Type I and III interferon fusion molecules and methods for their use

Abstract

Fusion molecules comprised of a type I interferon protein or a portion thereof and a type III interferon protein or a portion thereof, pharmaceutical compositions containing the fusion molecules, and methods for their use in inhibiting infection, inhibiting or treating cancer, inducing signaling of IFN-inducible gene transcription through the IFN-αR2 chain in subjects suffering from infections that degrade or downregulate the IFN-αR1 chain, and treating various diseases or disorders are provided.

Description

[Technical field]

[0001] This patent application claims the benefit of priority from U.S. Provisional Application No. 62 / 492,373, filed May 1, 2017, the contents of which are incorporated by reference in their entirety.

[0002] This invention was made with Government support under R01 AI057468 and R01 AI104669 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] background Interferons (IFNs) are fundamental cytokines in the establishment of a multifaceted antiviral response. Three distinct types of IFNs are currently recognized (types I, II, and III) based on their structural features, receptor utilization, and biological activity. Although all IFNs are important mediators of antiviral protection, their roles in antiviral defense vary. Type I IFNs (IFN-α / β / ω / ε / κ in humans) possess strong intrinsic antiviral activity and can induce a strong antiviral state in a wide variety of cells (Levy & Garcia-Sastre (2001) Cytokine Growth Factor Rev. 12(2-3):143-56; Samuel (2001) Clin. Microbiol. Rev. 14(4):778-809). Type I IFN receptor knockout mice are highly susceptible to many viral infections, and the essential role of type I IFN in inducing antiviral resistance has been clearly demonstrated using such animals (Mueller, et al. (1994) Science 264(5167):1918-21; Hwang, et al. (1995) Proc. Natl. Acad. Sci. USA 92(24):11284-8; Steinhoff, et al. (1995) J. Virol. 69(4):2153-8).In contrast, studies with IFN-γ and IFN-γ receptor knockout mice (Dalton, et al. (1993) Science 259(5102):1739-42; Huang, et al. (1993) Science 259(5102):1742-5; Lu, et al. (1998) Proc. Natl. Acad. Sci. USA 95(14):8233-8) as well as analyses of humans carrying inherited genetic mutations in the IFN-γ receptor (Dorman, et al. (2004) Lancet 364(9451):2113-21; Novelli & Casanova (2004) Cytokine Growth Factor Rev. 15(5):367-77) have revealed that antiviral activity is not the primary biological function of IFN-γ.

[0004] IFN-γ is classified as a Th1 type cytokine that stimulates cell-mediated immune responses that are critical for the development of host defense against pathogenic intracellular microorganisms such as Mycobacterium tuberculosis (Bach, et al. (1997) Annu. Rev. Immunol. 15:563-91; Boehm, et al. (1997) Annu. Rev. Immunol. 15:749-95; Pestka, et al. (1997) Cytokine Growth Factor Rev. 8(3):189-206). IFN-γ also plays a central role in the development of antitumor immune responses and can amplify the induction of antiviral activity by IFN-α or -β, or -λ. Thus, type I and type II IFNs often work together to activate various innate and adaptive immune responses that result in the induction of effective antitumor immunity and the elimination of viral infections (Biron (2001) Immunity 14(6):661-4; Le Bon & Tough (2002) Curr. Opin. Immunol. 14(4):432-6; Pestka, et al. (2004) Immunol. Rev. 202:8-32).

[0005] IFNs are members of a larger family of class II cytokines that also include six IL-10-related cytokines: IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 (Kotenko (2002) Cytokine Growth Factor Rev. 13(3):223-40; Renauld (2003) Nat. Rev. Immunol. 3(8):667-76; Pestka, et al. (2004) Annu. Rev. Immunol. 22:929-979) as well as several viral IL-10-related cytokines (Kotenko & Langer (2004) Int. Immunopharmacol. 4(5):593-608). IFNs and IL-10-related cytokines can be grouped into the same family because they all signal through receptors that share a common motif in their extracellular domains. These receptors include the class II cytokine receptor family (CRF2). As a result, IFN and IL-10-related cytokines are sometimes referred to as "CRF2 cytokines." The human type I IFN family is composed of 13 IFN-α species and the single species IFN-β, IFN-κ, IFN-ω, and IFN-ε (LaFleur, et al. (2001) J. Biol. Chem. 276(43):39765-71; Hardy, et al. (2004) Genomics 84(2):331-45; Langer, et al. (2004) Cytokine Growth Factor Rev. 15(1):33-48; Pestka, et al. (2004) Immunol. Rev. 202:8-32). Human type II IFN has only one species known as IFN-γ. Although the tertiary structure of IFN-γ is similar to that of IL-10, its primary structure is the most divergent from all the CRF2 ligands.The most recent additions to the CRF2 family, type III IFNs or IFN-lambda, demonstrate structural features of IL-10-related cytokines but also induce antiviral activity in a variety of target cells, supporting their functional classification as a new type of IFN (Kotenko, et al. (2003) Nat. Immunol. 4(1):69-77; Sheppard, et al. (2003) Nat. Immunol. 4(1):63-8). In humans, three distinct but closely related IFN-lambda proteins, IFN-lambda1, -lambda2, and -lambda3 (also known as IL-29, IL-28A, and IL-28B, respectively), were first identified. In 2013, the type III IFN family was expanded with an additional member, the IFN-λ4 protein, which shares only minor homology with IFN-λ1, -λ2, and -λ3 (Prokunina-Olson, et al. (2013) Nat. Genet. 45(2):164-71). Phylogenetically, the IFN-λ gene resides somewhere between the type I IFN family and the IL-10 gene family (Donnelly & Kotenko (2010) J. Interferon Cytokine Res. 30:555-64). Amino acid sequence comparisons show that type III IFNs exhibit approximately ∼5%–18% identity with either type I IFNs or IL-10-related cytokines.

[0006] IFN-lambda proteins bind to and signal through a receptor complex composed of a unique IFN-lambda R1 chain (also known as IL-28RA) and a common IL-10R2 chain (which is also part of the receptor complex for IL-10, IL-22, and IL-26) (Figure 1, and Kotenko, et al. (1997) EMBO J. 16(19):5894-903;Kotenko, et al. (2001) J. Biol. Chem. 276(4):2725-32;Xie, et al. (2000) J. Biol. Chem. 275(40):31335-9;Donnelly, et al. (2004) J. Leukoc. Biol. 76(2):314-21;Hor, et al. (2004) J. Biol. Chem. 279(32):33343-51; see Sheikh, et al. (2004) J. Immunol. 172(4):2006-10). In contrast, all type I IFNs exert their biological activities through a heterodimeric receptor complex composed of the IFN-αR1 (IFNAR1) and IFN-αR2 (IFNAR2) chains (see Figure 1), whereas type II IFNs (IFN-γ) engage the IFN-γR1 (IFNGR1) and IFN-γR2 (IFNGR2) chains to assemble their functional receptor complexes. IFN-λ does not use the IFN-α receptor complex for signaling, but signaling through either the IFN-λ or IFN-α receptor complexes results in activation of the same Jak-STAT signaling cascade (see Figure 1).

[0007] IFN-lambda first binds to the IFN-lambda R1 chain, and the binary complex formed by association of IFN-lambda with the IFN-lambda R1 chain induces a rapid conformational change that facilitates the recruitment of a second receptor chain, IL-10R2, to the complex. Once ternary complex assembly is complete, the receptor-associated Janus tyrosine kinases Jak1 and Tyk2 mediate transphosphorylation of the receptor chains that results in the formation of a phosphotyrosine-containing peptide motif on the intracellular domain (ICD) of the IFN-lambda R1 chain, which potentially provides a transient docking site for preformed cytoplasmic STAT proteins, including STAT1 and STAT2. Signaling through type I (IFN-α / β) or type III (IFN-lambda) IFN receptor complexes results in the formation of a transcription factor complex known as IFN-stimulated gene factor 3 (ISGF3). This complex is composed of three proteins, STAT1, STAT2, and IFN regulatory factor 9 (IRF-9) (also known as ISGF3γ or p48). Once assembled, ISGF3 then translocates to the nucleus where it binds to IFN-inducible response elements in the promoters of various IFN-inducible genes (ISGs). As a result, the biological activities induced by either type I or type III IFNs are quite similar, including induction of antiviral activity, upregulation of major histocompatibility complex (MHC) class I antigen expression on many cell types, and antitumor activity. Moreover, coadministration of IFN-α and IFN-λ has been shown to provide synergistic antitumor effects in mouse models of hepatocellular carcinoma (Lasfar, et al. (2008) Hepatology 48(4S):394A-395A; Lasfar, et al. (2016) Oncotarget 7(31):49259-49257). Summary of the Invention

[0008] SUMMARY OF THE PRESENT APPLICATION Aspects of the present invention relate to fusion molecules composed of a type I IFN protein or a portion thereof and a type III IFN protein or a portion thereof. In one non-limiting embodiment, the type I interferon protein molecule of the fusion molecule is interferon alpha, interferon alpha2, or interferon beta, or a portion thereof. In one non-limiting embodiment, the type III interferon protein of the fusion molecule is interferon lambda 1, interferon lambda 2, or interferon lambda 3, or a portion thereof. In some non-limiting embodiments, the fusion molecule further comprises a linker between the type I interferon protein or a portion thereof and the type III interferon protein or a portion thereof, and / or a signal peptide at the N-terminus of the fusion molecule.

[0009] Another aspect of the invention relates to a pharmaceutical composition comprising the fusion molecule and a pharma- ceutically acceptable carrier.

[0010] Another aspect of the invention relates to a method for inhibiting an infection in a subject, the method comprising administering to the subject an effective amount of a fusion molecule. In one non-limiting embodiment, the fusion molecule targets and inhibits infection in two or more cell types in the subject.

[0011] Another aspect of the invention relates to a method for inhibiting or treating cancer in a subject, the method comprising administering to the subject an effective amount of a fusion molecule.

[0012] Another aspect of the invention relates to a method for inducing signaling leading to the transcription of IFN-inducible genes through the IFN-αR2 chain in a subject suffering from an infection that degrades or downregulates the IFN-αR1 chain, the method comprising administering to the subject an effective amount of a fusion molecule.

