure. The antibody domains or binders of the disclosure can be linked in a Forward or Reverse orientation as summarized in Table 1. The corresponding sequences are provided in the Sequence Listing.Nucleic Acids, Nucleic acid constructs and Vectors

[0089] In one aspect, provided herein are various nucleic acid molecules, nucleic acid constructs and vectors including nucleotide sequences encoding and / or expressing the engineered polypeptides of the present disclosure.

[0090] The terms “nucleic acids” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA,genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set forth in 37 CFR §1.822 is used herein.

[0091] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules that are preferably between about 5 Kb and about 50 Kb, for example between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0092] The term “recombinant” nucleic acid molecule as used herein, refers to a nucleic acid molecule that has been altered through human intervention. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As nonlimiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site-specific recombination)) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature, 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence, and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence.

[0093] Methods for constructing a DNA sequence encoding the engineered polypeptides and expressing those sequences in a suitably transformed host include, but are not limited to, using a PCR-assisted mutagenesis technique. Mutations that consist of deletions or additions of amino acid residues to an engineered polypeptides can also be made with standard recombinant techniques. In the event of a deletion or addition, the nucleic acid moleculeencoding the engineered polynucleotides is optionally digested with an appropriate restriction endonuclease. The resulting fragment can either be expressed directly or manipulated further by, for example, ligating it to a second fragment. The ligation may be facilitated if the two ends of the nucleic acid molecules contain complementary nucleotides that overlap one another, but blunt-ended fragments can also be ligated. PCR-generated nucleic acids can also be used to generate various mutant sequences.

[0094] The complete amino acid sequence can be used to construct a back-tran slated gene. A DNA oligomer containing a nucleotide sequence coding for an engineered polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0095] In addition to generating polypeptides via expression of nucleic acid molecules that have been altered by recombinant molecular biological techniques, a subject single chain bispecific ligand in accordance with the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are generated by those of skill in the art.

[0096] Once assembled (by synthesis, site-directed mutagenesis or another method), the DNA sequences encoding an engineered polypeptide will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the engineered polypeptide in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0097] Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule as disclosed herein. As used herein, the term “expression cassette” refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or into a subject. As such, the term expression cassette may be used interchangeably with the term “expression construct.”

[0098] Also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acid molecules encoding any of the chimeric polypeptides and bispecific ligandsdisclosed herein. The nucleic acid molecules described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. In some embodiments of the present disclosure, the engineered polynucleotides can be expressed from vectors, preferably expression vectors. In some embodiments, the expression vectors are mammalian expression vectors. Examples of mammalian expression vectors are known to a person skilled in the art. Such vectors include but are not limited to pD649. In some embodiments, the VHHs and scFvs of the present disclosure and / or fusions thereof can be cloned into such expression vectors. Suitable vectors for use in eukaryotic are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al. , Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., "Molecular Cloning: A Laboratory Manual." 2nd ED. (1989).

[0099] In some embodiments the subject polypeptides, either alone or as a part of a chimeric polypeptide, such as those described above, can be obtained by expression of a nucleic acid molecule.

[0100] It should be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow the DNA encoding the engineered polypeptides or fragments thereof to be amplified in copy number. Such amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp,’- Mol. Cell Biol., 2, pp. 1304-19, 1982) or glutamine synthetase (“GS”) amplification (see, e.g., U.S. Pat. No. 5,122,464 and European published application 338,841).

[0101] The vectors can be useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operablylinked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses including lentivirus, adenoviruses, and adeno- associated viruses) are included also.

[0102] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0103] The expression constructs or vectors can be designed for expression of an engineered polypeptide thereof in host cells.

[0104] Vector DNA can be introduced into prokaryotic or eukaryotic ceils via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory' Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0105] The nucleic acid sequences encoding the engineered polypeptides, or agonists of the disclosure can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon optimization are known in the art. Codon usages within the coding sequence of the chimeric polypeptides and bispecific antibodies disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0106] Vectors suitable for use include T7 -based vectors for use in bacteria, the pMSXND expression vector for use in mammalian cells, and baculovirus-derived vectors for use in insect cells. In some embodiments nucleic acid inserts, which encode the subject engineered polypeptides, or agonists of the disclosure in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.

[0107] In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject chimeric polypeptide or bispecific antibody, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosenvector, the toxicity of the product coded for by the DNA sequences of this disclosure, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.

[0108] Within these parameters one of skill in the art may select various vector / expression control sequence / host combinations that will express the desired DNA sequences on fermentation or in large scale animal culture, for example, using CHO cells or COS 7 cells.

[0109] The choice of expression control sequence and expression vector, in some embodiments, will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Non-limiting examples of useful expression vectors for eukaryotic hosts, include, for example, vectors with expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Non-limiting examples of useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including col El, pCRI, pER32z, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Non-limiting examples of useful expression vectors for yeast cells include the 2p plasmid and derivatives thereof. Non-limiting examples of useful vectors for insect cells include pVL 941 and pFastBac™ 1.

[0110] In addition, any of a wide variety of expression control sequences can be used in these vectors. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, for example PL, the control regions of fd coat protein, the promoter for 3 -phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoA, the promoters of the yeast a-mating system, the polyhedron promoter of Baculovirus, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0111] A T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of anucleic acid molecule in a given tissue or cell type within the body. Skilled artisans will readily appreciate numerous promoters and other regulatory elements which can be used to direct expression of nucleic acids.

[0112] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resi stance (neoR) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells. Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0113] Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0114] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a subject engineered polypeptides, or agonists disclosed herein are also features of the disclosure. A cell of the disclosure is a transfected cell, e.g. , a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding an engineered polypeptide, has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered within the scope of the disclosure.

