Modified interferon-alpha-2 with reduced immunogenicity

Abstract

The present disclosure is directed to compositions comprising modified interferon-α2 polypeptides with interferon-α2 activity and reduced immunogenicity. In some embodiments, the genetically modified interferon-α2 polypeptides are hyperglycosylated, such as by the addition of a peptide sequence derived from GM-CSF with multiple O-glycosylation sites. The present disclosure also provides compositions comprising nucleic acid molecules encoding the modified interferon-α2. The present disclosure also provides compositions comprising recombinant protein-expressing cell lines comprising the nucleic acid molecules encoding the modified interferon-α2; wherein the recombinant protein-expressing cells comprise a plasmid or vector containing the nucleic acid molecules. Also disclosed are pharmaceutical compositions comprising modified interferon-α2 with interferon-α2 activity and reduced immunogenicity, as well as methods of using the pharmaceutical preparations for treating medical conditions in subjects.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Argentine Provisional Application No. 20190103715, filed December 17, 2019, entitled "Hyperglycosylated Interferon with Reduced Immunogencity," the entire contents of which are incorporated herein by reference.

[0002] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on December 11, 2020, is titled "EPV0027WO Sequence Patent-In_ST25" and is 54 KB in size. [Technical Field]

[0003] The present disclosure generally relates to the development of therapeutic molecules with pharmaceutical benefits for human application. More specifically, the disclosure relates to modified IFNα-2 polypeptides (including modified IFNα-2a, modified IFNα-2b, and modified IFNα-2c polypeptides) and related compounds and compositions. These modified IFNα-2 polypeptides exhibit proven antiviral biological activity, improved pharmacokinetic parameters relative to commercially available cytokines, and reduced immunogenicity. These modified IFNα-2 polypeptides and related compounds and compositions can be used in human therapy and treatment, including antiviral therapy. [Background technology]

[0004] Therapeutic recombinant proteins are part of everyday clinical practice and are used to treat a variety of diseases. They account for more than 20% of the pharmaceutical market, with a growth rate twice that of small molecule drugs. Protein therapeutics generally have few side effects and high efficacy. Indeed, in the treatment of many human diseases, from microbial infections to various cancers, arthritis, and autoimmune diseases, biopharmaceuticals offer the possibility of medical interventions not possible with other types of drugs.

[0005] However, clinical application of protein therapeutics requires overcoming numerous challenges, both operational and manufacturing, as well as product clinical limitations. These challenges include poor solubility, poor stability, short half-life in circulation, and poor retention of biological function when administered as therapeutic proteins. Furthermore, producing high-yield compositions containing pure proteins poses numerous challenges, making it difficult to achieve easily administered therapeutics. Thus, it is not surprising that efforts are underway to develop biopharmaceuticals that can elicit long-term, effective, and sustained biological responses.

[0006] These proteins (e.g., cytokines, growth factors, and monoclonal antibodies) are often composed of molecules nearly identical to those produced by the human body. However, numerous cases have been reported in which immune responses have been induced following administration of these drugs. Antibodies (ADAs) developed against these drugs affect protein activity and can have a variety of complex and serious effects, depending on factors such as the type of drug, its circulating duration, and its neutralizing activity. The most common effects include reduced therapeutic efficacy and hypersensitivity reactions, but they can also induce anaphylaxis and autoimmune diseases. The positive rate of induced antibodies ranges from less than 1% for drugs such as tissue plasminogen activator (Activase) to 70% for drugs such as OKT3 (an IgG2a monoclonal antibody).

[0007] The generation of neutralizing antibodies in response to administration of a therapeutic protein can occur as a result of a variety of factors, which can be broadly divided into extrinsic factors such as the route of administration, dose, formulation, presence of aggregates and / or contaminants, and / or the presence and / or type of glycosylation, and intrinsic factors such as the presence of immunogenic epitopes in the protein.

[0008] Adverse factors are fundamentally related to the design and quality of the manufacturing process. In this sense, the contamination of the product with pro-inflammatory or mutagenic nonspecific compounds, such as LPS (bacterial lipopolysaccharide), or the formation of aggregates in the product can generate signals important for the induction of an immune response. Furthermore, denaturation of therapeutics during formulation can result in a more immunogenic product than the intact one due to the presence of new epitopes that can be recognized by B lymphocytes, leading to the stimulation of an immune response accompanied by the induction of ADA. In many cases, these factors can be successfully avoided by developing careful manufacturing processes, both in the pre-production phase and in the final product purification phase. Furthermore, the incorporation of excipients that stabilize biotherapeutics has been shown to produce favorable results.

[0009] However, even when therapeutic agents are nearly identical to the patient's own proteins, controlling endogenous factors is extremely difficult because B lymphocyte activation contributes to antibody expression. B lymphocyte activation can be mediated by T cell cooperation or not, resulting in T cell-dependent or T cell-independent responses, respectively. T cell-independent responses result from the activation of specific groups of B lymphocytes and are stimulated by specific structural features of certain molecules, such as polymer repeats. The antibodies resulting from this T cell-independent activation are primarily low-affinity IgM types.

[0010] On the other hand, T cell-dependent activation is primarily related to the primary sequence of the protein. In T cell-dependent activation, once the molecule is endocytosed, processed, and the resulting peptides are presented on the surface of antigen-presenting cells (dendritic cells, macrophages, or B lymphocytes) in association with class II major histocompatibility complex (MHC) molecules, some sequences are expressed as T cell "helper" (T h ) (via cell surface receptors called TCRs (T cell receptors)). Once activated, these specific lymphocytes trigger an immune response that leads to B lymphocyte activation and the subsequent production of ADAs. In T cell-dependent responses, the antibodies produced are of the IgG type, have high affinity, and are produced for a longer period than in cases without T cell involvement. Currently, methodologies for assessing the immunogenicity of protein therapeutics are diverse, including computational and in silico immunogenicity prediction techniques, in vitro and ex vivo cell growth strategies, and the use of animal models. All of these assume that the immune response to most therapeutic proteins is dependent on T cells.

[0011] T cell-dependent B lymphocyte activation begins with the interaction of a group of B lymphocytes with a specific protein epitope via their cell surface antigen receptors (IgM / IgD), constituting the first sign of B lymphocyte activation. This signal promotes protein internalization, which is then processed into small peptide epitopes that are ultimately exposed within the "groove" of class II MHC molecules on the surface of B lymphocytes. B cells also co-express the CD40 molecule on their surface. T h When the (helper T) lymphocyte interacts with the composite epitope MHC class II and CD40 (on the surface of B lymphocytes) via the TCR and the ligand of the CD40 molecule (CD154), a second activation signal is initiated. This signal ultimately activates the B lymphocyte, and the T cell produces the cytokine IL-4 (T h lymphocyte type 2 response) or interferon gamma (T hThese cells mature the immune response by producing T-cell-mediated immune responses, such as B lymphocytes (type 1 lymphocytes). It is important to note that without the involvement of T cells to provide a second signal, B lymphocytes undergo programmed cell death (apoptosis). Therefore, attenuation of T-cell-mediated immune responses has become a focus of therapeutic recombinant protein research, a process known as "deimmunization."

[0012] In particular, in the case of treatment with IFNα or IFN-β, despite the cytokines being autologous, immune tolerance to autoantigens is broken in some patients, resulting in the production of anti-IFN antibodies. These antibodies may bind to IFN molecules ineffectively or may neutralize their activity by altering the pharmacokinetics of the cytokine or by blocking the binding domain to specific receptors on the target cell surface. In fact, numerous clinical studies have confirmed the development of anti-IFN-α antibodies in patients with chronic hepatitis C and neoplastic diseases treated with IFNα-2a or IFNα-2b.

[0013] Another major limitation of the use of IFNα-2 (including IFNα-2b) as a biotherapeutic is its short circulating half-life, which necessitates long-term treatment and, as a result, the aforementioned side effects. In this regard, PEGylation of the molecule has prolonged its plasma half-life, allowing for once-weekly administration and improving efficacy compared to the native molecule. PEGylation is often employed as a strategy to reduce the immunogenicity of recombinant proteins because it often causes erric impairment, which reduces antigen presentation. However, data suggest that 8% of patients with chronic hepatitis C who do not respond to PEGylated IFNα-2 and ribavirin therapy have anti-IFN neutralizing antibodies, while none of the patients who cleared HCV virus after IFN treatment had detectable levels of these antibodies.

[0014] Several strategies to improve plasma half-life target renal clearance, as renal excretion is the primary and most rapid pathway. Because the glomerular barrier filters proteins according to their charge and size, the starting point for reducing plasma clearance has been to alter their hydrodynamic volume. Therefore, to improve the pharmacokinetics of various biotherapeutics, N- and O-glycosylation engineering strategies have been implemented in recent years, enabling the generation of glycoproteins with very low glomerular filtration rates. This is due to the increased hydrodynamic radius of the glycosylated glycoproteins and the negatively charged terminal sialic acids of the glycosylated glycoproteins, which repel negatively charged glycosaminoglycans at the glomerular pore.

[0015] However, despite the positive results of this strategy (increased half-life), the introduction of a series of mutations into the protein's coding sequence to create consensus sites for N-glycosylation or large Ser / Tre-rich regions (where consensus sites are missing) required for O-glycosylation typically negatively impacts the therapeutic's biological activity. An example of this is the development of a hyperglycosylated version of wild-type IFNα-2b incorporating four N-glycosylation sites. This resulted in a 25-fold increase in the half-life of the modified cytokine (IFN-2b-4N) compared to the wild-type protein. However, in vitro bioactivity remained less than 80% of that of the wild-type protein.

[0016] Thus, there is a need in the art for interferon-derived protein therapeutics that not only have improved pharmacokinetic parameters and / or reduced immunogenicity, thereby improving safety among patient populations, but also retain biological activity, such as antiviral activity, and therapeutic efficacy, and are easy to manufacture and purify. Summary of the Invention

[0017] Thus, the present disclosure provides modified IFNα-2 polypeptides and related compositions that exhibit proven antiviral biological activity and have reduced immunogenicity and improved pharmacokinetic parameters relative to wild-type IFNα-2 and available commercial cytokines. Modified IFNα-2 polypeptides find use as human therapeutics for a variety of reasons, including better safety among patient populations, ease of manufacture and purification, reduced immunogenicity, improved pharmacokinetic profile, high relative antiviral activity, and low antiproliferative biological activity.