[0013] Yet another aspect of the present invention relates to a method for treating a disease or condition, comprising administering an effective amount of a fusion molecule to a subject in need of treatment, thereby treating the disease or condition in the subject. In a non-limiting embodiment, the disease or condition responds to interferon treatment. In a non-limiting embodiment, the disease or condition is a viral infection, a fungal infection, a bacterial infection, cancer, an inflammatory disease, or an autoimmune disease. [Brief description of the drawings]

[0014] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 depicts a model of the type III IFN (IFN-lambda) and type I IFN (IFN-α / β) receptor systems. IFN-lambda and type I IFNs use distinct heterodimeric receptor complexes. IFN-lambda engages its unique IFN-lambdaR1 and IL-10R2, whereas IFN-αR1 and IFN-αR2 form the active type I IFN receptor complex. Engagement of the IFN-α or IFN-lambda receptor results in phosphorylation of the receptor-associated JAK kinases JAK1 and Tyk2, followed by phosphorylation of STAT1 and STAT2, which interact with the DNA-binding protein IRF9, leading to the formation of a transcription complex named IFN-inducible gene factor 3 (ISGF3), which binds to IFN-inducible response elements (ISREs) and regulates the transcription of IFN-inducible genes (ISGs).

[0015] [Diagram 2] Figures 2A, 2B, 2C, and 2D show IHC staining of pSTAT1 (pTyr701) in the small intestine of wild-type (WT) 8-day-old pups or 8-week-old adult mice injected subcutaneously (SQ) with human IFN-αA / D (IFN-α; 1 μg (Figures 2A and 2B)) or mouse IFN-λ2 (IFN-λ; 1 μg (Figures 2C and 2D)). Black arrows indicate nuclear staining of pSTAT1 in IFN-treated epithelial cells, and white arrows indicate inflammatory cells within the lamina propria.

[0016] [Diagram 3]Figures 3A, 3B, and 3C show IHC staining for viral antigens in the airways of IAV-infected mice (Figure 3A) and IHC staining of pSTAT1 in the airways of WT adult mice injected SQ with IFN-α (1 μg; Figure 3B) or IFN-λ2 (1 μg; Figure 3C).

[0017] [Figure 4] Figures 4A and 4B show IHC staining of pSTAT1 in the airways of WT adult mice injected intranasally (IN) with IFN-α (1 μg; Figure 4A) or IFN-λ2 (1 μg; Figure 4B).

[0018] [Diagram 5] Figure 5 shows the synergistic effect of IFN-α and IFN-λ on tumor growth in vivo. Mice were injected with original tumor cells (control) or with tumor cells expressing either IFN-α, IFN-λ, or IFN-α+IFN-λ. Tumor survival was monitored. Results show the percentage of tumor-free mice.

[0019] [Figure 6] FIG. 6 shows the synergistic effect of IFN-α and IFN-λ on tumor growth in vivo. Mice were injected with 106 original BNL cells (hepatocellular carcinoma model) and tumors were allowed to form for 4-6 weeks. Approximately 90% of the tumors were surgically removed and mice were subsequently treated with either IFN-α, IFN-λ, or IFN-α+IFN-λ every 2 days for 2 weeks. Tumor size was monitored for 4-6 weeks. Tumor regression or change in tumor size is shown.

[0020] [Figure 7] Figure 7 shows the effect of local and systemic administration of IFN-α+IFN-λ on tumor size compared to mock administration. The results show that local administration of IFN-α+IFN-λ dramatically reduces tumor size.

[0021] [Figure 8]FIG. 8 shows that IFN-α and IFN-λ induce protection against human cytomegalovirus (HCMV) in ARPE-19 cells (a human retinal pigment epithelial cell line) when cells are pretreated with IFN 0.1, 6, 24, and 72 hours prior to HCMV infection.

[0022] [Figure 9] FIG. 9 shows that IFN-α and IFN-λ induce protection against vesicular stomatitis virus (VSV) in ARPE-19 cells when cells were pretreated with IFN 0.1, 6, 24, 72, and 96 hours prior to VSV infection.

[0023] [Figure 10] The differential kinetics of antiviral responses induced by type I and type III IFNs are illustrated in Figure 10. The combined use of type I and type III IFNs by itself provides a fast, long-lasting, efficient and broad-spectrum antiviral response.

[0024] [Figure 11] Figure 11 shows a schematic diagram of the structure of a fusion molecule between human type I IFN (IFN-I) and type III IFN (IFN-III) with the signal peptide (SP) from type I IFN. A linker sequence is depicted between the two IFN proteins.

[0025] [Figure 12] 12 shows the amino acid sequence of the human (h)IFN-α-hIFN-λ1 fusion molecule (SEQ ID NO:51). The N-terminal signal peptide sequence, a glycine / serine-rich linker, is boxed.

[0026] [Figure 13] 13 shows the amino acid sequence of the hIFN-α-hIFN-λ3 fusion molecule (SEQ ID NO:52). The N-terminal signal peptide sequence, a glycine / serine-rich linker, is boxed.

[0027] [Figure 14] 14 shows the amino acid sequence of the hIFN-β-hIFN-λ3 fusion molecule (SEQ ID NO:53). The N-terminal signal peptide sequence, a glycine / serine-rich linker, is boxed.

[0028] [Figure 15] FIG. 15 provides a schematic diagram of reporter CHO cell lines expressing chimeric receptor complexes for either human type I or type III IFN.

[0029] [Figure 16] FIG. 16 shows that treatment of a reporter CHO cell line with a fusion IFN protein of the invention (10 ng / ml) results in STAT1 activation as determined by electrophoretic mobility shift assay (EMSA).

[0030] [Figure 17] Figure 17 depicts a model demonstrating the ability of the fusion IFN molecules to signal through the combined type I and type III IFN receptor complex in the presence of the negative regulator USP18. IFN-induced USP18 protein inhibits type I IFN signaling by competing with JAK1 for binding to IFN-αR2. JAK1 molecules associated with IFN-λR1 boost signaling through both type I and type III IFN receptors in the combined IFN receptor complex that is oligomerized by the IFN fusion molecule.

[0031] [Figure 18]Figure 18 depicts a model demonstrating the ability of fusion IFN molecules to signal through the combined type I and type III IFN receptor complex in the absence of the IFN-αR1 chain. Infection suppresses type I IFN signaling by targeting the IFN-αR1 chain for downregulation and degradation. IFN fusion molecules induce clustering of the remaining receptor subunits, allowing cross-activation of receptor-associated JAK kinases and induction of the IFN signaling cascade.

[0032] [Figure 19] Figure 19 demonstrates that the fusion IFN molecules of the invention have the ability to induce enhanced signaling through type I and type III combined IFN receptor complexes when all four receptor subunits are present. Also demonstrated herein is the ability of the fusion IFN molecules of the invention to signal through type I and type III combined IFN receptor complexes when only two (or three) receptor subunits are present. The IFN fusion molecules induce clustering of the remaining receptor subunits, allowing cross-activation of receptor-associated JAK kinases and induction of the IFN signaling cascade.

[0033] [Figure 20]Figures 20A, 20B and 20C show that the fusion IFN molecule IFN-β-IFNλ2 suppressed in vivo tumor growth. Mouse breast cancer E0771 cells were engineered to secrete either mouse IFN-λ2, IFN-β or IFN-β-IFNλ2 (IFN-β-λ2) fusion molecules. The tumorigenicity of modified E0771 cells was assessed in syngeneic C57BL / 6 mice. Mice were injected with 50,000 E0771 cells as indicated into the mammary fat pad and tumor development was monitored. The results of the two experiments are shown in Figures 20A and 20B. Three of five mice (FIG. 20A) and four of six mice (FIG. 20B) implanted with E0771 cells expressing fusion IFN-β-λ2 molecules did not develop tumors (TF-tumorless), whereas all mice implanted with E0771 cells expressing only IFN molecules developed tumors. In FIG. 20B, mice were also injected with E0771 cells expressing only IFN molecules at a 50:50 percent ratio. FIG. 20C depicts the tumor volume in each individual mouse at day 26 after tumor cell implantation. FIG. 20D shows abdominal skin thickening and increased abdominal volume (ascites) in mice bearing tumors expressing a combination of single IFN molecules, and the lack of these signs in mice bearing tumors expressing fusion IFN-β-λ2 molecules. FIG. 20E shows signs of anemia in tumors and other tissues (liver) of mice bearing tumors expressing the single IFN molecule combination, and the lack of these signs in mice bearing tumors expressing the fusion IFN-β-λ2 molecule.

[0034] [Figure 21] 21 shows the amino acid sequence of the mouse (m)IFN-α-mIFN-λ2 fusion molecule (SEQ ID NO:54). The N-terminal signal peptide sequence, a glycine / serine-rich linker, is boxed.

[0035] [Figure 22]22 shows the amino acid sequence of the mIFN-β-mIFN-λ2 fusion molecule (SEQ ID NO:55). The N-terminal signal peptide sequence, a glycine / serine-rich linker, is boxed.

[0036] [Figure 23] FIG. 23 shows the results of immunoblotting, using anti-His antibody, of supernatants from HEK293 cells expressing single or fusion mIFN molecules containing a C-terminal 6×His tag for detection and purification.

[0037] [Figure 24] FIG. 24 shows the antiviral activity of His-tagged single or fusion mIFN molecules produced in HEK293 cells tested against mouse intestinal epithelial cells (mIEC).

[0038] [Diagram 25] FIG. 25 shows upregulation of MHC class I antigens in mIEC in response to His-tagged single or fusion mIFN molecules produced in HEK293 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description of the Invention Disclosed herein are fusion molecules comprising interferon proteins or portions thereof, pharmaceutical compositions containing the fusion molecules, and methods for their use in inhibiting infections, inhibiting or treating cancer, inducing transcriptional signaling of IFN-inducible genes through the IFN-αR2 chain, and treating various diseases or conditions in subjects suffering from infections that degrade or downregulate the IFN-αR1 chain.

[0040] For purposes of the present invention, the terms "fusion protein" and "fusion molecule" are used interchangeably and are meant to cover polypeptides, proteins, and / or molecules made of portions from different sources. Such fusion molecules are created through the joining of two or more genes or fragments thereof that originally code for separate proteins or portions thereof. Translation of these fusion genes or portions thereof results in a single or multiple polypeptides with functional properties derived from each of the original proteins. In one non-limiting embodiment, the fusion molecules or proteins are artificially created by recombinant DNA technology for use in biological research or therapy.

[0041] For purposes of the present invention, "portion thereof" refers to a fragment of less than the full-length interferon protein, said fragment retaining at least a portion of the functional activity of the full-length protein and / or binding to at least one of the receptor subunits.

[0042] Type I IFNs, IFN-α / β, are used in the clinic to treat a variety of pathological conditions, including viral infections, cancer, and multiple sclerosis (IFN-beta). However, the use of IFN-α / β remains problematic due to low efficacy and a number of significant side effects. Type III IFNs or IFN-λ have been shown to possess antiviral and antitumor activity comparable to type I IFNs in mouse models of viral infection and cancer. Although type I and type III IFNs have similar biological activities, they utilize unique IFN type-specific receptor complexes for signaling (Figure 1). The receptors for type I and type III IFNs have demonstrated distinct patterns of cell type and tissue distribution, with these IFNs targeting both overlapping and distinct IFN type-specific cell populations.

