The present invention provides evolved interferon-alpha polypeptides, and conjugates thereof, and nucleic acids encoding the polypeptides. The invention also includes compositions comprising these polypeptides, conjugates, and nucleic acids; cells containing or expressing the polypeptides, conjugates, and nucleic acids; methods of making the polypeptides, conjugates, and nucleic acids; and methods of using the polypeptides, conjugates, and nucleic acids.

Description of the Invention

Molecules and Methods of the Invention

Molecules of the invention (e.g., polypeptides of the invention, conjugates of the invention, and nucleic acids encoding said polypeptides) are useful for the treatment of diseases and disorders which are responsive to treatment by interferon-alpha, particularly diseases associated with viral infection, such as, for example, infection by HCV.

Patients with chronic HCV infection have viral loads typically in the range of 10.sup.4-10.sup.7 copies of HCV RNA/ml of serum prior to treatment. Upon treatment with IFN-alpha, viral load in these patients characteristically undergoes two distinct log-linear phases of decline (Neumann A. U., et al. (1998) Science 282:103-107). The initial rapid drop in viral load that occurs within the first two days of IFN-alpha therapy is believed to be due to interferon-alpha mediated reduction in virus production in the infected liver cells and concomitant protection of naive cells against infection. The rate of viral production reaches a new steady state at about two days, at which time a second less rapid log-linear phase of viral clearance is observed. This second phase of viral clearance is generally believed to be due in part to T-cell mediated killing of infected liver cells (Neumann, et al., supra). IFN-alpha is believed to play a key role in this biological response through the stimulation of antigen specific T cells to differentiate into T.sub.H1 cells. Furthermore, the mode of action of Ribavirin is believed to be due to augmentation of the T.sub.H1 response, and is thought to be the mechanistic basis of its efficacy in combination therapy with IFN-alpha. HCV-infected patients who are non-responsive to interferon-alpha therapies currently in use (generally termed "non-responders") exhibit much shallower viral load clearance profiles.

Although the present invention is not intended to be limited by a particular theory of underlying mechanism, it is proposed that antiviral activity in surrogate assay systems (such as those described in more detail herein) may be predictive of interferon-alpha efficacy, in for example the first phase of viral clearance. Exemplary antiviral assays, described in the Examples section, monitor the effectiveness of IFN-alpha in protecting against the cytopathic effect of Encephalomyocarditis Virus (EMCV) HeLa human cervical carcinoma cells as a surrogate system for effectiveness against HCV in human liver cells. Other useful virus/cell assay systems include EMCV in WISH human amnion-derived cells, EMCV in HuH7 human liver-derived cells, Vesicular Stomatitis Virus (VSV) in HuH7 cells, Vaccinia Virus (VV) in HeLa cells, Yellow Fever Virus (YFV) in HepG2 human hepatocarcinoma cells, as well as Human Immunodeficiency Virus (HIV) in primary CD4+ T-cells. Example 2 and FIG. 3 (see Original Patent) shows antiviral activities of representative polypeptides of the invention in the EMCV/HeLa antiviral assay.

Other surrogate assay systems useful for monitoring effectiveness against HCV in infected hepatocytes include HCV replicon systems, as described, for example, by Lohmann V., et al., (1999) Science 285(5424):285-293; Randall G. and Rice C. M. (2001) Curr Opin Infect Dis 14(6):743-7477; and Bartenschlager, R. (2002) Nature Reviews/Drug Discovery 1:911. An example of a useful in vivo host system for monitoring HCV antiviral efficacy is a chimeric human liver SCID mouse, as described by Mercer, et al. (2001) Nature Medicine 7(8):927-933.

It is furthermore proposed, without being limited by theory, that enhancement of T.sub.H1 differentiation and/or suppression of T.sub.H2 differentiation by IFN-alpha may be a contributing factor to interferon-alpha efficacy, for example, in the second phase of viral clearance. According to this theory, evolved IFN-alphas with increased potency in these biological activities (i.e., enhancement of T.sub.H1 differentiation and/or suppression of T.sub.H2 differentiation) would be predicted to have increased efficacy relative to, for example, currently approved therapeutic interferon-alpha molecules administered at the same dosage. An exemplary assay, described in the Examples section herein, monitors the enhancement of T.sub.H1 differentiation and/or suppression of T.sub.H2 differentiation by IFN-alpha on naive T.sub.H0 cells, by measuring production of cytokines associated with the T.sub.H1-phenotype (e.g., IFN-gamma) and/or the T.sub.H2-phenotype (e.g., IL-5, IL4) via ELISA or via intracellular staining and FACS sorting.