[0018] In some embodiments, the present disclosure provides a modified interferon-α2 polypeptide with reduced immunogenicity. In some embodiments, the modified interferon-α2 is a modified interferon-α2b polypeptide, an interferon-α2a polypeptide, or an interferon-α2c polypeptide. In some embodiments, the modified interferon-α2 polypeptide comprises a substitution of one or more amino acids occupying a position selected from the group consisting of: 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157 in native human interferon-α2, wherein the substitution comprises changing the amino acid at that position to an amino acid selected from the group consisting of alanine, glycine, or threonine. In embodiments, the modified interferon-α2 polypeptide comprises a substitution of one or more amino acids occupying a position selected from the group consisting of the following positions in native human interferon-α2: 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein such mutations reduce the immunogenicity of the modified interferon-α2 compared to native human interferon-α2. In the above polypeptide embodiments, the modified interferon-α2 polypeptide can be isolated, synthetic, or recombinant.

[0019] In some embodiments, the modified interferon-α2 also comprises the addition of amino acids that comprise one or more N- or O-glycosylation sites. In some embodiments, the modified interferon-α2 also comprises the addition of amino acids that comprise one or more N- or O-glycosylation sites, and these added amino acids comprise a sequence having at least 80%, at least 90%, or at least 95% homology to APARSPSTQPWE or a fragment thereof. In some embodiments, the added amino acids comprise the addition of the amino acid sequence APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof. In some embodiments, the fragment of APARSPSTQPWE is at least 7, at least 8, at least 9, and / or at least 10 amino acids in length. Such amino acid additions may be added to the N-terminus and / or C-terminus of the modified interferon-α2 polypeptides disclosed herein. In the above polypeptide embodiments, the modified interferon-α2 polypeptide may be isolated, synthetic, or recombinant.

[0020] In embodiments, the present disclosure is directed to a modified interferon-α2b polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence comprising at least 60, 70, 80, 90, or 95% identity to SEQ ID NO: 12, and further comprising at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. In embodiments, the substitutions comprise the mutations L9A, F47A, L117A, F123A, and L128A. In embodiments, the substitutions comprise the following mutations: L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In embodiments, the substitutions comprise the following mutations: L9A, F47A, N65A, L66A, L117A, F123A, and L128A. In embodiments, the substitutions comprise the following mutations: L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A.

[0021] In embodiments, the present disclosure is directed to a modified GMOP-interferon-α2b polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence comprising at least 60, 70, 80, 90, or 95% identity to SEQ ID NO: 10, and further comprising at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. In embodiments, the substitutions comprise the mutations L23A, F61A, L131A, F137A, and L142A. In embodiments, the substitutions comprise the following mutations: L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In embodiments, the substitutions comprise the following mutations: L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In embodiments, the substitutions comprise the following mutations: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A.

[0022] In embodiments, the present disclosure is directed to modified interferon-α2a polypeptides having interferon-α2 activity, the polypeptides comprising an amino acid sequence comprising at least 60, 70, 80, 90, or 95% identity to SEQ ID NO: 22 and further comprising at least five amino acid substitutions selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acid at said positions to alanine, glycine, or threonine. In embodiments, the substitutions comprise the mutations L9A, F47A, L117A, F123A, and L128A. In embodiments, the substitutions comprise the following mutations: L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In embodiments, the substitutions comprise the following mutations: L9A, F47A, N65A, L66A, L117A, F123A, L128A. In embodiments, the substitutions comprise the following mutations: L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A.

[0023] In embodiments, the present disclosure is directed to a modified GMOP-interferon-α2a polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence comprising at least 60, 70, 80, 90, or 95% identity to SEQ ID NO:21, and further comprising at least five amino acid substitutions at any of positions 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171 selected from the set consisting of: changing the amino acid at said positions to alanine, glycine, or threonine. In embodiments, the substitutions comprise the mutations L23A, F61A, L131A, F137A, and L142A. In embodiments, the substitutions comprise the following mutations: L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In embodiments, the substitutions comprise the following mutations: L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In embodiments, the substitutions comprise the following mutations: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A.

[0024] In some embodiments, the present disclosure is directed to a modified interferon-α2c polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence comprising at least 60, 70, 80, 90, or 95% identity to SEQ ID NO: 24, and further comprising at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said positions to alanine, glycine, or threonine. In some embodiments, the substitutions comprise the mutations L9A, F47A, L117A, F123A, and L128A. In some embodiments, the substitutions comprise the following mutations: L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In embodiments, the substitutions comprise the following mutations: L9A, F47A, N65A, L66A, L117A, F123A, L128A. In embodiments, the substitutions comprise the following mutations: L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A.

[0025] In embodiments, the disclosure is directed to a modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence comprising at least 60, 70, 80, 90, or 95% identity to SEQ ID NO: 23, and further comprising at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions comprise changing the amino acids at the positions to alanine, glycine, or threonine. In embodiments, the substitutions comprise the mutations L23A, F61A, L131A, F137A, and L142A. In embodiments, the substitutions comprise the following mutations: L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In embodiments, the substitutions comprise the following mutations: L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In embodiments, the substitutions comprise the following mutations: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A.

[0026] In embodiments, the modified interferon-α2 polypeptide is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20. In embodiments, the modified interferon-α2 polypeptide is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. In embodiments, the genetically modified interferon-α2 is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6.

[0027] In some embodiments, the modified interferon-α2 polypeptides disclosed herein have antiviral activity comparable to that of native human interferon-α2. In some embodiments, the modified interferon-α2 has a relative antiviral activity of 10-90% compared to that of native human interferon-α2.

[0028] In embodiments, the present disclosure is directed to polynucleotides (e.g., DNA or RNA) encoding one or more of the modified polypeptides of the present disclosure. In embodiments of polynucleotides disclosed herein, the polynucleotides may be isolated, synthetic, or recombinant. In embodiments, expression cassettes, plasmids, expression vectors, and recombinant viruses comprising such polynucleotides are provided. In embodiments, microorganisms or cells comprising expression cassettes, plasmids, vectors, or recombinant viruses of the present disclosure are provided. In embodiments, the present disclosure is directed to characterized cell lines comprising nucleic acids encoding one or more modified interferon-α2 polypeptides of the present invention, which also exhibit reduced immunogenicity. In embodiments, the cell lines are suitable for producing modified interferon-α2 with reduced immunogenicity. Preferably, the cell lines are selected from the group consisting of CHO-K1, HEK293, NS0, BHK, Sp2 / 0, CAP, and CAP / T.

[0029] In some embodiments, the present disclosure is directed to pharmaceutical compositions comprising one or more modified IFN-α2 polypeptides, nucleic acids, cells, and / or vectors disclosed herein, and optionally a pharmaceutically acceptable excipient and / or carrier. In some embodiments, the pharmaceutical compositions disclosed herein comprising at least one or more modified IFN-α2 polypeptides, nucleic acids, cells, and / or vectors can be used to treat a variety of diseases, including melanoma (including malignant melanoma), chronic hepatitis C (including patients with liver decompensation), acute and chronic hepatitis B, acute and chronic non-A and non-B hepatitis, Kaposi's sarcoma (including AIDS-associated Kaposi's sarcoma), multiple sclerosis, genital warts, leukemia (including hairy cell leukemia), lymphoma (including follicular lymphoma), condyloma acuminata, and viral infections (including SARS-COV-2 infection, ZIKV infection, CHIKV infection, and influenza A infection).

[0030] In embodiments, the present disclosure directly relates to methods of preventing or treating one or more medical conditions in a subject, including administering one or more modified interferon-α2 compounds or compositions of the present disclosure, and methods of preventing or treating one or more medical conditions in a subject by administering one or more modified interferon-α2 compounds or compositions of the present disclosure, such as melanoma, melanoma (including malignant melanoma), chronic hepatitis C (including patients with compensated liver disease), acute and chronic hepatitis B, acute and chronic non-A, non-B hepatitis, Kaposi's sarcoma (including AIDS-associated Kaposi's sarcoma), multiple sclerosis, genital warts, leukemia (including hairy cell leukemia), lymphoma (including follicular lymphoma), condyloma acuminata, and other viral infections (including SARS-CoV-2 infection, ZIKV infection, CHIKV infection, and influenza A infection).

[0031] In aspects, the present disclosure provides uses of one or more modified interferon-α2 compounds or compositions of the present disclosure for the manufacture of a medicament for the treatment of melanoma (including malignant melanoma), chronic hepatitis C (including patients with compensated liver disease), acute and chronic hepatitis B, acute and chronic non-A non-B hepatitis, Kaposi's sarcoma (including AIDS-associated Kaposi's sarcoma), multiple sclerosis, genital warts, leukemia (including hairy cell leukemia), lymphoma (including follicular lymphoma), condyloma acuminata, viral infections (including SARS-COV-2 infection, ZIKV infection, CHIKV infection, or influenza A infection), and compositions described herein. [Brief explanation of the drawings]