[0115] The precise components of the expression system are not critical. For example, an engineered polypeptide as disclosed herein can be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell e.g., an Sf21 cell), or mammalian cells e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0116] The expressed polypeptides can be purified from the expression system using routine biochemical procedures, and can be used, e.g., as therapeutic agents, as described herein.

[0117] In some embodiments, engineered polypeptides obtained will be glycosylated or unglycosylated depending on the host organism used to produce the chimeric polypeptides orbispecific antibodies. If bacteria are chosen as the host then the chimeric polypeptide or bispecific antibody produced will be unglycosylated. Eukaryotic cells, on the other hand, will glycosylate the chimeric polypeptides or bispecific antibodies, although perhaps not in the same way as native polypeptides is glycosylated. The engineered polypeptides produced by the transformed host can be purified according to any suitable methods known in the art. Produced engineered polypeptides can be isolated from inclusion bodies generated in bacteria such as E. coli, or from conditioned medium from either mammalian or yeast cultures producing a given engineered polypeptide using cation exchange, gel filtration, and or reverse phase liquid chromatography.

[0118] In addition or alternatively, another exemplary method of constructing a DNA sequence encoding the engineered polypeptides of the disclosure is by chemical synthesis. This includes direct synthesis of a peptide by chemical means of the protein sequence encoding for a engineered polypeptide exhibiting the properties described. This method can incorporate both natural and unnatural amino acids at positions that affect the binding affinity of the engineered polypeptide with the target protein. Alternatively, a gene which encodes the desired engineered polypeptide can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired engineered polypeptide, and preferably selecting those codons that are favored in the host cell in which the engineered polypeptides will be produced. In this regard, it is well recognized in the art that the genetic code is degenerate-that an amino acid may be coded for by more than one codon. For example, Phe (F) is coded for by two codons, TIC or TTT, Tyr (Y) is coded for by TAC or TAT and his (H) is coded for by CAC or CAT. Trp (W) is coded for by a single codon, TGG. Accordingly, it will be appreciated by those skilled in the art that for a given DNA sequence encoding a particular engineered polypeptide, there will be many DNA degenerate sequences that will code for that engineered polypeptide. For example, it will be appreciated that in addition to the DNA sequences for engineered polypeptides provided in the Sequence Listing, there will be many degenerate DNA sequences that code for the engineered polypeptides disclosed herein. These degenerate DNA sequences are considered within the scope of this disclosure. Therefore, “degenerate variants thereof’ in the context of this disclosure means all DNA sequences that code for and thereby enable expression of a particular engineered polypeptide.

[0119] The DNA sequence encoding the subject engineered polypeptide, whether prepared by site directed mutagenesis, chemical synthesis or other methods, can also include DNAsequences that encode a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the engineered polypeptide. It can be prokaryotic, eukaryotic or a combination of the two. In general, the inclusion of a signal sequence depends on whether it is desired to secrete the engineered polypeptide as disclosed herein from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be included.

[0120] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double- stranded or single-stranded (e.g., either a sense or an antisense strand).

[0121] The nucleic acid molecules are not limited to sequences that encode polypeptides; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.

[0122] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found as such in the natural state. Thus, this disclosure encompasses recombinant molecules, such as those in which a nucleic acid sequence (for example, a sequence encoding a engineered polypeptide) is incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).Recombinant Cells and cell cultures

[0123] The engineered polypeptides and recombinant nucleic acid molecules of the disclosure can be introduced into a cell, such as, for example, a eukaryotic cell, to produce a recombinant cell, e.g., an engineered cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell isa mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell.

[0124] Accordingly, some embodiments of the disclosure relate to methods for making a recombinant cell, including (a) providing a host cell capable of protein expression; and transducing the provided host cell with a recombinant nucleic acid molecule of the disclosure to produce a recombinant cell. Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle- mediated nucleic acid delivery, and the like.

[0125] Accordingly, in some embodiments, a nucleic acid molecule of the disclosure can be introduced into a host cell by viral or non-viral delivery vehicles known in the art to produce a recombinant cell. For example, the nucleic acid molecule can be stably integrated in the recombinant cell’s genome, or can be episomally replicating, or present in the recombinant cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is present in the recombinant cell as a mini-circle expression vector for transient expression. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activatorlike effector nucleases).

[0126] The nucleic acid molecules of the disclosure can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In a non- limiting example, baculoviral virus or adeno- associated virus (AAV) can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range ofspecies and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0127] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as genedelivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0128] Accordingly, in some embodiments, provided herein are recombinant cells, e.g., host cells, expressing the engineered polypeptides of the disclosure. In some embodiments, the recombinant cells are prokaryotic cells. In some embodiments, the recombinant cells are eukaryotic cells. Non-limiting examples of recombinant cells that can be used include 293 variants (Expi293F™, Expi293™ GnTI-, 293F, 293S, etc.), ExpiCHO-S™, High Five cells, and E. coli.

[0129] In some embodiments, host cells can be genetically engineered e.g., transduced or transformed or transfected) with, for example, a vector construct of the present disclosure that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0130] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. As discusses above, techniques for transforming a wide variety of the above-mentioned cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.Pharmaceutical Compositions

[0131] In some embodiments, the engineered polypeptides, nucleic acids, recombinant cells, and cell cultures of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include one or more of the engineered polypeptides, nucleic acids, recombinant cells of the disclosure. In some embodiments, the compositions are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions of the disclosure include a pharmaceutically acceptable excipient and one or more the following: engineered polypeptides, nucleic acids, recombinant cells of the disclosure.