The therapeutic efficacy of IFN-alpha molecules tends to be diminished in part due to dose-limiting toxicities, e.g. thrombocytopenia and neutropenia. Although the present invention is not intended to be limited by a particular theory of underlying mechanism, it is proposed that such toxicity may be associated with anti-proliferative effects of IFN-alpha on platelet and neutrophil precursors, and that antiproliferative activity in surrogate assay systems (such as those described herein) may be predictive of the relative toxicity of an interferon-alpha molecule. Thus, dose-limiting toxicities associated with IFN-alpha therapy may be diminished in IFN-alpha molecules that exhibit reduced antiproliferative activity relative to, for example, currently approved therapeutic interferon-alpha molecules, such as ROFERON.RTM.-A (Interferon alfa-2a, recombinant; Hoffmann-La Roche Inc.), INTRON.RTM. A (Interferon alfa-2b, recombinant; Schering Corporation), and INFERGEN.RTM. (interferon alfacon-1; InterMune, Inc.). An exemplary antiproliferative activity assay, described in the Examples section herein, monitors the effect of IFN-alpha on the proliferation of human Daudi lymphoid cells. Alternatively, or in addition, dose-limiting toxicities may be reduced as a result of administering more therapeutically active molecules, which would permit dosing in lower concentrations or at lower frequency than currently approved molecules.

It is an object of the invention to provide novel interferon-alpha polypeptides, and nucleic acids which encode the polypeptides. Polypeptides of the invention are useful for the treatment of diseases and disorders which are responsive to treatment by interferon-alpha, particularly diseases associated with viral infection, such as, for example, infection by HCV. Some polypeptides of the invention exhibit an interferon-alpha activity, such as, for example, antiviral activity, antiproliferative activity, and/or T.sub.H1 differentiation activity. Some polypeptides of the invention exhibit one or more of the following properties: increased or decreased antiviral activity compared to a reference IFN-alpha polypeptide; increased or decreased T.sub.H1 differentiation activity compared to a reference IFN-alpha polypeptide; increased or decreased antiproliferative activity compared to a reference IFN-alpha polypeptide. The reference IFN-alpha polypeptide may comprise a sequence of a non-naturally occurring interferon-alpha, such as IFN-alpha Con1 (SEQ ID NO:333), or may comprise a sequence of a naturally-occurring (i.e., wild-type) interferon-alpha polypeptide. Examples of sequences of naturally occurring interferon-alpha polypeptides include sequences of human IFN-alpha polypeptides, such as, for example, huIFN-alpha 2b (SEQ ID NO:321), huIFN-alpha 2a (SEQ ID NO:322), huIFN-alpha 2c (SEQ ID NO:322 with position 34=Arg), huIFN-alpha 8b (SEQ ID NO:327), huIFN-alpha 8a (SEQ ID NO:327 with positions 98=Val, 99=Leu, 100=Cys, and 101=Asp), huIFN-alpha 8c (SEQ ID NO:327 with position 161=Asp and amino acids at positions 162-166 deleted), huIFN-alpha 14a (SEQ ID NO:329), huIFN-alpha 14c (SEQ ID NO:329 with position 152=Leu), or a sequence of any other naturally occurring human interferon alpha polypeptide, such as those listed in Allen G. and Diaz M. O. (1996), supra.

In another aspect, the invention provides interferon-alpha polypeptides which exhibit enhanced efficacy in clearing a virus from virus-infected cells, compared to a reference interferon-alpha molecule, such as one currently employed as a therapeutic (such as, for example, ROFERON-A, INTRON A, or INFERGEN). Exemplary viruses include, but are not limited to, viruses of the Flaviviridae family, such as, for example, Hepatitis C Virus, Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus, Dengue Virus, and Bovine Viral Diarrhea Virus; viruses of the Hepadnaviridae family, such as, for example, Hepatitis B Virus; viruses of the Pico aviridae family, such as, for example, Encephalomyocarditis Virus, Human Rhinovirus, and Hepatitis A Virus; viruses of the Retroviridae family, such as, for example, Human Immunodeficiency Virus, Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, and Rous Sarcoma Virus; viruses of the Coronaviridae family, such as, for example, SARS coronavirus; viruses of the Rhabdoviridae family, such as, for example, Rabies Virus and Vesicular Stomatitis Virus, viruses of the Paramyxoviridae family, such as, for example, Respiratory Syncytial Virus and Parainfluenza Virus, viruses of the Papillomaviridae family, such as, for example, Human Papillomavirus, and viruses of the Herpesviridae family, such as, for example, Herpes Simplex Virus. Such enhanced efficacy may arise from enhanced antiviral activity, enhanced T.sub.H1-differentiation activity, or both, relative to the reference molecule. For example, some interferon-alpha polypeptides of the invention may be particularly useful in clearing viruses or viral strains that show poor response to treatment with interferon-alpha molecules currently in use, such as, for example, Genotype 1 of HCV.