[0032] The present disclosure may be better understood with reference to the following figures. [Figure 1A]Figure 1A shows the in silico immunogenicity analysis of GMOP-IFNα-2b. EpiMatrix-predicted 9-mer hits for eight common HLA class II alleles are aligned along the GMOP-IFN2b sequence. Peptides with an EpiMatrix "Z" score of 1.64 or higher (top 5%) are considered potential epitopes (gray bars). Peptides with a score of 2.32 or higher (top 1%) are highly likely to bind to MHC (black bars). Clusters identified by EpiMatrix are boxed with the respective scores. Published epitopes determined by experimental methods (bars below the map) overlap with those defined here. Figure 1A shows the predicted MHC class II binding clusters of GMOP-IFN predicted by EpiMatrix. A total of six binding clusters were predicted. [Figure 1B] Figure 1B shows the in silico immunogenicity analysis of GMOP-IFNα-2b. EpiMatrix-predicted 9-mer hits for eight common HLA class II alleles are aligned along the GMOP-IFN2b sequence. Peptides with an EpiMatrix "Z" score of 1.64 or higher (top 5%) are considered potential epitopes (gray bars). Peptides with a score of 2.32 or higher (top 1%) are highly likely to bind to MHC (black bars). Clusters identified by EpiMatrix are boxed with the respective scores. Published epitopes determined by experimental methods (bars below the map) overlap with those defined here. Figure 1B shows the impact of 10 selected mutations on the overall immunogenic potential of GMOP-IFN. [Figure 2]Figure 2 shows the EpiMatrix MHC-binding cluster immunogenicity scores. GMOP-IFN-2b and its deimmunized variants (GMOP-IFN-VAR1, GMOP-IFN-VAR2, GMOP-IFN-VAR3, and GMOP-IFN-VAR4) are mapped onto the cluster immunogenicity score according to their individual EpiMatrix scores. The EpiMatrix cluster immunogenicity score represents the deviation of estimated epitope content from the baseline expected value based on random peptide standards. MHC-binding cluster scores of +10 or higher are considered potentially immunogenic, while lower scores are considered less likely to be immunogenic. Several positive control peptides and proteins are also ranked by their immunogenicity EpiMatrix scores, from highest (+80) to lowest (-50). [Figure 3] Figure 3 shows the purity assessment of different modified GMOP-IFNα-2b polypeptides by denaturing SDS-PAGE gel after one-step immunoaffinity chromatography. Purity levels of over 94% were achieved. Lane 1 contains protein molecular weight markers. Lane 2 contains nonglycosylated IFN-α2b. Lane 3 contains wild-type IFN-α2b. Lane 4 contains GMOP-IFN-α2b. Lane 5 contains GMOP-IFN-α2b-VAR1. Lane 6 contains GMOP-IFN-α2b-VAR2. Lane 7 contains GMOP-IFN-α2b-VAR3. Lane 8 contains GMOP-IFN-α2b-VAR4. [Figure 4]Figure 4 depicts an isoelectric focusing assay. The charge-based heterogeneity of the modified GMOP-IFN variants was analyzed by IEF followed by Coomassie blue staining. Different sialylated forms were distinguished for each protein variant, revealing seven isoforms for GMOP-IFN and 11 electrophoretic bands for GMOP-IFN-VAR2 and 3. The deimmunized variants of GMOP-IFN showed a high content of glycan structures attached to O-glycosylation sites. Lane 1 contains wild-type IFN-α2b. Lane 2 contains GMOP-IFN-α2B. Lane 3 contains GMOP-IFN-α2B-VAR2. Lane 4 contains GMOP-IFN-α2B-VAR3. The sialic acid content increases from the top to the bottom of the gel. [Figure 5-1] Figure 5-1 depicts a sandwich ELISA measuring IFN-γ secretion by T cells after incubation with IFN-pulsed dendritic cells. Data were obtained from 20 donors. The stimulation index (SI) was defined as the ratio of cytokine concentrations from protein-challenged samples divided by cytokine concentrations from excipient-treated samples. Differences between treatments were assessed by one-way analysis of variance (ANOVA). Differences were considered statistically significant when p<0.05. A post-hoc Tukey's multiple comparison test was then applied. The modified GMOP-IFN-α molecule demonstrated reduced immunogenicity compared to the original molecule. [Figure 5-2]Figure 5-2 depicts a sandwich ELISA measuring IFN-γ secretion by T cells after incubation with IFN-pulsed dendritic cells. Data were obtained from 20 donors. The stimulation index (SI) was defined as the ratio of cytokine concentrations from protein-challenged samples divided by cytokine concentrations from excipient-treated samples. Differences between treatments were assessed by one-way analysis of variance (ANOVA). Differences were considered statistically significant when p<0.05. A post-hoc Tukey's multiple comparison test was then applied. The modified GMOP-IFN-α molecule demonstrated reduced immunogenicity compared to the original molecule. [Figure 6] Figure 6 shows an HLA-DR antibody blocking assay to study HLA restriction of IFN-derived peptide presentation by DCs. A progressive decrease in the IFN-γ stimulation index (SI) was observed when two different blocking Ab concentrations were evaluated. SI was normalized to the untreated control (vehicle). IFN-derived peptides are presented in the context of HLA-DR molecules. [Figure 7] Figure 7 is a graph depicting the pharmacokinetic plasma profile of IFN-α2 at different times after subcutaneous injection in Wistar rats. Plasma protein concentrations were plotted against time. Data points represent the mean ± SEM of four animals in each group. [Figure 8] Figure 8 shows sandwich ELISA tests performed using supernatants from the production line of each GMOP-IFN-α2b variant. The supernatants corresponding to GMOP-IFN-α2b-VAR1 and GMOP-IFN-α2b-VAR4 were pure, while those corresponding to GMOP-IFN-α2b-VAR2 and GMOP-IFN-α2b-VAR3 were diluted 1 / 20 to allow preliminary quantification of each protein. All supernatants showed the presence of the target cytokines. [Figure 9]Figure 9 shows data from a preliminary antiviral activity test performed on cell line culture supernatants producing different deimmunized mutants of GMOP-IFN-α2b. The absorbance data were plotted on a logarithmic scale as a function of the corresponding activity value of IFN-α2b (standard) and the dilution value of the sample, and a biological activity value (AB) was calculated for each molecule by comparison. All supernatants showed antiviral activity, although at different levels. [Figure 10] Figure 10 shows the antiviral biological evaluation of purified GMOP-IFN-2b and its two purified deimmunized variants, GMOP-IFN-2b-VAR1 and GMOP-IFN-2b-VAR4. Quantitation of the specific activity of each molecule was determined by comparison with an international standard (NIBSC). Relative antiviral activity values ​​in percent were calculated. [Figure 11] Figure 11 shows the antiviral biological evaluation of two purified deimmunized variants of GMOP-IFN-2b, GMOP-IFN-2b-VAR2 and GMOP-IFN-2b-VAR3. Quantitation of the specific activity of each molecule was determined by comparison with an international standard (NIBSC). Relative antiviral activity values ​​in percent were calculated. Detailed Description of the Invention

[0033] [General matters] The following description of specific embodiments is merely exemplary in nature and is in no way intended to limit the scope of the present disclosure, its application, or uses, which, of course, may vary. The present disclosure is described in conjunction with non-limiting definitions and terms contained herein. These definitions and terms are not intended to serve as limitations on the scope or practice of the present disclosure, but are presented for exemplary and explanatory purposes only. Although processes and compositions are described as using a particular order of individual steps or specific materials, it is understood that the steps or materials may be interchangeable, such that the description of the present disclosure can include multiple steps or components arranged in numerous ways, as would be readily understood by one skilled in the art.

[0034] Reference is now made in detail to various embodiments of the modified IFNα-2 polypeptides (including modified IFNα-2b, IFN-α2a, and IFN-α2c polypeptides) disclosed herein, nucleic acids encoding such modified IFNα-2 polypeptides, expression cassettes, plasmids, expression vectors, recombinant viruses, or cells containing such nucleic acids, and modified IFNα-2 polypeptide pharmaceutical compositions and formulations, which have demonstrated antiviral biological activity, improved pharmacokinetic parameters relative to commercially available cytokines, and reduced immunogenicity. As described, these various compounds and compositions find use in the treatment of various viral infections, such as chronic hepatitis B, chronic hepatitis C, and condyloma acuminata, as well as hairy cell leukemia, malignant melanoma, AIDS-related Kaposi's sarcoma, and follicular non-Hodgkin's lymphoma.

[0035] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. As used in this specification and the appended claims, the singular forms include plural references unless the context clearly dictates otherwise. All documents cited herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0036] [Definition] To facilitate a better understanding of the present disclosure, a number of terms and phrases are defined below. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with the meaning in the context of the relevant art and the present disclosure, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined as such herein.

[0037] The terms used herein are for the purpose of describing particular embodiments / aspects and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural, including "at least one," unless the context clearly dictates otherwise. "Or" means "and / or." As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used herein, the terms "comprise" and / or "comprising," or "includes" and / or "including," specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but it will be further understood that they do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and groups thereof. The term "or combinations thereof" means combinations including at least one of the aforementioned elements.

[0038] As used herein, the term "biological sample" refers to a sample of tissue, cells, secretions, etc. from an organism.

[0039] As used herein, the term "medical condition" includes any condition or disease that manifests as one or more physical and / or psychological symptoms for which treatment and / or prevention is desirable, including, but not limited to, previously identified diseases and other newly identified disorders.

[0040] As used herein, the term "immune response" refers to the concerted action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by such cells or the liver, leading to the selective damage, destruction, or elimination from the body of cancer cells, metastatic tumor cells, malignant melanoma, invading pathogens, pathogen-infected cells or tissues, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0041] As used herein, the terms "effective amount," "therapeutically effective amount," and the like, of a composition containing a modified interferon-α2 compound or composition of the present disclosure refer to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., prevention or reduction of symptoms associated with the disease being treated. The amount of a compound or composition of the present disclosure administered to a subject will depend on the type and severity of the disease and individual characteristics, such as general health, age, sex, weight, and tolerance to drugs. It will also depend on the extent, severity, and type of disease. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. The compounds and compositions of the present disclosure can also be administered in combination with each other or with one or more additional therapeutic compounds.

[0042] As used herein, the term "T cell epitope" refers to an MHC ligand or protein determinant that is 7 to 30 amino acids in length and that can specifically bind to a human leukocyte antigen (HLA) molecule and interact with a specific T cell receptor (TCR). Generally, T cell epitopes are linear and do not express specific three-dimensional properties. T cell epitopes are not affected by the presence of denaturing solvents. The ability to interact with T cell epitopes can be predicted by in silico methods (De Groot AS et al., (1997), AIDS Res Hum Retroviruses, 13(7):539-41; Schafer JR et al., (1998), Vaccine, 16(19):1880-4; De Groot AS et al., (2001), 30 Vaccine, 19(31):4385-95; De Groot AR et al., (2003), Vaccine, 21(27-30):4486-504, all of which are incorporated herein by reference in their entireties.

[0043] As used herein, the term "T cell epitope cluster" refers to a polypeptide comprising between about 4 and about 40 MHC-binding motifs. In specific embodiments, a T cell epitope cluster comprises between about 5 and about 35 MHC-binding motifs, between about 8 and about 30 MHC-binding motifs; and between about 10 and about 20 MHC-binding motifs.

[0044] As used herein, the term "immunostimulatory T cell epitope polypeptide" refers to a molecule that can induce an immune response, for example, a humoral immune response, a T cell-mediated immune response, or an innate immune response.

[0045] As used herein, the term "B-cell epitope" refers to a protein determinant capable of specific binding to an antibody. B-cell epitopes usually consist of chemically active surface groupings composed of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0046] As used herein, the term "subject" refers to any living organism in which an immune response can be elicited. The term "subject" includes, but is not limited to, non-human primates such as humans, chimpanzees, other apes, and monkeys; domestic animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; and laboratory animals including rodents such as mice, rats, and guinea pigs. The term does not denote a particular age or sex. Thus, it is intended to encompass adults, newborns, and fetuses of both sexes.

