Fusion polypeptide and method of use

Abstract

To provide a fusion polypeptide and a use method.SOLUTION: The invention discloses a fusion polypeptide including fragments derived from first and second isoforms of an interferon lambda family, a nucleic acid for encoding the fusion polypeptide, a vector and host cell including the nucleic acid, and methods for producing and using a composition in treatment of an interferon lambda related disease, disorder and state. In one embodiment, the invention provides the fusion polypeptide having a first fragment derived from a first interferon lambda isoform, and a second fragment derived from a second interferon lambda isoform, the first and second fragments are fused on a fusion portion for forming a proximate polypeptide, the fusion portion includes a sequence of almost 6 amino acid which is equal to a corresponding sequence in the first and second interferon lambda isoforms.SELECTED DRAWING: None

Description

Background Art

[0001] IL28B, together with IL28A and IL29, represents a subset (type III) of the interferon family. Their expression can be induced by viral infection in various human cell types (Nat Immunol. 2003; 4(1): 69-77, Nat Immunol. 2003; 4(1): 63-68), and they can subsequently bind to and signal through a heterodimeric receptor complex composed of IL28R and IL10R2. The repertoire of genes induced by IL28B, IL28A, and IL29 is essentially the same as that induced by interferon alpha. Among them are, in particular, OAS1 and MX1, which bind to and signal through a heterodimeric receptor complex composed of IFNAR1 and IFNAR2 (Gastroenterology. 2006; 131(6): 1887-1898). The antiviral activity induced by type III interferons, including IL28B, IL28A, and IL29, has been demonstrated against encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), influenza (Eur J Immunol. 2004;34(3):796 - 805, J Virol. 2006;80(9):4501 - 4509), hepatitis B virus (HBV), and hepatitis C virus (HCV) (J Virol. 2005;79(6):3851 - 3854). However, the magnitude of the antiviral response to type III interferons is often smaller than that to interferon alpha in many cell types. The significant difference between type III interferons and the interferon alpha system is the pattern of receptor distribution. The receptor for interferon alpha is ubiquitously expressed, while the IL28R component of the type III interferon receptor is present only in a limited subset of cells, including hepatocytes (Cytokine 2005, 31, 109 - 118). Functional IL28R is significantly absent in most hematopoietic cells (Hepatology. 2006;44(4):896 - 906). Preclinical toxicology studies have shown that pegylated IL29 peptide, unlike pegylated interferon alpha, does not induce inhibition of bone marrow stem cell colony formation or induce antiviral and antiproliferative activity in peripheral blood leukocytes (Ann NY Acad Sci. 2009;1182 :80 - 87).

[0002] Approximately 150 million people worldwide are chronically infected with hepatitis C virus (HCV), which is a major cause of cirrhosis, hepatocellular carcinoma, and liver transplantation (WHO. Hepatitis C fact sheet, No. 164). HCV is considered a curable disease in most patients. The current first-line treatment regimen consists of pegylated interferon alpha in combination with small molecule antiviral agents such as ribavirin (Health Technology Assessment 2004; 8: 39), telaprevir, and boceprevir (Ther Adv Gastroenterol 2012; 5(2): 139-151). Unfortunately, treatment with pegylated interferon alpha is not always well tolerated and results in poor patient compliance. The main toxicities associated with pegylated interferon alpha include influenza-like symptoms such as headache, fatigue, and asthenia; neuropsychiatric abnormalities such as depression, anxiety, and irritability; and more importantly, but not limited to, hematological disorders such as neutropenia and anemia. Approximately 350 million people worldwide are chronically infected with hepatitis B virus (HBV). Current treatments include pegylated interferon alpha and small molecule antiviral agents such as lamivudine, adefovir, tenofovir, telbivudine, and entecavir. Treatment of HBV with pegylated interferon alpha results in similar toxicities as in the case of HCV.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 1

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Non-Patent Document 3

Non-Patent Document 4

[0004] Therefore, there is a significant need for new antiviral drugs for hepatitis C and other diseases that are better tolerated and / or more effective than existing treatments. The present invention addresses this need and provides related advantages as well. The present invention encompasses modified human interleukin - 28B (IL28B) and human interleukin - 29 (IL29) fusion polypeptides. The present invention also provides methods for the production of the fusion polypeptides in prokaryotic systems such as E. coli. Further, the present invention discloses the pharmaceutical use of the fusion polypeptides in the treatment of viral infections (including but not limited to hepatitis C, hepatitis B, and influenza); autoimmune diseases (including but not limited to multiple sclerosis); and various types of cancer (including but not limited to hepatocellular carcinoma).

[0005] In one aspect, the present invention provides a fusion polypeptide comprising a first fragment derived from a first interferon lambda isoform and a second fragment derived from a second interferon lambda isoform, wherein the first and second fragments are fused together at a fusion site to form a proximity polypeptide, and the fusion site comprises at least about 6 amino acids of a sequence identical to the corresponding sequence in the first and second interferon lambda isoforms.

[0006] In some cases, the fusion polypeptide can retain the secondary structure of the first or second isoform. In some cases, the fusion polypeptide can lack any additional T-epitopes compared to the first or second isoform. In further cases, the fusion polypeptide can lack any additional B-epitopes compared to the first or second isoform.

[0007] In some examples, the first interferon lambda isoform can be the IL29 isoform. In some examples, the second interferon lambda isoform can be the IL28B isoform. In further examples, the first interferon lambda isoform can be the IL29 isoform and the second interferon lambda isoform can be the IL28B isoform.

[0008] In some examples, the fusion site can comprise at least about 8 amino acids of a sequence identical to the corresponding sequence in the first and second interferon lambda isoforms.

[0009] In another aspect, the present invention has the structure of Formula I: (S1)-(Helix A)-(S2)-(Helix C)-(S3)-(Helix D)-(S4)-(Helix E)-(S5)-(Helix F)-(S6) and has a. Helix D comprises an amino acid sequence that shows at least 90% homology to a fragment having residues of approximately V98 to Q112 of IL28B (SEQ ID NO: 2) or approximately V89 to Q103 of IL29 (SEQ ID NO: 1), b. Helix E comprises an amino acid sequence that shows at least 90% homology to a fragment having residues of approximately R130 to E145 of IL28B (SEQ ID NO: 2) or approximately R121 to E136 of IL29 (SEQ ID NO: 1), c. Each of S1, S2, S3, S4, S5 and S6 is independently a spacer sequence having between 1 and approximately 50 amino acid residues A fusion polypeptide, i. Helix A comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately P27 to L44 of IL28B (SEQ ID NO: 2), Helix C comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately R56 to A80 of IL29 (SEQ ID NO: 1), and Helix F comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately G139 to A161 of IL29 (SEQ ID NO: 1), or ii. Helix A comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately P20 to L37 of IL29 (SEQ ID NO: 1), Helix C comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately R63 to A87 of IL28B (SEQ ID NO: 2), and Helix F comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately G148 to A170 of IL28B (SEQ ID NO: 2), or iii. Helix A comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately P27 to L44 of IL28B (SEQ ID NO: 2), Helix C comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately R63 to A87 of IL28B (SEQ ID NO: 2), and Helix F comprises an amino acid sequence that shows at least 95% homology to a fragment having residues of approximately G139 to A161 of IL29 (SEQ ID NO: 1), or iv. The helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1), the helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1), and the helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G148 to A170 of IL28B (SEQ ID NO: 2), or v. The helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2), the helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1), and the helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G148 to A170 of IL28B (SEQ ID NO: 2), or vi. The helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1), the helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2), and the helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1), and provided is a fusion polypeptide characterized by this.

[0010] In some cases, the helix A may be identical to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2). In other cases, the helix A may be identical to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1).

[0011] In some cases, the helix C may be identical to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2). In other cases, the helix C may be identical to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1).

[0012] In some cases, helix F can be identical to a fragment having residues approximately G148 - A170 of IL28B (SEQ ID NO: 2). In other cases, helix F can be identical to a fragment having residues approximately G139 - A161 of IL29 (SEQ ID NO: 1).

[0013] In some cases, S2 can further include helix B.

[0014] In some cases, the fusion polypeptide can further include at least one modification to the amino acid residues corresponding to IL28B (SEQ ID NO: 2), and the at least one modification is selected from the group consisting of dV2, dP3, dV4, dA5, dR6, dL7, dR8, G9K, A10P, L11T, P12T, D13T, A14G, R15K, A20G, Q21R, Q31A, A32S, R35K, K37R, L45K, D48N, C49W, K50S, R52S, R54P, L55V, R58G, T59N, Q64L, T88A, dD90, dT91, D92P, G96E, R114Q, T127P, C168S, C175S, P3G, V4P, A5V, R6P, L7T, and R8S.

[0015] In some cases, the fusion polypeptide can also include at least one modification to the amino acid residues corresponding to IL29 (SEQ ID NO: 1), and the at least one modification is selected from the group consisting of R14Q, L57Q, A81T, 82aD, 82bT, G83D, E87G, Q105R, P118T, and D162E.

[0016] In some cases, the fusion polypeptide can include a fusion site that includes at least about 6 amino acids of a sequence that is identical to the corresponding sequence in IL28B (SEQ ID NO: 2) and IL29 (SEQ ID NO: 1). In further cases, the fusion site can include at least about 8 amino acids of a sequence that is identical to the corresponding sequence in IL28B (SEQ ID NO: 2) and IL29 (SEQ ID NO: 1). In some examples, the fusion site can include at least about 6 - 25 amino acids of a sequence that is identical to the corresponding sequences of at least two interferon lambda isoforms.

[0017] In some cases, the fusion polypeptide can retain the secondary structure of IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). In some cases, the fusion polypeptide can lack any additional T - epitopes compared to the case of IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). In some cases, the fusion polypeptide can lack any additional B - epitopes compared to the case of IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1).

[0018] In some cases, the fusion polypeptide can exhibit at least 90% sequence homology to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). In further cases, the fusion polypeptide can exhibit at least 95% sequence homology to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1).

[0019] In some cases, the N - terminus of the fusion polypeptide can be further modified by polyethylene glycol (PEG). In some examples, the polyethylene glycol can be monomethoxy PEG propionaldehyde. In some examples, the polyethylene glycol can have a molecular weight of about 12Kd - 40Kd. In further cases, the pegylated fusion polypeptide can exhibit an extended in vivo half - life compared to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1).

[0020] In some cases, the fusion polypeptide can exhibit enhanced chemical stability compared to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1).

[0021] In some examples, the fusion polypeptide can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0022] In yet another aspect, the present invention provides a host cell expressing the fusion polypeptide. In some cases, the host cell can be a prokaryotic cell. In some examples, the prokaryotic cell can be E. coli. In other cases, the host cell can be a eukaryotic cell.

[0023] In one aspect, the present invention provides a method for treating viral infection in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide. In some cases, the viral infection can be imparted by a virus selected from the group consisting of hepatitis B, hepatitis C, and influenza.

[0024] In another aspect, the present invention provides a method for treating inflammation in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide. In some cases, the inflammation can be multiple sclerosis.

[0025] In yet another aspect, the present invention provides a method for treating cancer in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide. In some cases, the cancer can be selected from colon cancer, melanoma, and hepatocellular carcinoma.

[0026] In one aspect, the present invention provides a pharmaceutical composition comprising at least one of the fusion polypeptides and a pharmaceutically acceptable excipient.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising at least one of the fusion polypeptides and a second therapeutic agent.

[0028] In yet another aspect, the present invention provides a vector comprising a polynucleotide encoding a fusion polypeptide.