Some polypeptides of the invention exhibit an increased ratio of (antiviral activity/antiproliferative activity) compared to the reference IFN-alpha molecule, and/or an increased ratio of (T.sub.H1 differentiation activity/antiproliferative activity) compared to the reference IFN-alpha molecule. Polypeptides exhibiting such properties may be particularly effective in treatment of viral infections, such as, for example, infection by a virus listed above. Some such polypeptides may, for example, provide enhanced therapeutic efficacy over currently-approved interferon-alpha molecules in the treatment of HCV, in one or both phases of the biphasic viral clearance profile, and/or may exhibit reduced toxicity. Some such polypeptides may provide enhanced therapeutic efficacy over currently-approved interferon-alpha molecules in the treatment of Genotype 1 HCV.

It is another object of the invention to provide conjugates, such conjugates comprising one or more non-polypeptide moiety linked to a polypeptide of the invention, which conjugate exhibits an interferon-alpha activity (such as one or more of the activities listed above), and which optionally exhibits other desirable properties, such as increased serum half-life and/or functional in vivo half-life, and/or decreased antigenicity, compared to the non-conjugated polypeptide. Some such conjugates may exhibit enhanced efficacy in clearing a virus from cells infected with the virus, compared to a reference interferon-alpha molecule, such as an interferon-alpha conjugate currently employed as a therapeutic (such as, for example, PEGASYS.RTM. (Peginterferon alfa-2a; Hoffmann-La Roche, Inc.) or PEG-INTRON.RTM. (peginterferon alfa-2b; Schering Corporation). Exemplary viruses include, but are not limited to, viruses of the Flaviviridae family, such as, for example, Hepatitis C Virus, Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus, Dengue Virus, and Bovine Viral Diarrhea Virus; viruses of the Hepadnaviridae family, such as, for example, Hepatitis B Virus; viruses of the Pico aviridae family, such as, for example, Encephalomyocarditis Virus, Human Rhinovirus, and Hepatitis A Virus; viruses of the Retroviridae family, such as, for example, Human Immunodeficiency Virus, Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, and Rous Sarcoma Virus; viruses of the Coronaviridae family, such as, for example, SARS coronavirus; viruses of the Rhabdoviridae family, such as, for example, Rabies Virus and Vesicular Stomatitis Virus, viruses of the Paramyxoviridae family, such as, for example, Respiratory Syncytial Virus and Parainfluenza Virus, viruses of the Papillomaviridae family, such as, for example, Human Papillomavirus, and viruses of the Herpesviridae family, such as, for example, Herpes Simplex Virus. Such enhanced efficacy may arise from enhanced antiviral activity, enhanced T.sub.H1-differentiation activity, or both, relative to the reference molecule. For example, some interferon-alpha conjugates of the invention may be particularly useful in clearing viruses or viral strains that show poor response to treatment with interferon-alpha molecules currently in use, such as, for example, Genotype 1 of HCV.

Some conjugates of the invention exhibit an increased ratio of (antiviral activity/antiproliferative activity) compared to the reference IFN-alpha molecule, and/or an increased ratio of (T.sub.H1 differentiation activity/antiproliferative activity) compared to the reference IFN-alpha molecule. Conjugates exhibiting such properties may be particularly effective in treatment of viral infections, such as infection by a virus listed above, such as, for example, HCV. Some such conjugates may, for example, provide enhanced therapeutic efficacy over currently-approved interferon-alpha molecules in the treatment of HCV, in one or both phases of the biphasic viral clearance profile, and/or may exhibit reduced toxicity. Some such conjugates may provide enhanced therapeutic efficacy over currently-approved interferon-alpha molecules in the treatment of Genotype 1 HCV.