[0043] While type I IFN receptors are expressed in most cell types, IFN-λR1 has demonstrated a more restricted pattern of expression, primarily in epithelial cells of the respiratory tract, gastrointestinal tract, and reproductive tract, with a limited response to type III IFNs (Sommereyns, et al. (2008) PLoS Pathog. 4:e1000017; Lasfar, et al. (2006) Cancer Res. 66:4468-4477). The unique functional tissue specificity of the IFN-λ response is due to the cell type-restricted pattern of IFN-λR1 expression; while all cells express receptors for type I IFNs, IFN-λR1 is expressed primarily in epithelial cells and specific subsets of immune cells (Kotenko and Durbin (2017) J. Biol. Chem. 292(18):7295-7303). Indeed, it has been demonstrated that both type I and type III IFN systems are capable of providing efficient, comparable, and independent in vivo antiviral defense against infections targeting epithelial tissues in which receptors for both types of IFN are expressed (Sheppard, et al. (2003) Nat. Immunol. 4:63-68; Kotenko, et al. (2003) Nat. Immunol. 4:69-77; Doyle, et al. (2006) Hepatology 44:896-906; Ank, et al. (2006) J. Virol. 80:4501-4509). However, it has been demonstrated that the antiviral defense of intestinal epithelial cells against gastrointestinal (GI) viruses primarily relies on the action of the type III IFN antiviral system (Pott, et al. (2011) Proc. Natl. Acad. Sci. USA 108:7944-49; Lin, et al. (2016) PLOS Path. 12:e1005600).These studies revealed that, unlike influenza A virus infection in the lungs, mice lacking a functional IFN-lambda receptor complex had impaired control of oral rotavirus (RV) infection; the type I IFN system alone was able to provide only weak protection against rotavirus infecting intestinal epithelial cells. Importantly, mice lacking both type I and type III IFN receptors were more susceptible to RV infection than mice lacking either IFN receptor (Lin, et al. (2016) PLOS Path. 12:e1005600). Consequently, systemic administration of IFN-lambda or type I IFN could induce an antiviral state in intestinal epithelial cells that led to the suppression of rotavirus replication. However, only type I IFN protected against hepatotropic viruses. Thus, type I and type III IFN systems have unique functions in the overall antiviral protection. Type I IFN appears to be required for antiviral defense in the liver, endothelial cells, fibroblasts, and immune cells, where type III IFN has minimal or no activity. In contrast, the type III IFN system is required for effective antiviral defense in the intestinal epithelium that is independent of and does not overlap with the type I IFN antiviral system, while both types of IFN can provide efficient and independent antiviral defense in the lung (Mordstein, et al (2010) J. Virol. 84:5670-7).

[0044] Demonstrating that type I and type III IFN target distinct organs and tissues was confirmed by viral studies using transgenic reporter mice with a luciferase reporter gene controlled by the Mx2 promoter that is specifically and uniquely induced by type I and type III IFN (Pulverer, et al. (2010) J. Virol. 84:8626). Using such reporter mice, it was shown that the strongest expression of luciferase was induced in organs with mucosal surfaces, such as the stomach, small and large intestine, lungs, and salivary glands. In contrast, type I IFN responses were strong in the liver, spleen, and kidneys. Minimal or no responses were detected in the GI tract and salivary glands to intravenously administered type I IFN, while the lungs responded to type I IFN. Interestingly, in addition to targeting different organs, type I and type III IFN demonstrated differential kinetics of response. Whole-body live imaging showed that luciferase expression peaked at 3 hours in response to IFN-β, 6 hours in response to IFN-α, and 9 hours in response to IFN-λ injection. Indeed, the response to IFN-λ was early, clearly detectable at 3 hours in the lungs and GI tract, and could persist for 24 hours. Thus, the predominant target organs of type I and type III IFNs and the kinetics of their action are clearly distinct.

[0045] Intestinal epithelial cells respond only to IFN-λ in adult animals, but not in neonates, a cohort susceptible to rotavirus-induced diarrhea. Both IFNLR and IFNAR are essential for optimal protection against rotavirus during neonatal infection, and both IFN pathways are active in the neonatal intestine (Lin, et al. (2016) PLOS Path. 12:e1005600). In Figures 2A-2D, IFN signaling was detected 30 min after subcutaneous (SQ) injection of IFN-α or IFN-λ. Formalin-fixed paraffin-embedded (FFPE) tissues were stained with a monoclonal antibody against tyrosine phosphorylated STAT1 (pSTAT1). Nuclear localization of pSTAT1 is evidence of signaling through either IFNAR or IFNLR. In those sections from the intestine of adult mice treated with IFN, nuclear staining of hematopoietic cells in the lamina propria (white arrow) is present only in intestinal epithelial cells (black arrow) after treatment with IFN-α and after IFN-λ treatment. However, in neonates, intestinal epithelial cells responded well to either IFN-α or IFN-λ as measured by nuclear localized pSTAT1. All cell lines derived from the intestine tested are fully sensitive to both types of IFN. Thus, although type I and type III IFN pathways appear to be redundant in cultured cells, the effects in vivo are specific to certain anatomic compartments. Thus, co-administration of type I and type III IFN via fusion molecules such as the present invention will be specifically effective, since type I and type III IFN target different sets of cells in the GI tract to obtain efficient antiviral defense in the GI tract, specifically against viruses that can replicate in different cell types.

[0046] Similarly, subcutaneous (SQ) administration of IFN-α led to STAT1 activation in alveolar epithelium but not in airway (tracheal, bronchial) epithelium, whereas IFN-λ acted preferentially on columnar airway-lining cells (see Fig. 3A-3C). As shown, influenza virus replicates in both cell types (Fig. 3A-3C). Administration of either IFN-α or IFN-λ by intranasal injection induced STAT1 phosphorylation and nuclear translocation in a subset of alveoli cells (Fig. 4). However, similar to SQ injection, respiratory epithelial cells lining airways responded only to type III IFN (Fig. 4). These results demonstrate that the actions of type I and type III IFNs are strictly compartmentalized not only in the GI tract but also in the respiratory tract. Therefore, if type I and type III IFNs preferentially target different levels of the respiratory tree in vivo, it is expected that the most effective therapeutic approach will involve co-treatment with both IFN types via fusion molecules such as those of the present invention to suppress viral replication in both alveolar and bronchial epithelia. Moreover, due to the increased affinity of the fusion IFN molecules compared to each IFN acting in combination, it is expected that the IFN fusion molecules will induce enhanced IFN signaling in cells expressing all four IFN receptor subunits. In addition, where many viruses suppress IFN-mediated antiviral responses in infected cells by downregulating selected IFN receptor subunits or targeting them for degradation (Figure 18) (Sen, et al. (2017) J. Virol. JVI.01394-17), the ability of IFN fusion molecules to signal through the remaining type I and type III IFN receptor subunits in the combined type I and type III IFN receptor complex (Figures 18 and 19) would still allow induction of IFN signaling in infected cells.Similarly, type I IFN signaling is quickly downregulated by the IFN-induced negative regulator USP18, which displaces JAK1 from association with IFN-αR2, leading to the termination of type I IFN signaling. It is expected that IFN fusion molecules will still be able to engage and activate IFN-αR2 by bringing IFN-λR1-associated JAK1 into the tetrameric IFN receptor complex, leading to IFN-αR2 phosphorylation and thus induction of type I IFN signaling in cells expressing USP18 ( FIG. 17 ).

[0047] In addition, different cell types express different levels of specific IFN receptor subunits. For example, intestinal epithelial cells express low levels of IFN-αR2, but IFN-αR1 is expressed at higher levels (Mahlakoiv, et al. (2015) PLoS Pathog. 11(4):e1004782). In cells expressing low levels of one or two IFN receptor subunits, IFN fusion molecules will still be able to bind and engage the remaining IFN receptor subunits and induce IFN signaling (Figure 19).

[0048] Moreover, each IFN type has unique biological characteristics. For instance, expression of IFN-λ receptors demonstrates a tissue-restricted pattern. Epithelial cells are the primary targets for type III IFNs. Thus, type III IFNs can be expected to play a crucial role in host defense, as epithelia are the main barrier for primary viral infections. Many viruses enter the host through the upper gastrointestinal, respiratory, or genitourinary tracts, or through damaged skin. Current data demonstrate that type III IFNs are highly active in airway and intestinal epithelial cells, and in keratinocytes, suggesting that their antiviral potency may be strongest in epithelial-like tissues. Moreover, although respiratory polarized epithelial cells respond to type I IFNs only at their basolateral, not their apical, surfaces, there is evidence suggesting that the cells are still sensitive to type III IFNs at their apical (luminal) side. This unique feature makes IFN-λ-based therapy effective following intranasal delivery to prevent, inhibit, or treat respiratory viral infections. Furthermore, type I IFN is uniquely positioned to defend submucosal tissues, since many viruses can rapidly spread into the submucosal tissue and infect fibroblast-like cells. Intranasal delivery of type I / type III IFN combinations via the fusion molecules of the present invention may provide antiviral defense to uninfected airway epithelia through the action of IFN-λ on the apical surface of intact cells. At airway sites where viral infection has already occurred, the integrity of the epithelial barrier may be compromised, and type I IFN (e.g., co-delivered IFN-α or IFN-β) may gain access to the basolateral surface of epithelial cells, as well as to underlying connective tissue, immune cells, and endothelial cells within blood vessels where type III IFN is not effective (see Figures 2-4).

[0049] Microarray experiments demonstrated that type I and type III IFNs induce nearly identical sets of genes. However, similar to in vivo experiments with Mx2-luciferase reporter mice, the kinetics of IFN-induced gene expression is different, resulting in a longer-lasting antiviral state in response to type III IFN, whereas the antiviral state induced by type I IFN declines rapidly 24 hours after treatment (see Figure 10). Thus, administration of IFN fusion molecules would deliver the fast antiviral action of type I IFN with the long-lasting antiviral effect of type III IFN in the same molecule. As mentioned above, the transient action of type I IFN is mediated by the IFN-inducible negative regulator USP18. However, the fusion IFN molecule is expected to overcome this negative regulatory mechanism and provide a series of signaling through the type I IFN receptor (Figure 17).