[0132] The present disclosure also provides pharmaceutical compositions including the engineered polypeptides nucleic acids, and / or recombinant cells disclosed herein. In some embodiments, the pharmaceutical compositions include, in addition to the engineered polypeptides nucleic acids, and / or recombinant cells disclosed herein, a pharmaceutically acceptable excipient or carrier.

[0133] The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions.

[0134] In some embodiments, the engineered polypeptides of the disclosure are prepared with carriers that will protect the engineered polypeptides against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, poly orthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. NO.4,522,811. As described in greater detail below, the recombinant polypeptides of the present disclosure may also be modified to achieve extended duration of action such as by PEGylation, acylation, Fc fusions, linkage to molecules such as albumin, etc. In some embodiments, the recombinant polypeptides can be further modified to prolong their half-life in vivo and / or ex vivo. Non-limiting examples of known strategies and methodologies suitable for modifying the recombinant polypeptides of the disclosure include (1) chemical modification of a recombinant polypeptide described herein with highly soluble macromolecules such as polyethylene glycol (“PEG”) which prevents the recombinant polypeptides from contacting with proteases; and (2) covalently linking or conjugating a recombinant polypeptide described herein with a stable protein such as, for example, albumin. Accordingly, in some embodiments, the recombinant polypeptides of the disclosure can be fused to a stable protein, such as, albumin. For example, human albumin is known as one of the most effective proteins for enhancing the stability of polypeptides fused thereto and there are many such fusion proteins reported.

[0135] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0136] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the common methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0137] Oral compositions, if used, generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound (e.g., engineered polypeptides, or agonists of the disclosure) can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel™, or com starch; a lubricant such as magnesium stearate or Sterotes™; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0138] In the event of administration by inhalation, the subject engineered polypeptides, or agonists of the disclosure are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0139] Systemic administration of the engineered polypeptides, or agonists of the disclosure can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0140] In some embodiments, the engineered polypeptides, or agonists of the disclosure can also be prepared in the form of suppositories (<?.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0141] In some embodiments, the engineered polypeptides, or agonists of the disclosure can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. I. Health Syst. Pharm. 53: 151- 160, 1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).IV. METHODS OF THE DISCLOSUREMethods for Identifying Surrogate Cytokine Agonists

[0142] Provided herein, in the present disclosure, are also methods for identifying surrogate cytokine agonists wherein the methods include providing nanohodies or scFvs against (<?.g., having binding affinity for) a first target cytokine receptor and against a second target cytokine receptor; and linking a nanobody or scFv against the first target cytokine receptor with a nanobody or scFv against the second target cytokine receptor thereby identifying a surrogate cytokine agonist.

[0143] In some embodiments, the method further comprises screening for induction of downstream signaling activity. In some embodiments, the screening can be for the induction of STAT1, STAT2, STAT3, STAT5, STAT6, Akt, S6, or ERK activity or combinations thereof.

[0144] An exemplary workflow of the methods for identifying surrogate cytokine agonists is as follows. However, the discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0145] First a collection of small, single Ig-domain VHH and / or scFv binders are generated against (e.g., having binding affinity for) a target receptor or ECD antigen. For example, in some embodiments, single Ig-domain VHH and / or scFv binders are generated against human IFNAR1 and IFNAR2. Bactrian camels can be immunized by the appropriate antigens expressed as Fc fusions. Following the isolation of peripheral blood cells, phage-displayed VHH libraries can be constructed and subjected to bio-panning for binding to receptor ECDs. VHH clones can be recombinantly expressed and ELISA-based screening can be used to identify receptor- specific binders. In some embodiments, the abilities of the binders areassessed for binding to NK cells. Bispecific ligands can then be generated by fusing two binders. In some embodiments, the binders are fused by peptide linkers of varying amino acid lengths. In some embodiments, no linkers are used. In some embodiments, the binders are linked in the forward orientation. In some embodiments, the binders are linked in the reverse orientation. The binders can be utilized in a pairwise combinatorial manner. In some embodiments, these small protein constructs can be rapidly produced by gene synthesis, expressed through a transient transfection and purified. This approach rapidly generates a small library of compounds. In some embodiments, the protein constructs are between 20-50 kDa. The compounds, or surrogate dimerizing ligands are screened for the induction of downstream signaling activity as shown, for example in FIGS. 1-2, or in the Examples provided below.

[0146] This same general workflow can be followed for other cytokine systems. This same approach and strategy could also be applied towards any cell surface receptor pairs across both cytokine, receptor tyrosine kinase (RTK), and other dimeric systems such as IgSF family of receptors. The same platform can be also used to create surrogate agonists against trimeric receptors including death receptors, such as TNF receptor- 1, CD95 (Fas), TRAMP, TRAIL- Rl, or TRAIL-R2. The same platform could also be used for the generation and screening of agonists for naturally-occurring as well as non-naturally occurring combinations of receptors. An example of a non-naturally occurring receptor is detailed throughout the disclosure as well as in the Examples pertaining to the IFNAR1 / IFNAR2 combination.Methods for Identifying Surrogate Agonists For Cell Surface Receptors

[0147] Also provided herein are methods for identifying surrogate agonists for cell surface receptors. The cell surface receptors can be dimeric or trimeric receptors. Examples of dimeric receptors include, but are not limited to cytokine receptors, RTK receptors, and IgSF family. Examples of trimeric receptors include death receptors such as such as TNF receptor- 1, CD95 (Fas), TRAMP, TRAIL-R1, or TRAIL-R2. The cell surface receptors can be naturally- occurring or non-naturally occurring. The methods of the disclosure can also pertain, as taught supra, to creating agonists that bring together non-natural combinations of receptor components.