It is another object of the invention to provide a method of inhibiting viral replication in virus-infected cells, the method comprising administering to the virus-infected cells a polypeptide or conjugate of the invention in an amount effective to inhibit viral replication in said cells. The invention also provides a method of reducing the number of copies of a virus in virus-infected cells, comprising administering to the virus-infected cells a polypeptide or conjugate of the invention in an amount effective to reduce the number of copies of the virus in said cells. The virus may be a virus of the Flaviviridae family, such as, for example, Hepatitis C Virus, Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus, Dengue Virus, or Bovine Viral Diarrhea Virus; a virus of the Hepadnaviridae family, such as, for example, Hepatitis B Virus; a virus of the Picornaviridae family, such as, for example, Encephalomyocarditis Virus, Human Rhinovirus, or Hepatitis A Virus; a virus of the Retroviridae family, such as, for example, Human Immunodeficiency Virus, Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, or Rous Sarcoma Virus; a virus of the Coronaviridae family, such as, for example, SARS coronavirus; a virus of the Rhabdoviridae family, such as, for example, Rabies Virus or Vesicular Stomatitis Virus; a virus of the Paramyxoviridae family, such as, for example, Respiratory Syncytial Virus or Parainfluenza Virus; a virus of the Papillomaviridae family, such as, for example, Human Papillomavirus; or a virus of the Herpesviridae family, such as, for example, Herpes Simplex Virus. The virus may for example be an RNA virus, such as HCV, a DNA virus, such as HBV, or a retrovirus, such as HIV. The cells may be in culture or otherwise isolated from a mammal (i.e., in vitro or ex vivo), or may be in vivo, e.g., in a mammal (e.g. such as a SCID mouse model as described by Mercer, et al. (2001) Nature Medicine. 7(8): 927-933), in a primate, or in man.

The invention also provides a method of enhancing T.sub.H1 differentiation of T.sub.H0 cells, comprising administering to a population comprising T.sub.H0 cells a polypeptide or conjugate of the invention in an amount effective to increase the production of a cytokine associated with the T.sub.H1-phenotype (e.g., IFN-gamma) and/or decrease the production of a cytokine associated with the T.sub.H2-phenotype (e.g., IL-4 or IL-5) in said population. The population may be in culture or otherwise isolated from a mammal (i.e., in vitro or ex vivo), or may be in vivo, e.g., in a mammal, in a primate, or in man.

The invention also provides a method of inhibiting proliferation of a cell population, comprising contacting the cell population with a polypeptide, variant, or conjugate of the invention in an amount effective to decrease proliferation of the cell population. The cell population may be in culture or otherwise isolated from a mammal (i.e., in vitro or ex vivo), or may be in vivo, e.g., in a mammal, a primate, or man.

These and other objects of the invention are discussed in more detail below.

Polypeptides of the Invention

The invention provides novel interferon-alpha polypeptides, collectively referred to herein as "polypeptides of the invention". The term "polypeptide(s) of the invention" is intended throughout to include variants of the polypeptide sequences disclosed herein. Also included in this invention are fusion proteins comprising polypeptides of the invention, and conjugates comprising polypeptides of the invention.

Fragments of human interferon-alpha nucleic acids were recursively recombined to form libraries of shufflants comprising recombinant polynucleotides, from which polypeptides of the invention were derived. Methods for obtaining libraries of recombinant polynucleotides and/or for obtaining diversity in nucleic acids used as the substrates for recursive sequence recombination are described infra.

Exemplary polypeptides of the invention include polypeptides comprising the sequences identified herein as -- see Original Patent.

The invention also includes isolated or recombinant polypeptides which each comprise a sequence which differs in 0 to 16 amino acid positions from a sequence selected from SEQ ID NO:1--SEQ ID NO:319, e.g., differs in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions, e.g., differs in 0-16 amino acid positions, 0-15 amino acid positions, in 0-14 amino acid positions, in 0-13 amino acid positions, in 0-12 amino acid positions, 0-11 amino acid positions, in 0-10 amino acid positions, in 0-9 amino acid positions, in 0-8 amino acid positions, in 0-7 amino acid positions, in 0-6 amino acid positions, in 0-5 amino acid positions, in 0-4 amino acid positions, in 0-3 amino acid positions, in 0-2 amino acid positions or in 0-1 amino acid positions. Some such polypeptides comprise a sequence which differs in 0 to 16 amino acid positions from a sequence selected from SEQ ID -- see Original Patent.