[0050] IFN-induced regulatory mechanisms differentially affect type I and type III IFN signaling. For example, IFN-inducible ISG15, which uncouples ubiquitin protease 43 (UBP43, also known as USP18), has been shown to compete with Jak1 for binding to the intracellular domain of IFN-αR2, effectively suppressing IFN-α signaling ( FIG. 17 ; Malakhova, et al. (2006) EMBO J. 25:2358-67), whereas USP18 has minimal activity with respect to the type III IFN receptor. Due to the action of USP18, cells treated with IFN become insensitive to type I IFN within 6 hours of IFN treatment, with a refractory period lasting up to 72 hours. In contrast, cells are constantly responsive to type III IFN. Thus, antiviral resistance can be maintained in cells in the presence of type III IFN, but type I IFN is more rapid in inducing early expression of antiviral mediators in cells.

[0051] Infection also targets IFN-αR1 or other IFN receptors for degradation or downregulation, thereby suppressing their type I IFN-mediated biological activity (Sen, et al. (2017) J. Virol. JVI.01394-17). Oligomerization of both type I and type III IFN receptor subunits within one receptor complex, triggered by binding of the IFN fusion molecule, is expected to result in cross-activation of the IFN-αR2-associated JAK1 kinase by kinases associated with the IFN-λ receptor subunit, allowing signaling through the IFN-αR2 subunit to continue in the absence of the IFN-αR1 subunit (FIG. 18). As a result, the fusion molecules of the present invention are expected to be useful in methods for inducing transcription of IFN-inducible genes through the IFN-αR2 chain in subjects suffering from infections that degrade or downregulate the IFN-αR1 chain.

[0052] Similarly, the absence of another IFN receptor subunit, such as IFN-lambda R1 or IFN-alpha R2, or any combination of two receptor subunits, due to virus-induced downregulation or degradation of the IFN receptor subunits will still render the virus-infected cell responsive to an IFN fusion molecule (FIG. 19). Consequently, the fusion molecules of the present invention are expected to be useful in methods for inducing transcription of IFN-inducible genes through any two IFN receptor subunits in subjects suffering from infections that degrade or downregulate one or two IFN receptor chains.

[0053] Finally, viruses develop their own IFN-specific countermeasures to circumvent IFN-mediated antiviral defenses. For example, some viruses may be more vulnerable to type III IFN. Several poxviruses, including variola virus, the causative agent of smallpox, encode secreted IFN antagonists that cannot suppress type III IFN (Huang, et al. (2007) Proc. Natl. Acad. Sci. USA 104:9822-27). Thus, type III IFN may be more effective in treating poxvirus infections.

[0054] Specifically, for biodefense purposes, when the identity of a bioagent may remain unknown for a period of time, there is a clear unmet need for the development of a wide variety of fast-acting and long-acting antiviral drugs. In addition, many viruses infect multiple organs and cell types. Thus, antiviral therapeutics capable of inducing antiviral responses at the level of the whole body are particularly useful for effective defense against such viruses. A growing body of evidence demonstrates that each type of IFN has unique functional features, targets overlapping but distinct groups of organs and tissues for induction of an antiviral state, and demonstrates different kinetics of their action. Thus, type I and type III IFN antiviral systems have evolved to coordinately induce antiviral protection against distinct viral pathogens, and both systems are essential for effective antiviral defense. Therefore, a combination of type I and type III IFN is essential to obtain the highest level of antiviral response in multiple organs. Moreover, application of fast-acting IFN-β with slower but longer-acting IFN-λ3, IFN-λ1, or IFN-λ2 will provide the highest level of fast and long-lasting antiviral defense that is wide-spread and covers the entire organism. The IFN combination therapy of the present invention is specifically effective against viruses that can replicate in multiple organs or multiple cell types.

[0055] In addition, type I and type III IFNs have now been found to have synergistic antitumor effects in mouse models of cancer (see Figures 5 and 6). In one non-limiting embodiment, local administration showed synergistic effects (see Figure 7). Consequently, the present invention provides a combination therapy involving co-administration of type I and type III IFNs. In one embodiment, the type I IFN is IFN-α (e.g., IFN-α2) or IFN-β. In another embodiment, the type III IFN is IFN-λ, particularly IFN-λ1, IFN-λ2, or IFN-λ3.

[0056] Importantly, the study presented in Figure 20 revealed that the fusion IFN-β-FN-λ2 molecule was much more potent than a single IFN molecule, as well as the combination of IFN-β and IFN-λ2, in suppressing the growth of breast tumor cells. Four out of six mice implanted with breast tumor cells expressing the fusion IFN-β-FN-λ2 protein remained tumor-free, whereas all mice injected with a mixture of tumor cells expressing either IFN-β or FN-λ2 developed tumors. The IFN-β-FN-λ2-expressing tumors that developed in two mice were smaller in size than the tumors in mice implanted with a mixture of tumor cells expressing either IFN-β or FN-λ2. Thus, the IFN fusion molecule displayed enhanced synergistic antitumor activity compared to type I and type III IFN combination treatment. Moreover, increased abdominal volume (ascites) and signs of severe anemia in tumors and other tissues (liver) were observed in mice bearing tumors expressing the single IFN molecule combination. Similar symptoms and signs were not observed in mice bearing tumors expressing the fusion IFN-β-λ2 molecule (Figures 20D and 20E). Anemia is a known side effect of type I IFN treatment due to type I IFN induced suppressed hematopoiesis and antiangiogenic effects. The lack of these side effects in mice bearing tumors expressing the IFN fusion molecules of the present invention is indicative of a safer profile for the fusion molecules, as they may lack or impose less side effects compared to type I IFN treatment or even compared to combined IFN type I and III treatment. In addition, these data demonstrate that the IFN fusion molecules have unique biological activities and profiles, which are distinct from those of mixtures of type I and type III IFN.

[0057] Given that treatment of mice with either IFN-α or IFN-λ alone leads to activation of antiviral responses (see Figures 8 and 9), simultaneous administration of type I and type III IFNs is expected to provide long-lasting, efficient and broad-spectrum antiviral responses, especially in overlapping but distinct organs, tissues and cell types (Figure 10). Thus, combinations of type I and type III IFNs are also of use in the prevention and / or treatment of viral infections. To facilitate the co-delivery of type I and type III IFNs, a non-limiting embodiment of the present invention provides fusion molecules of IFN-α or IFN-β with IFN-λ (see, for example, Figures 11-14 and 21-22). The fusion molecule of IFN-α2 and IFN-λ1 retains the functional activity of both type I and type III IFN, as demonstrated by the ability of the fusion molecule to cause STAT1 activation in reporter cell lines that respond to either type I or type III IFN (FIGS. 15 and 16). In this assay, STAT1 activation was assessed in a reporter cell line in which intracellular signaling is mediated by the intracellular domain(s) of the IFN-γ receptor, and was not accompanied by tetramerization of type I and type III IFN receptor subunits by the IFN fusion molecule, as would be expected to occur in normal human cells (FIG. 19). STAT1 activation was measured in an EMSA that assessed IFN-induced STAT1 dimerization and binding of STAT1 dimers to a radiolabeled DNA probe. Formation of STAT1 dimers requires phosphorylation of STAT1 on Tyr701. Although Tyr phosphorylation of STAT1 and STAT2, leading to the formation of the ISGF3 transcription complex, is a hallmark and unique feature of both type I and type III IFN-induced JAK-STAT signaling cascades, several additional post-translational modifications, including methylation, acetylation, Ser phosphorylation, etc., have been reported and may affect the kinetics and magnitude of ISG expression, as well as the preferential selection for specific subsets of ISGs.ISGF3 complexes are clearly activated by IFN fusion molecules, as they induce antiviral defenses that depend on ISGF3 activation (Figure 24). However, because some of the secondary posttranslational modifications in ISGF3 may differ between type I and type III IFN signaling, it is expected that ISGF3 activated in response to fusion molecules may not be identical to ISGF3 induced in response to single IFN molecules and may therefore give rise to alternative profiles of ISG expression in terms of kinetics, magnitude, or ISG subset selectivity.

[0058] There are certain advantages to using IFN fusion molecules over combinations of separate IFN proteins.

[0059] For example, fusion molecules provide for simplified therapeutic production and delivery of a single therapeutic agent.

[0060] Furthermore, as mentioned above, studies using Mx2 luciferase reporter mice have demonstrated that type I IFN preferentially targets the liver, whereas type III IFN has unique activity in the GI tract and airway epithelium. Nevertheless, various human hepatocyte-like cell lines and cell lines derived from the GI tract and respiratory tract respond to both type I and type III IFN. Moreover, freshly isolated mouse hepatocytes and intestinal epithelial cells respond to both types of IFN. Thus, the little or weak in vivo responsiveness of selected organs and tissues to individual IFNs may be the result of the differential deliverability of type I and type III IFN. Thus, the fusion molecule will deliver both IFN-α and IFN-λ to all tissues and organs.

[0061] In addition, because type I and type III IFNs target distinct cell types that only partially overlap, administration of type I and type III IFNs as a fusion molecule provides a wide variety of cellular effects. Furthermore, there is cross-regulation of signaling between type I and type III IFNs in cells that respond to both types of IFNs; for example, the action of both types of IFNs is essential to make neutrophils fully functional in controlling invasive fungal growth (Espinosa, et al., (2017) Sci. Immunol. (16) pii: eaan5357). As a result, administration of type I and type III IFNs as a fusion molecule will also be useful in this application.

[0062] As mentioned above, IFN fusion molecules are expected to overcome the negative regulatory effect of IFN-inducible USP18 protein, allowing sustained signaling through the type I IFN pathway. IFN-λR1 expression is restricted to distinct cell types, whereas IL-10R2 is ubiquitously expressed. Thus, fusion molecules will engage three receptor subunits in type I IFN-responsive cells. This receptor should have a higher affinity that can make signaling through the trimeric IFN receptor complex stronger than through the heterodimeric type I IFN receptor complex. Similarly, various epithelial cells that respond primarily only to type III IFN still express lower levels of one or both type I IFN receptor subunits. Lower levels of expression of type I IFN receptor subunit(s) will be involved, resulting in some complexes composed of three or four IFN receptor subunits. These complexes are high affinity receptor complexes and may lead to stronger IFN signaling in epithelial cells.

[0063] Furthermore, the fusion molecule is useful for intranasal delivery. As mentioned above, the apical surface of airway epithelial cells mainly responds to type III IFN. Therefore, intranasal delivery of type I and type III IFN fusion molecules provides antiviral protection to non-infected airway epithelial cells through the action of IFN-λ on the apical surface of intact cells. When the apical IFN-λ receptor complex is internalized after binding to the IFN fusion molecule and then recycled together with the fusion molecule to the basolateral surface of epithelial cells, the type I IFN portion of the IFN fusion molecule can access the basolateral surface of the top layer of airway lining epithelial cells as well as the underlying connective tissue.