[0029] In a further aspect, the present invention provides a method for producing a fusion polypeptide, the method comprising expressing a vector in a cell under conditions suitable for protein expression, thereby producing the fusion polypeptide. The present invention also provides, for example, the following items. (Item 1) A fusion polypeptide comprising a first fragment derived from a first interferon lambda isoform and a second fragment derived from a second interferon lambda isoform, wherein the first fragment and the second fragment are fused together at a fusion site to form a proximity polypeptide, and the fusion site comprises at least about 6 amino acids of a sequence identical to the corresponding sequence in the first interferon lambda isoform and the second interferon lambda isoform. (Item 2) The fusion polypeptide according to Item 1, which retains the secondary structure of the first isoform or the second isoform. (Item 3) The fusion polypeptide according to Item 1, which lacks any additional T-epitopes as compared to the case of the first isoform or the second isoform. (Item 4) The fusion polypeptide according to Item 1, which lacks any additional B-epitopes as compared to the case of the first isoform or the second isoform. (Item 5) The fusion polypeptide according to item 1, wherein the first interferon lambda isoform is an IL29 isoform. (Item 6) The fusion polypeptide according to item 1, wherein the second interferon lambda isoform is an IL28B isoform. (Item 7) The fusion polypeptide according to item 1, wherein the first interferon lambda isoform is an IL29 isoform and the second interferon lambda isoform is an IL28B isoform. (Item 8) The fusion polypeptide according to item 1, wherein the fusion site comprises at least about 8 amino acid sequences identical to the corresponding sequences in the first interferon lambda isoform and the second interferon lambda isoform. (Item 9) Formula I: (S1)-(Helix A)-(S2)-(Helix C)-(S3)-(Helix D)-(S4)-(Helix E)-(S5)-(Helix F)-(S6) having the structure of a. Helix D comprises an amino acid sequence showing at least 90% homology to a fragment having residues of about V98 - Q112 of IL28B (SEQ ID NO: 2) or about V89 - Q103 of IL29 (SEQ ID NO: 1), b. Helix E comprises an amino acid sequence showing at least 90% homology to a fragment having residues of about R130 - E145 of IL28B (SEQ ID NO: 2) or about R121 - E136 of IL29 (SEQ ID NO: 1), c. Each of S1, S2, S3, S4, S5 and S6 is independently a spacer sequence having 1 to about 50 amino acid residues A fusion polypeptide, i. Helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2), Helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1), Helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1), or ii. Helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1), Helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2), Helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G148 to A170 of IL28B (SEQ ID NO: 2), or iii. Helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2), Helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2), Helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1), or iv. Helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1), Helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1), Helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G148 to A170 of IL28B (SEQ ID NO: 2), or v. The helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2), the helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1), and the helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G148 to A170 of IL28B (SEQ ID NO: 2), or vi. The helix A comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1), the helix C comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2), and the helix F comprises an amino acid sequence showing at least 95% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1), characterized in that it is a fusion polypeptide. (Item 10) The fusion polypeptide according to item 9, wherein the helix A is identical to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2). (Item 11) The fusion polypeptide according to item 9, wherein the helix A is identical to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1). (Item 12) The fusion polypeptide according to item 9, wherein the helix C is identical to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2). (Item 13) The fusion polypeptide according to item 9, wherein the helix C is identical to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1). (Item 14) The fusion polypeptide according to item 9, wherein the helix F is identical to a fragment having residues from about G148 to A170 of IL28B (SEQ ID NO: 2). (Item 15) The fusion polypeptide according to item 9, wherein the helix F is identical to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1). (Item 16) The fusion polypeptide according to item 9, wherein S2 further comprises helix B. (Item 17) The fusion polypeptide according to item 1 or 9, further comprising at least one modification to the amino acid residue corresponding to IL28B (SEQ ID NO: 2), wherein the at least one modification is selected from the group consisting of dV2, dP3, dV4, dA5, dR6, dL7, dR8, G9K, A10P, L11T, P12T, D13T, A14G, R15K, A20G, Q21R, Q31A, A32S, R35K, K37R, L45K, D48N, C49W, K50S, R52S, R54P, L55V, R58G, T59N, Q64L, T88A, dD90, dT91, D92P, G96E, R114Q, T127P, C168S, C175S, P3G, V4P, A5V, R6P, L7T and R8S. (Item 18) The fusion polypeptide according to item 1 or 9, further comprising at least one modification to the amino acid residue corresponding to IL29 (SEQ ID NO: 1), wherein the at least one modification is selected from the group consisting of R14Q, L57Q, A81T, 82aD, 82bT, G83D, E87G, Q105R, P118T and D162E. (Item 19) The fusion polypeptide according to item 9, further comprising a fusion site comprising at least about 6 amino acids that are identical to the corresponding sequences in IL28B (SEQ ID NO: 2) and IL29 (SEQ ID NO: 1). (Item 20) The fusion polypeptide according to item 19, wherein the fusion site comprises at least about 8 amino acids that are identical to the corresponding sequences in IL28B (SEQ ID NO: 2) and IL29 (SEQ ID NO: 1). (Item 21) The fusion polypeptide according to item 1 or 9, wherein the fusion site comprises at least about 6 - 25 amino acids that are identical to the corresponding sequences of the at least two interferon lambda isoforms. (Item 22) The fusion polypeptide according to item 1 or 9, which retains the secondary structure of IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). (Item 23) The fusion polypeptide according to item 1 or 9, lacking any additional T-epitopes compared to the case of IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). (Item 24) The fusion polypeptide according to item 1 or 9, lacking any additional B-epitopes compared to the case of IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). (Item 25) The fusion polypeptide according to item 1 or 9, showing at least 90% sequence homology to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). (Item 26) The fusion polypeptide according to item 1 or 9, showing at least 95% sequence homology to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). (Item 27) The fusion polypeptide according to item 1 or 9, wherein the N-terminus of the fusion polypeptide is further modified by polyethylene glycol (PEG). (Item 28) The fusion polypeptide according to item 27, wherein the polyethylene glycol is monomethoxy PEG propionaldehyde. (Item 29) The fusion polypeptide according to item 27, wherein the polyethylene glycol has a molecular weight of about 12 Kd to 40 Kd. (Item 30) The fusion polypeptide according to item 27, wherein the pegylated fusion polypeptide shows an extended in vivo half-life compared to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). (Item 31) The fusion polypeptide according to item 1 or 9, showing enhanced chemical stability compared to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 1). (Item 32) The fusion polypeptide according to item 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. (Item 33) A host cell expressing the fusion polypeptide according to any one of items 1 to 32. (Item 34) The host cell according to item 33, which is a prokaryotic cell or a eukaryotic cell. (Item 35) A method for treating viral infection in a mammal in need of treatment for viral infection, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide according to any one of items 1 to 32. (Item 36) The method according to item 35, wherein the viral infection is caused by a virus selected from the group consisting of hepatitis B, hepatitis C, and influenza. (Item 37) A method for treating inflammation in a mammal in need of treatment for inflammation, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide according to any one of items 1 to 32. (Item 38) The method according to item 37, wherein the inflammation is multiple sclerosis. (Item 39) A method for treating cancer in a mammal in need of treatment for cancer, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide according to any one of items 1 to 32. (Item 40) The method according to item 39, wherein the cancer is selected from colon cancer, melanoma, and hepatocellular carcinoma. (Item 41) A pharmaceutical composition comprising at least one of the fusion polypeptides according to any one of items 1 to 32 and a pharmaceutically acceptable excipient. (Item 42) A pharmaceutical composition comprising at least one of the fusion polypeptides according to any one of items 1 to 32 and a second therapeutic agent. (Item 43) A vector comprising a polynucleotide encoding the fusion polypeptide according to any one of items 1 to 32. (Item 44) A method for producing the fusion polypeptide according to any one of items 1 to 32, comprising the step of expressing the vector according to item 43 in a cell under conditions suitable for protein expression, thereby producing the fusion polypeptide.

[0030] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which shows and describes merely illustrative embodiments of the present disclosure. As will be appreciated, the present disclosure is capable of other and different embodiments, and some of the details thereof are capable of modification in various obvious respects without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive. Incorporation by reference

[0031] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0032] The novel features of the invention are set forth in detail in the appended claims. A more complete understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that illustrates embodiments in which the principles of the invention are utilized and to the accompanying drawings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0050] Before describing the embodiments of the present invention, it should be understood that such embodiments are presented merely as examples, and various alternatives of the embodiments of the present invention described herein may be used in the practice of the present invention. Thus, those skilled in the art will be able to conceive of numerous variations, changes, and substitutions without departing from the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the practice or testing of the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Thus, those of ordinary skill in the art will envision numerous variations, changes, and substitutions without departing from the present invention.

[0052] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0053] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers modified by any other suitable means, such as by disulfide bond formation, glycosylation, lipid attachment, acetylation, phosphorylation, or conjugation to labeling components.

[0054] The term "amino acid" refers to any natural and / or non-natural or synthetic amino acid, including but not limited to both D or L optical isomers, as well as amino acid analogs and peptidomimetics. Amino acids are named using the standard one-letter or three-letter codes.

[0055] The term "natural L-amino acid" means the L-enantiomeric forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S) and threonine (T).

[0056] The term "non-natural origin", when applied to a sequence and used herein, means a polypeptide or polynucleotide sequence that does not have, is not complementary to, or does not have a high degree of homology with the corresponding wild-type or natural origin sequence present in a mammal. For example, a polypeptide or fragment of non-natural origin can share an amino acid sequence identity of 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or less or even lower when properly aligned and compared to a natural sequence.

[0057] The terms "hydrophilic" and "hydrophobic" refer to the degree of affinity a substance has for water. Hydrophilic substances have a strong affinity for water and tend to dissolve in, mix with, or be wetted by water, while hydrophobic substances are substantially lacking in affinity for water and tend to avoid and not absorb water, and tend not to dissolve in, mix with, or be wetted by water. Amino acids can be characterized based on their hydrophobicity. Several scales have been developed. An example is the scale developed by Hopp, TP et al., Proc Natl Acad Sci U S A (1981) 78:3824, and by Levitt, M et al., J Mol Biol (1976) 104:59. Examples of "hydrophilic amino acids" are arginine, lysine, threonine, alanine, asparagine and glutamine. Hydrophilic amino acids, aspartic acid, glutamic acid and serine and glycine are of particular interest. Examples of "hydrophobic amino acids" are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine and valine.

[0058] A "fragment", when applied to a protein, is a truncated form of a native biologically active protein that may or may not retain at least a portion of the therapeutic and / or biological activity. A "variant", when applied to a protein, is a protein having sequence homology to a native biologically active protein that retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein can share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared to a reference biologically active protein. As used herein, the term "biologically active protein portion" includes proteins that have been modified, either deliberately, such as by site-directed mutagenesis, synthesis of the coding gene, insertion, etc., or incidentally by mutation.

[0059] "Conjugated", "linked", "fused", and "fusion" are used interchangeably herein. These terms refer to connecting two or more chemical elements, sequences, or components together by any means, including chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are in proximity and in the reading phase or in-frame. "In-frame fusion" refers to connecting two or more open reading frames (ORFs) in a manner that maintains the correct reading frame of the original ORFs to form a continuous, longer ORF. Thus, the resulting "fusion polypeptide" is a single protein containing two or more fragments corresponding to polypeptides encoded by the original ORFs (segments that would not normally be connected in this way if they were native). A "fusion site" refers to the sequence where two or more fragments are connected together. In some cases, the fusion site can be a sequence that is identical in two or more fragments. For example, the fusion site can be a sequence of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids that is identical in the connected fragments. In specific examples, the fusion site can be a sequence of about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids that is identical in the connected fragments.