The invention also includes isolated or recombinant polypeptides which each comprise a sequence which differs in 0 to 16 amino acid positions from a sequence selected from SEQ ID -- see Original Patent.

The invention includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:1 (clone M32), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:1, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:44, SEQ ID NO:69, SEQ ID NO:116, SEQ ID NO:163, SEQ ID NO:201, SEQ ID NO:207, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:260, SEQ ID NO:290, or SEQ ID NO:302. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:1, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:1, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:44, SEQ ID NO:260, or SEQ ID NO:302. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:1, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:1, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:44.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:2 (clone M34), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:2, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:293. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:2, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:2, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:49, or SEQ ID NO:293. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:2, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:2, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:49, or SEQ ID NO:293.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:6 (clone M43), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:6, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:293. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:6, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:6, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:293. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:6, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:6, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:49, or SEQ ID NO:293.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:7 (clone M41), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:7, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:37, or SEQ ID NO:313. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:7, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:7, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:37, or SEQ ID NO:313. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:7, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:7, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:7 or SEQ ID NO:313.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:9 (clone M12), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:9, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:73, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:294, or SEQ ID NO:305. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:9, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:9, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:73, SEQ ID NO:221, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:294, or SEQ ID NO:305.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:10 (clone M26), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:10, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence -- see Original Patent.

In another aspect, the invention includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:18 (clone M46), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:18, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence -- see Original Patent.