[0064] Moreover, the fusion molecule will have higher efficacy due to higher affinity binding to the receptor. It is expected that the fusion molecule will induce oligomerization of type I and type III IFN receptor complex, resulting in increased affinity of the fusion molecule for this combined cell surface receptor complex, which will translate into stronger antiviral activity (Figure 19). In other words, the fusion IFN molecule binds to the tetrameric receptor complex, whereas the single IFN molecule binds to the homodimeric receptor complex (Figure 19). Therefore, it is expected that the fusion molecule should have higher affinity for the receptor than the single IFN molecule. In addition, because USP18 only displaces Jak1 from the type I receptor complex, the combined type I and type III IFN receptor complex (Figure 17) brought together by the IFN fusion molecule will still have Jak1 associated with the IFN-lambda receptor. This Jak1 can phosphorylate (activate) the intracellular domains of both the IFN-lambda R1 and IFN-α R2 chains, allowing signaling through both type I and type III IFN receptors to proceed normally even in the presence of the negative regulator USP18. In cases where the IFN-α R1 chain is downregulated by infection, IFN fusion molecules should be able to induce signaling through the IFN-α R2 chain as well (Figure 18). Other negative regulators such as SOCS1 and SOCS3 also show preferential inhibition of type I vs. type III IFN signaling pathways. Since the fusion molecules bind and cluster the four receptor subunits simultaneously, the absence of one or even two of them should not block the ability of the fusion IFN molecule to signal through the remaining two or three receptor subunits due to proteasomal degradation or due to the action of inhibitory molecules.In other words, the presence of any two of the four receptor subunits should be sufficient for the fusion molecule to induce downstream signaling cascades and biological activity, whereas removal or blocking of only one receptor subunit in either type I or type III IFN receptor complexes leads to competitive inhibition of signaling by single IFN molecules. The increased affinity of fusion molecules to tetrameric receptor complexes and lower sensitivity to inhibitory signals should make IFN fusion molecules more biologically potent than combinations of IFNs. It is also possible that IFN receptor subunits in tetrameric complexes have different affinities for common downstream signaling participants such as JAK kinases and STATs, and will compete for a limited amount of these signaling molecules. Thus, signaling in tetrameric IFN receptor complexes can be diverged toward signaling pathways specific to one type of IFN. In addition, IFN-lambda signaling appears to involve JAK2 tyrosine kinase during signaling events (Lee, et al. (2012) Int. J. Mol. Med. 30:945-952). Fusion molecules would therefore bring JAK2 kinase into the bound IFN receptor complex and may modulate type I IFN signaling. There are also reports that type III IFNs reduce the permeability of the blood-brain barrier, independent of STAT1 activation (Lazear, et al. (2015) Sci. Transl. Med. 7:284ra259). Thus, fusion IFN molecules are expected to involve pathways that are complimentary or additional to canonical JAK-STAT signaling. As mentioned above, in addition to canonical STAT1 and STAT2 Tyr phosphorylation, post-translational modifications of the ISGF3 transcription complex may also differ in response to IFN fusion molecules versus single IFNs, which may affect the kinetics, magnitude, and subset selectivity of ISG expression and subsequent biological activity.Dysregulated or over-exacerbated type I IFN activity is associated with autoimmune diseases such as lupus as well as chronic viral infections. In addition to the subset of antiviral ISGs induced by both type I and type III IFNs, type I IFNs, but not type III IFNs, also induce a group of ISGs that encode proinflammatory mediators (Galani, et al. (2017) Immunity 46(5):875-890.e6). As mentioned above, subtle changes in the post-translational modification of ISGF3 in response to IFN fusion molecules can abolish or reduce the expression of this group of proinflammatory ISGs in response to fusion molecules. Moreover, type III IFNs were reported to stimulate the development and proliferation of immunosuppressive T regulatory cells through their action on dendritic cells (Mennechet, et al. (2006) Blood 107(11):4417-23). Dendritic cells respond to both types of IFNs, with type I IFN exerting immunostimulatory activity on these cells. Therefore, IFN fusion molecules are expected to alter the responsiveness of dendritic cells to IFNs, leading to a more balanced and better-tuned activation of the cells, thus avoiding their excessive activation by type I IFNs.

[0065] As indicated, the present invention encompasses fusion molecules comprised of a type I IFN protein or portion thereof and a type III IFN protein or portion thereof. The fusion molecule can have either type I or type III at the N-terminus (i.e., type I-type III fusion or type III-type I fusion). Non-limiting exemplary fusion molecules are depicted in Figures 12-14 and 21-22. The fusion molecule may or may not include a signal peptide at its N-terminus. As known in the art, a "signal peptide" is a peptide that is usually present at the N-terminus of newly synthesized secretory or membrane proteins that directs the protein across or into the cell membrane (plasma membrane in prokaryotes and endoplasmic reticulum membrane in eukaryotes). It is then usually removed. When a signal peptide is included, it can originate from either type I or type III IFN protein or can be obtained from any known protein known to be secreted. Non-limiting exemplary signal peptide sequences are listed in Table 1. In some embodiments, the fusion molecule of the invention has an N-terminal signal peptide. In certain embodiments, the signal peptide is set forth in SEQ ID NO:1. [Table 1-1] [Table 1-2]

[0066] Type I IFN proteins for use in the fusion molecules of the present invention include, but are not limited to, IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-ε (epsilon), and IFN-ω (omega) or portions thereof. The genetics and structures of these proteins are well known in the art and are described, for example, in Samarajiwa, et al. (2006) The Interferons: Characterization and Application, Wiley-VCH, pages 3-34. In some embodiments, the type I IFN is a mammalian type I IFN. In some embodiments, the type I IFN is a human type I IFN. In certain embodiments, the type I IFN is an IFN-α protein (e.g., IFN-α2) or an IFN-β protein (e.g., IFN-β1) or portions thereof. IFN-β protein is produced in large amounts by fibroblasts and is known to exert antiviral activity. Two types of IFN-β have been described, IFN-β1 (IFNB1) and IFN-β3 (IFNB3). IFN-α proteins are produced by white blood cells.These are mainly involved in the innate immune response to viral infections and are classified into 13 subtypes: IFNA1 (GENBANK accession number BAM72353), IFNA2 (GENBANK accession number NP_000596), IFNA4 (GENBANK accession number NP_066546), IFNA5 (GENBANK accession number NP_002160), IFNA6 (GENBANK accession number NP_066282), IFNA7 (GENBANK accession number NP_066401), and IFNA Human type I IFN proteins include IFN-ω (GENBANK Accession No. NP_002168), IFN-κ (GENBANK Accession No. NP_064509), IFN-ε (GENBANK Accession No. NP_795372). Genes encoding type I IFN proteins were found together in a cluster on chromosome 9. Specific examples of commercially available IFN products include IFN-γ1b (ACTIMMUNE®), IFN-β1a (AVONEX®, and REBIF®), IFN-β1b (BETASERON®), IFN alfacon-1 (INFERGEN®), IFN-α2 (INTRON A®), IFN-α2a (ROFERON-A®), pegylated interferon alpha-2a (PEGASYS®), and pegylated interferon alpha-2b (PEG-INTRON®), each of which find use in the present invention. Non-limiting exemplary mature type I IFN proteins are listed in Table 2. [Table 2]

[0067] Type III IFN proteins are also known in the art and described in US 7,820,793, which is incorporated herein by reference in its entirety. Type III IFNs include IFN-lambda1, IFN-lambda2, and IFN-lambda3 (also called IL29, IL28A, and IL28B, respectively), and portions thereof. These IFNs signal through a receptor complex composed of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). In one embodiment, the type III IFN is a mammalian type III IFN. In another embodiment, the type III IFN is a human type III IFN. In some embodiments, the type III IFN is IFN-lambda1 (GENBANK Accession Nos. NP_742152 and 3OG6_A), IFN-lambda2 (GENBANK Accession Nos. NP_742150 and AAN86126), or IFN-lambda3 (GENBANK Accession Nos. NP_742151 and AAN86127). Exemplary mature IFN-lambda proteins are listed in Table 3. In certain embodiments, the type III IFN is IFN-lambda1. Some of the type I IFN proteins, such as IFN-β, IFN-ε, IFN-κ, and type III IFN proteins, such as IFN-lambda1, IFN-lambda-2, and IFN-lambda-3, have unpaired Cys residues that can be substituted to improve folding and purification of the fusion molecule. Variants of type I and type III IFNs (which have lower affinity for their corresponding receptors) can also be used for the generation of fusion IFN proteins (which reduce their signaling capabilities through their individual heterodimeric IFN-receptor complexes but preserve their synergistic activity through the tetrameric bound IFN-receptor complex). Non-limiting exemplary type III IFN sequences are presented in Table 3. [Table 3-1] [Table 3-2]

[0068] While the type I IFN and type III IFN proteins, or portions thereof, may be directly linked to each other, in some embodiments, the type I IFN and type III IFN proteins, or portions thereof, are linked to each other by a linker. In this respect, the fusion molecule of the invention may have the structure type I IFN-linker-type III IFN or type III IFN-linker-type I IFN (N→C-terminal orientation). The linker for use in the fusion molecule of the invention is preferably flexible and has a length ranging from 5 to 50 amino acids, or more preferably from 10 to 30 amino acids. In some embodiments, the linker element is a glycine / serine linker, i.e. a peptide linker substantially composed of the amino acids glycine and serine. The amino acids threonine or alanine may also be used in the linker. It will be clear to the skilled person that in the case where the IFN at the N-terminus of the fusion molecule is already terminated, for example with a Gly, such Gly may form the first Gly of the linker in the linker sequence. Likewise, in the case where the C-terminus of IFN starts with, for example, Pro, such a Pro residue may form the final Pro of the linker in the linker sequence. Non-limiting examples of individual linker sequences are listed in Table 4. In a specific embodiment, the linker of the fusion molecule of the present invention is represented by SEQ ID NO: 36. [Table 4]

[0069] While it has been demonstrated that fusion molecules of type I and type III interferons are useful for exerting antitumor activity and activating antiviral responses, it is contemplated that type I and / or type III interferons may also be fused with type II interferons to modulate immune and inflammatory responses and suppress or treat fungal and bacterial infections. Type II interferons, also known as IFN-γ, are antiparallel homodimers that bind to the IFN-γ receptor (IFNGR) complex. IFN-γ has several antiviral and antitumor effects and enhances the effects of type I and type III IFNs. IFN-γ recruits white blood cells to the site of infection, resulting in increased inflammation. It also stimulates macrophages to kill engulfed bacteria. IFN-γ also regulates Th2 responses. In one embodiment, the type II IFN is a mammalian type II IFN. In another embodiment, the type II IFN is a human type II IFN. Human type II IFN is known in the art under GENBANK accession number NP_000610 and is described as follows: It has a mature amino acid sequence of DPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ (SEQ ID NO:49).