[0047] As used herein, the term "MHC complex" refers to a protein complex that is capable of binding to a specific repertoire of polypeptides known as HLA ligands and transporting the ligands to the cell surface.

[0048] As used herein, the term "MHC ligand" refers to a polypeptide capable of binding to one or more specific MHC alleles. The term "HLA ligand" is interchangeable with the term "MHC ligand." Cells expressing MHC / ligand complexes on their surface are called "antigen-presenting cells" (APCs).

[0049] As used herein, the term "T cell receptor" or "TCR" refers to a protein complex expressed by T cells that is capable of engaging a specific repertoire of MHC / ligand complexes displayed on the surface of APCs.

[0050] As used herein, the term "MHC binding motif" refers to a pattern of amino acids in a protein sequence that predicts binding to a particular MHC allele.

[0051] As used herein, the term "EpiBar®" refers to a 9-mer peptide predicted to react with at least four different HLA alleles.

[0052] As used herein, the term "immune synapse" refers to the protein complex formed by the simultaneous binding of a given T cell epitope to both a cell surface MHC complex and a TCR.

[0053] The term "polypeptide" refers to a polymer of amino acids and does not denote a specific length. Thus, peptides, oligopeptides, and proteins are included in the definition of polypeptide. As used herein, a polypeptide is said to be "isolated" or "purified" when it is substantially free of cellular material when isolated from recombinant and non-recombinant cells, or when it is free of chemical precursors or other chemicals when chemically synthesized. However, a polypeptide of the present disclosure (e.g., a modified IFNα-2 polypeptide) can be spliced, linked, or inserted into another polypeptide with which it is not normally associated in a cell (e.g., a heterologous polypeptide) and still be "isolated" or "purified." When a polypeptide is recombinantly produced, it can also be substantially free of culture medium; for example, culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.

[0054] The terms "polynucleotide" and "nucleic acid sequence" are used interchangeably to refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and, unless otherwise limited, include known analogs of natural nucleotides (e.g., peptide nucleic acids) that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. The term "polynucleotide" is not intended to limit the present invention to polynucleotides composed of DNA. Those skilled in the art will recognize that polynucleotides can be composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present invention also encompass sequences in all forms, including, but not limited to, single-stranded and double-stranded forms. As used herein, the terms "encode" or "encoded," when used in the context of a particular polynucleotide, mean containing the information necessary to direct translation of a polynucleotide sequence into a specific polypeptide. The information that encodes a polypeptide is specified by the use of codons. A polynucleotide encoding a polypeptide may contain untranslated sequences (e.g., introns) within the translated region of the nucleic acid, or may be free of intervening untranslated sequences (e.g., cDNA).

[0055] As used herein, the terms "natural interferon," "natural human interferon-α2b" (hIFN-α2b), "natural human interferon-α2a" (hIFN-α2a), "natural human interferon-α2c" (hIFN-α2c), "wild-type interferon," "native interferon," or variants thereof refer to cytokines (e.g., polypeptides, nucleic acids, etc.) that have not been artificially modified or mutated and are in the state they are in in nature (i.e., wild-type).

[0056] As used herein, the term "amino acid substitution" means changing one amino acid in the primary sequence of a native (ie, wild-type) protein, such as hIFN-α2, for another amino acid.

[0057] As used herein, the terms "modified interferon-α2," "modified interferon-α2," "glycosylated-modified interferon-α2," "human modified interferon-α2 with reduced immunogenicity," "modified GMOP-interferon-α2," "modified IFN-α2," "modified GMOP-IFN-α2," "modified interferon-α2," "modified GMOP-interferon-α2," "modified IFN-α-2," "modified GMOP-IFN-α2," "modified interferon-2," "modified GMOP-interferon-2," "modified IFN-2," "modified GMOP-IFN-2," or variants thereof refer to modified interferon α2 molecules that contain alterations in the amino acid or nucleic acid sequence compared to the appropriate native interferon, in embodiments including at least one glycosylation site, with or without the GMOP amino acid sequence. In embodiments, the molecules have reduced immunogenicity compared to native human interferon.

[0058] As used herein, the term "GMOP" refers to the amino acid sequence of a peptide derived from human granulocyte-macrophage colony-stimulating factor (GM-CSF) that contains four potential O-glycosylation sites (SEQ ID NO:26), and the nucleic acid sequence encoding the GMOP peptide (SEQ ID NO:25). "GMOP" may refer to the GMOP amino acid and / or nucleic acid sequence itself, or as a component of a larger amino acid and / or nucleic acid sequence.

[0059] As used herein, the term "hyperglycosylated" refers to a molecule that contains four or more additional glycosylations relative to that of native interferon-α2. Preferably, the glycosylation-modified interferon-α2 of the present disclosure is hyperglycosylated, consisting of four to six additional glycosylations than are present in native interferon.

[0060] As used herein, the term "O-glycosylation site" refers to a serine or threonine residue within an amino acid sequence that is susceptible to O-glycosylation. The "location" of an "O-glycosylation site" is indicated by the location of a serine or threonine residue in the amino acid sequence that is susceptible to O-glycosylation. The Ser or Thr residue in the sequence may be glycosylated by an O-type enzyme, such as an O-glycosyltransferase. While several O-glycosylation sites for specific proteins are known, it is understood that there are no known consensus recognition sequences for O-glycosyltransferases.

[0061] As used herein, the term "N-glycosylation site" refers to an Asn-Xaa-Ser / Thr tripeptide, where X can be any residue except a proline residue. The "position" of an "N-glycosylation site" is indicated by the position occupied by the amino acid residue in the amino acid sequence of native human interferon-alpha 2b that is replaced by Asn, or it is an asparagine in the consensus sequence. The Asn residue in the consensus sequence may be subject to glycosylation by an N-type enzyme.

[0062] As used herein, the term "PEGylation" refers to the addition of one or more PEG (polyethylene glycol) polymer chains to a molecule (e.g., a polypeptide). PEGylation can be achieved by covalent and / or non-covalent attachment of the PEG polymer chains to the molecule and / or by covalent and / or non-covalent amalgamation. "PEGylated" refers to a molecule that has undergone PEGylation (i.e., has one or more PEG polymer chains attached to the molecule).

[0063] As used herein, the term "Z-score" refers to how many standard deviations an element is from the mean. The Z-score can be calculated using the following formula: Z=(X-μ) / σ where Z is the Z-score, X is the value of a certain element, μ is the population mean, and σ is the standard deviation.

[0064] The following abbreviations and / or acronyms are used in this application:

[0065] ADA (antibody developed against) APC antigen presenting cells DMSO dimethyl sulfoxide DR antibody Antigen D related antibody EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay HLA human leukocyte antigen IFN Interferon MHC major histocompatibility complex PBMC peripheral blood mononuclear cells RPMI Roswell Park Memorial Institute Medium TCR T cell receptor T eff Effector T cells T h Helper T cells T Reg regulatory T cells

[0066] [Modified IFN-α2 Polypeptides and Nucleic Acids] In several aspects, the present disclosure provides modified IFNα-2 polypeptides (including modified IFNα-2b polypeptides, modified IFNα-2a polypeptides, and IFNα-2c polypeptides) that have proven antiviral biological activity, improved pharmacokinetic parameters over wild-type and commercially available IFNα-2 cytokines (e.g., INTRON-A, PEGINTRON, SYLATRON), and reduced immunogenicity, and therefore find use in human therapy, including human antiviral therapy.

[0067] In some embodiments, the present disclosure provides modified interferon-α2 polypeptides or nucleic acids having interferon-α2 activity (e.g., antiviral activity) and reduced immunogenicity. In some embodiments, the modifications made in the native amino acid sequence of human interferon-α2 to obtain the modified interferon-α2 of the present disclosure occur as a result of modifications of amino acids encoding native human interferons or modifications of genes encoding native human interferons (e.g., hIFN-α2a, hIFN-α2b, and hIFN-α2c, etc.). In some embodiments, modifications made to the native amino acid sequence of human interferon-α2, optionally with a GMOP peptide sequence (or fragment thereof) attached to the N- and / or C-terminus of the human interferon-α2 sequence to obtain the recombinant GMOP-interferon-α2 of the present disclosure, are the result of modifications of the amino acids encoding the native human interferon or the gene encoding it, such as modifications to wild-type GMOP-IFN-α2a, wild-type GMOP-IFN-α2b, and wild-type GMOP-IFN-α2c. Furthermore, the modifications are introduced in a manner that reduces the immunogenicity of the amino acid sequence compared to native human interferon while maintaining its biological activity (e.g., antiviral biological activity).

[0068] In some embodiments, the modified interferon-α2 polypeptides and related modified interferon-α2 compounds and compositions of the present disclosure have reduced immunogenicity compared to native interferon-α2. Mutations that reduce the immunogenicity of modified interferon-α2 compared to native interferon-α2 were identified by EpiMatrix® analysis. EpiMatrix® is a proprietary computer algorithm developed by EpiVax (Providence, Rhode Island) that is used to screen protein sequences for the presence of putative T cell epitopes. The input sequence is parsed into 9-mer frames, with each frame overlapping the last frame by 8 amino acids. Each resulting frame is scored for predicted binding affinity against a panel of eight common class II HLA alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, DRB1*1501). Raw scores are normalized against the scores of a large sample of randomly generated peptides. The resulting "Z" score is reported. In a 3D model, any 9-mer peptide with an allele-specific EpiMatrix® Z-score greater than 1.64, theoretically in the top 5% of any sample, is considered a putative T cell epitope.