[0148] The methods for identifying surrogate agonists include assembling one or more antibody domains to form ligands. The ligands can be monospecific or bispecific. The ligands can be monospecific but include 3 identical binding sites so as to homodimerize three copies of the receptor (in case of trimeric receptors). In some embodiments, the antibody domains areVHHs. In some embodiments the antibody domains are scFvs.

[0149] In some embodiments, the methods further comprise employing a screening of the differential induction of interferon stimulated genes (ISGs) as a metric for surrogate IFN activity. The biased induction of ISGs can be used a metric for identifying agonists that force non-natural combinations of receptors. This methodology can be a powerful technique for drug discovery.Methods for modulating IFN -mediated signaling and / or for the treatment of a health condition

[0150] Administration of any one of the therapeutic compositions described herein, e.g., engineered polypeptides, nucleic acids, recombinant cells, cell cultures, and pharmaceutical compositions, can be used in the treatment of relevant health conditions, such as proliferative diseases (e.g., cancers), autoimmune diseases, and chronic infections (e.g., viral infections). In some embodiments, the engineered polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more health conditions or diseases associated with cell signaling mediated by IFN. Exemplary health conditions or diseases can include, without limitation, cancers, immune diseases, and infection. In some embodiments, the infection is a viral infection. In some embodiments, the individual is a patient under the care of a physician.

[0151] Accordingly, in one aspect, some embodiments of the disclosure relate to methods modulating IFN-mediated signaling. In some embodiments, the methods include administering to the subject a composition including one or more of the following: (a) an engineered polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; (c) a recombinant cell as disclosed herein; and (d) a pharmaceutical composition as disclosed herein. In another aspect, some embodiments of the disclosure relate to methods for the treatment of a health condition in a subject in need thereof, the methods include administering to the subject a composition including one or more of the following: (a) an engineered polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; (c) a recombinant cell as disclosed herein; and (d) a pharmaceutical composition as disclosed herein. In some embodiments, the methods include administering a therapeutically effective amount or number of (i) a engineered polypeptide of the disclosure, (ii) a recombinant nucleic acid molecule of the disclosure, (iii) a recombinant cell of the disclosure, and / or (iv) apharmaceutical composition of the disclosure.

[0152] A therapeutically effective amount generally refers to an amount of a therapeutic composition that is sufficient to promote a particular effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a health condition, e.g., a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0153] The efficacy of a treatment including a disclosed therapeutic composition for the treatment of a health condition (e.g., disease) can be determined by the skilled clinician. However, a treatment is considered effective treatment if at least any one or all of the signs or symptoms of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0154] Non-limiting exemplary embodiments of the embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the administered composition results in an induced downstream signaling activity in the subject. In some embodiments, the downstream signaling activity is induced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% compared to a reference subject that has not been administered with the composition.

[0155] In some embodiments, the downstream signaling activity includes STAT1, STAT2, STAT3, STAT5, STAT6, Akt, S6, or ERK activity, or a combination of any thereof. In some embodiments, the downstream signaling activity includes activation of innate and / or adaptive immune responses. In some embodiments, the downstream signaling activity includes differential induction of an interferon stimulated gene (ISG) as a metric for surrogate IFN activity. In some embodiments, the ISG is selected from the group consisting of MX1, OAS1, IFIT1, IFITM1, TRAIL, CXCL10, ISG15, CH25CH, cGAS, BST2, and NCOA7ISG. In someembodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject has or is suspected of having a health disease associated with IFN-mediated signaling. In some embodiments, the health condition is a cancer, an immune disease, or an infection. In some embodiments, the infection is a viral infection.Additional therapies

[0156] As discussed supra, any one of the compositions disclosed herein, e.g., engineered polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be administered to a subject in need thereof as a sole therapy e.g., monotherapy). In addition or alternatively, in some embodiments of the disclosure, the engineered polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be administered to the subject as a first therapy in combination with one or more additional therapies, e.g., at least one, two, three, four, or five additional therapies. Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. Other suitable therapies include therapeutic agents such as chemotherapeutics, anti -cancer agents, and anti-cancer therapies.

[0157] Administration “in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order. Accordingly, in some embodiments, the methods of the disclosure include administration of a composition disclosed herein to a subject individually as a sole therapy e.g., monotherapy). In some embodiments, a composition of the disclosure is administered to a subject as a first therapy in combination with a second therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.IV. KITS

[0158] In one aspect, also provided herein are kits for the practice of a method described herein. A kit can include instructions for use thereof and one or more of the engineered polypeptides, nucleic acids, recombinant cells, and pharmaceutical compositions disclosed herein as described and provided herein. For examples, provided herein, in some embodiments, are kits that include one or more engineered polypeptides of the disclosure. In some embodiments, provided herein are kits that include one or more nucleic acids, recombinant cells, and / or pharmaceutical compositions of the disclosure. In some embodiments, the kits of disclosure further include written instructions for preparing the engineered polypeptides, nucleic acids, recombinant cells, and pharmaceutical compositions of the disclosure and using the same.

[0159] In some embodiments, the kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer one any of the provided engineered polypeptides, nucleic acids, recombinant cells, and pharmaceutical compositions to a subject in need thereof. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for modulating cell signaling mediated by IFN, or treating a health condition in a subject in need thereof.