In another aspect, the invention includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:24 (clone M42), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:24, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:32, or SEQ ID NO:43. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:24, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:24, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:24, or SEQ ID NO:32.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:26 (clone M31), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:26, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:42, SEQ ID NO:49, or SEQ ID NO:293. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:26, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:26, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:42, or SEQ ID NO:293.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:30 (clone M19), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:30, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:10, SEQ ID NO:30, SEQ ID NO:40, or SEQ ID NO:71. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:30, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:30, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:30 or SEQ ID NO:71.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:46 (clone M30), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:46, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:77, SEQ ID NO:213, SEQ ID NO:216, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:46, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:46, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:77, SEQ ID NO:241, SEQ ID NO:250, SEQ ID NO:310, or SEQ ID NO:312.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:87 (clone M21), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:87, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:87, SEQ ID NO:261, or SEQ ID NO:299.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:88 (clone M45), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:88, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:66, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:145, SEQ ID NO:149, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:202, SEQ ID NO:223, SEQ ID NO:284, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:88, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:88, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:66, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:284, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:88, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:88, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:88 or SEQ ID NO:288.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:89 (clone M10), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:89, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:89 or SEQ ID NO:298.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:94 (clone M33), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:94, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:57, SEQ ID NO:94, SEQ ID NO:237, SEQ ID NO:255, or SEQ ID NO:273.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:103 (clone M38), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:103, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:58, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:145, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:223, SEQ ID NO:244, SEQ ID NO:276, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:309, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:103, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:103, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:58, SEQ ID NO:85, SEQ ID NO:93, SEQ ID NO:98, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:145, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:309, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:103, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:103, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:58, SEQ ID NO:85, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:145, SEQ ID NO:199, SEQ ID NO:295, or SEQ ID NO:296.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:107 (clone M9), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:107, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:107 or SEQ ID NO:301.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:124 (clone M35), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:124, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:58, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:93, SEQ ID NO:98, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:145, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:309, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:124, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:124, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:85, SEQ ID NO:93, SEQ ID NO:98, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:145, SEQ ID NO:199, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:124, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:124, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:98, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:145, or SEQ ID NO:295.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:127 (clone M16), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:127, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:127, SEQ ID NO:197, SEQ ID NO:261, or SEQ ID NO:268.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:140 (clone M40), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:140, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:108, SEQ ID NO:140, SEQ ID NO:272, or SEQ ID NO:278.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:141 (clone M37), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:141, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:141 or SEQ ID NO:160.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:148 (clone M3) that is, comprises a sequence which is at least 95% identical to SEQ ID NO:148, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:148, SEQ ID NO:170, or SEQ ID NO:206. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:148, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:148, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:148 or SEQ ID NO:170.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:167 (clone M27), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:167, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:65, SEQ ID NO:102, SEQ ID NO:118, SEQ ID NO:143, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:181, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:271, or SEQ ID NO:315. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:167, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:167, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:181, or SEQ ID NO:315. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:167, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:167, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:181, or SEQ ID NO:315.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:191 (clone M36), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:191, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:133 or SEQ ID NO:191.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:195 (clone M29), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:195, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:131, SEQ ID NO:137, SEQ ID NO:150, SEQ ID NO:195, SEQ ID NO:234, or SEQ ID NO:278. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:195, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:195, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:108, SEQ ID NO:195, or SEQ ID NO:278. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:195, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:195, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:195 or SEQ ID NO:278.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:208 (clone M02), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:208, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:67 or SEQ ID NO:208.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:232 (clone M23), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:232, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:12, SEQ ID NO:167, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:262, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:232, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:232, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:232 or SEQ ID NO:262.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:234 (clone M28), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:234, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:105, SEQ ID NO:139, SEQ ID NO:188, SEQ ID NO:195, SEQ ID NO:234, SEQ ID NO:278, or SEQ ID NO:285. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:234, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:234, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:105, SEQ ID NO:139, SEQ ID NO:234, SEQ ID NO:278, or SEQ ID NO:285. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:234, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:234, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:105 or SEQ ID NO:234.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:258 (clone M8), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:258, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:99 or SEQ ID NO:258.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:260 (clone M07), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:260, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:44, SEQ ID NO:69, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:145, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:201, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:218, SEQ ID NO:228, SEQ ID NO:237, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:260, SEQ ID NO:276, or SEQ ID NO:290. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:260, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:260, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:44, SEQ ID NO:69, SEQ ID NO:116, SEQ ID NO:201, SEQ ID NO:207, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:260, or SEQ ID NO:290. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:260, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:260, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:18 or SEQ ID NO:260.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:262 (clone M04), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:262, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:12, SEQ ID NO:39, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:232, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:262, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:262, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:12, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:232, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:266 (clone M05), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:266, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:79, SEQ ID NO:90, SEQ ID NO:114, SEQ ID NO:215, SEQ ID NO:229, SEQ ID NO:245, SEQ ID NO:266, or SEQ ID NO:274. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:266, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:266, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:79, SEQ ID NO:90, SEQ ID NO:215, SEQ ID NO:229, SEQ ID NO:245, SEQ ID NO:266, or SEQ ID NO:274.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:293 (clone M25), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:293, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:293. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:293, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:293, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:49, or SEQ ID NO:293. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:293, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:293, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:49, or SEQ ID NO:293.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:296 (clone M14), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:296, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:145, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:202, SEQ ID NO:223, SEQ ID NO:244, SEQ ID NO:284, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:309, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:296, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:296, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:58, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:93, SEQ ID NO:98, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:145, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:284, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:309, or SEQ ID NO:314. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:296, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:296, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:145, SEQ ID NO:184, SEQ ID NO:199, SEQ ID NO:290, SEQ ID NO:295, SEQ ID NO:296, or SEQ ID NO:314.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:297 (clone M20), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:297, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:12, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:297, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:297, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:12, SEQ ID NO:39, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:297, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:297, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:298 (clone M39), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:298, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:53, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:118, SEQ ID NO:143, SEQ ID NO:150, SEQ ID NO:212, SEQ ID NO:227, SEQ ID NO:271, SEQ ID NO:292, or SEQ ID NO:298. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:298, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:298, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, or SEQ ID NO:298. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:298, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:298, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:102 or SEQ ID NO:298.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:303 (clone M17), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:303, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:74, SEQ ID NO:235, SEQ ID NO:256, or SEQ ID NO:303. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:303, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:303, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:74, SEQ ID NO:256, or SEQ ID NO:303. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:303, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:303, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:20, SEQ ID NO:256, or SEQ ID NO:303.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:306 (clone M18), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:306, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:65, SEQ ID NO:71, SEQ ID NO:153, SEQ ID NO:159, SEQ ID NO:166, SEQ ID NO:200, SEQ ID NO:205, SEQ ID NO:230, SEQ ID NO:306, or SEQ ID NO:308. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:306, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:306, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:65, SEQ ID NO:153, SEQ ID NO:166, SEQ ID NO:200, SEQ ID NO:205, SEQ ID NO:230, SEQ ID NO:306, or SEQ ID NO:308. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:306, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:306, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:5, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:205, SEQ ID NO:230, SEQ ID NO:306, or SEQ ID NO:308.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:310 (clone M24), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:310, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:12, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:310, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:310, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:310, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:310, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:39, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312.