[0070] Like type I and type III IFN, IFN-γ has a signal peptide, the sequence of which is: MKYTSYILAFQLCIVLGSLGCYCQ (SEQ ID NO: 50). Fusion molecules encompassing type II IFN can have the structure (N→C terminal orientation) of type II IFN-linker-type I IFN; type I IFN-linker-type II IFN; type II IFN-linker-type III IFN; type III IFN-linker-type II IFN-linker-type III IFN; type II IFN-linker-type I IFN-linker-type III IFN; type III IFN-linker-type II IFN-linker-type I IFN; or type I IFN-linker-type III IFN-linker-type II IFN.

[0071] The fusion molecules of the present invention can be produced by conventional recombinant expression techniques using known expression systems, including, but not limited to, E. coli, yeast, baculovirus, insect, plant, or mammalian protein expression systems. The fusion molecules can be recovered and purified from the recombinant cell culture in any effective manner, such as ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. See, for example, Lin, et al. (1986) Meth. Enzymol. 119: 183-192. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Additionally, methods that may be used for the production and isolation of the fusion molecules of the present invention are disclosed in US 6,433,145.

[0072] In addition, the polypeptides of the present invention can be chemically synthesized using any available technique (see, e.g., Creighton (1983) Proteins: Structures and Molecular Principles, WH Freeman & Co., NY; Hunkapiller, et al. (1984) Nature 310:105-111). For example, fusion molecules or fragments of fusion molecules can be synthesized using a peptide synthesizer.

[0073] The present invention also encompasses fusion molecules that are co- or post-translationally modified, by way of example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc. Any of the myriad chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin, etc.

[0074] Additional post-translational modifications encompassed by the present invention include, by way of example only, N-linked or O-linked carbohydrate chains, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. Fusion molecules may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic, or affinity label, to allow for detection and isolation of the protein.

[0075] Also provided by the present invention are chemically modified derivatives of the fusion molecules of the present invention, which may provide additional advantages such as increased solubility, stability, and circulation time of the polypeptide, or reduced immunogenicity (see US 4,179,337). Chemical moieties for derivatization may be selected from water-soluble polymers, such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The polypeptides may be modified at random positions within the molecule or at predetermined positions within the molecule and may include one, two, three, or more attached chemical moieties.

[0076] The polymers may be of any molecular weight, branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term "about" indicates that in preparations of polyethylene glycol, some molecules are heavier and some are lighter than the stated molecular weight). Other sizes may be used depending on the desired therapeutic profile (e.g., the desired duration of sustained release, the effect, if any, on biological activity, ease of processing, the degree or lack of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or analogs). For example, polyethylene glycol may be about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500 , 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0077] As mentioned above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in US 5,643,575; Morpurgo, et al. (1996) Appl. Biochem. Biotechnol. 56:59-72; Vorobjev, et al. (1999) Nucleosides Nucleotides 18:2745-2750; and Caliceti, et al. (1999) Bioconjug. Chem. 10:638-646.

[0078] The polyethylene glycol molecule (or other chemical moiety) should be attached to the fusion molecule after considering the effect on the functional or antigenic domain of the protein. There are many attachment methods available to those skilled in the art, see for example EP 0 401 384, which teaches the coupling of PEG to G-CSF, and Malik, et al. (1992) Exp. Hematol. 20:1028-1035, which describes the PEGylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently attached through amino acid residues via reactive groups such as free amino or carboxyl groups. Reactive groups are groups to which activated polyethylene glycol molecules may be attached. Amino acid residues with free amino groups may include lysine residues and N-terminal amino acid residues; those with free carboxyl groups may include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Sulfhydryl groups may also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or at a lysine group.

[0079] As suggested above, polyethylene glycol may be attached to a protein via linkage to any of a number of amino acid residues. For example, polyethylene glycol may be linked to a protein via a covalent bond to a lysine, histidine, aspartic acid, glutamic acid, or cysteine ​​residue. One or more reaction chemistries may be employed to attach polyethylene glycol to individual amino acid residues of a protein (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) or to more than one type of amino acid residue of a protein (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof).

[0080] On the other hand, proteins chemically modified at the N-terminus may also be specifically desired. Using polyethylene glycol as an illustration, a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), ratios of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of PEGylation reaction to be performed, and the method of obtaining the N-terminally PEGylated selected protein may be selected. The method of obtaining the N-terminally PEGylated preparation (i.e., separating this moiety from other mono-PEGylated moieties, if necessary) may be by purification of the N-terminally PEGylated material from a population of PEGylated protein molecules. Selected proteins chemically modified with N-terminal modifications may be accomplished by reductive alkylation, taking advantage of the differential reactivity (lysine vs. N-terminus) of the various types of primary amino groups available for derivatization of a particular protein. Under appropriate reaction conditions, substantially selective derivatization at the N-terminus of a protein with a carbonyl group-containing polymer is achieved.

[0081] As shown above, the pegylation of the fusion molecule of the present invention can be accomplished by several means. For example, polyethylene glycol can be attached to protein either directly or via an intermediate linker. Linkerless systems for attaching polyethylene glycol to protein are described in Delgado et al. (1992) Crit. Rev. Thera. Drug Carrier Sys. 9:249-304; Francis, et al. (1998) Intern. J. Hematol. 68:1-18; US 4,002,531; US ​​5,349,052; WO 95 / 06058; and WO 98 / 32466.

[0082] The number of polyethylene glycol moieties attached to the fusion molecules of the invention (i.e., the degree of substitution) can also vary. For example, a pegylated protein of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of internal substitution can range from 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20, etc. polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are described, for example, in Delgado, et al. (1992) Crit. Rev. Thera. Drug Carrier Sys. 9:249-304.

[0083] As shown, the fusion molecules of the present invention can be used for the treatment of various cancers, viral diseases, and other indications. In a non-limiting embodiment, the fusion molecules are used in indications where IFN-α or IFN-λ are used, such as, but not limited to, fungal infections, bacterial infections, autoimmune conditions, and inflammation. Consequently, the present invention also provides a method for preventing or treating a disease or condition by administering an effective amount of a type I and type III IFN fusion molecule to a subject in need of treatment. In a particular embodiment, the disease or condition is a disease or condition that responds to IFN-α or IFN-λ.

[0084] "Responding" as used herein is meant to encompass any cellular response or any response by a subject to administration of an interferon, said interferon being indicative of an interferon that is useful in preventing, ameliorating, reducing, or eliminating one or more symptoms or symptoms associated with a disease.

[0085] For purposes of the present invention, a "subject" is intended to include mammals, such as humans, non-human primates (e.g., baboons, orangutans, monkeys), mice, pigs, cows, goats, cats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; or non-mammals, including, by way of example, non-mammalian vertebrates such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates.

[0086] According to the method of the present invention, an "effective amount" refers to a dosage or amount of a fusion molecule or a pharmaceutical composition comprising the fusion molecule sufficient to produce a desired result. The desired result may include an objective or subjective improvement in the subject receiving the dosage or amount. In particular, an effective amount is an amount that prevents, ameliorates, reduces, or eliminates one or more signs or symptoms associated with a disease or illness. Treatment may include the treatment of an existing condition or the prophylaxis of an anticipated future infection, including, but not limited to, common recurrent infections such as influenza, and situations requiring emergency prophylaxis, such as a biological weapon attack.

[0087] In some non-limiting embodiments, the method of the present invention is useful for treating viral infections, such as, but not limited to, chronic hepatitis C infection, chronic hepatitis B infection, and AIDS; cancers, such as, but not limited to, hairy cell leukemia, malignant melanoma, hepatocellular carcinoma, follicular lymphoma, AIDS-related Kaposi's sarcoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, basal cell carcinoma, multiple myeloma, carcinoid tumor, bladder cancer, cutaneous T-cell lymphoma, and renal cell carcinoma; autoimmune diseases, such as, but not limited to, Crohn's disease, multiple sclerosis, and condyloma acuminata; inflammation; bacterial infections, and fungal infections.In certain non-limiting embodiments, the fusion molecules and methods of the present invention are useful for treating viral infections or cancers.In another non-limiting embodiment, the fusion molecules and methods of the present invention are useful for targeting and suppressing infection in two or more cell types in a subject.

[0088] Any effective amount of the fusion molecule of the invention may be administered to a subject in need thereof, for example, a subject having a disease or disorder, or a subject at risk of developing a disease or disorder. As a general proposition, the total pharmacologic effective amount administered parenterally per dose will be in the range of about 1 μg / kg / day to 10 mg / kg / day of patient weight, but as noted above, this will be subject to therapeutic discretion. More preferably, the dose is at least 0.01 mg / kg / day, and most preferably, for humans, between about 0.01 and 1 mg / kg / day. When administered continuously, the composition is typically administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either by 1 to 4 injections per day or by continuous subcutaneous infusion, for example, using a minipump. An intravenous bag solution may also be used. The length of treatment needed to observe changes, and the interval following treatment for a response to occur, can vary depending on the desired effect.

[0089] For therapeutic purposes, the fusion molecules of the present invention are preferably provided as pharmaceutical compositions containing the fusion molecules mixed with a pharmaceutical acceptable carrier. The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject, including animals or humans. A pharmaceutical composition generally comprises an effective amount of an active agent and a carrier, including, for example, a pharmaceutical acceptable carrier, such as a non-toxic solid, semi-solid, or liquid filler, diluent, any type of encapsulating material, or formulation auxiliary.

[0090] Pharmaceutical compositions containing the fusion molecules of the invention may be administered by any effective route, for example, orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops, or transdermal patch), buccally, or as an oral or nasal spray.

[0091] The term "parenteral" as used herein refers to any effective parenteral mode of administration, including modes of administration such as intravenous, intramuscular, intraperitoneal, substernal, subcutaneous, and intraarticular injection and infusion.