[0060] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide from the amino to the carboxyl terminus direction, where residues adjacent to each other in the sequence are proximal in the primary structure of the polypeptide. A "partial sequence" is a linear sequence of a part of a polypeptide that is known to include additional residues in one or both directions.

[0061] The terms "polynucleotide", "nucleic acid", "nucleotide" and "oligonucleotide" are used interchangeably. These refer to polymeric forms of nucleotides of any length, which are either deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, locus (loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can contain modified nucleotides such as methylated nucleotides and nucleotide analogs. When present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide constituents. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling constituent.

[0062] The terms "gene" and "gene fragment" are used interchangeably herein. These refer to a polynucleotide containing at least one open reading frame that can encode a specific protein after transcription and translation. A gene or gene fragment can be genomic or cDNA as long as the polynucleotide contains at least one open reading frame, which can cover the entire coding region or a segment thereof. A "fusion gene" is a gene composed of at least two heterologous polynucleotides linked together.

[0063] "Identity" or "identical" or "sequence identity" refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When determining sequence identity, similarity or homology between two different amino acid sequences using a program such as Emboss Needle or BestFit, the default settings can be used or an appropriate scoring matrix such as blosum45 or blosum80 can be selected to optimize the identity, similarity or homology score. Preferably, homologous polynucleotides are polynucleotides that hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, even more preferably 99% sequence identity compared to these sequences. When sequences of equal length are optimally aligned, homologous polypeptides preferably have at least 80% or at least 90% or at least 95% or at least 97% or at least 98% sequence identity or at least 99% sequence identity.

[0064] The terms "percent identity" and "% identity", when applied to polynucleotide sequences, refer to the percentage of residues that match between at least two polynucleotide sequences aligned using a standardization algorithm. Such algorithms can optimize the alignment between two sequences and thus insert gaps into the sequences being compared in a standardized and reproducible manner to achieve a more meaningful comparison of the two sequences. Percent identity can be measured over the length of the entire defined polynucleotide sequence or over a shorter length, for example, a fragment obtained from a larger defined polynucleotide sequence, such as a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues. Such lengths are merely exemplary, and it is understood that any fragment length supported by the sequences shown in this specification, tables, figures or sequence listings may be used to describe the lengths over which percent identity can be measured.

[0065] "Percent (%) sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in the query sequence that are identical to the amino acid residues of a second reference polypeptide sequence or a portion thereof, after aligning the sequences to achieve maximum percent sequence identity without considering any conservative substitutions as part of the sequence identity, and introducing gaps as necessary. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. Percent identity can be measured over the length of the defined polypeptide sequence as a whole, or over a shorter length, for example, a fragment obtained from a larger defined polypeptide sequence, such as a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are merely exemplary, and it is understood that lengths can be described that are used to measure percent identity using any fragment lengths supported by the sequences shown in this specification, tables, figures or sequence listings.

[0066] "Host cell" includes individual cells or cell cultures that can or have been recipients of the present vector. Host cells include the progeny of a single host cell. The progeny need not be identical to the original parent cell (either morphologically or in total DNA complement of the genome) due to natural, accidental or deliberate mutations. Host cells include cells transfected in vitro with the vectors of the invention. In some cases, the host cell is a prokaryotic cell. In some examples, the prokaryotic cell is E. coli. In other cases, the host cell is a eukaryotic cell.

[0067] A "vector" is preferably a nucleic acid molecule that replicates itself in a suitable host and transfers an inserted nucleic acid molecule into and / or among host cells. This term includes vectors that function primarily for the insertion of DNA or RNA into cells, vectors that function primarily for the replication of vectors that replicate DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Vectors that provide two or more of the above functions are also included. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide(s) when introduced into a suitable host cell. An "expression system" usually implies a suitable host cell composed of an expression vector that can function to produce the desired expression product.

[0068] The terms "effective amount" or "therapeutically effective amount" refer to an amount of a compound described herein that is sufficient to achieve the intended use, which includes, but is not limited to, the disease treatments defined below. The therapeutically effective amount can vary depending on the intended use (in vitro or in vivo), or the subject being treated and the disease state, e.g., the body weight and age of the subject, the severity of the disease state, the mode of administration, and others, which can be readily determined by one of ordinary skill in the art. This term also applies to the dosage that would induce a specific response, e.g., induction of a target gene and / or apoptosis, in target cells. The specific dosage will vary depending on the particular compound selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue into which it is administered, and the physical delivery system by which it is delivered.

[0069] As used herein, the terms "treatment", "treating", "alleviating", or "remitting" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic and / or prophylactic benefits. A therapeutic benefit means eradication or remission of the underlying disorder being treated. Also, a therapeutic benefit is achieved by eradication or remission of one or more of the physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder. For prophylactic benefits, the composition can be administered to a subject at risk of developing a particular disease or to a subject reporting one or more of the physiological symptoms of the disease, even if the disease has not yet been diagnosed in the subject.

[0070] "Therapeutic effect", as the term is used herein, encompasses the above-described therapeutic and / or prophylactic benefits. A prophylactic effect includes delay or elimination of the appearance of a disease or condition, delay or elimination of the onset of symptoms of a disease or condition, slowing, halting, or reversal of the progression of a disease or condition, or any combination thereof.

[0071] The terms "co-administration", "administered in combination with", and their grammatical equivalents, as used herein, encompass the administration of two or more agents to an animal such that both agents and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0072] The term "pharmaceutically acceptable salt" refers to salts derived from various organic and inorganic counterions well-known in the art. By way of example only, when the molecule contains an acidic functionality, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and others; when the molecule contains a basic functionality, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate (methanesulfonate), ethanesulfonate, acetate, maleate, oxalate, phosphate, and others. In a compound having two or more basic moieties, two or more of the basic moieties can be converted to a salt form, such as, but not limited to, bis or tris salts. Alternatively, a compound having two or more basic moieties can form a salt with only one of the basic moieties.

[0073] The terms "antagonist" and "inhibitor" are used interchangeably and refer to a compound having the ability to inhibit the biological function of a target protein by inhibiting either the activity or the expression of the target protein. Thus, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein. Preferred antagonists herein are compounds that interact specifically (e.g., bind) with the target but also inhibit the biological activity of the target protein by interacting with other members of the signaling pathway of which the target protein is a member, and are specifically included within this definition. Preferred biological activities inhibited by the antagonist are related to tumor development, growth, or spread.

[0074] As used herein, the term "agonist" refers to a compound that has the ability to elicit or enhance the biological function of a target protein, either by inhibiting or enhancing the activity or expression of the target protein. Thus, the term "agonist" is defined in the context of the biological role of the target polypeptide. Preferred agonists herein are compounds that interact specifically (e.g., bind) with the target, but also include within this definition compounds that elicit or enhance the biological activity of the target polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member.

[0075] As used herein, "agent" or "biologically active agent" refers to a biological, pharmaceutical or chemical compound or other moiety. Non-limiting examples include simple or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, vitamin derivatives, carbohydrates, toxins or chemotherapeutic compounds. A variety of compounds can be synthesized, for example, small molecules and oligomers (e.g., oligopeptides and oligonucleotides), as well as synthetic organic compounds based on a variety of core structures. In addition, various natural sources such as plant or animal extracts etc. can provide compounds for screening.

[0076] "Anticancer agent", "antineoplastic agent" or "chemotherapeutic agent" refers to any agent useful in the treatment of a neoplastic condition. One class of anticancer agents includes chemotherapeutic agents. "Chemotherapy" refers to the administration of one or more chemotherapeutic drugs and / or other agents to a cancer patient by a variety of methods including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal or inhalation or suppository forms.

[0077] The term "cell proliferation" refers to the phenomenon where the number of cells changes as a result of division. This term also encompasses cell growth where the cell morphology changes (e.g., size increases) in concert with the proliferation signal.

[0078] The terms "selective inhibition" or "selectively inhibit" refer to a biologically active agent that, by direct or indirect interaction with a target, has the ability to preferentially reduce target signaling activity compared to off-target signaling activity.

[0079] The term "in vivo" refers to an event that occurs within the body of a subject.

[0080] The term "in vitro" refers to an event that occurs outside the body of a subject. For example, an in vitro assay encompasses any assay that is performed outside of the subject assay. An in vitro assay encompasses cell-based assays in which living or dead cells are used. An in vitro assay also encompasses cell-free assays in which intact cells are not used. Nomenclature of Polypeptides

[0081] Polypeptides are named herein interchangeably using polypeptide nomenclature, organic chemical nomenclature, chemical formulas, amino acid sequences, or mixtures thereof. For example, substitutions in an analog of IL28B can be indicated as "original amino acid-position-substituted amino acid".

[0082] Thus, the designation "C175S IL28B" or "Cys175Ser IL28B" means that the IL28B analog contains a substitution of cysteine with serine at the analog amino acid position corresponding to the amino acid at position 175 in IL28B (SEQ ID NO: 2) when the analog and IL28B are aligned as described below ("alignment"). Multiple substitutions can be separated by a comma (with a space after the comma) and can be enclosed in parentheses to clarify that they belong to the same analog. Thus, "(C168S, C175S) IL28B" means that the IL28B analog contains two substitutions of cysteine with serine at the analog amino acid positions corresponding to the amino acids at positions 168 and 175 in IL28B (SEQ ID NO: 2).

[0083] The extension in an analog of IL28B can be described with reference to SEQ ID NO:2 by adding position numbers (continuing with positive numbers at the C-terminus and negative numbers at the N-terminus) to the compound in question using its exact sequence, or more simply, by adding the amino acids of the extension in question. Thus, M-IL28B is named as the polypeptide of SEQ ID NO:2 having M at position -1 with reference to SEQ ID NO:2.

[0084] An insertion in an analog of IL29 can be described as "amino acid position number before insertion - index - inserted amino acid". The amino acid position number before insertion refers to the amino acid position in IL29 (SEQ ID NO:1) immediately before the gap that occurs when the insertion analog and IL29 are aligned as described below ("alignment"). The index is in lowercase alphabetical order, for example, the first inserted amino acid is "a", the second inserted amino acid is "b", etc. Thus, "82aD IL29" names an analog of IL29 having an insertion of glycine after amino acid position 82 in IL29 (SEQ ID NO:1).

[0085] A deletion in an analog of IL28B can be described as "des deleted amino acid - deleted amino acid position" or "d deleted amino acid - deleted amino acid position". The deleted amino acid position refers to the amino acid position in IL28B (SEQ ID NO:2) in the gap that occurs when the analog and IL28B are aligned as described below ("alignment"). Thus, "des V2 IL28B" names an analog of IL28B having a deletion of the valine residue at position 2 in IL28B (SEQ ID NO:2).

[0086] If necessary, the alignment of the two amino acid sequences can be done by using the Needle program of the EMBOSS package (http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The Needle program executes a global alignment algorithm (J. Mol. Biol. 1970, 48:443-453). The substitution matrix used is BLOSUM62, the gap opening penalty is 50, and the gap extension penalty is 0.5. Fusion polypeptide, host cell and vector

[0087] The present invention relates to a fusion polypeptide useful for treating disease states in mammals. The fusion polypeptide can comprise a first fragment derived from a first isoform of a protein family and a second fragment derived from a second isoform. In some cases, both the first and second isoforms can be members of the same protein family. In other cases, the first and second isoforms can belong to different protein families. In some examples, the fusion polypeptide can comprise fragments derived from the first and second interferon lambda isoforms. Examples of interferon lambda isoforms include, but are not limited to, various isoforms of IL28A, IL28B and IL29. In some examples, the first interferon lambda isoform is an IL28B isoform, including but not limited to those set forth in SEQ ID NO: 2. In some examples, the second interferon lambda isoform is an IL29 isoform, including but not limited to those set forth in SEQ ID NO: 1. In a further example, the first interferon lambda isoform is an IL28B isoform and the second interferon lambda isoform is an IL29 isoform.