[0069] Peptides containing clusters of putative T cell epitopes are more likely to test positive in in vitro and in vivo assays. The results of the initial EpiMatrix® analysis are further screened for the presence of putative T cell epitope "clusters" using a second proprietary algorithm known as the Clusterimer® algorithm. The Clusterimer® algorithm identifies subregions within any amino acid sequence that contain a statistically unusually high number of putative T cell epitopes. A typical T cell epitope "cluster" is approximately 9 to 30 amino acids in length and, taking into account affinity for multiple alleles and affinity for multiple 9-mer frames, can contain approximately 4 to 40 putative T cell epitopes. For each epitope cluster, an aggregate EpiMatrix® score is calculated by summing the scores of the putative T cell epitopes and subtracting a correction factor based on the expected score of a randomly generated cluster of the same length as the candidate epitope cluster. An EpiMatrix® cluster score greater than +10 is considered significant. In embodiments, the modified interferon-α2 molecules disclosed herein contain one or more modifications (e.g., alterations, substitutions, or mutations) in the T cell epitope clusters to reduce their immunogenicity. For example, modified interferon-α2 mutations for the modified interferon-α2 molecules disclosed herein are selected to reduce the immunogenicity of the molecule but also to not significantly reduce its biological activity, such as antiviral activity, and / or not affect binding to receptors involved in the biological activity of interferon. In embodiments, such modifications for the modified interferon-α2 molecules disclosed herein are selected to not disrupt the structure or function of the native interferon and include substituting one or more amino acids occupying selected positions in native human interferon-α2 with alanine, threonine, or glycine.

[0070] Many of the most reactive T cell epitope clusters share a feature called an "EpiBar®." As previously mentioned, an EpiBar® is a single 9-mer frame predicted to be reactive to at least four different HLA alleles. In embodiments, the modified interferon-α2 molecules of the present disclosure can contain one or more modifications (e.g., alterations, substitutions, or mutations) within the EpiBar® of native interferon-α2. In embodiments, the modifications of the modified interferon-α2 molecule reduce the immunogenicity of the modified interferon-α2 molecule compared to native IFN-α2. In embodiments, the modifications of the modified interferon-α2 molecule also do not disrupt the structure or function of native interferon-α2 activity. For example, modified interferon-α2 mutations are selected that do not significantly reduce its biological activity, such as its antiviral activity, and / or do not affect binding to receptors involved in interferon biological activity. In embodiments, such modifications to the modified interferon-α2 molecules of the present disclosure are selected so as not to disrupt the structure or function of the native interferon, and include substitution of one or more amino acids occupying selected positions in native human interferon-α2 with alanine, threonine, or glycine.

[0071] In some embodiments, the contribution of each amino acid in these identified cluster regions to HLA binding was assessed using the OptiMatrix tool (part of the EpiVax ISPRI Deimmunization Toolkit). OptiMatrix first examines the "critical" residues that contribute most to MHC binding affinity across multiple 9-mer frames and multiple HLA alleles. The program then iteratively replaces all 19 alternative amino acids at any position in the protein sequence (operator-defined inputs can limit the list to naturally conserved variants) and reanalyzes the predicted immunogenicity of the resulting sequence. To avoid adverse effects on protein structure and, therefore, biological activity, a comprehensive literature search for critical residues was also performed to identify amino acids that were not candidates for modification. In some embodiments, the modifications to the modified interferon-α2 molecule reduce the immunogenicity of the modified interferon-α2 molecule compared to native IFN-α2. In some embodiments, the modifications to the modified interferon-α2 molecule do not further disrupt the structure or function of native interferon-α2 activity. For example, modified interferon-α2 mutations are selected that do not significantly reduce its biological activity, such as its antiviral activity, and / or do not affect its binding to receptors involved in the biological activity of interferon. In embodiments, such modifications to the modified interferon-α2 molecules of the present disclosure are selected so as not to disrupt the structure or function of the native interferon, and include substitution of one or more amino acids occupying selected positions in native human interferon-α2 with alanine, threonine, or glycine.

[0072] In embodiments, the modified interferon-α2 polypeptide comprises a substitution of one or more amino acids occupying a position selected from the group consisting of: 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157 in naturally occurring human interferon-α2 (including interferon-α2b (SEQ ID NO: 12), interferon-α2a (SEQ ID NO: 22), and interferon-α2c (SEQ ID NO: 24)). In embodiments, the modified interferon-α2 polypeptide comprises a substitution of one or more amino acids occupying a position selected from the group consisting of: 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157 in naturally occurring human interferon-α2 (including interferon-α2b (SEQ ID NO: 12), interferon-α2a (SEQ ID NO: 22), and interferon-α2c (SEQ ID NO: 24)). wherein the substitution comprises changing the amino acid at that position to an amino acid selected from the group consisting of alanine, glycine, or threonine. In embodiments, the modified interferon-α2 polypeptide comprises a substitution of one or more amino acids occupying positions selected from the group consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157 in native human interferon-α2 (including interferon-α2b (SEQ ID NO: 12), interferon-α2a (SEQ ID NO: 22), and interferon-α2c (SEQ ID NO: 24)). Such mutations reduce the immunogenicity of the modified interferon-α2 polypeptide compared to native human interferon-α-2. In embodiments, the modified interferon-α2 molecule is a modified interferon-α-2b polypeptide. In embodiments, the modified interferon-α2 polypeptide is a modified IFN-α2a polypeptide. In some embodiments, the modified interferon-α2 polypeptide is a modified IFN-α2c polypeptide. In some embodiments, the modified interferon-α2 polypeptides described herein are hyperglycosylated.In the polypeptide embodiments described above, the modified interferon-α2 polypeptide may be isolated, synthetic, or recombinant.

[0073] In some embodiments, the modified interferon-α2 polypeptides described herein are hyperglycosylated. Glycosylation of certain eukaryotic proteins occurs at specific positions along the polypeptide backbone, and generally, two types of glycosylation exist: O-glycosylation, in which oligosaccharides are attached to the "-OH" (hydroxyl) group of serine or threonine residues, and N-glycosylation, in which oligosaccharides are attached to asparagine residues. In particular, N-glycosylation occurs via the consensus sequence Asn-X-Ser / Thr, where X is any amino acid other than proline. All oligosaccharides attached to proteins via N-linkages share a pentasaccharide core consisting of three mannose residues and two N-acetylglucosamine residues. The sugars attached to this pentasaccharide core can exhibit a wide variety of oligosaccharide patterns. The presence or absence of these oligosaccharides may affect the physical properties of proteins and be important for their function, stability, secretion, and intracellular location. In embodiments, the modified interferon-α2 polypeptide comprises the addition of amino acids that comprise one or more sites for N- or O-glycosylation. In some embodiments, the modified interferon-α2 polypeptide as described herein comprises GMOP, a peptide sequence having the sequence APARSPSTQPWE (SEQ ID NO:26), or a fragment thereof, linked to a modified interferon-α2 sequence. In some embodiments, the fragment of the sequence APARSPSTQPWE is at least 7, at least 8, at least 9, and / or at least 10 amino acids in length. GMOP is derived from the N-terminal region of a 14-amino acid peptide (SEQ ID NO:26) and is a granulocyte colony-stimulating factor and human macrophage-CSF (hGM-CSF), a growth factor that stimulates the proliferation and maturation of hematopoietic progenitor cells of various cell lineages. It is secreted by many cell types (endothelial cells, fibroblasts, macrophages, T cells, mast cells) in response to specific signals and acts in a paracrine manner. hGM-CSF is a monomeric glycoprotein that, in its mature form, consists of 127 amino acids and exhibits a molecular weight between 14.5 and 32 kDa. This molecular heterogeneity is due to two N-glycosylation sites, N44 and N54, and four O-glycosylation sites, S22, S24, S26, and T27, in the N-terminal region (corresponding to S5, S7, S9, and T10, respectively, of the mature form of hGM-CSF). The first seven amino acids of mature hGM-CSF (APARSPS) form a linear epitope recognized by an anti-hGM-CSF monoclonal antibody (mAb CC1H7). The interaction between this epitope and the corresponding paratope exhibits affinity changes with ionic strength, providing operational advantages for the development of immunochemical techniques such as enzyme-linked immunosorbent assays (ELISAs), immunoaffinity chromatography, and Western blotting (Perotti, Oggero, Etcheverrigaray, Kratje, AR057215A1).

[0074] The addition of the GMOP sequence adds 4 to 6 O-glycosylation sites to the molecule of the invention. In this manner, modified interferon-α2 to which one or more GMOP peptide sequences (APARSPSTQPWE) or fragments thereof have been added is referred to as modified GMOP-interferon-α2 (e.g., it may also be referred to as GMOP-IFN-α2, etc.). The addition of this peptide sequence can be accomplished using any technique known in the art. In embodiments, the GMOP peptide sequence or tag (APARSPSTQPWE) or fragments thereof can be located at the amino-terminal end of the modified interferon-α2 polypeptide sequence and / or the carboxyl-terminal end of the modified interferon-α2 sequence. In embodiments, the fragment of APARSPSTQPWE is at least 7, at least 8, at least 9, and / or at least 10 amino acids in length. In a preferred embodiment, the GMOP peptide sequence (SEQ ID NO: 26) is added to the N-terminus of the modified interferon-α2 sequence.

[0075] In embodiments, the modified interferon-α2 also includes the addition of amino acids that comprise one or more sites for N- or O-glycosylation, wherein these added amino acids consist of one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE or a fragment thereof. In embodiments, the modified interferon-α2 disclosed herein includes the addition of one or more of the amino acid sequence APARSPSTQPWE (SEQ ID NO:26) or a fragment thereof. In embodiments, the modified interferon-α2 includes the addition of amino acids that comprise one or more sites for N- or O-glycosylation, wherein these added amino acids consist of one or more sequences having at least 70%, 80%, or 90% homology to APARSPSTQPWE (SEQ ID NO:26) or a fragment thereof, wherein the amino acids at positions 5, 7, 9, and 10 of the sequence of SEQ ID NO:26 are not substituted. In embodiments, the above amino acids containing one or more sites for N- or O-glycosylation (e.g., one or more sequences above having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE or a fragment thereof) may be added to the N-terminus and / or C-terminus of the modified interferon-α2 polypeptides disclosed herein. In embodiments, the fragments of APARSPSTQPWE are at least 7, at least 8, at least 9, and / or at least 10 amino acids in length. In the above polypeptide embodiments, the modified interferon-α2 polypeptide may be isolated, synthetic, or recombinant.