[0160] For example, any of the above-described kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents for ex vivo production of the recombinant cell populations. In some embodiments, any of the above-described kits can further include negative control polypeptides, positive control polypeptides.

[0161] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container.

[0162] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette,flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0163] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0164] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0165] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.EXAMPLESEXAMPLE 1Type I Interferon (IFN) Systems

[0166] A collection of small, single Ig-domain VHH binders against IFNAR1 and IFNAR2 was generated. Human IFN ARI and IFNAR2 antigens were expressed as Fc fusions and purified, then were used to immunize Bactrian camels. Following isolation of peripheral blood cells, phage-displayed VHH libraries were constructed and subjected to three rounds of bio-panning for binding to receptor ECDs. ELISA-based screening of recombinantly expressed VHH clones identified 10 IFNAR1 binders and 13 IFNAR2 binders (see, e.g., Table 2 below)

[0167] Subsequently, surrogate agonists in the Type I interferon (IFN) system were generated using a collection of VHH and scFv binders to human IFNAR1 and IFNAR2, fused via 2-AA. or 5-AA linkers (FIG. 1A). A subset of binders were selected for SPR analysis, andbound to their corresponding receptor with high affinities (FIGs. 2A, 2B, and Table 2 below).TABLE 2: Summary of exemplary binders described in the present disclosure. IFNAR1-VHHs and IFNAR2-VHHs bind to IFNAR1 or IFNAR2 with various affinity. Binding constants were determined from kinetic fitting and summarized in Table 2.Note: * (N / D) = not detectable. ** ( — ) = not determined.

[0168] Despite their high affinities, an initial 66-member screening matrix (11 IFNAR1binders x 6 IFNAR2 binders) in the IFNAR1-IFNAR2 orientation produced only 12 active hits, yielding an approximate 18% hit rate (FIG. 1 A). These 12 hits were then expressed in the reverse (IFNAR2-IFNAR1) orientation and screened, none of which had measurable activity. A subset of active molecules, termed “Human Interferon Surrogates” (HIS)l-7, were selected for further studies (FIG. 1A). The HIS ligands induced dose-dependent pSTATl activation, the hallmark STAT activated by Type I IFNs, on YT-1 and A549 (lung epithelial) cell lines, as well as on human PBMCs, exhibiting partial agonist Emax relative to the natural cytokine human IFNw (FIGs. 1B-1D). Since Type I IFNs also activate additional STATs, STAT1- STAT6 phosphorylation was profiled. A reduced pSTATl activation relative to IFNco but equivalent pSTAT2 and pSTAT3 activation on both on the NK cell line YT-1 and A549 cells were observed (FIGs. 1E-1F). Thus, the surrogate IFN ligands display signaling bias for pSTAT activation relative to human IFNw.

[0169] Since Type I IFNs are a critical viral defense mechanism, the surrogate ligands were tested as to whether they exhibited antiviral activity on A549 cells infected with Sendai virus (SeV). All HIS ligands showed similar inhibition of SeV replication as IFNw, despite their reduced pSTATl activation (FIG. 1G). HIS agonists also inhibited SARS-CoV-2 replication in A549 cells expressing human ACE2 receptor, as measured with an antiviral assay using recombinant SARS-CoV-2 engineered to express nanoluciferase (Hou et al., 2020) (FIG. 1H). Additionally, HIS agonists inhibited SARS-CoV-2 replication in primary human airway cells (FIG. 2C). After 24 hours pretreatment, a potent dose-dependent antiviral effect on SARS- CoV-2 replication was observed. Interestingly, the antiviral potency of HIS ligands varied based on the identity of the IFNAR1 binder. Whereas all 4 ligands using the “3F11” scFv (HIS1-HIS4) exhibited potent antiviral activity, 2 / 3 ligands with the “Al” VHH (HIS5-7) (VHH “Al” was identified from a commercially available yeast-displayed VHH library (https: / / www.kerafast.com / item / 1770 / yeast-display-nanobody-library-nblib) had poor activity (FIGs. 1 A and 1H) suggesting that the magnitude and / or composition of the generated antiviral response is guided by VHH or scFv structure-activity relationships.

[0170] Type I IFNs exhibit antiviral ability by inducing interferon stimulated genes (ISGs), and the surrogate IFN ligands compared with IFNw showed biased induction of ISGs. Specifically, the surrogates maintained high levels of antiviral gene expression but induced lower levels of pro-inflammatory and pro-apoptotic gene expression (FIGs. II and 2D). In human primary airway epithelial cells, HIS ligands induced high levels of the antiviral genes MX! and OAS1 with minimal induction of pro-inflammatory genes CXCL9 and CXCL10 (FIG.1J). Moreover, HIS agonists effectively inhibited SeV replication in PBMCs while barely inducing pro-inflammatory cytokine expression (FIGs. IK- IL).

[0171] Another functional property of type I IFNs is anti-proliferative activity, less pro- apoptotic gene induction by HIS agonists was observed. Consistent with this ISG bias, HIS agonists did not suppress cell proliferation as much as IFNw in primary airway epithelial cells (FIG. IM). Taken together, these ligands have biased ISG induction, which contributes to preserved antiviral activity but restrained anti-proliferative and pro-inflammatory effects. Collectively, these data demonstrate that surrogate IFN agonists are exquisitely potent antiviral agents against SARS-CoV-2 and could be further explored as potential medical countermeasures for COVID-19, as well as for other viruses.EXAMPLE 2General Materials and MethodsCamel Immunization