[0088] In some cases, the fragments can be fused together at the fusion site, thereby forming a proximity polypeptide. In some examples, the fusion site can include a sequence of at least about 6 amino acids that is identical to the corresponding sequence present in the first and second isoforms. In further examples, the fusion site can include a sequence of at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids that is identical to the corresponding sequence present in the first and second isoforms. When the first and second isoforms are interferon lambda isoforms, respectively, the fusion site can include a sequence of at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids that is identical to the corresponding sequence present in the first and second interferon lambda isoforms.

[0089] Although not wishing to be bound by any particular theory, having a fusion site that shares sequence identity for the first and second isoforms is particularly advantageous for avoiding new epitopes and thus unwanted immunogenicity resulting from the fusion event. In general, T cells are rigorously selected for survival and undergo both positive and negative selection to produce T cells that recognize self-major histocompatibility complex (MHC) molecules but not native peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, while MHC class II molecules present longer peptides, 13 to 17 amino acids in length. Thus, although the fusion polypeptide is non-native as a whole, it can be non-immunogenic or have insufficient immunogenicity if all possible peptide sequences representing T cell epitopes are native. By selecting a fusion site in the center of an amino acid fragment having at least 6 contiguous amino acids that are identical in both fusion partners (to avoid dominant non-native MHC class I-binding 9mer T epitopes), at least 10 contiguous amino acids that are identical in both fusion partners (to avoid most non-native MHC class I-binding T epitopes), or at least 16 contiguous amino acids that are identical in both fusion partners (to avoid most non-native MHC class II in addition to MHC class I-binding T epitopes), a fusion polypeptide with minimal immunogenicity can be produced in a predictable manner.

[0090] In some cases, the fusion polypeptide can have the structure of Formula I: (S1)-(Helix A)-(S2)-(Helix C)-(S3)-(Helix D)-(S4)-(Helix E)-(S5)-(Helix F)-(S6) (wherein each of Helix A, Helix C, Helix D, Helix E and Helix F is independently an alpha helix and each of S1, S2, S3, S4, S5 and S6 is independently a spacer sequence) can have.

[0091] In some cases, Helix A can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about P27 to L44 of the IL28B peptide (SEQ ID NO: 2) or from about P20 to L37 of the IL29 peptide (SEQ ID NO: 1). In some examples, Helix A can be identical to a fragment having residues from about P27 to L44 of the IL28B peptide (SEQ ID NO: 2). In other examples, Helix A can be identical to a fragment having residues from about P20 to L37 of the IL29 peptide (SEQ ID NO: 1).

[0092] In some cases, Helix C can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about R63 to A87 of the IL28B peptide (SEQ ID NO: 2) or from about R56 to A80 of the IL29 peptide (SEQ ID NO: 1). In some examples, Helix C can be identical to a fragment having residues from about R63 to A87 of the IL28B peptide (SEQ ID NO: 2). In other examples, Helix C can be identical to a fragment having residues from about R56 to A80 of the IL29 peptide (SEQ ID NO: 1).

[0093] In some cases, Helix D can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about V98 to Q112 of the IL28B peptide (SEQ ID NO: 2) or from about V89 to Q103 of the IL29 peptide (SEQ ID NO: 1). In some examples, Helix D can be identical to a fragment having residues from about V98 to Q112 of the IL28B peptide (SEQ ID NO: 2). In other examples, Helix D can be identical to a fragment having residues from about V89 to Q103 of the IL29 peptide (SEQ ID NO: 1).

[0094] In some cases, Helix E can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about R130 to E145 of the IL28B peptide (SEQ ID NO: 2) or about R121 to E136 of the IL29 peptide (SEQ ID NO: 1). In some examples, Helix E can be identical to a fragment having residues of about R130 to E145 of the IL28B peptide (SEQ ID NO: 2). In other examples, Helix E can be identical to a fragment having residues of about R121 to E136 of the IL29 peptide (SEQ ID NO: 1).

[0095] In some cases, Helix F can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about G148 to A170 of IL28B (SEQ ID NO: 2) or about G139 to A161 of IL29 (SEQ ID NO: 1). In some examples, Helix F can be identical to a fragment having residues of about G148 to A170 of IL28B (SEQ ID NO: 2). In other examples, Helix F can be identical to a fragment having residues of about G139 to A161 of IL29 (SEQ ID NO: 1).

[0096] In some cases, each of S1, S2, S3, S4, S5 and S6 can independently have from 1 to about 5, from 1 to about 10, from 1 to about 15, from 1 to about 20, from 1 to about 30, from 1 to about 40, from 1 to about 50, from 1 to about 60, from 1 to about 80 or from 1 to about 100 amino acid residues. In some examples, S2 can further comprise Helix B.

[0097] In some embodiments, helix A can comprise an amino acid sequence that exhibits at least about 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2), helix C can comprise an amino acid sequence that exhibits at least about 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1), and helix F can comprise an amino acid sequence that exhibits at least about 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1). For example, helix A can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues from about P27 to L44 of IL28B (SEQ ID NO: 2), helix C can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues from about R56 to A80 of IL29 (SEQ ID NO: 1), and helix F can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1).

[0098] In other embodiments, helix A can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having the residues of about P20-L37 of IL29 (SEQ ID NO: 1), helix C can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having the residues of about R63-A87 of IL28B (SEQ ID NO: 2), and helix F can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having the residues of about G148-A170 of IL28B (SEQ ID NO: 2). For example, helix A can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having the residues of about P20-L37 of IL29 (SEQ ID NO: 1), helix C can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having the residues of about R63-A87 of IL28B (SEQ ID NO: 2), and helix F can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having the residues of about G148-A170 of IL28B (SEQ ID NO: 2).

[0099] In yet other embodiments, Helix A can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having the residues of about P27 to L44 of IL28B (SEQ ID NO: 2), Helix C can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having the residues of about R63 to A87 of IL28B (SEQ ID NO: 2), and Helix F can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having the residues of about G139 to A161 of IL29 (SEQ ID NO: 1). For example, Helix A can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having the residues of about P27 to L44 of IL28B (SEQ ID NO: 2), Helix C can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having the residues of about R63 to A87 of IL28B (SEQ ID NO: 2), and Helix F can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having the residues of about G139 to A161 of IL29 (SEQ ID NO: 1).

[0100] In some embodiments, Helix A can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about P20 - L37 of IL29 (SEQ ID NO: 1), Helix C can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about R56 - A80 of IL29 (SEQ ID NO: 1), and Helix F can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about G148 - A170 of IL28B (SEQ ID NO: 2). For example, Helix A can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues of about P20 - L37 of IL29 (SEQ ID NO: 1), Helix C can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues of about R56 - A80 of IL29 (SEQ ID NO: 1), and Helix F can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues of about G148 - A170 of IL28B (SEQ ID NO: 2).

[0101] In other embodiments, helix A can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about P27-L44 of IL28B (SEQ ID NO: 2), helix C can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about R56-A80 of IL29 (SEQ ID NO: 1), and helix F can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues of about G148-A170 of IL28B (SEQ ID NO: 2). For example, helix A can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues of about P27-L44 of IL28B (SEQ ID NO: 2), helix C can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues of about R56-A80 of IL29 (SEQ ID NO: 1), and helix F can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues of about G148-A170 of IL28B (SEQ ID NO: 2).

[0102] In yet other embodiments, Helix A can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1), Helix C can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2), and Helix F can comprise an amino acid sequence that exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1). For example, Helix A can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues from about P20 to L37 of IL29 (SEQ ID NO: 1), Helix C can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues from about R63 to A87 of IL28B (SEQ ID NO: 2), and Helix F can comprise an amino acid sequence that exhibits at least 95% homology to a fragment having residues from about G139 to A161 of IL29 (SEQ ID NO: 1).

[0103] In some cases, the fusion polypeptide can retain the secondary structure of the first and / or second isoform. The secondary structure of an isoform can be defined by the number and / or order of secondary units, including but not limited to alpha-helices and beta-sheets. In some examples, the fusion polypeptide can comprise the same number and order of secondary units as the first and / or second isoform. When the first and second isoforms are interferon lambda isoforms, respectively, the fusion polypeptide can retain the secondary structure of the IL28B peptide (SEQ ID NO: 2) or the IL29 peptide (SEQ ID NO: 1).

[0104] In some cases, the fusion polypeptide may be substantially devoid of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating a protein with low immunogenicity has been previously disclosed; see, for example, WO98 / 52976, WO02 / 079232 and WO00 / 3317, which are hereby incorporated by reference. Assays for human T cell epitopes have been described (Stickler, M. et al. (2003) J Immunol Methods 281:95-108). Peptide sequences that can oligomerize without generating T-cell epitopes or non-human sequences are of particular interest. This is achieved by examining tandem repeats of these sequences for the presence of T-cell epitopes and non-human 6-15-mer, particularly 9-mer sequences, and subsequently modifying the design of the XTEN sequence to eliminate or disrupt the epitope sequence. A decrease in the number of epitopes capable of binding to MHC receptors is accompanied by a concomitant decrease in the potential for T cell helper function along with T cell activation, a decrease in B cell activation or upregulation, and a decrease in antibody production. A low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as, for example, TEPITOPE (Sturniolo, T. et al. (1999) Nat Biotechnol 17:555-61).

[0105] In some cases, the fusion polypeptide may lack any additional T-epitopes compared to the case of the IL28B peptide (SEQ ID NO: 2) or the IL29 peptide (SEQ ID NO: 1). In further cases, the fusion polypeptide can have fewer T-epitopes compared to the case of the IL28B peptide (SEQ ID NO: 2) or the IL29 peptide (SEQ ID NO: 1).

[0106] In some cases, the fusion polypeptide may lack any additional B-epitopes compared to the case of the IL28B peptide (SEQ ID NO: 2) or the IL29 peptide (SEQ ID NO: 1). In further cases, the fusion polypeptide can have fewer B-epitopes compared to the case of the IL28B peptide (SEQ ID NO: 2) or the IL29 peptide (SEQ ID NO: 1).

[0107] In various cases, the fusion polypeptide can further comprise at least one modification to an amino acid residue corresponding to the IL28B peptide (SEQ ID NO: 2), and the at least one modification is selected from the group consisting of dV2, dP3, dV4, dA5, dR6, dL7, dR8, G9K, A10P, L11T, P12T, D13T, A14G, R15K, A20G, Q21R, Q31A, A32S, R35K, K37R, L45K, D48N, C49W, K50S, R52S, R54P, L55V, R58G, T59N, Q64L, T88A, dD90, dT91, D92P, G96E, R114Q, T127P, C168S, C175S, P3G, V4P, A5V, R6P, L7T and R8S.

[0108] In various cases, the fusion polypeptide can further comprise at least one modification to an amino acid residue corresponding to the IL29 peptide (SEQ ID NO: 1), and the at least one modification is selected from the group consisting of R14Q, L57Q, A81T, 82aD, 82bT, G83D, E87G, Q105R, P118T and D162E.

[0109] In some cases, the fusion polypeptide can further comprise a fusion site comprising a sequence of at least about 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids that is identical to the corresponding sequence in IL28B (SEQ ID NO: 2) and IL29 (SEQ ID NO: 1). In other cases, the fusion site can comprise a sequence of about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids that is identical to the corresponding sequence in IL28B (SEQ ID NO: 2) and IL29 (SEQ ID NO: 1).