[0076] [Modified interferon-α2b polypeptides and nucleic acids] In embodiments, the modified interferon-α2 polypeptide of the present disclosure is a modified interferon-α2b polypeptide that has interferon-α2b activity and reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In embodiments, the modified interferon-α2b polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2b (SEQ ID NO: 12) and has one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157. In embodiments, the modified interferon-α2b polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and has one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitution comprises changing the amino acid at said position to alanine, glycine, or threonine. In embodiments, the modified interferon-α2b polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and has at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157. In several embodiments, the modified interferon-α2b polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2b (SEQ ID NO: 12) and has at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine.In the above polypeptide embodiments, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0077] In embodiments, modified interferon-α2b polypeptides with interferon-α2b activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and have amino acid substitutions at positions 9, 47, 117, 123, and 128, which change the amino acids at those positions to alanine, glycine, or threonine. In embodiments, modified interferon-α2b polypeptides with interferon-α2b activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and have the mutations L9A, F47A, L117A, F123A, and L128A. In the above polypeptide embodiments, the modified interferon-α2b polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In the above polypeptide embodiments, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0078] In embodiments, modified interferon-α2b polypeptides with interferon-α2b activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and have amino acid substitutions at positions 9, 47, 117, 123, 128, 147, and 157, which change the amino acids at those positions to alanine, glycine, or threonine. In embodiments, modified interferon-α2b polypeptides with interferon-α2b activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and have the mutations L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In the above polypeptide embodiments, the modified interferon-α2b polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In the above polypeptide embodiments, the modified interferon-α2b polypeptide can be isolated, synthetic, or recombinant.

[0079] In embodiments, modified interferon-α2b polypeptides with interferon-α2b activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and have amino acid substitutions at positions 9, 47, 65, 66, 117, 123, and 128, where the substitutions change the amino acids at those positions to alanine, glycine, or threonine. In embodiments, modified interferon-α2b polypeptides with interferon-α2b activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2b (SEQ ID NO: 12) and have the mutations L9A, F47A, N65A, L66A, L117A, F123A, and L128A. In the above polypeptide embodiments, the modified interferon-α2b polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In the above polypeptide embodiments, the modified interferon-α2b polypeptide can be isolated, synthetic, or recombinant.

[0080] In several embodiments, the modified interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2b (SEQ ID NO: 12) and has amino acid substitutions at positions 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, which substitutions change the amino acids at those positions to alanine, glycine, or threonine. In some embodiments, the modified interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2b (SEQ ID NO: 12) and has the mutations L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A. In some embodiments of the above polypeptides, the modified interferon-α2b polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In some embodiments of the above polypeptides, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0081] In embodiments, the modified interferon-α2b polypeptide having interferon-α2b activity is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20. In embodiments, the modified interferon-α2b polypeptide having interferon-α2b activity is selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 18. In embodiments, the modified interferon-α2b polypeptide comprises the amino acid sequence of SEQ ID NO: 14, and in embodiments, the modified interferon-α2b polypeptide comprises the amino acid sequence of SEQ ID NO: 20. In preferred embodiments, the modified interferon-α2b polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In embodiments, the modified interferon-α2b polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In the above polypeptide embodiments, the modified interferon-α2b polypeptide has a reduced immunogenicity or tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In the above polypeptide embodiments, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0082] In embodiments, modified interferon-α2b polypeptides disclosed herein with interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have a relative antiviral activity of between 5% and 95% compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In embodiments, modified interferon-α2b polypeptides disclosed herein with interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. In embodiments, modified interferon-α2b polypeptides disclosed herein with interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have a relative antiviral activity of between 20% and 80% compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12.

[0083] In some embodiments, the modified interferon-α2b polypeptides disclosed herein that have interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have a rate of antiproliferative biological activity of between 0% and 50%. In some embodiments, the modified interferon-α2b polypeptides that have interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have a rate of antiproliferative biological activity of less than 10%. In some embodiments, the modified interferon-α2b polypeptides that have interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have a rate of antiproliferative biological activity of less than 5%.

[0084] In some embodiments, the modified interferon-α2b polypeptides disclosed herein having interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have an apparent plasma clearance rate (Cl ) of between 5 mL / h and 200 mL / h. app In embodiments, modified interferon-α2b polypeptides having interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 115 mL / h. app In embodiments, the modified interferon-α2b polypeptides having interferon-α2b activity, such as the modified interferon-α2b polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 50 mL / h. app )

[0085] In embodiments, the present disclosure provides polynucleotides or nucleic acids (e.g., DNA, including cDNA, or RNA, including mRNA) encoding modified interferon-α2b polypeptides having interferon-α2b activity, such as the modified interferon-α2b polypeptides described above. For example, in embodiments, the present disclosure provides nucleic acids encoding one or more modified interferon-α2b polypeptides selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20. In embodiments, the present disclosure provides nucleic acids encoding one or more modified interferon-α2b polypeptides selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18. In embodiments, the nucleic acid encoding one or more modified interferon-α2b polypeptides comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19. In embodiments, the nucleic acid encoding one or more modified interferon-α2b polypeptides comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:17. In embodiments, the nucleic acid encoding the modified interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO:13. In embodiments, the nucleic acid encoding the modified interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO: 19. In preferred embodiments, the nucleic acid encoding the modified interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO: 15. In preferred embodiments, the nucleic acid encoding the modified interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO: 17.

[0086] In embodiments, the modified interferon-α2b comprises the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein the added amino acids comprise one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE, or a fragment thereof. In embodiments, the modified interferon-α2b disclosed herein comprises the addition of one or more of the amino acid sequence APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof. In embodiments, the modified interferon-α2b comprises the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein the added amino acids comprise one or more sequences having at least 70%, 80%, or 90% homology to APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof, wherein the amino acids at positions 5, 7, 9, and 10 of SEQ ID NO: 26 are not substituted. In embodiments, the amino acids (e.g., a sequence having at least 60%, 70%, 80%, 90%, or 95% homology to the sequence APARSPSTQPWE) or a fragment thereof containing one or more N- or O-glycosylation sites may be added to the N- and / or C-terminus of the modified interferon-α2b polypeptide disclosed herein. In embodiments, the fragment of APARSPSTQPWE is at least 5, at least 6, at least 7, at least 8, at least 9, and / or at least 10 amino acids in length. In the above polypeptide embodiments, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0087] In embodiments, a vector or plasmid is provided that includes a nucleic acid of the present disclosure encoding one or more modified interferon-α2b polypeptides of the present disclosure, for example, a nucleic acid (e.g., DNA or RNA) encoding a modified interferon-α2b polypeptide having a sequence including (consisting essentially of, or consisting of) one or more of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19. In embodiments, the present disclosure is directed to a cell that includes a vector or plasmid of the present disclosure.

[0088] [Modified GMOP-interferon-α2b polypeptides and nucleic acids] In some embodiments, the modified interferon-α2 polypeptides of the present disclosure, including the modified IFNα-2b polypeptides described above, are modified GMOP-interferon-α2b polypeptides having interferon-α2b activity and reduced immunogenicity or a reduced propensity to elicit an immune response compared to wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In some embodiments, the modified IFNα-2b polypeptides include additions of amino acids that comprise one or more sites for N- or O-glycosylation, and these added amino acids include one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE or a fragment thereof. In some embodiments, the modified IFNα-2b as disclosed herein includes one or more additions of the amino acid sequence APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof. In some embodiments, the modified IFNα-2b comprises the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein these additional amino acids consist of one or more sequences having at least 70%, 80%, or 90% identity to APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof, where the amino acids at positions 5, 7, 9, and 10 of SEQ ID NO: 26 are not substituted. In some embodiments, the aforementioned amino acids comprising one or more N- or O-glycosylation sites (e.g., the one or more sequences having at least 60%, 70%, 80%, 90%, or 95% identity to APARSPSTQPWE) or a fragment thereof may be added to the N-terminus and / or C-terminus of the modified IFNα-2b polypeptide disclosed herein. In some embodiments, the fragment of the sequence APARSPSTQPWE is at least 5, at least 6, at least 7, at least 8, at least 9, and / or at least 10 amino acids in length.

[0089] In several embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171. In several embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2b (SEQ ID NO: 10) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitution comprises changing the amino acid at said position to alanine, glycine, or threonine. In several embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171. In embodiments, a modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions change the amino acid at said position to alanine, glycine, or threonine. In the above polypeptide embodiments, the modified GMOP-interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0090] In some embodiments, the modified GMOP-interferon-α2b polypeptide with interferon-α2b activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 131, 137, and 142, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2b polypeptide with interferon-α2b activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has the mutations L23A, F61A, L131A, F137A, and L142A. In the above polypeptide embodiments, the modified GMOP-interferon-α2b polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or the wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In the above polypeptide embodiments, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0091] In several embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% homology to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 131, 137, 142, 161, and 171, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has the mutations L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2b polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In some embodiments of the above polypeptides, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0092] In embodiments, a modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and comprises one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 79, 80, 131, 137, and 142, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has the mutations L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2b polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In some embodiments of the above polypeptides, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0093] In several embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2b (SEQ ID NO: 10) and has the following mutations: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2b polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In some embodiments of the above polypeptides, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0094] In embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. In embodiments, the modified GMOP-interferon-α2b polypeptide having interferon-α2b activity is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6. In embodiments, the modified GMOP-interferon-α2b polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In embodiments, the modified GMOP-interferon-α2b polypeptide consists of the amino acid sequence of SEQ ID NO: 8. In embodiments, the modified GMOP-interferon-α2b polypeptide consists of the amino acid sequence of SEQ ID NO: 4. In embodiments, the modified GMOP-interferon-α2b polypeptide comprises the amino acid sequence of SEQ ID NO: 6. In the above polypeptide embodiments, the modified interferon-α2b polypeptide has reduced immunogenicity and a reduced tendency to elicit an immune response compared to wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In the above polypeptide embodiments, the modified interferon-α2b polypeptide may be isolated, synthetic, or recombinant.

[0095] In embodiments, modified GMOP-interferon-α2b polypeptides having interferon-α2b activity, such as the modified GMOP-interferon-α2b polypeptides described above, have a relative antiviral activity of 5% to 95% compared to the wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In embodiments, modified GMOP-interferon-α2b polypeptides having interferon-α2b activity, such as the modified GMOP-interferon-α2b polypeptides described above, have a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10). In several embodiments, modified GMOP-interferon-α2b polypeptides having interferon-α2b activity, such as the modified GMOP-interferon-α2b polypeptides described above, have a relative antiviral activity of between 20% and 80% compared to wild-type interferon-α2b polypeptide (SEQ ID NO: 12) and / or wild-type GMOP-interferon-α2b (SEQ ID NO: 10).