The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:312 (clone M01), that is, comprises a sequence which is at least 95% identical to SEQ ID NO:312, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:12, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:232, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 6 amino acid positions from the sequence SEQ ID NO:312, that is, comprises a sequence which is at least 96% identical to SEQ ID NO:312, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312. The invention also includes an isolated or recombinant polypeptide which comprises a sequence which differs in 0 to 3 amino acid positions from the sequence SEQ ID NO:312, that is, comprises a sequence which is at least 98% identical to SEQ ID NO:312, and which exhibits an interferon-alpha activity; such as, for example, a polypeptide comprising the sequence SEQ ID NO:77, SEQ ID NO:216, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:250, SEQ ID NO:264, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:310, or SEQ ID NO:312.

Other modifications contemplated for polypeptides of the invention include those described below and in the section entitled "INTERFERON-ALPHA CONJUGATES"  -- see Original Patent.

Formulaations and Routes of Administration

Therapeutic formulations of the polypeptide or conjugate of the invention are typically administered in a composition that includes one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions may be prepared in a manner known per se in the art to result in a polypeptide pharmaceutical that is sufficiently storage-stable and is suitable for administration to humans or animals.

Drug Form

The polypeptide or conjugate of the invention can be used "as is" and/or in a salt form thereof. Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure.

Excipients

"Pharmaceutically acceptable" means a carrier or excipient that at the dosages and concentrations employed does not cause any untoward effects in the patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000)).

Mix of Drugs

The composition of the invention may be administered alone or in conjunction with other therapeutic agents. Ribavirin, for example, is often co-administered with IFN-alpha and has been shown to increase efficacy in antiviral treatments, such as HCV treatment. A variety of small molecules are being developed against both viral targets (viral proteases, viral polymerase, assembly of viral replication complexes) and host targets (host proteases required for viral processing, host kinases required for phosphorylation of viral targets such as NS5A and inhibitors of host factors required to efficiently utilize the viral IRES). Other cytokines may be co-administered, such as for example IL-2, IL-12, IL-23, IL-27, or IFN-gamma. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the polypeptide or conjugate of the invention, either concurrently or in accordance with another treatment schedule. In addition, the polypeptide, conjugate or composition of the invention may be used as an adjuvant to other therapies.

Patients

A "patient" for the purposes of the present invention includes both humans and other mammals. Thus the methods are applicable to both human therapy and veterinary applications.

Types of Composition and Administration Route

The pharmaceutical composition comprising the polypeptide or conjugate of the invention may be formulated in a variety of forms, e.g. as a liquid, gel, lyophilized, or as a compressed solid. The preferred form will depend upon the particular indication being treated and will be apparent to one skilled in the art.

The administration of the formulations of the present invention can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner. The formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps (e.g., subcutaneous osmotic pumps) or implantation. In some instances the formulations may be directly applied as a solution or spray.

Parenterals

An example of a pharmaceutical composition is a solution designed for parenteral administration. Although in many cases pharmaceutical solution formulations are provided in liquid form, appropriate for immediate use, such parenteral formulations may also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

Parenterals may be prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additional possibilities are phosphate buffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typically added in amounts of about 0.2%-1% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount between 0.1% and 25% by weight, typically 1% to 5%, taking into account the relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, .alpha.-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e. <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as "wetting agents") may be present to help solubilize the therapeutic agent as well as to protect the therapeutic polypeptide against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the polypeptide. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic.RTM. polyols, polyoxyethylene sorbitan monoethers (Tween.RTM.-20, Tween.RTM.-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxymethylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.

Sustained Release Preparations

Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the polypeptide or conjugate, the matrices having a suitable form such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the ProLease.RTM. technology or Lupron Depot.RTM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for long periods such as up to or over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37.degree. C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

Oral Administration

For oral administration, the pharmaceutical composition may be in solid or liquid form, e.g. in the form of a capsule, tablet, suspension, emulsion or solution. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of the active ingredient. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but can be determined by persons skilled in the art using routine methods.

Solid dosage forms for oral administration may include capsules, tablets, suppositories, powders and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as is normal practice, additional substances, e.g. lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

The polypeptides or conjugates may be admixed with adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, they may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhere herein.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants such as wetting agents, sweeteners, flavoring agents and perfuming agents.