[0110] In some cases, the fusion site can include a sequence of at least about 2 - 30, about 3 - 25, about 4 - 20, about 5 - 15, or about 6 - 10 amino acids that is identical to the corresponding sequence of at least two interferon lambda isoforms.

[0111] In some cases, the fusion polypeptide can exhibit at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to IL28B (SEQ ID NO: 2). For example, the fusion polypeptide can exhibit at least about 90% sequence identity to IL28B (SEQ ID NO: 2). Additionally, the fusion polypeptide can exhibit at least about 95% sequence identity to IL28B (SEQ ID NO: 2).

[0112] In some cases, the fusion polypeptide can exhibit about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less than 99% sequence identity to IL28B (SEQ ID NO: 2). For example, the fusion polypeptide can exhibit less than about 80% sequence identity to IL28B (SEQ ID NO: 2). Additionally, the fusion polypeptide can exhibit less than about 50% sequence identity to IL28B (SEQ ID NO: 2).

[0113] In some cases, the fusion polypeptide can exhibit at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to IL29 (SEQ ID NO: 1). For example, the fusion polypeptide can exhibit at least about 90% sequence identity to IL29 (SEQ ID NO: 1). Additionally, the fusion polypeptide can exhibit at least about 95% sequence identity to IL29 (SEQ ID NO: 1).

[0114] In some cases, the fusion polypeptide can exhibit sequence homology of less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% to IL29 (SEQ ID NO: 1). For example, the fusion polypeptide can exhibit sequence homology of less than about 90% to IL29 (SEQ ID NO: 1). Further, the fusion polypeptide can exhibit sequence homology of less than about 50% to IL29 (SEQ ID NO: 1).

[0115] In some cases, the fusion polypeptide can be further modified. The modification can occur at the N-terminus, C-terminus or any reactive amino acid side chain.

[0116] In some embodiments, the fusion polypeptide can be further modified with polyethylene glycol (PEG). Examples of polyethylene glycol include, but are not limited to, monomethoxy PEG maleimide, monomethoxy PEG iodoacetamide or monomethoxy PEG propionaldehyde. Further, the polyethylene glycol can have a molecular weight of about 1 Kd to 200 Kd, about 5 Kd to 200 Kd, about 5 Kd to 150 Kd, about 8 Kd to 150 Kd, about 8 Kd to 100 Kd, about 10 Kd to 100 Kd, about 10 Kd to 50 Kd, about 12 Kd to 50 Kd or about 12 Kd to 40 Kd.

[0117] In some cases, the pegylated fusion polypeptide can exhibit a longer in vitro half-life compared to the first and second members of the protein family. For example, the fusion polypeptide can exhibit a longer in vitro half-life compared to IL28B (SEQ ID NO: 1) or IL29 (SEQ ID NO: 2).

[0118] In some cases, the fusion polypeptide can exhibit enhanced chemical stability compared to the first and second members of the protein family. For example, the fusion polypeptide can exhibit enhanced chemical stability compared to IL28B (SEQ ID NO: 2) or IL29 (SEQ ID NO: 2).

[0119] The fusion polypeptide can contain an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0120] The present invention also provides a host cell that expresses the fusion protein disclosed herein. The host cell includes individual cells, cell cultures, or cell lines. The host cell includes the progeny of a single host cell. The host cell can be transfected with a heterologous sequence containing the vector of the present disclosure. The host cell can be a prokaryote or a eukaryote, such as a bacterial cell, a fungal cell, an animal cell, an insect cell, a plant cell, or the like. Examples of bacterial host cells include microorganisms belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and others. For example, as bacterial host cells, Escherichia coli XL1-Blue, XL2-Blue, DH1, MC1000, KY3276, W1485, JM109, HB101, No.49, i W3110, NY49, G1698, BL21, or TB1 can be mentioned, but not limited thereto. Other bacterial host cells include Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Pseudomonas putida, Pseudomonas sp. D-0110, and others, but not limited thereto.

[0121] Yeast host cells can include microorganisms belonging to the genera Kluyveromyces, Trichosporon, Saccharomyces, Schizosaccharomyces, Schwanniomyces, Pichia, Candida, etc., such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Candida utilis, and others.

[0122] Examples of eukaryotic cells include animal cells such as mammalian cells. For example, host cells include, but are not limited to, Chinese hamster ovary cells (CHO), monkey cells, such as COS cells, HepG2 cells, A549 cells, and any cells available through ATCC or other depository institutions.

[0123] The host cells of the present disclosure can be grown in culture and in any apparatus used for growing cultures, including fermenters. These can be grown as a monolayer or attached to a surface. Alternatively, the host cells can be grown in suspension. The cells can be grown in a serum-free culture medium. The medium can be a commercially available medium, such as, but not limited to, Opti-CHO (Invitrogen, catalog #12681) supplemented with glutamine, such as 8 mM L-glutamine.

[0124] The host cells of the present disclosure can contain heterologous sequences for effecting the expression of the present fusion polypeptide. The heterologous sequences are preferably self-replicating nucleic acid molecules and can include vectors that transfer and insert nucleic acid molecules into and / or among host cells. Vectors can include vectors that function primarily for the insertion of DNA or RNA into cells, vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Vectors that provide two or more of the above-described functions are also included. An expression vector is a polynucleotide that can be transcribed and translated into a polypeptide(s) when introduced into a suitable host cell.

[0125] The heterologous sequences encoding the fusion protein of the present invention can be expressed by single or multiple vectors. The nucleic acid sequences can be arranged in any order, either in a single operon or in separate operons placed in one or more vectors. Optionally, two or more expression vectors each containing one or more heterologous sequences operably linked in a single operon can be used. Linkage refers to connecting two or more chemical elements or components together by any means, including chemical conjugation or recombinant means. Operably linked refers to an arrangement in which the components so described are in a relationship that enables them to function in their intended manner. For example, a promoter sequence is linked or operably linked to a coding sequence if the promoter sequence facilitates the transcription of the coding sequence. The present vectors can maintain their replicability episomally or as an integral part of the host cell genome.

[0126] The heterologous sequences of the present disclosure can be placed under the control of a single regulatory element. In some cases, the heterologous nucleic acid sequences are regulated by a single promoter. In other cases, the heterologous nucleic acid sequences are placed within a single operon. In still other cases, the heterologous nucleic acid sequences are placed within a single reading frame.

[0127] The preparation of the present nucleic acid can be carried out by various routine recombinant techniques and synthetic procedures. Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor (1989) (Maniatis) as well as T. J. Silhavy, M. L. Bennan and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly described, the present nucleic acid can be prepared from genomic DNA fragments, cDNA and RNA, all of which can be directly extracted from cells or recombinantly produced by various amplification processes including, but not limited to, PCR and rt-PCR.

[0128] The direct chemical synthesis of nucleic acids typically involves the sequential addition of 3'- and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing nucleotide polymer chain, with each addition typically being effected by a nucleophilic attack of the terminal 5'-hydroxyl group of the growing chain at the 3'-position of the added monomer, which is a phosphorus derivative such as a phosphotriester, phosphoramidite or the like. Such methodologies are known to those skilled in the art and are described in the relevant texts and literature (e.g., Matteuci et al., Tet. Lett. 21, 719 (1980); U.S. Patent No. 4,500,707, Caruthers et al.; and U.S. Patents Nos. 5,436,327 and 5,700,637, Southern et al.).

[0129] Regulatory elements include, for example, promoters and operators, which can also be engineered to increase the expression of one or more heterologous sequences encoding glycoproteins. A promoter is a nucleotide sequence that initiates and controls the transcription of a nucleic acid sequence by an RNA polymerase enzyme. An operator is a nucleotide sequence adjacent to a promoter that functions to control the transcription of a desired nucleic acid sequence. The operator contains a protein-binding domain to which a specific repressor protein can bind. In the absence of a suitable repressor protein, transcription is initiated by the promoter. In the presence of a suitable repressor protein, the repressor protein binds to the operator, thereby inhibiting transcription from the promoter.

[0130] In some embodiments of the present disclosure, the promoter used in the expression vector is inducible. In other embodiments, the promoter used in the expression vector is constitutive. In some embodiments, one or more nucleic acid sequences are operably linked to an inducible promoter, and one or more other nucleic acid sequences are operably linked to a constitutive promoter. Non-limiting examples of promoters suitable for use in eukaryotic host cells include, but are not limited to, the CMV immediate early promoter, the HSV thymidine kinase promoter, the early or late SV40 promoter, the LTR derived from retrovirus, and the mouse metallothionein-I promoter.

[0131] Genes in the expression vector will typically also encode a ribosome binding site to direct the translation (i.e., synthesis) of any encoded mRNA gene product. Other regulatory elements that can be used in an expression vector include transcriptional enhancer elements and transcriptional terminators. See, for example, Bitter et al., Methods in Enzymology, Vol. 153: 516-544 (1987).

[0132] An expression vector may be suitable for use in a particular type of host cell and may not be suitable otherwise. However, one of ordinary skill in the art can readily determine, by routine experimentation, whether a particular expression vector is suitable for a given host cell. For example, an expression vector can be introduced into a host organism, which can then be monitored for viability and the expression of any genes contained in the vector.

[0133] The expression vector can also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for the selection or other identification of host cells carrying the expression vector. Non-limiting examples of selectable markers suitable for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

[0134] This vector can be introduced into host cells stably or transiently by various established techniques. For example, one method involves calcium chloride treatment, where the expression vector is introduced by calcium precipitates. Other salts, such as calcium phosphate, can also be used following a similar procedure. Additionally, electroporation (i.e., the application of an electric current to increase the permeability of cells to nucleic acids) can be used. Other transformation methods include microinjection, DEAE-dextran-mediated transformation, and heat shock in the presence of lithium acetate. Host cells can also be transfected using lipid complexes, liposomes, and dendrimers.

[0135] After introduction of the heterologous sequence into the host cell, various methods can be implemented to identify the host cells into which this vector has been introduced. One exemplary selection method involves subculturing individual cells to form individual colonies, followed by testing for the expression of the desired protein product. Another method requires the selection of host cells containing the heterologous sequence based on the phenotypic traits conferred by the expression of a selectable marker gene contained within the expression vector. Those skilled in the art can identify the genetically modified host cells using these or other methods available in the art.

[0136] For example, the introduction of various heterologous sequences of the present disclosure into host cells can be confirmed by methods such as PCR, Southern blot, or Northern blot hybridization. For example, nucleic acids can be prepared from the resulting host cells, and the specific sequence of interest can be amplified by PCR using primers specific to the sequence of interest. The amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, followed by staining with ethidium bromide, SYBR Green solution, or others, or detecting DNA by UV detection. Alternatively, a nucleic acid probe specific to the sequence of interest can be used in the hybridization reaction. The expression of a specific gene sequence can be confirmed by detecting the corresponding mRNA by RT-PCR or Northern blot hybridization associated with reverse transcription, or by immunoassay using an antibody reactive with the encoded gene product. Exemplary immunoassays include, but are not limited to, ELISA, radioimmunoassay, and sandwich immunoassay.

[0137] Furthermore, the introduction of various heterologous sequences of the present disclosure into host cells can be confirmed by the enzymatic activity of the enzyme encoded by the heterologous sequence. The enzyme can be assayed by various methods known in the art. Generally, enzymatic activity can be confirmed by the formation of the product of the enzymatic reaction under test or the conversion of the substrate. The reaction can be carried out in vitro or in vivo.