[0092] The composition may also be suitably administered by sustained release systems.Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules.Sustained release matrices include polylactides (US 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, et al. (1983) Biopolymers 22:547-556), poly(2-hydroxyethyl methacrylate) (Langer, et al. (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech. 12:98-105), ethylene vinyl acetate or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

[0093] In preferred embodiments, the compositions of the present invention are formulated in a biodegradable polymer drug delivery system, for example as described in US 4,938,763; US 5,278,201; US ​​5,278,202; US 5,324,519; US 5,340,849; US 5,487,897; WO01 / 35929; WO00 / 24374; and WO00 / 06117. In certain preferred embodiments, the compositions of the present invention are formulated using the ATRIGEL Biodegradable System from Atrix Laboratories, Inc. (Fort Collins, CO).

[0094] Examples of biodegradable polymers that can be used in the formulation of the compositions of the present invention include, but are not limited to, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials.Preferred polymers are those that have lower crystallinity and are more hydrophobic.These polymers and copolymers are more soluble in biocompatible solvents than highly crystalline polymers such as polyglycolides and chitin, which also have a high degree of hydrogen bonding. Preferred materials with the desired solubility parameters are polylactide, polycaprolactone, and their copolymers with glycolide, which have more amorphous regions that enhance solubility. In certain preferred embodiments, a biodegradable polymer that may be used in formulating the composition is poly(lactide-co-glycolide).

[0095] Polymer properties such as molecular weight, hydrophobicity, and lactide / glycolide ratio were They may be modified to obtain a desired drug release profile (see, e.g., Ravivarapu, et al. (2000) J. Pharmaceut. Sci. 89:732-741).

[0096] It is also preferred that the solvent for the biodegradable polymer is non-toxic, water-miscible, and otherwise biocompatible. Examples of such solvents include, but are not limited to, C2-C6 alkanols such as N-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, glycerol propylene glycol, butanol, C1-C15 alcohols, diols, triols, and tetraols; C3-C15 alkyl ketones such as acetone, diethyl ketone, and methyl ethyl ketone; C3-C15 esters such as methyl acetate, ethyl acetate, and ethyl lactate; alkyl ketones such as methyl ethyl ketone, C1-C15 amides such as dimethylformamide, dimethylacetamide, and caprolactam. ; C3-C20 ethers such as tetrahydrofuran or solketal; Tween, triacetin, propylene carbonate, decyl methyl sulfoxide, dimethyl sulfoxide, oleic acid, 1-dodecylazacycloheptan-2-one, other preferred solvents are benzyl alcohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate, glycerin, glycofurol, isopropyl myristate, isopropyl palmitate, oleic acid, polyethylene glycol, propylene carbonate, and triethyl citrate. The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin, and propylene carbonate due to their solvating ability and compatibility.

[0097] In addition, the compositions and formulations containing biodegradable polymers of the present invention may also include release-rate modification agents and / or pore formers. Examples of release-rate modification agents include, but are not limited to, fatty acids, triglycerides, other similar hydrophobic compounds, organic solvents, plasticizing compounds, and hydrophilic compounds. Suitable release rate modification agents include, for example, esters of mono-, di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate, dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate, glycerol triacetate, di(n-butyl) sebacate, and the like; polyhydroxy alcohols, such as propylene glycol, polyethylene glycol, glycerin, sorbitol, and the like; fatty acids; triesters of glycerol, such as triglycerides, epoxidized soybean oil, and other epoxidized vegetable oils; sterols, such as cholesterol; alcohols, such as C6-C12 alkanols, 2-ethoxyethanol, and the like. The release rate modification agents may be used alone or in combination with other such agents. Suitable combinations of release rate modifiers include, but are not limited to, glycerin / propylene glycol, sorbitol / glycerin, ethylene oxide / propylene oxide, butylene glycol / adipic acid, etc. Preferred release rate modifiers include, but are not limited to, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, and hexanediol. Suitable pore formers that may be used in the polymer composition include, but are not limited to, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, polymers such as hydroxypropylcellulose, carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone. Solid crystals that will provide a constant pore size, such as salts or sugars, are preferred.

[0098] The sustained release composition also includes the polypeptide encapsulated in liposome. The liposome containing the polypeptide of the present invention is prepared by the method known in the art (DE 3,218,121; Epstein, et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; JP 83-118008; US 4,485,045; US 4,544,545; and EP 102,324). Typically, the liposomes are small (about 200-800 angstroms), unilamellar, with lipid content greater than about 30 mol. percent cholesterol, the selected percentage being adjusted for effective polypeptide therapeutic.

[0099] The fusion molecules of the invention may be administered in combination with other known antiviral, immunomodulatory, and antiproliferative therapies, such as IL-2, KDI, ribavirin, and temozolomide.

[0100] The present invention also provides a pharmaceutical pack or kit including one or more containers filled with one or more of the components of the pharmaceutical composition of the present invention. A notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products may be associated with such container(s), which notice reflects approval by said agency of manufacture, use, or sale for human administration. In addition, the fusion molecules of the present invention may be employed in conjunction with other therapeutic compounds.

[0101] The following non-limiting examples further illustrate the invention. example

[0102] Example 1: Several IFN fusion molecules were generated and tested in various assays. These molecules include: i) hIFN-α-hIFN-λ1 (Figure 12; SEQ ID NO: 51); ii) hIFN-α-hIFN-λ3 (Figure 13; SEQ ID NO: 52); iii) hIFN-β-hIFN-λ3 (Figure 14; SEQ ID NO: 53); iv) mIFN-α-mIFN-λ2 (Figure 21; SEQ ID NO: 54); and v) mIFN-β-mIFN-λ2 (Figure 22; SEQ ID NO: 55). Mammalian expression plasmids encoding these fusion molecules were generated by using standard PCR and DNA cloning techniques. Some of the constructs, specifically the fusion mIFN-β-mIFN-λ2, as well as the constructs encoding single mIFN-β and mIFN-λ2, were generated without or with a 6xHis tag for protein detection and purification. Expression plasmids were transiently transfected into HEK293 cells and their expression levels were assessed either by immunoblotting with His antibodies (Figure 23) and / or by biological assays (Figures 16, 24 and 25).

[0103] Example 2: IFN fusion molecules were tested for their ability to induce IFN-specific biological activities, including i) upregulation of MHC class I antigen expression (Figure 25); ii) induction of antiviral defense (Figure 24); and STAT1 activation (Figure 16). Human IFN fusion molecules were tested against human retinal pigment epithelial cells or CHO reporter cell lines expressing human type I or type II chimeric IFN receptor complexes (Figure 15). These two separate reporter cell lines respond exclusively and specifically to either human type I IFN or human type III IFN, whereas the parental CHO cells do not respond to either type of human IFN. These cell lines were therefore used to demonstrate that IFN fusion molecules retain both type I and type III IFN biological activities (Figure 16). In these experiments, reporter cells were treated with recombinant human IFN-α or human IFN-λ1, or with conditioned medium from HEK293 cells transiently transfected with a plasmid expressing a human IFN-α-IFN-λ1 fusion molecule; with conditioned medium from HEK293 cells transfected with an empty plasmid (mock transfected), which served as a negative control. Reporter cells were treated with IFN for 15 min, harvested and lysed; and STAT1 activation was assessed by electrophoretic mobility shift assay (EMSA). In this assay, STAT1 dimers, which only form upon STAT1 Tyr701 phosphorylation, bind to a specific radiolabeled DNA probe. The presence of a DNA:protein complex is indicative of STAT1 phosphorylation and activation. STAT1 activation can also be detected by immunoblotting using a specific Tyr701 STAT1 antibody. For this assay, reporter or unmodified human or mouse cell lines or primary cells are treated with IFN samples for 15 min, lysed, and immunoblotted. Because mouse type III IFN can signal through the human type III IFN receptor, CHO reporter cells were used to demonstrate induction of type III IFN signaling by mouse IFN fusion molecules.Mouse fibroblasts, which respond only to type I IFN, were used to demonstrate induction of type I IFN signaling by mouse IFN fusion molecules. Human retinal pigment epithelial ARPE-19 cells, which respond to human type I and type III IFN, and mouse lung epithelial MLE cells and mouse intestinal epithelial cells (mIEC), which respond to mouse type I and type III IFN, were also used in these assays. All IFN fusion molecules were tested for their ability to stimulate STAT1 activation on appropriate cell lines in either EMSA and / or immunoblotting, and shown to retain both type I and type III IFN activity. The fusion IFN molecules will be purified to homogeneity, protein concentrations will be determined, and their potency in the STAT1 activation assay will be compared to that of single recombinant IFN molecules.

[0104] Example 3: For antiviral assays, either ARPE-19 cells or mIECs were used. The species appropriate cells were pretreated for 24 with serial (1:3) dilutions of HEK293 conditioned medium containing IFN fusion molecules or each IFN alone or recombinant IFN. The cells were then challenged with vesicular stomatitis virus (VSV) and cell viability was measured by crystal violet staining (Figure 24). Upon purification of IFN fusion molecules, additional antiviral assays will be performed to compare the antiviral potency of IFN fusion molecules with that of single recombinant IFN molecules.

[0105] Example 4: IFN fusion molecules were also tested for their ability to upregulate cell surface MHC class I antigen expression. For these assays, appropriate cells were treated with recombinant IFN or with HEK293 conditioned medium containing IFN fusion molecules or each IFN alone. After 72 hours, cells were harvested and the level of MHC class I antigen expression was determined by flow cytometry. IFN-untreated cells or cells treated with conditioned medium from mock-transfected HEK293 cells were used as controls. Upon purification of IFN fusion molecules, additional assays will be performed to compare the potency of IFN fusion molecules with that of single recombinant IFN molecules for upregulating MHC class I antigen expression.

[0106] Example 5: For animal tumor growth experiments, mouse breast cancer E0771 cells were generated that constitutively express either single mIFN-β or mIFN-λ2, or the fusion mIFN-β-mIFN-λ2 molecule. The cells were stably transfected with the corresponding expression vectors, and G418-resistant cell populations expressing comparable levels of the IFN molecules were selected. No changes were observed in the in vitro growth kinetics of the modified cells. Next, 10 μg / ml of IFN-β was implanted into the center of the right #4 inguinal mammary fat pad of 8-week-old syngeneic wild-type C57BL / 6 (E0771) female nulliparous mice. 5 Parental or modified E0771 breast cancer cells (resuspended in 50% Matrigel) were injected into mice. To compare the efficacy of IFN combinations to that of IFN fusion molecules, a cohort of mice was injected with a 50:50 mixture of tumor cells expressing either mIFN-β or mIFN-λ2. Primary tumor volumes were assessed and recorded every other day. On day 29 (FIG. 20A) or day 26 (FIGS. 20B and 20C), animals were sacrificed for final tumor evaluation and histological and immunological analysis.