[0096] In some embodiments, the modified GMOP-interferon-α2b polypeptides disclosed herein with interferon-α2b activity, such as those described above, have a rate of antiproliferative biological activity of between 0% and 50%. In some embodiments, the modified GMOP-interferon-α2b polypeptides with interferon-α2b activity, such as those described above, have a rate of antiproliferative biological activity of less than 10%. In some embodiments, the modified GMOP-interferon-α2b polypeptides with interferon-α2b activity, such as those described above, have a rate of antiproliferative biological activity of less than 5%.

[0097] In embodiments, the modified GMOP-interferon-α2b polypeptides disclosed herein that have interferon-α2b activity, such as the modified GMOP-interferon-α2b polypeptides described above, have an apparent plasma clearance rate (Cl ) of between 5 mL / h and 200 mL / h. app In embodiments, modified GMOP-interferon-α2b polypeptides having interferon-α2b activity, such as the modified GMOP-interferon-α2b polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 115 mL / h. app In embodiments, a modified GMOP-interferon-α2b polypeptide having interferon-α2b activity, such as the modified GMOP-interferon-α2b polypeptides described above, has an apparent plasma clearance rate (Cl ) of less than 50 mL / h. app )

[0098] In embodiments, the present disclosure provides polynucleotides or nucleic acids (e.g., DNA, including cDNA, or RNA, including mRNA) encoding modified GMOP-interferon-α2b polypeptides having interferon-α2b activity, such as the modified GMOP-interferon-α2b polypeptides described above. For example, in embodiments, the present disclosure provides nucleic acids encoding one or more modified GMOP-interferon-α2b polypeptides selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In embodiments, the present disclosure provides nucleic acids encoding one or more modified GMOP-interferon-α2b polypeptides selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6. In embodiments, the nucleic acid encoding one or more modified GMOP-interferon-α2b polypeptides comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. The nucleic acid encoding one or more modified GMOP-interferon-α2b polypeptides comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5. In embodiments, the nucleic acid encoding the modified GMOP-interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO: 1. In embodiments, the nucleic acid encoding the modified GMOP-interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO: 7. In preferred embodiments, the nucleic acid encoding the modified GMOP-interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO: 3. In preferred embodiments, the nucleic acid encoding the modified GMOP-interferon-α2b polypeptide comprises the nucleic acid sequence of SEQ ID NO: 5.

[0099] In embodiments, provided are vectors or plasmids comprising a nucleic acid of the invention encoding one or more modified GMOP-interferon-α2b polypeptides of the present disclosure, for example, a nucleic acid (e.g., DNA or RNA) encoding at least one modified GMOP-interferon-α2b polypeptide having a sequence including (or consisting essentially of) one or more of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7. In embodiments, the present disclosure is directed to cells comprising a vector or plasmid of the present disclosure.

[0100] [Modified interferon-α2a polypeptides and nucleic acids] In embodiments, the modified interferon-α2a polypeptides of the present disclosure are modified interferon-α2a polypeptides that have interferon-α2a activity and reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In embodiments, the modified interferon-α2a polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2a (SEQ ID NO: 22) and has one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157. In embodiments, the modified interferon-α2a polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and has one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In embodiments, the modified interferon-α2a polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and has at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157. In several embodiments, the modified interferon-α2a polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2a (SEQ ID NO: 22) and has at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine.In the above polypeptide embodiments, the modified interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0101] In embodiments, modified interferon-α2a polypeptides with interferon-α2a activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and have amino acid substitutions at positions 9, 47, 117, 123, and 128, where the substitutions change the amino acids at those positions to alanine, glycine, or threonine. In embodiments, modified interferon-α2a polypeptides with interferon-α2a activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and have the mutations L9A, F47A, L117A, F123A, and L128A. In the above polypeptide embodiments, the modified interferon-α2a polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In the above polypeptide embodiments, the modified interferon-α2a polypeptide can be isolated, synthetic, or recombinant.

[0102] In embodiments, modified interferon-α2a polypeptides with interferon-α2a activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and have amino acid substitutions at positions 9, 47, 117, 123, 128, 147, and 157, where the substitutions change the amino acids at those positions to alanine, glycine, or threonine. In embodiments, modified interferon-α2a polypeptides with interferon-α2a activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and have the mutations L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In the above polypeptide embodiments, the modified interferon-α2a polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In the above polypeptide embodiments, the modified interferon-α2a polypeptide can be isolated, synthetic, or recombinant.

[0103] In embodiments, modified interferon-α2a polypeptides with interferon-α2a activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and have amino acid substitutions at positions 9, 47, 65, 66, 117, 123, and 128, where the substitutions change the amino acids at those positions to alanine, glycine, or threonine. In embodiments, modified interferon-α2a polypeptides with interferon-α2a activity comprise an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2a (SEQ ID NO: 22) and have the mutations L9A, F47A, N65A, L66A, L117A, F123A, and L128A. In the above polypeptide embodiments, the modified interferon-α2a polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In the above polypeptide embodiments, the modified interferon-α2a polypeptide can be isolated, synthetic, or recombinant.

[0104] In several embodiments, the modified interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2a (SEQ ID NO: 22) and has amino acid substitutions at positions 9, 47, 117, 123, 128, 147, and 157, wherein the substitutions consist of changing the amino acids at those positions to alanine, glycine, or threonine. In some embodiments, the modified interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2a (SEQ ID NO: 22) and has the mutations L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A. In some embodiments of the above polypeptides, the modified interferon-α2a polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In some embodiments of the above polypeptides, the modified interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0105] In embodiments, the modified interferon-α2a polypeptide having interferon-α2a activity is selected from the group consisting of SEQ ID NOs: 31-34. In embodiments, the modified interferon-α2a polypeptide having interferon-α2a activity is selected from the group consisting of SEQ ID NOs: 32 and 33. In embodiments, the modified interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 31. In embodiments, the modified interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 34. In a preferred embodiment, the modified interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 32. In embodiments, the modified interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 33. In the above polypeptide embodiments, the modified interferon-α2a polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In the above polypeptide embodiments, the modified interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0106] In embodiments, modified interferon-α2a polypeptides having interferon-α2a activity, such as the modified interferon-α2a polypeptides described above, have a relative antiviral activity of between 5% and 95% compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In embodiments, modified interferon-α2a polypeptides having interferon-α2a activity, such as the modified interferon-α2a polypeptides described above, have a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. In embodiments, modified interferon-α2a polypeptides having interferon-α2a activity, such as the modified interferon-α2a polypeptides described above, have a relative antiviral activity of between 20% and 80% compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22.

[0107] In some embodiments, the modified interferon-α2a polypeptides disclosed herein with interferon-α2a activity, such as those described above, have a rate of antiproliferative biological activity of between 0% and 50%. In some embodiments, the modified interferon-α2a polypeptides with interferon-α2a activity, such as those described above, have a rate of antiproliferative biological activity of less than 10%. In some embodiments, the modified interferon-α2a polypeptides with interferon-α2a activity, such as those described above, have a rate of antiproliferative biological activity of less than 5%.

[0108] In embodiments, the modified interferon-α2a polypeptides disclosed herein that have interferon-α2a activity, such as the modified interferon-α2a polypeptides described above, have an apparent plasma clearance rate (Cl ) of between 5 mL / h and 200 mL / h. app In embodiments, modified interferon-α2a polypeptides having interferon-α2a activity, such as the modified interferon-α2a polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 115 mL / h. app In embodiments, modified interferon-α2a polypeptides having interferon-α2a activity, such as the modified interferon-α2a polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 50 mL / h. app )

[0109] In some embodiments, the present disclosure provides polynucleotides or nucleic acids (e.g., DNA, including cDNA, or RNA, including mRNA) encoding modified interferon-α2a polypeptides having interferon-α2a activity, such as the modified interferon-α2a polypeptides described above. For example, in some embodiments, the present disclosure provides a nucleic acid encoding a modified interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 22 and has the following amino acid substitutions: L9A, F47A, L117A, F123A, and L128A. In some embodiments, the present disclosure provides a nucleic acid encoding a modified interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 22 and has the following amino acid substitutions: L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In embodiments, the disclosure provides a nucleic acid encoding a modified interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 22 and has the following amino acid substitutions: L9A, F47A, N65A, L66A, L117A, F123A, and L128A. In embodiments, the disclosure provides a nucleic acid encoding a modified interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 22 and has the following amino acid substitutions: L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A.

[0110] In embodiments, the modified interferon-α2a comprises the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein these added amino acids comprise one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE, or a fragment thereof. In embodiments, the modified interferon-α2a disclosed herein comprises the addition of one or more of the amino acid sequence APARSPSTQPWE (SEQ ID NO:26) or a fragment thereof. In embodiments, the modified interferon-α2a comprises the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein these added amino acids comprise one or more sequences having at least 70%, 80%, or 90% homology to APARSPSTQPWE (SEQ ID NO:26) or a fragment thereof, wherein the amino acids at positions 5, 7, 9, and 10 of SEQ ID NO:26 are not substituted. In embodiments, the amino acids containing one or more N- or O-glycosylation sites (e.g., the one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE) or fragments thereof may be added to the N- and / or C-terminus of the modified interferon-α2a polypeptides disclosed herein. In embodiments, the fragments of APARSPSTQPWE are at least 5, at least 6, at least 7, at least 8, at least 9, and / or at least 10 amino acids in length. In the above polypeptide embodiments, the modified interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0111] In some embodiments, a nucleic acid of the present disclosure encoding one or more modified interferon-α2a polypeptides of the present disclosure is provided, for example, but not limited to, a vector or plasmid comprising a nucleic acid (e.g., DNA or RNA) encoding at least one modified interferon-α2a polypeptide. In some embodiments, the present disclosure is directed to a cell comprising a vector or plasmid of the present disclosure.

[0112] [Modified GMOP-interferon-α2a polypeptides and nucleic acids] In some embodiments, the modified interferon-α2 polypeptides disclosed herein are modified GMOP-interferon-α2a polypeptides that have interferon-α2a activity and have reduced immunogenicity or a decreased tendency to elicit an immune response compared to wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or wild-type GMOP-interferon-α2a (SEQ ID NO: 21). In some embodiments, the modified IFNα-2a polypeptides include the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein these added amino acids comprise one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE or a fragment thereof. In some embodiments, the modified IFNα-2a disclosed herein includes the addition of one or more of the amino acid sequence APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof. In some embodiments, the modified IFNα-2a also includes the addition of amino acids containing one or more N- or O-glycosylation sites, wherein these added amino acids comprise one or more sequences having at least 70%, 80%, or 90% identity to APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof, and the amino acids at positions 5, 7, 9, and 10 of SEQ ID NO: 26 are not substituted. In some embodiments, the amino acids containing one or more N- or O-glycosylation sites (e.g., the one or more sequences having at least 60%, 70%, 80%, 90%, or 95% identity to APARSPSTQPWE or a fragment thereof) may be added to the N-terminus and / or C-terminus of the modified IFNα-2a polypeptide disclosed herein. In some embodiments, the fragment of APARSPSTQPWE is at least 5, at least 6, at least 7, at least 8, at least 9, and / or at least 10 amino acids in length.