[0172] Human IFNAR1 ECD (residues 28-436), and IFNAR2 ECD (residues 27-243) were expressed as Fc fusions in HEK293F cells and purified by protein A affinity chromatography. Purified receptor ECDs were mixed with Freund's adjuvant, then individually injected into healthy Bactrian camels (Camelus bactrianus). After the seventh immunization, antiserum titer reached l.OxlO5(indicating a strong immune response) and 100 mL of peripheral blood was collected for phage display library construction. All camel experiments were performed in compliance with ethics guidelines approved by Shanghai Science and Technology Committee (STCSM).VHH library construction

[0173] Following isolation of peripheral blood lymphocytes (PBLs) from immunized camels, RNA was extracted, cDNAs were reverse transcribed, and VHHs were amplified by two-step nested PCR. Purified VHH fragments were subcloned into the phage-display phagemid pMECS and used to construct the phage display libraries. The quality of libraries was evaluated by size and insertion rate. Insertion rate was calculated by randomly screening 24 clones per library and determining insertion size by PCR amplification.VHH library selection

[0174] VHHs specific for IFNAR1 and IFNAR2 were selected from phage-display libraries using target proteins and enriched by three consecutive rounds of bio-panning with the infection of VCSM13 helper phages. Three hundred individual colonies were randomlyselected from the enriched pool and positive clones were identified using periplasmic extract ELISA (PE-ELISA).Protein expression

[0175] VHH were fused using a 2-8 AA linker, and VHH-scFv were fused via an 8-AA or a Gly-Ser linker. VHH and scFv fusions were cloned into a pD649 mammalian expression vector (ATUM DNA 2.0), which carries an HA secretion signal peptide and a C-terminal 6- His tag. Proteins were expressed in Expi293F cells (Thermo Fisher Scientific) for 5-7 days according to manufacturer protocols, isolated using Ni2+ affinity chromatography, then further fractionated over a Superdex 200 increase column equilibrated with 20 mM HEPES (pH 7.4) and 150 mM NaCl.Cell culture

[0176] CD45+YT-1 cells (Kuziel et al., 1993) and human PBMC were isolated from LRS chambers (Stanford Blood Center), were maintained at 37°C in a 5% CO2 humidified chamber, and cultured in complete RPMI medium (RPMIc) containing 10% FBS and supplemented with 25mM HEPES, 2mM pyruvate, 4mM GlutaMAX, non-essential amino acids, and penicillin-streptomycin (all cell cultured reagents were purchased from Gibco). Prior to stimulation for pERK and pAkt studies, cells were starved in serum-free RPMI for 1-2 hours. Primary cells were rested overnight without cytokine before measuring signaling.

[0177] Normal human primary bronchial / tracheal epithelial cells were purchased from ATCC (PCS-300-010) and grown in Airway Epithelial Cell Basal Media (ATCC PCS-300- 030) supplemented with Bronchial / Tracheal Epithelial Cell Growth Kit components (ATCC PCS-300-040) following manufacturer’s instructions. A549 cells were maintained in complete DMEM medium containing 10% FBS and supplemented with 25 mM HEPES, 2mM sodium pyruvate, 4mM GlutaMAX, and penicillin-streptomycin.

[0178] RNA-seq experiments.- T cells were pre-activated for 4d with a-CD3 / CD28, washed, and rested overnight without stimulation. The following day, CD8+T cells were purified using MACS (CD8+T cell isolation kit, Miltenyi Biotec), then stimulated with lOOnM natural cytokine (IFN) or surrogate ligand for 24 hours, at 37°C. Total RNA from 1-2 million cells per condition was extracted using an RNeasy micro kit (Qiagen). For each condition, three (3) biological replicates, representing samples from 3 independent donors, were performed. cDNA library preparation and RNA sequencing were performed by Novogene. cDNA libraries were loaded onto an Illumina NovaSeq 6000 sequencer, PE150 platform. Reference genome and gene model annotation files were downloaded from the genome website browser(NCBUUCSC / Ensembl) directly. Paired-end clean reads were aligned to the reference genome using STAR software, and differential expression analysis was conducted using the DESeq2 R package (Love et al., 2014). Data (raw and processed) are deposited under GEO accession record GSE183436.A549-hACE2 SARS-CoV-2 antiviral assay

[0179] 96- well plates were seeded with 20,000 A549-hACE2 cells / well. A549 is a human lung epithelial cell line stably expressing the SARS-CoV-2 receptor, hACE2, to facilitate efficient infection for antiviral assays (Hou et al., 2020). Culture medium was removed 24 hr. post-seeding, and a 9-point agonist dose-response (top concentration lOOOnM, 10-fold steps) was prepared in “infection medium” (DMEM (Gibco), 5% fetal bovine serum (Hyclone), lx anti / anti (antibiotic, antimycotic, Gibco). Cells were transported to Biosafety Level 3 after 24 hours, treatment with agonists, at which point cells were infected with recombinant SARS- CoV-2 engineered to express nanoluciferase at a multiplicity of infection of 0.25. After incubation for 1 hour at 37°C, input virus was removed, cells were washed once with infection medium and I OOLIL fresh infection medium was added. As a positive control, a similar doseresponse of recombinant human IFNw was employed. As a negative control, the monomeric hlFNARl- specific VHH “Al” was employed, which should not facilitate the dimerization of the type I interferon receptor subunits. After 48 hours of infection, levels of virus replication were measured by Promega NanoGio assay measured on a Promega GloMax Luminometer. Similarly treated uninfected sister plates were generated in order to gauge potential cytotoxicity by Promega CellTiter Gio assay read on a Promega GloMax Luminometer.NK cell culture and stimulation

[0180] PBMCs were isolated by ficoll density gradient centrifugation and resuspended in RPMI supplemented with 10% FBS, 1% L-glutamine, 1% HEPES, 1% MEM Non-Essential Amino Acids Solution, 1 % sodium pyruvate, and 1 % penicillin streptomycin. NK cells were stimulated with hIL-18 (100 ng / mL, R&D), hIL-15 (20 ng / mL, R&D), and hIL-12 (10 ng / mL, BioLegend) for 18 hours, washed 3 times, then cultured in cRPMI for 2 days.