[0138] In another aspect, the present invention provides a method of producing a fusion polypeptide to achieve desired pharmacokinetic, pharmacological, or pharmaceutical properties. In some cases, the fusion polypeptide can be produced by expressing a vector in a cell under conditions suitable for protein expression.

[0139] Factors such as incubation time, temperature, and medium, although not limited thereto, the conditions suitable for protein expression can depend on the cell type and will be readily determined by those skilled in the art. Method of treatment

[0140] In one aspect, the present invention provides a method of using the fusion polypeptide of the present invention for treating a disease state in a mammal, including but not limited to states associated with interferon lambda receptor (IFNλR1 and / or IL10R2) dysfunction.

[0141] In some cases, the present invention provides a method of treating a viral infection in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide of the present invention. In some examples, the viral infection can be conferred by hepatitis B or hepatitis C. In other examples, the viral infection can be conferred by influenza. Further examples of viral infections include, but are not limited to, human T-lymphotropic virus type 1 (HTLV-1) and human papillomavirus (HPV).

[0142] In other cases, the present invention provides a method of treating an inflammatory disorder in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide of the present invention. In some cases, the inflammatory disorder can be multiple sclerosis. In other cases, the inflammatory disorder can be an autoimmune disease. Examples of autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, celiac disease, Crohn's disease, diabetes mellitus type 1, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, lupus erythematosus, multiple sclerosis, myasthenia gravis, opsoclonus-myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus, polyarthritis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, Reiter's syndrome, Takayasu arteritis, temporal arteritis (also known as "giant cell arteritis"), warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia universalis, Chagas disease, chronic fatigue syndrome, autonomic neuropathy, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, and vulvodynia. Other disorders include bone resorption disorders and thrombosis.

[0143] In further cases, the present invention provides a method of treating cancer in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of the fusion polypeptide of the present invention. In some cases, the cancer can be hepatocellular carcinoma. In other cases, the cancer can be acute myeloid leukemia, thymus, brain, lung, squamous cell, skin, eye, retinoblastoma, intraocular melanoma, oral and oropharynx, bladder, gastric, stomach, pancreas, bladder, breast, cervical, head, neck, kidney, renal, liver, ovary, prostate, colorectal, esophagus, testis, gynecological, thyroid, CNS, PNS, AIDS-related (e.g., lymphoma and Kaposi's sarcoma) or virus-induced cancer.

[0144] Moreover, the fusion polypeptides described herein can be used for the treatment of synovitis, lupus, acute disseminated encephalomyelitis (ADEM), Addison's disease, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, celiac disease, Crohn's disease, type 1 diabetes mellitus, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, inflammatory bowel disease, erythematosus, myasthenia gravis, opsoclonus-myoclonus syndrome (OMS), optic neuritis, ord thyroiditis, osteoarthritis, retinitis uveitis, pemphigus, polyarthritis, primary biliary cirrhosis, Reiter's syndrome, Takayasu arteritis, temporal arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia universalis, Chagas disease, chronic fatigue syndrome, autonomic neuropathy, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, vulvodynia, appendicitis, arteritis, arthritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, cholecystitis, chorioamnionitis, colitis, conjunctivitis, cystitis, dacryadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epiglottitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, hepatitis, hidradenitis, ileitis, iritis, laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, phlebitis, interstitial pneumonia, proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendinitis, tonsillitis, uveitis, vaginitis, vasculitis or vulvitis.

[0145] In some cases, the mammal is a human. In other cases, the mammal can be a mouse, rat, cat, dog, rabbit, pig, sheep, horse, cow, goat, shrew, hamster, guinea pig, monkey, or any other mammal. Many such mammals can be subjects known in the art as preclinical models for certain diseases or disorders, including solid tumors and / or other cancers (e.g., Talmadge et al., 2007, Am. J. Pathol. 170:793; Kerbel, 2003, Canc. Biol. Therap. 2(4 Suppl 1):S134; Man et al., 2007, Canc. Met. Rev. 26:737; Cespedes et al., 2006, Clin. TransL Oncol. 8:318).

[0146] In another aspect, the present invention provides a method of using the fusion polypeptides of the present invention for treating a disease state in a mammal, the method comprising formulating or administering the fusion polypeptide in combination with a second agent. In some cases, the second agent can be an antiviral agent. Such agents include, but are not limited to, telaprevir, boceprevir, semiprevir, sofosbuvir, daclastavir, asunaprevir, lamivudine, adefovir, entecavir, tenofovir, telbivudine, interferon alpha, and pegylated interferon alpha. In other cases, the second agent can be an agent that acts to reduce the symptoms of inflammatory conditions such as encephalomyelitis, asthma, and other diseases described herein. Such agents include non-steroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid; ibuprofen; naproxen; indomethacin; nabumetone; tolmetin; and the like. Corticosteroids are used to reduce inflammation and suppress the activity of the immune system. The most commonly prescribed drug of this type is prednisone. Chloroquine (Aralen) or hydroxychloroquine (Plaquenil) can also be very useful in some lupus individuals. These are most frequently prescribed for the skin and joint symptoms of lupus. Azathioprine (Imuran) and cyclophosphamide (Cytoxan) tend to suppress inflammation and the immune system. Other agents such as methotrexate and cyclosporine are used to control the symptoms of lupus. Anticoagulants are used to prevent the rapid clotting of blood. These range from very low doses of aspirin, which prevent platelet adhesion, to heparin / coumadin.

[0147] In a further example, the agent can be an anti-cancer agent (e.g., a chemotherapeutic agent). Chemotherapeutic drugs can be selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antihormonal drugs, angiogenesis inhibitors and antiandrogen drugs. Non-limiting examples of chemotherapeutic agents, cytotoxic agents and non-peptide small molecules include Gleevec® (imatinib mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib) and adriamycin as well as hosts of chemotherapeutic agents, but are not limited thereto. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.;aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, Casodex (trademark), chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin, etc., antibiotics; methotrexate and 5-fluorouracil (5-FU), etc., antimetabolites; denopterin, methotrexate, pteropterin, trimetrexate, etc., folic acid analogs; fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc., purine analogs; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, calusterone, etc., androgens, drostanolone propionate, epithioestanol, mepitiostane, testolactone, etc., pyrimidine analogs; aminoglutethimide, mitotane, trilostane, etc., anti-adrenal drugs; folinic acid, etc., folic acid supplements; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline;Diaziquone; Elfomithine; Elliptinium acetate; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK.R (trademark); Razoxane; Schizophyllan; Spirogermanium; Tenazone acid; Triazicone; 2,2’,2’’-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Taxanes, for example, Paclitaxel (TAXOL (trademark), Bristol-Myers Squibb Oncology, Princeton, N.J.) and Docetaxel (TAXOTERE (trademark), Rhone-Poulenc Rorer, Antony, France); Retinoic acid; Esperamicin; Capecitabine; and any pharmaceutically acceptable salts, acids or derivatives of the foregoing. Suitable chemotherapy cell conditioners include, for example, anti-estrogen drugs such as Tamoxifen, (Nolvadex (trademark)), Raloxifene, Aromatase inhibitor 4(5)-imidazole, 4-Hydroxytamoxifen, Trioxifene, Keoxifene, LY 117018, Onapristone and Toremifene (Fareston); and anti-androgen drugs such as Flutamide, Nilutamide, Bicalutamide, Leuprolide and Goserelin; Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as Cisplatin and Carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda;Ibandronate; Camptothecin-11 (CPT-11); Topoisomerase inhibitor RFS 2000;Also included are antihormonal agents that act to modulate or inhibit hormonal action in tumors, such as difluoromethylornithine (DMFO). Optionally, the fusion polypeptides of the invention may be Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, acridine carboxamide, Adecatumumab, 17-N-allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, anti-CD22 immunotoxin, anti-neoplastic agent, anti-tumorigenic herb, apaziquone, atiprimod, azathioprine, Belotecan, bendamustine, BIBW 2992, Biricodar, Brostallicin, bryostatin, buthionine sulfoximine, CBV (chemotherapy), caliculin, cell cycle non-specific anti-neoplastic agent, dichloroacetic acid, discodermolide, elsamitrucin, enocitabine, epothilone, eribulin, everolimus, exatecan, exisulind, ferruginol, folotyn, fosfestrol, ICE chemotherapy regimen, IT-101, imexon, imiquimod, indolocarbazole, irofulven, laniquidar, larotaxel, lenalidomide, lucanthone, lurtotecan, mafosfamide, mitozolomide, nafoxidine, nedaplatin, olaparib, ortataxel, PAC-1, pawpaw, pixantrone, proteasome; It can be used in combination with commonly prescribed anticancer drugs such as inhibitors, rebeccamycin, resiquimod, rubitecan, SN-38, salinosporamide A, sapacitabine, Stanford V, swainsonine, talaporfin, tariquidar, tegafur-uracil, temodar, tesetaxel, triplatin tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uramustine, vadimezan, vinflunine, ZD6126, and zosuquidar. Pharmaceutical composition

[0148] The pharmaceutical composition of the present invention typically comprises the active ingredient of the present invention (e.g., a fusion polypeptide, a PEG-modified fusion polypeptide) or a pharmaceutically acceptable salt and / or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including but not limited to inert solid diluents and fillers, diluents, sterile aqueous solutions, various organic solvents, osmotic activators, solubilizers, and adjuvants. The following are non-limiting exemplary pharmaceutical compositions and methods for preparing the same.

[0149] The pharmaceutical composition can be in a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained-release formulations, solutions, suspensions; parenteral injection as a sterile solution, suspension, or emulsion; topical administration as an ointment or cream; or rectal administration as a suppository. The pharmaceutical composition can be in a unit dosage form suitable for a single administration of an exact dosage. The pharmaceutical composition can further comprise a fusion polypeptide and / or a PEG-modified fusion polypeptide according to the present invention as an active ingredient, and can comprise conventional pharmaceutical carriers or excipients. Furthermore, it can comprise other medicinal or pharmaceutical agents, carriers, adjuvants, etc.

[0150] Exemplary parenteral dosage forms include solutions or suspensions of the active polypeptide and / or PEG-modified polypeptide in sterile aqueous solutions, such as aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered with salts, such as histidine and / or phosphate, as needed.

[0151] In some cases, the present invention provides a pharmaceutical composition for injection containing the polypeptide of the present invention or a PEG-modified polypeptide and a pharmaceutical excipient suitable for injection. The components and amounts of the agents in the composition are as described herein.

[0152] Forms that can incorporate the novel compositions of the present invention for administration by injection include aqueous or oily suspensions, or elixirs, mannitol, dextrose or sterile aqueous solutions and similar pharmaceutical media, together with emulsions with sesame oil, corn oil, cottonseed oil or peanut oil.

[0153] Aqueous solutions in physiological saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol and others (and suitable mixtures thereof), cyclodextrin derivatives and vegetable oils can also be used. Suitable fluidity can be maintained, for example, in the case of dispersions, by the use of coatings such as lecithin for maintaining the required particle size, or by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and others.

[0154] The sterile injectable solutions are prepared by incorporating the required amount of the polypeptide of the invention and / or the PEG-modified polypeptide into a suitable solvent having the various other ingredients enumerated above, and are subsequently sterile filtered, if required. Generally, the dispersion systems are prepared by incorporating various sterilized active ingredients into a sterile medium containing a basic dispersion medium and the required other ingredients derived from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, certain desirable preparation methods are vacuum drying and lyophilization techniques which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0155] In some cases, the invention provides a pharmaceutical composition for oral administration, containing the fusion polypeptide of the invention and / or the PEG-modified fusion polypeptide of the invention and a pharmaceutical excipient suitable for oral administration.