Pulmonary Delivery

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the polypeptide or conjugate dissolved in water at a concentration of, e.g., about 0.01 to 25 mg of conjugate per mL of solution, preferably about 0.1 to 10 mg/mL. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure), and/or human serum albumin ranging in concentration from 0.1 to 10 mg/ml. Examples of buffers that may be used are sodium acetate, citrate and glycine. Preferably, the buffer will have a composition and molarity suitable to adjust the solution to a pH in the range of 3 to 9. Generally, buffer molarities of from 1 mM to 50 mM are suitable for this purpose. Examples of sugars which can be utilized are lactose, maltose, mannitol, sorbitol, trehalose, and xylose, usually in amounts ranging from 1% to 10% by weight of the formulation.

The nebulizer formulation may also contain a surfactant to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts will generally range between 0.001% and 4% by weight of the formulation. An especially preferred surfactant for purposes of this invention is polyoxyethylene sorbitan monooleate.

Specific formulations and methods of generating suitable dispersions of liquid particles of the invention are described in WO 94/20069, U.S. Pat. No. 5,915,378, U.S. Pat. No. 5,960,792, U.S. Pat. No. 5,957,124, U.S. Pat. No. 5,934,272, U.S. Pat. No. 5,915,378, U.S. Pat. No. 5,855,564, U.S. Pat. No. 5,826,570 and U.S. Pat. No. 5,522,385 which are hereby incorporated by reference.

Formulations for use with a metered dose inhaler device will generally comprise a finely divided powder. This powder may be produced by lyophilizing and then milling a liquid conjugate formulation and may also contain a stabilizer such as human serum albumin (HSA). Typically, more than 0.5% (w/w) HSA is added. Additionally, one or more sugars or sugar alcohols may be added to the preparation if necessary. Examples include lactose maltose, mannitol, sorbitol, sorbitose, trehalose, xylitol, and xylose. The amount added to the formulation can range from about 0.01 to 200% (w/w), preferably from approximately 1 to 50%, of the conjugate present. Such formulations are then lyophilized and milled to the desired particle size.

The properly sized particles are then suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. This mixture is then loaded into the delivery device. An example of a commercially available metered dose inhaler suitable for use in the present invention is the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C., USA.

Formulations for powder inhalers will comprise a finely divided dry powder containing conjugate and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50% to 90% by weight of the formulation. The particles of the powder shall have aerodynamic properties in the lung corresponding to particles with a density of about 1 g/cm.sup.2 having a median diameter less than 10 micrometers, preferably between 0.5 and 5 micrometers, most preferably of between 1.5 and 3.5 micrometers. An example of a powder inhaler suitable for use in accordance with the teachings herein is the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass., USA.

The powders for these devices may be generated and/or delivered by methods disclosed in U.S. Pat. No. 5,997,848, U.S. Pat. No. 5,993,783, U.S. Pat. No. 5,985,248, U.S. Pat. No. 5,976574, U.S. Pat. No. 5,922,354, U.S. Pat. No. 5,785,049 and U.S. Pat. No. 5,654,007.

Mechanical devices designed for pulmonary delivery of therapeutic products, include but are not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those of skill in the art. Specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo., USA; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo., USA; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C., USA; the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass., USA the "standing cloud" device of Nektar Therapeutics, Inc., San Carlos, Calif., USA; the AIR inhaler manufactured by Alkermes, Cambridge, Mass., USA; and the AERx pulmonary drug delivery system manufactured by Aradigm Corporation, Hayward, Calif., USA.

Kits

The present invention also provides kits including the polypeptides, conjugates, polynucleotides, expression vectors, cells, methods, compositions, and systems, and apparatuses of the invention. Kits of the invention optionally comprise at least one of the following of the invention: (1) an apparatus, system, system component, or apparatus component as described herein; (2) at least one kit component comprising a polypeptide or conjugate or polynucleotide of the invention; a plasmid expression vector encoding a polypeptide of the invention; a cell expressing a polypeptide of the invention; or a composition comprising at least one of any such component; (3) instructions for practicing any method described herein, including a therapeutic or prophylactic method, instructions for using any component identified in (2) or any composition of any such component; and/or instructions for operating any apparatus, system or component described herein; (4) a container for holding said at least one such component or composition, and (5) packaging materials.
 

Claim 1 of 18 Claims

1. An isolated or recombinant polypeptide comprising a sequence which differs in 0 to 8 amino acid positions from the sequence SEQ ID NO:312, which polypeptide exhibits antiviral activity.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.