[0107] Example 6: Additional studies will be performed in another breast tumor mouse model (i.e., 4T1 breast cancer, which forms breast tumors when implanted in the mammary fat pad of BALB / c mice, a mouse strain syngeneic to the 4T1 cancer cells), and in a mouse model of melanoma in which B16 melanoma cells are implanted SQ into C57BL / 6 syngeneic mice. The experiments will follow the same protocol described above as for the E0771 breast cancer model. Cancer cells will be engineered to constitutively express single mIFN-β or mIFN-λ2, or a fusion mIFN-β-mIFN-λ2 molecule. Eight-week-old syngeneic mice will be implanted with parental and modified tumors, and primary tumor volumes will be assessed and recorded every other day. To compare the efficacy of IFN combinations against the efficacy of IFN fusion molecules, a group of mice will be implanted with a 50:50 mixture of tumor cells expressing either mIFN-β or mIFN-λ2. Primary tumors will grow to 1 cm in either group of mice. 3When volume was reached, all animal cohorts will be sacrificed for final tumor evaluation and histological and immunological analysis. Lung metastases, if any, will also be quantified. Primary tumors, as well as lung, bone, brain, and other major organs, will be weighed, half snap-froze, and the other half fixed for further biochemical and histological analysis to study proliferation (Ki67), apoptosis (Tunnel), microvessel (CD31), and PASR staining. Primary tumors will also be assessed by a combination of Nanostring, FACS, and IHC-based methods to explore the cellular frequency of PMN, DC, MPh, NK cells, and T cells in the tumor and at the tumor border. When primary tumors are thereby removed, parts of them will also be used to examine their borders by IF, and then the tumor and tumor-infiltrating cells will be isolated by enzymatic digestion to profile F4 / F80+MPh, GR1+neutrophils, CD11+DC, as well as T cells, myofibroblasts and endothelial cells (PECAM+cells). Leukocyte (DC, MPh, NK cells and T cells) infiltration and DC maturation status at the tumor site will be quantified by staining immune cells with specific markers such as CD86 (Alexa 350 labelled) for DC, F4 / F80 (Alexa 405 labelled) for MPh, and CD4+ (PE-Cy7 labelled) and CD8+ (Alexa 649 labelled) for T cells, followed by FACS analysis. Moreover, tumor-associated cytokines and chemokines will be quantified by RT-PCR, and protein expression measurements will be analysed by custom-made MSD-cytokine arrays or ELISA. Blood will be collected to test for various tumor-associated cytokines, chemokines, and adipokines associated with inflammatory responses, as well as stromal stimulation. If a subset of animals did not develop primary tumors, these tumor-free mice will be injected with the original tumor to test for the development of antitumor immunity in these mice.The occurrence of anti-tumor response will also be evaluated to test the tumoricidal activity of splenocytes in tumor-bearing mice. After tumor formation or the lack of it, a cohort of mice will be sacrificed to isolate splenocytes, which will be used to evaluate the activation and proliferation of tumor-induced CTLs by measuring IFN-γ production by splenocytes co-cultured with tumor cells in vitro. In addition, the tumor cell killing ability of splenocytes will be evaluated by carrying out a cytotoxicity assay. In said assay, splenocytes. 51 Co-culture with Cr-labeled tumor cells 51 Cr release will be measured. Unrelated tumor cells (B16 for E0771 and vice versa) will be used as controls.

[0108] Example 7: We intend to test recombinant IFN fusion molecules that are >95% pure and endotoxin-free as cancer therapeutics. We intend to utilize the mouse models of breast cancer and melanoma growth described above. Mice will be injected with parental cancer cells and tumors will grow to ~0.5cm 3 Tumor-bearing mice will be IV-injected with various doses of IFN fusion molecules, single IFN, or a combination of single IFN. The effect of IFN treatment on tumor progression and metastasis formation will be monitored as described above. Various histological and immunological assays described above will also be performed.

[0109] Example 8: The antiviral potential of the IFN fusion molecules will be tested using a mouse model of influenza A infection and compared to the efficacy of single IFNs or their 50:50 combination. As a prophylaxis, mice will be administered 1 LD 50Influenza A virus strains PR8, WSN, Udorn or other strains will be injected SQ or intranasally (IN) at various doses (0.1, 0.3, 1, 3, 10 μg / 1 adult-20 mg 8-week-old mouse; PBS will be used as a control mock treatment). Survival and weight loss will be monitored daily. In addition, in separate experiments, virus titers and lung histopathology will be determined 3, 6, and 9 days after infection. Morbidity will be assessed by examination of histopathology. IHC staining for viral antigens will determine if treatments have altered the pattern of virus dissemination. The optimal IFN treatment to enhance survival after infection will be determined. In this experiment, influenza A virus (1 LD 50 The effect of post-infection treatment with strains PR8, WSN, Udorn or other strains will be assessed using multiple dosing regimens. As described above, mice will be treated with IFN fusion molecules, single IFNs, or combinations thereof injected SQ or intranasally (IN) at various doses (0.1, 0.3, 1, 3, 10 μg / 1 adult-20 mg 8-week-old mouse; PBS will be used as a control mock treatment). Infected mice will be treated according to the following schedule: days 1, 3, 5; days 1-4; days 2, 4, 6; days 2-5. Mice will be analyzed as described above to determine antiviral protection as well as disease.

[0110] Example 9: Mx2-luciferase reporter mouse strains will be used to assess the in vivo induction of ISGs. Mx2-luciferase transgenic reporter mice have a luciferase reporter gene controlled by the IFN-induced Mx2 promoter. This system allows sensitive in vivo monitoring by the use of live imaging of the whole body by the use of a Caliper IVIS 200 live animal imaging system. Adult 8-week-old Mx2-luciferase reporter mice will be treated with IFN fusion molecules, single IFN or combinations of these injected SQ or IN at various doses (0.1, 0.3, 1, 3, 10 μg / adult-20 mg 8-week-old mouse; PBS will be used as control mock treatment). To determine the duration and intensity of IFN stimulation by live imaging, luciferase expression will be monitored 30 min after IFN treatment, every hour for 8 hours, and 16, 24, and 48 hours after IFN treatment.

[0111] Example 10: IHC of various tissues obtained from mice treated with IFN will be performed to measure the levels and localization of pSTAT1. STAT1 is a transcription factor that is specifically activated (Tyr phosphorylated, pSTAT1) by type I and type III IFN (Figure 1) and localizes to the cell nucleus after activation. For these experiments, wild-type mice will be treated with IFN fusion molecules, single IFN or combinations of these injected SQ or IN at various doses (0.1, 0.3, 1, 3, 10 μg / 1 adult-20 mg 8-week-old mice; PBS will be used as a control mock treatment). Cohorts of animals will be sacrificed 5 min, 10 min, 15 min, 30 min after IFN treatment, hourly for 8 hours, and 16, 24, and 48 hours after IFN treatment. Various tissues including lung, large and small intestine, kidney, spleen, and liver will be dissected, formalin fixed, and paraffin embedded. After antigen retrieval and blocking of endogenous peroxidase activity performed on deparaffinized 5 micron sections, sections will then be stained with pSTAT1 antibody (Tyr701). After immunostaining, tissue sections will be counterstained with Mayer's hematoxylin and Scott's bluing buffer. Nuclear localization of pSTAT1 will be examined in various tissues and cell types to assess the tissue distribution, cellular targets, intensity and duration of IFN signaling triggered in response to individual IFN treatment regimes using single IFNs, their combinations, or IFN fusion molecules.

Claims

1. A fusion molecule comprising a type I interferon protein or a fragment thereof and a type III interferon protein or a fragment thereof, the fusion molecule is capable of inducing enhanced signaling through a bound type I and type III interferon (IFN) receptor complex when all four receptor subunits that make up the type I and type III IFN receptor complex are present; The fusion molecule further comprises a linker between the type I interferon protein or fragment thereof and the type III interferon protein or fragment thereof, the type I interferon protein is a human type I interferon protein selected from interferon alpha or interferon beta; the type III interferon protein is a human type III interferon protein selected from interferon lambda 1, interferon lambda 2, or interferon lambda 3; The fusion molecule, wherein the type I interferon protein fragment and the type III interferon protein fragment are fragments shorter than the full-length interferon protein while maintaining functional activity relative to the full-length protein.

2. The fusion molecule of claim 1 , wherein the type I interferon protein or fragment thereof is interferon alpha or a fragment thereof.

3. The fusion molecule of claim 2 , wherein the interferon alpha or a fragment thereof is interferon alpha 2 or a fragment thereof.

4. The fusion molecule of claim 1 , wherein the type I interferon protein or fragment thereof is interferon beta or a fragment thereof.

5. The type III interferon protein or fragment thereof is interferon lambda 1 or a fragment thereof; or The type III interferon protein or fragment thereof is interferon lambda 2 or a fragment thereof; or The fusion molecule of claim 1 , wherein the type III interferon protein or a fragment thereof is interferon lambda 3 or a fragment thereof.

6. The fusion molecule of claim 1 , further comprising a signal peptide at its N-terminus.

7. comprises a mature fragment of a Type I or Type III interferon protein; or The fusion molecule of claim 1 , comprising the entire sequence encompassing the signal peptide of a type I or III interferon protein.

8. A pharmaceutical composition comprising the fusion molecule of any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

9. A fusion molecule according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in a method for treating a disease or condition responsive to interferon treatment, the method comprising administering an effective amount of the fusion molecule according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 to a subject in need of treatment, thereby treating the disease or condition in the subject.

10. A fusion molecule according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in a method for treating a disease or condition in a subject, the method comprising administering to the subject an effective amount of the fusion molecule according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8, thereby treating the disease or condition in the subject, wherein the disease or condition is a viral infection, a fungal infection, a bacterial infection, cancer, an inflammatory disease, or an autoimmune disease.

11. A fusion molecule according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in a method for inhibiting an infectious disease in a subject, the method comprising administering to a subject an effective amount of the fusion molecule according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8.

12. The fusion molecule or pharmaceutical composition for use according to claim 11, wherein the fusion molecule targets two or more cell types in a subject.

13. Two or more cell types are present in the lung or respiratory tract of the subject; or Two or more cell types are present in the intestine of the subject; or The fusion molecule or pharmaceutical composition for use according to claim 12, wherein the two or more cell types are present in multiple organs of a subject.

14. A fusion molecule according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in a method for suppressing or treating cancer in a subject, the method comprising administering to a subject an effective amount of the fusion molecule according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8.

15. A fusion molecule according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in a method for inducing transcriptional signaling of IFN-inducible genes through the IFN-αR2 chain in a subject suffering from an infection that degrades or downregulates the IFN-αR1 chain, the method comprising administering to the subject an effective amount of the fusion molecule according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8.

Images