[0181] NKL cells were cultured in cRPMI containing 100IU human IFN, with media and IFN changes every other day.

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Claims

CLAIMSWHAT IS CLAIMED IS.

1. An engineered polypeptide comprising a single-chain multispecific ligand that comprises a first antigen-binding moiety specific for IFNAR1 and a second antigen-binding moiety, wherein: the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1- VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10.

2. An engineered polypeptide comprising a single-chain multispecific ligand that comprises a first antigen-binding moiety and a second antigen-binding moiety specific for INFAR2, wherein: the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2- VHH9, IFNAR2-VHH10, IFNAR2-VHH11 , IFNAR2-VHH12, and IFNAR2- VHH13.

3. The engineered polypeptide of claim 1, wherein the second antigen-binding moiety is specific for IFNAR2, and wherein the second antigen-binding moiety comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2-VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

4. The engineered polypeptide of claim 2, wherein the first antigen-binding moiety is specific for IFNAR1, and wherein the first antigen-binding moiety comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1 -VHH6, IFNAR1 -VHH7, IFNAR1 -VHH8, IFNAR1 -VHH9, and IFNAR1 -VHH10.

5. An engineered polypeptide comprising a single-chain bispecific ligand that comprises a first antigen-binding moiety specific for IFNAR1 and a second antigen- binding moiety specific for INFAR2, wherein:(a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH1, IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH5, IFNAR1-VHH6, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1- VHH9, and IFNAR1-VHH10; and / or(b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH1, IFNAR2-VHH2, IFNAR2-VHH3, IFNAR2-VHH4, IFNAR2-VHH5, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH8, IFNAR2- VHH9, IFNAR2-VHH10, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2- VHH13.

6. The engineered polypeptide of claim 5, wherein:(a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and / or(b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH4, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH9, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

7. An engineered polypeptide comprising a single-chain bispecific ligand that comprises a first antigen-binding moiety specific for IFNAR1 and a second antigen- binding moiety specific for INFAR2, wherein:(a) the first antigen-binding moiety specific for IFNAR1 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR1-VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1-VHH10; and(b) the second antigen-binding moiety specific for IFNAR2 comprises all three CDRs from an IFNAR2-VHH identified as such in FIG. 3B.

8. An engineered polypeptide comprising a single-chain bispecific ligand that comprises a first antigen-binding moiety specific for IFNAR1 and a second antigen- binding moiety specific for INFAR2, wherein:(a) the first antigen-binding moiety specific for IFNAR1 comprises all three CDRs from an IFNAR2-VHH identified as such in FIG. 3A; and(b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH4, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH9, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

9. An engineered polypeptide comprising a single-chain bispecific ligand that comprises a first antigen-binding moiety specific for IFNAR1 and a second antigen-binding moiety specific for INFAR2, wherein:(a) the first antigen-binding moiety specific for IFNAR1 comprises all three CDRs from a VHH selected from the group consisting of IFNAR1 -VHH2, IFNAR1-VHH3, IFNAR1-VHH4, IFNAR1-VHH7, IFNAR1-VHH8, IFNAR1-VHH9, and IFNAR1- VHH10; and(b) the second antigen-binding moiety specific for IFNAR2 comprises all three complementary determining regions (CDRs) from a VHH selected from the group consisting of IFNAR2-VHH4, IFNAR2-VHH6, IFNAR2-VHH7, IFNAR2-VHH9, IFNAR2-VHH11, IFNAR2-VHH12, and IFNAR2-VHH13.

10. The engineered polypeptide any one of claims 1-9, wherein the CDR sequences are indicated in FIGs. 3A-3B.

11. The engineered polypeptide of any one of claims 1-9, wherein the CDR sequences of the IFNAR1 are selected from the group consisting of SEQ ID NO: 58-78 and 161-169.

12. The engineered polypeptide of any one of claim 1-9, wherein the CDR sequences of the IFNAR2 are selected from the group consisting of SEQ ID NO: 79-99 and 170-187.

13. The engineered polypeptide of any one of claims 1-12, wherein at least one of the CDRs comprises one, two, three, four, or five substitutions.

14. The engineered polypeptide of any one of claims 1-13, wherein the first antigen-binding moiety further comprises a framework region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 100-127.

15. The engineered polypeptide of any one of claims 1-14, wherein the first antigen-binding moiety comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7 and 15-23.

16. The engineered polypeptide of any one of claims 1-15, wherein the first antigen-binding moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7 and 15-23, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.

17. The engineered polypeptide of any one of claims 1-15, wherein the second antigenbinding moiety further comprises a framework region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 128-155.

18. The engineered polypeptide of any one of claims 1-17, wherein the first antigen-binding moiety comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-14 and 24-29.

19. The engineered polypeptide of any one of claims 1-18, wherein the second antigenbinding moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-14 and 24-29, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.

20. The engineered polypeptide of any one of claims 1-19, wherein the single-chain bispecific ligand is a dimerizing-ligand for an IFNAR1 / IFNAR2 receptor heterodimer.

21. The engineered polypeptide of any one of claims 1-20, wherein the engineered polypeptide is capable of inducing phosphorylation of STAT1, STAT2, and / or STAT3 in vitro.