[0156] In some cases, the invention provides a solid pharmaceutical composition for oral administration, containing (i) an effective amount of the polypeptide of the invention or the PEG-modified polypeptide; optionally (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for oral administration. In some embodiments, the composition further contains (iv) an effective amount of a third agent.

[0157] In some cases, the pharmaceutical composition can be a liquid pharmaceutical composition suitable for oral consumption. The pharmaceutical compositions of the invention suitable for oral administration can be presented as separate dosage forms, such as capsules, cachets, tablets, or liquids or aerosol sprays, each containing a predetermined amount of the active ingredient as a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, a water-in-oil emulsion or an oil-in-water liquid emulsion. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of associating the active ingredient with a carrier which constitutes one or more necessary ingredients. Generally, the compositions are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and subsequently shaping the product into the desired form, if required.

[0158] Since water may promote the degradation of some polypeptides, the present invention further encompasses anhydrous pharmaceutical compositions and dosage forms containing an active ingredient. For example, in order to simulate long-term storage as a means of determining characteristics such as the shelf life of a formulation or stability over time, water can be added in the pharmaceutical art (e.g., 5%). The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low-moisture-containing components and low-moisture or low-humidity conditions. When substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is anticipated, the pharmaceutical compositions and dosage forms of the present invention containing lactose can be made anhydrous. The anhydrous pharmaceutical compositions can be prepared and stored such that their anhydrous nature is maintained. Thus, the anhydrous compositions can be packaged using materials known to prevent exposure to water so that they can be included in a suitable formulary kit. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, other unit-dose containers, blister packs, and strip packs.

[0159] The fusion polypeptide can be combined with a pharmaceutical carrier in a tight mixture according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of the preparation desired for administration. In the preparation of compositions for oral dosage forms, for oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols, for example, any of the usual pharmaceutical media such as water, glycols, oils, alcohols, flavorings, preservatives, colorants, etc. can be used as the carrier; or in the case of oral solid preparations, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrants can be used, and in some embodiments, lactose is not used. For example, suitable carriers include, for solid oral preparations, powders, capsules, and tablets. If desired, tablets can be coated by standard aqueous or non-aqueous techniques.

[0160] Examples of binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.

[0161] Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrose, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof.

[0162] Disintegrants can be used in the compositions of the present invention to provide tablets that disintegrate when exposed to an aqueous environment. Too much disintegrant can result in tablets that can disintegrate in the bottle. Too little may be insufficient for disintegration to occur and thus can alter the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a sufficient amount of disintegrant, not too little or too much so as to detrimentally alter the release of the active ingredient(s), can be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used can vary based on the type of formulation and mode of administration and can be readily discernible by one of ordinary skill in the art. About 0.5 to about 15 weight percent disintegrant or about 1 to about 5 weight percent disintegrant can be used in the pharmaceutical composition. Disintegrants that can be used to form the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to, agar-agar, alginic acid, Calcium acid, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, Examples include, but are not limited to, potato or tapioca starch, other starches, pregelatinized starch, other starches, clays, other algins, other celluloses, gums, or mixtures thereof.

[0163] Lubricants that can be used in the formation of the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, or mixtures thereof. Additional lubricants include, for example, colloidal silica, coagulated aerosols of synthetic silica, or mixtures thereof. The lubricant can optionally be added in an amount of less than about 1 weight percent of the pharmaceutical composition.

[0164] If an aqueous suspension and / or elixir is desired for oral administration, the active ingredient therein can be combined with emulsifying and / or suspending agents along with various sweetening or flavoring agents, coloring or dyeing agents, and, if necessary, diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof.

[0165] The tablets may or may not be coated and may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer-lasting effect over a longer period. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used. Formulations for oral use can be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium such as peanut oil, liquid paraffin or olive oil.

[0166] Surfactants that can be used in the formation of the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to, hydrophilic surfactants, lipophilic surfactants and mixtures thereof. That is, mixtures of hydrophilic surfactants can be used, mixtures of lipophilic surfactants can be used, or mixtures of at least one hydrophilic surfactant and at least one lipophilic surfactant can be used.

[0167] Surfactants with lower HLB values are more lipophilic or hydrophobic and have greater solubility in oil, while surfactants with higher HLB values are more hydrophilic and have greater solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be compounds with HLB values greater than about 10, and anionic, cationic or zwitterionic compounds for which the HLB scale is generally not applicable. Similarly, lipophilic (i.e., hydrophobic) surfactants are compounds with HLB values equal to or less than about 10. However, the HLB value of a surfactant is only a rough general guide commonly used to enable the formulation of industrial pharmaceuticals and cosmetic emulsions.

[0168] The hydrophilic surfactant can be either ionic or non-ionic. Suitable ionic surfactants include alkylammonium salts; fusidate salts; fatty acid derivatives of amino acids, oligopeptides and polypeptides; glyceride derivatives of amino acids, oligopeptides and polypeptides; lecithin and hydrogenated lecithin; lysophosphatidylcholine and hydrogenated lysophosphatidylcholine; phospholipids and their derivatives; lysophospholipids and their derivatives; carnitine fatty acid ester salts; salts of alkyl sulfates; fatty acid salts; sodium docusate; acyl lactylate; mono- and diacetyl tartrate esters of mono- and diglycerides; succinylated mono- and diglycerides; citrate esters of mono- and diglycerides; and mixtures thereof, but are not limited thereto.

[0169] Among the above groups, the ionic surfactants include, by way of example: lecithin, lysophosphatidylcholine, phospholipids, lysophospholipids and their derivatives; carnitine fatty acid ester salts; salts of alkyl sulfates; fatty acid salts; sodium docusate; acylactylate; mono- and diacetyl tartrate esters of mono- and diglycerides; succinylated mono- and diglycerides; citrate esters of mono- and diglycerides; and mixtures thereof.

[0170] The ionic surfactant can be an ionized form of lecithin, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP-phosphatidylethanolamine, lactylate of fatty acid, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglyceride, mono / diacetylated tartrate ester of mono / diglyceride, citrate ester of mono / diglyceride, cholylsarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, teracesyl sulfate, doxate, lauroyl carnitine, palmitoyl carnitine, myristoyl carnitine and salts and mixtures thereof.

[0171] Examples of hydrophilic nonionic surfactants include, but are not limited to, alkyl glucosides; alkyl maltosides; alkyl thioglucosides; lauryl macrogol glycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkyl phenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acid monoesters and polyethylene glycol fatty acid diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic ester transfer reaction products of polyols by at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, their derivatives, and analogs; polyoxyethylated vitamins and their derivatives; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic ester transfer reaction products of polyols by at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol can be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide.

[0172] Other hydrophilic nonionic surfactants include, without limitation, PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6 caprylate / caprate glyceride, PEG-8 caprylate / caprate glyceride, polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phytosterol, PEG-30 soy sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10 oleate, Tween 40, Tween 60, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonylphenol series, PEG 15-100 octylphenol series, and poloxamer.

[0173] Suitable lipophilic surfactants include, by way of example only, fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acid esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and diglycerides; hydrophobic ester transfer reaction products of polyols by at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins / vitamin derivatives; and mixtures thereof. Among this group, preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters and mixtures thereof, or are hydrophobic ester transfer reaction products of polyols by at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils and triglycerides.

[0174] In one embodiment, the composition can include a solubilizer to ensure excellent solubilization and / or dissolution of the compounds of the present invention and, in addition, to minimize precipitation of the compounds of the present invention. This can be particularly important for compositions for parenteral use, for example, compositions for injection. A solubilizer can be added to increase the solubility of hydrophilic drugs and / or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

[0175] Examples of suitable solubilizers include, but are not limited to: ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol and its isomers, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrin and cyclodextrin derivatives, etc., alcohols and polyols; tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG, etc., ethers of polyethylene glycol having an average molecular weight of about 200 to about 6000; 2-pyrrolidone, 2-piperidone, ε-caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone, etc., amides and other nitrogen-containing compounds; ethyl propionate, tributyl citrate, triethyl acetyl citrate, tributyl acetyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, ε-caprolactone and its isomers, δ-valerolactone and its isomers, β-butyrolactone and its isomers, etc., esters; and dimethylacetamide, dimethyl isosorbide, N-methylpyrrolidone, mono-octanoin, diethylene glycol monoethyl ether and water, etc., other solubilizers known in the art.

[0176] Mixtures of solubilizing agents can also be used. By way of example, but not limited to, triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrin, ethanol, polyethylene glycol 200-100, glycolfurole, transcutol, propylene glycol and dimethylisosorbide. Particularly preferred solubilizing agents include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycolfurole and propylene glycol.

[0177] The amount of solubilizing agent that can be included is not particularly limited. The amount of a given solubilizing agent can be limited to an amount acceptable to the organism, which can be readily determined by one of ordinary skill in the art. In certain situations, for example, to maximize the concentration of the drug, it may be advantageous to include an amount of solubilizing agent far in excess of the amount acceptable to the organism and to remove the excess solubilizing agent using conventional techniques such as distillation or evaporation prior to administering the composition to the subject. Thus, when present, the solubilizing agent can be in a weight ratio of 10%, 25%, 50%, 100% or up to about 200% by weight relative to the combined weight of the drug and other excipients. If desired, very small amounts of solubilizing agent can also be used, such as 5%, 2%, 1% or even less. Typically, the solubilizing agent can be present in an amount of about 1% to about 100% by weight, more typically about 5% to about 25% by weight.

[0178] The composition can further comprise one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, antifoaming agents, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants and mixtures thereof.

[0179] In addition, an acid or a base can be incorporated into the composition to facilitate processing, enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium bicarbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS), and others. Bases that are salts of pharmaceutically acceptable acids such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinonesulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, and others are also suitable. Salts of polybasic acids such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used. When the base is a salt, the cation can be any convenient and pharmaceutically acceptable cation such as ammonium, an alkali metal, an alkaline earth metal, or others. Examples include, but are not limited to, sodium, potassium, lithium, magnesium, calcium, and ammonium.

[0180] Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and others. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acid, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acid, formic acid, fumaric acid, gluconic acid, hydroquinonesulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, and others.

[0181] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are presented by way of example only. Thus, those skilled in the art will envision numerous variations, changes, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. The following claims are intended to define the scope of the present invention, and it is intended that methods and structures within such claims and their equivalents be covered thereby.

Examples

[0182] The examples and preparations shown below further illustrate and exemplify the fusion polypeptides of the present invention and methods of using and preparing the same. It should be understood that the scope of the present invention is in no way limited by the scope of the following examples and preparations.

[0183] (Example 1) Cloning and Expression of IL28B / IL29 Fusion Polypeptide Fusion polypeptides with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 were designed and expressed in E. coli. Briefly, the gene encoding the fusion polypeptide was inserted between the Nde1 and BamH1 restriction sites of the expression vector pET11c and expression was carried out under the control of the phage T7 promoter. This vector was transformed into E. coli BL21(DE3). Cells were grown in LB medium supplemented with 100 μg / ml ampicillin until an OD 450 of 0.4 - 0.6 was reached. Expression was induced by addition of 1 mM IPTG at 37 °C for 12 hours. Cells were collected by centrifugation, suspended in PBS and sonicated. The cell homogenate was centrifuged. SDS-PAGE analysis was performed to demonstrate successful expression in the insoluble inclusion body fractions of the fusion polypeptides, e.g., SEQ ID NO: 8 and SEQ ID NO: 12 (Figures 1 & 2 respectively).

[0184] (Example 2) Refolding and purification of the IL28B / IL29 fusion polypeptide The fusion polypeptides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 and 17 were refolded and purified as follows. Inclusion body pellets were solubilized in 50 mM Tris pH 8.0, 6 M guanidine, 10 mM DTT and clarified by centrifugation. The solubilized inclusion bodies were then dialyzed overnight at 4°C against 50 mM Tris pH 7.8, 1 M arginine, 2 mM GSH, 1 mM GSSG (MWCO: 3000). The refolded fusion polypeptides were purified by cation exchange chromatography using SP BB (GE Healthcare) (50 mM NaOAc, pH 5.5, 0 - 1 M NaCl), followed by hydrophobic interaction chromatography using Butyl Sepharose Fast Flow resin (GE Healthcare) (50 mM NaOAc, 1 - 0 M (NH4)2SO4). Further purification was achieved by cation exchange chromatography using SP HP resin (GE Healthcare) (50 mM NaOAc, pH 5.5, 0 - 1 M NaCl). SDS-PAGE analysis showed that in some cases, the fusion polypeptides (e.g., SEQ ID NO: 8) did not yield a visibly purified protein by this method (Figure 3), while in other cases, the fusion polypeptides (e.g., SEQ ID NO: 12) were demonstrated to have been successfully refolded and purified (Figure 4).

[0185] (Example 3) PEGylation of the IL28B / IL29 fusion polypeptide at the N-terminus

[0186] The purified fusion polypeptide of SEQ ID NO: 3 was concentrated to 1 mg / mL and buffer-exchanged into 50 mM NaOAc, pH 5.5, 10 mM NaCNBH3. Monomethoxy PEG propionaldehyde (20 Kd, NOF) was added (5 molar equivalents relative to the IL28B analog) and the reaction mixture was incubated overnight at room temperature. The resulting PEGylated fusion polypeptide (Compound A) was then purified by cation exchange chromatography using SP HP (50 mM NaOAc, pH 5.5, 0 - 1 M NaCl) (Figure 5).

[0187] The fusion polypeptides of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 were each pegylated at the N-terminus with 20Kd monomethoxy PEG using the method described above, yielding Compound B, Compound C, Compound D, Compound E and Compound F, respectively (Figure 5).

[0188] (Example 4) Pegylation of the IL28B / IL29 fusion polypeptide at the cysteine thiol moiety

[0189] The purified fusion polypeptide of SEQ ID NO: 14 was concentrated to 1 mg / mL and buffer-exchanged into PBS at pH 7.0. Methoxypolyethylene glycol maleimide (20Kd, NOF) was added (20 molar equivalents relative to the fusion polypeptide), and the reaction mixture was incubated at 4 °C overnight. This pegylated the fusion polypeptide at the thiol moiety of C168 to obtain Compound G, which was subsequently purified by cation exchange chromatography using SP HP (50 mM NaOAc, pH 5.5, 0 - 1 M NaCl) (Figure 6).

[0190] Pegylation of SEQ ID NO: 15 was performed in the same manner to obtain Compound H (Figure 6).

[0191] (Example 5) Induction of interferon-stimulated genes by the IL28B / IL29 fusion polypeptide In an antiviral gene induction assay, the antiviral effect of the IL28B / IL29 fusion polypeptide was evaluated. This assay measured the induction of interferon-stimulated genes (ISGs) in Hep G2 cells (ATCC HB-8065) after addition of the IL28B / IL29 fusion polypeptide.

[0192] In a 6-well plate, 5×10 5Hep G2 cells were seeded at a cell / well density. Twenty-four hours after seeding the cells, drug treatment was initiated by replacing the cell culture medium with fresh medium containing the test protein at a concentration of 0.1 ng / ml, 1 ng / ml, 10 ng / ml, or 100 ng / ml. Cells were collected at 3, 12, 24, 48, or 72 hours after the start of drug treatment. As a control, cells were stimulated with human IFNα (PeproTech, 300-02AB) or IL-29 (SEQ ID NO: 1) positive control, or used as an unstimulated negative control. All treatments were performed in triplicate.

[0193] Next, cells were analyzed for viability by MTT assay, which showed that drug treatment had no effect on cell growth and viability. Total RNA was isolated from cell pellets and treated with DNase free of RNase. 2 μg of total RNA was used as a template for cDNA synthesis using PrimeScript RT Master Mix (Takara, RR036) and oligo(dT) as a primer. ISG gene induction was evaluated by real-time PCR using SYBR Premix Ex Taq (Takara, RR820) in LightCycler 480 (Roche Applied Science). Each PCR reaction was performed in triplicate and the mean value was used for calculation. The data shown were normalized to GAPDH or β-actin and shown as fold induction of unstimulated cells.

[0194] 1. Dose-dependence As an example, at 12 hours after the start of treatment, both the reference IL-29 peptide (SEQ ID NO: 1) and the four IL-28B / IL-29 fusion polypeptides (SEQ ID NOs: 3, 5, 7, and 12) showed significant dose-dependent induction of Mx and OAS in Hep G2 cells (Figures 7 & 8, respectively). At a concentration of 10 ng / ml or higher, Mx expression increased 200 - 400-fold and OAS levels increased 30 - 60-fold. Furthermore, the IL-28B / IL-29 fusion polypeptides were shown to induce antiviral gene expression at levels equal to or slightly higher than the reference IL-29 protein.

[0195] 2. Time-dependence Induction of Mx and OAS expression was observed after 3 hours of drug treatment and reached maximum induction after 12 hours (Figures 9 & 10, respectively).

[0196] 3. When the analog is denatured, the Mx and OAS induction activities are lost To confirm the effects observed in the above experiments, the IL-28B / IL-29 fusion polypeptide was denatured by heating at 95°C for 5 minutes. Recombinant human growth hormone was used as a negative control in the repeated experiments. The results showed that when the IL-28B / IL-29 fusion polypeptide was first denatured, the antiviral gene induction was significantly reduced, while no significant effect on Mx and OAS expression was seen with recombinant human growth hormone, indicating that the activity seen in the above experiments was specific to the IL-28B / IL-29 fusion polypeptide (Figures 11 & 12, respectively).

[0197] 4. Pegylated IL-28B / IL-29 fusion polypeptide shows similar biological activity Further examination of the pegylated IL-28B / IL-29 fusion polypeptide showed that it possesses similar antiviral gene induction activity compared to the non-modified polypeptide (Figures 13 & 14, respectively).

[0198] (Example 6) Inhibition of HCV replication in Huh-7.5.1 cells by pegylated IL-28B / IL-29 fusion polypeptide HCV is a single-stranded positive-sense RNA virus that does not replicate in conventional cell cultures due to its restricted tropism. The development of an infection system using cell culture-derived infectious HCV (HCVcc) has greatly facilitated the testing of the complete viral replication cycle and drug discovery efforts regarding the overall infectious viral life cycle.

[0199] To test the ability of the pegylated IL-28B / IL-29 fusion polypeptide to inhibit HCV replication, genotype 2a HCV genomic RNA was transcribed in vitro from plasmid pJFH-1 and used for transfection of Huh-7.5.1 cells. HCVcc was collected from the supernatant of the cell culture medium, and a high-titer virus stock was prepared by propagation in Huh-7.5.1 cells. To determine the virus titer (focus-forming units, FFU / ml), Huh-7.5.1 cells were seeded in an 8-well chamber slide at 2×10 4 cells / well, infected with different amounts of the virus stock solution, and the number of positive foci was counted after immunostaining using anti-HCV core antigen (Pierce Antibodies, Thermo Scientific, clone C7-50, MA1-080).

[0200] For in vitro drug efficacy testing, Huh-7.5.1 cells were seeded in an 8-well chamber slide at a density of 2×10 4 cells / well in complete DMEM medium. After 24 hours, the cells were infected with JFH-1 HCVcc at 0.1×M.O.I., and after 4 hours, drug treatment was initiated by replacing the cell culture medium with fresh medium containing the test protein at a concentration of 0 ng / ml, 1 ng / ml, 10 ng / ml, or 100 ng / ml; the culture medium was replaced with fresh medium containing the same test protein daily. The entire treatment was performed in triplicate. After 48 hours from the start of drug treatment, the cells were immunostained against the HCV core antigen. The total number of positive foci in each well was counted under a fluorescence microscope using a 10× objective lens. The results showed that the derivatives PEG-NO:16 (N-terminal 20K pegylated SEQ ID NO:16) and PEG-NO:17 (N-terminal 20K pegylated SEQ ID NO:17) were similarly potent in inhibiting HCV replication compared to PEG-IFNa and reference PEG-IL-29 (SEQ ID NO:1) (Figure 15).

[0201] (Example 7) The IL-28A / IL29 fusion polypeptide inhibits influenza A virus replication in A549 cells The ability of an IL-28B / IL29 fusion polypeptide to inhibit influenza virus replication was examined in H3N2-infected A549 cells (human adenocarcinoma alveolar basal epithelial cells). A549 cells were pretreated with the test protein for 24 hours and subsequently infected with H3N2 virus for 90 minutes; 72 hours later, the cells were fixed and immunostained with an anti-NP antibody followed by anti-mouse HRP; the drug efficacy was evaluated by ELISA measuring the absorbance value of each well at OD490nm.

[0202] In a 96-well plate, A549 cells were seeded at a concentration of 3×10 4 cells / well in complete DMEM medium. Twenty-four hours after seeding the cells, the cell culture medium was replaced with fresh medium containing the test protein at a concentration of 0.5 ng / ml, 5 ng / ml, 50 ng / ml or 500 ng / ml. Twenty-four hours later, the cell culture medium was replaced with fresh medium containing 30×TCID 50 / 50 μl of H3N2 (A3 / Brisbane) virus. After 90 minutes, the cell culture medium was replaced with fresh medium without virus. As controls, cells were untreated without infection (CV), or infected without treatment (VV). All treatments were replicated 3 times. IFNa2b, reference IL29 (SEQ ID NO: 1), SEQ ID NO: 17, N-terminal 20K pegylated fusion polypeptide SEQ ID NO: 16 (PEG-NO: 16) and N-terminal 20K pegylated fusion polypeptide SEQ ID NO: 17 (PEG-NO: 17) as well as CV and VV were examined (Figure 16).

[0203] Seventy-two hours after the start of virus infection, the cells were fixed with ice-cold acetone and immunostained with a mouse anti-NP monoclonal antibody followed by rabbit anti-mouse-HRP. The OD490nm of each well was scored using a plate reader. The results showed that the IL28B / IL29 fusion polypeptides SEQ ID NO: 16 and SEQ ID NO: 17 and their respective N-terminal 20K pegylated derivatives were effective in inhibiting influenza virus replication (Figure 17).

Chemical formula

Chem.

Chem.

Claims

1. A fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO:

17.

2. The fusion polypeptide according to claim 1, wherein the N-terminus of the fusion polypeptide is further modified by polyethylene glycol (PEG).

3. A host cell expressing the fusion polypeptide according to claim 1 or claim 2.

4. Use of the fusion polypeptide according to claim 1 or claim 2 in the manufacture of a medicament for treating viral infection in a mammal in need of treatment for viral infection.

5. A pharmaceutical composition comprising the fusion polypeptide according to claim 1 or claim 2 and a pharmaceutically acceptable excipient.

6. A vector comprising a polynucleotide encoding the fusion polypeptide according to claim 1 or claim 2.

7. A method for producing the fusion polypeptide according to claim 1 or claim 2, the method comprising expressing the vector according to claim 6 in a cell under conditions suitable for protein expression, thereby producing the fusion polypeptide.

8. A composition comprising the fusion polypeptide according to claim 1 or claim 2 for treating viral infection in a mammal in need of treatment for viral infection.

Images