22. The engineered polypeptide of any one of claims 1-21, wherein the engineered polypeptide is capable of inducing phosphorylation of STAT1, STAT2, and / or STAT3 or a combination thereof in vivo.

23. The engineered polypeptide of any one of claims 1-21, wherein the engineered polypeptide is capable of inhibiting viral replication.

24. The engineered polypeptide of claim 23, wherein the engineered polypeptide is capable of inhibiting viral replication in vitro.

25. The engineered polypeptide of claim 23, wherein the engineered polypeptide is capable of inhibiting viral replication in vivo.

26. The engineered polypeptide of claim 23, wherein the engineered polypeptide is capable of inhibiting viral replication in a cell without inducing the expression of pro-inflammatory cytokines.

27. The engineered polypeptide of claim 23, wherein the engineered polypeptide is capable of inhibiting viral replication in a cell without inducing the expression of anti-proliferative cytokines.

28. The engineered polypeptide of any one of claims 1-27, wherein the engineered polypeptide is capable of inhibiting SARS-CoV-2 replication.

29. The engineered polypeptide of claim 28, wherein the engineered polypeptide is capable of inhibiting SARS-CoV-2 replication in vitro.

30. The engineered polypeptide of claim 28, wherein the engineered polypeptide is capable of inhibiting SARS-CoV-2 replication in vivo.

31. The engineered polypeptide of claim 28, wherein the engineered polypeptide is capable of inhibiting SARS-CoV-2 replication in a cell without inducing the expression of pro- inflammatory cytokines.

32. The engineered polypeptide of claim 28, wherein the engineered polypeptide is capable of inhibiting SARS-CoV-2 replication in a cell without inducing the expression of antiproliferative cytokines.

33. The engineered polypeptide of any one of claims 26-27 or 31-32, wherein the cell is a mammalian cell.

34. The engineered polypeptide of claim 33, wherein the mammalian cell is a human cell.

35. The engineered polypeptide of any one of claims 1-34, wherein the engineered polypeptide further comprises a linker inserted between the first antigen-binding moiety and the second antigen-binding moiety.

36. The engineered polypeptide of claim 35, wherein the linker is a peptide linker.

37. The engineered polypeptide of any one of claims 1-36, wherein the engineered polypeptide is an IFN agonist.

38. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the engineered polypeptide of any one of claims 1-37.

39. The recombinant nucleic acid molecule of claim 38, wherein the nucleic acid sequence is operably linked to a heterologous nucleic acid sequence.

40. The recombinant nucleic acid molecule of any one of claims 38-39, wherein the nucleic acid molecule is incorporated into an expression cassette or an expression vector.

41. A recombinant cell comprising a recombinant nucleic acid molecule of any one of claims 38-40.

42. The recombinant cell of claim 41 , wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell.

43. The recombinant cell of claim 42, wherein the eukaryotic cell is a mammalian cell.

44. A cell culture comprising at least one recombinant cell of any one of claims 41-43, and a culture medium.

45. A pharmaceutical composition comprising one or more pharmaceutically acceptable excipients and:(a) an engineered polypeptide according to any one of claims 1-37;(b) a nucleic acid molecule according to any one of claims 38-40; and / or(c) a recombinant cell according to any one of claims 41-43.

46. A method for modulating IFN-mediated signaling, the method comprises administering to the subject a composition comprising:(a) an engineered polypeptide according to any one of claims 1-37;(b) a nucleic acid molecule according to any one of claims 38-40;(c) a recombinant cell according to any one of claims 41-43; and / or(d) a pharmaceutical composition according to claim 45.

47. A method for the treatment of a health condition in a subject in need thereof, the method comprises administering to the subject a composition comprising:(a) an engineered polypeptide according to any one of claims 1-37;(b) a nucleic acid molecule according to any one of claims 38-40;(c) a recombinant cell according to any one of claims 41-43; and / or(d) a pharmaceutical composition according to claim 45.

48. The method of any one of claims 46-47, wherein the administered composition results in an induced downstream signaling activity.

49. The method of claim 48, wherein the downstream signaling activity comprises STAT1, STAT2, STAT3, STAT5, STAT6, Akt, S6, or ERK activity, or a combination of any thereof.

50. The method of claim 48, wherein the downstream signaling activity comprises activation of innate and / or adaptive immune responses.

51. The method of any one of claims 49-50, wherein the downstream signaling activity comprises differential induction of an interferon stimulated gene (1SG) as a metric for surrogate IFN activity.

52. The method of claim 51 , wherein the ISG is selected from the group consisting of MX1, OAS1, IFIT1, IFITM1, TRAIL, CXCL10, ISG15, CH25CH, cGAS, BST2, and NCOA71SG.

53. The method of any one of claims 46-52, wherein the subject is a mammal.

54. The method of claim 53, wherein the mammal is a human.

55. The method of any one of claims 46-54, wherein the subject has or is suspected of having a health disease associated with IFN-mediated signaling.

56. The method of any one of claims 46-55, wherein the health condition is a proliferative disease, a cancer, an immune disease, or an infection.

57. The method of claim 56, wherein the infection is a viral infection.

58. The method of claim 56, wherein the infection is a SARS-CoV-2 infection.

59. A kit for modulating IFN-mediated signaling and / or for treating a health condition, the kit comprising:(a) an engineered polypeptide according to any one of claims 1-37;(b) a nucleic acid molecule according to any one of claims 38-40;(c) a recombinant cell according to any one of claims 41-43; and / or d) a pharmaceutical composition according to claim 45.

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