[0113] In several embodiments, the modified GMOP-interferon-α2a polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171. In several embodiments, the modified GMOP-interferon-α2a polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitution consists of changing the amino acid at that position to alanine, glycine, or threonine. In embodiments, the modified GMOP-interferon-α2a polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171. In embodiments, the modified GMOP-interferon-α2a polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2a (SEQ ID NO: 21). and at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein said substitutions comprise changing the amino acids at said positions to alanine, glycine, or threonine. In the above polypeptide embodiments, the modified GMOP-interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0114] In some embodiments, the modified GMOP-interferon-α2a polypeptide with interferon-α2a activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 131, 137, and 142, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2a polypeptide with interferon-α2a activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has the mutations L23A, F61A, L131A, F137A, and L142A. In embodiments of the above polypeptides, the modified GMOP-interferon-α2a polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or wild-type GMOP-interferon-α2a (SEQ ID NO: 21). In embodiments of the above polypeptides, the modified GMOP-interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0115] In several embodiments, the modified GMOP-interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 131, 137, 142, 161, and 171, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has the mutations L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2a polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or wild-type GMOP-interferon-α2a (SEQ ID NO: 21). In some embodiments of the above polypeptides, the modified GMOP-interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0116] In several embodiments, the modified GMOP-interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 79, 80, 131, 137, and 142, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has the mutations L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2a polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or wild-type GMOP-interferon-α2a (SEQ ID NO: 21). In some embodiments of the above polypeptides, the modified GMOP-interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0117] In several embodiments, the modified GMOP-interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In embodiments, the modified GMOP-interferon-α2a polypeptide having interferon-α2a activity comprises an amino acid sequence having at least 70% homology to wild-type GMOP-interferon-α2a (SEQ ID NO: 21) and has the following mutations: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A. In these polypeptide embodiments, the modified GMOP-interferon-α2a polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or wild-type GMOP-interferon-α2a (SEQ ID NO: 21). In these polypeptide embodiments, the modified GMOP-interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0118] In embodiments, the modified GMOP-interferon-α2a polypeptide having interferon-α2a activity is selected from the group consisting of SEQ ID NOs: 27-30. In embodiments, the modified GMOP-interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 28 and SEQ ID NO: 29. In embodiments, the modified GMOP-interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 27. In embodiments, the modified GMOP-interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In embodiments, the modified GMOP-interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 28. In embodiments, the modified GMOP-interferon-α2a polypeptide comprises the amino acid sequence of SEQ ID NO: 29. In embodiments of the above polypeptides, the modified interferon-α2a polypeptide has a reduced tendency to be immunogenic or to elicit an immune response compared to wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or wild-type GMOP-interferon-α2a (SEQ ID NO: 21). In the above polypeptide embodiments, the modified interferon-α2a polypeptide may be isolated, synthetic, or recombinant.

[0119] In embodiments, modified GMOP-interferon-α2a polypeptides having interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have a relative antiviral activity of 5% to 95% compared to the wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or the wild-type GMOP-interferon-α2a polypeptide (SEQ ID NO: 21). In embodiments, modified GMOP-interferon-α2a polypeptides having interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or the wild-type GMOP-interferon-α2a polypeptide (SEQ ID NO: 21). In several embodiments, modified GMOP-interferon-α2a polypeptides having interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have a relative antiviral activity of between 20% and 80% compared to the wild-type interferon-α2a polypeptide (SEQ ID NO: 22) and / or the wild-type GMOP-interferon-α2a polypeptide (SEQ ID NO: 21).

[0120] In embodiments, modified GMOP-interferon-α2a polypeptides disclosed herein that have interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have a rate of antiproliferative biological activity of between 0% and 50%. In embodiments, modified GMOP-interferon-α2a polypeptides that have interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have a rate of antiproliferative biological activity of less than 10%. In embodiments, modified GMOP-interferon-α2a polypeptides that have interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have a rate of antiproliferative biological activity of less than 5%.

[0121] In embodiments, the modified GMOP-interferon-α2a polypeptides disclosed herein that have interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have an apparent plasma clearance rate (Cl ) of between 5 mL / h and 200 mL / h. app In embodiments, modified GMOP-interferon-α2a polypeptides having interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 115 mL / h. app In embodiments, modified GMOP-interferon-α2a polypeptides having interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 50 mL / h. app )

[0122] In some embodiments, the present disclosure provides polynucleotides or nucleic acids (e.g., DNA, including cDNA, or RNA, including mRNA) encoding modified GMOP-interferon-α2a polypeptides having interferon-α2a activity, such as the modified GMOP-interferon-α2a polypeptides described above. For example, in some embodiments, the present disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 21 and has the following amino acid substitutions: L23A, F61A, L131A, F137A, and L142A. In some embodiments, the present disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 21 and has the following amino acid substitutions: L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In embodiments, the disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 21 and has the following amino acid substitutions: L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In embodiments, the disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 21 and has the following amino acid substitutions: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A.

[0123] In some embodiments, a nucleic acid of the present invention encoding one or more modified GMOP-interferon-α2a polypeptides of the present disclosure is provided, for example, but not limited to, a vector or plasmid comprising a nucleic acid (e.g., DNA or RNA) encoding at least one modified GMOP-interferon-α2a polypeptide. In some embodiments, the present disclosure is directed to a cell comprising a vector or plasmid of the present disclosure.

[0124] [Modified interferon-α2c polypeptides and nucleic acids]In embodiments, the modified interferon-α2 polypeptide of the present disclosure is a modified interferon-α2c polypeptide that has interferon-α2c activity and has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In embodiments, the modified interferon-α2c polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2c (SEQ ID NO: 24) and has one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157. In some embodiments, the modified interferon-α2c polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In some embodiments, the modified interferon-α2c polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157. In several embodiments, the modified interferon-α2c polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2c (SEQ ID NO: 24) and has at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions include changing the amino acid at said position to alanine, glycine, or threonine.In the above polypeptide embodiments, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0125] In embodiments, the modified interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has amino acid substitutions at positions 9, 47, 117, 123, and 128, which change the amino acids at said positions to alanine, glycine, or threonine. In embodiments, the modified interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has the mutations L9A, F47A, L117A, F123A, and L128A. In the above polypeptide embodiments, the modified interferon-α2c polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In the above polypeptide embodiments, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0126] In some embodiments, the modified interferon-α2c polypeptide has interferon-α2c activity and comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has amino acid substitutions at positions 9, 47, 117, 123, 128, 147, and 157, where the substitutions change the amino acids at those positions to alanine, glycine, or threonine. In some embodiments, the modified interferon-α2c polypeptide has interferon-α2c activity and comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has the mutations L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In the above polypeptide embodiments, the modified interferon-α2c polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In the above polypeptide embodiments, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0127] In some embodiments, the modified interferon-α2c polypeptide has interferon-α2c activity and comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has amino acid substitutions at positions 9, 47, 65, 66, 117, 123, and 128, where the substitutions change the amino acids at those positions to alanine, glycine, or threonine. In some embodiments, the modified interferon-α2c polypeptide has interferon-α2c activity and comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 24) and has the mutations L9A, F47A, N65A, L66A, L117A, F123A, and L128A. In the above polypeptide embodiments, the modified interferon-α2c polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In the above polypeptide embodiments, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0128] In several embodiments, the modified interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2c (SEQ ID NO: 24) and has amino acid substitutions at 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions consist of changing the amino acids at those positions to alanine, glycine, or threonine. In some embodiments, the modified interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type interferon-α2c (SEQ ID NO: 24) and has the mutations L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A. In some embodiments of the above polypeptides, the modified interferon-α2c polypeptide has reduced immunogenicity or a decreased tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In some embodiments of the above polypeptides, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0129] In embodiments, the modified interferon-α2c polypeptide having interferon-α2c activity is selected from the group consisting of SEQ ID NOs: 39-42. In embodiments, the modified interferon-α2c polypeptide having interferon-α2c activity is selected from the group consisting of SEQ ID NOs: 40 and 41. In embodiments, the modified interferon-α2c polypeptide comprises the amino acid sequence of SEQ ID NO: 39. In embodiments, the modified interferon-α2c polypeptide comprises the amino acid sequence of SEQ ID NO: 42. In a preferred embodiment, the modified interferon-α2c polypeptide comprises the amino acid sequence of SEQ ID NO: 40. In embodiments, the modified interferon-α2c polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In the above polypeptide embodiments, the modified interferon-α2c polypeptide has a reduced tendency to be immunogenic or to elicit an immune response compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In the above polypeptide embodiments, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0130] In embodiments, modified interferon-α2c polypeptides disclosed herein having interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have a relative antiviral activity of between 5% and 95% compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In embodiments, modified interferon-α2c polypeptides having interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. In embodiments, modified interferon-α2c polypeptides having interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have a relative antiviral activity of between 20% and 80% compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24.

[0131] In some embodiments, the modified interferon-α2c polypeptides disclosed herein that have interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have a rate of antiproliferative biological activity of between 0% and 50%. In some embodiments, the modified interferon-α2c polypeptides that have interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have a rate of antiproliferative biological activity of less than 10%. In some embodiments, the modified interferon-α2c polypeptides that have interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have a rate of antiproliferative biological activity of less than 5%.

[0132] In some embodiments, the modified interferon-α2c polypeptides disclosed herein having interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have an apparent plasma clearance rate (Cl ) of between 5 mL / h and 200 mL / h. app In some embodiments, the modified interferon-α2c polypeptides having interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 115 mL / h. app In embodiments, the modified interferon-α2c polypeptides having interferon-α2c activity, such as the modified interferon-α2c polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 50 mL / h. app )

[0133] In some embodiments, the present disclosure provides polynucleotides or nucleic acids (e.g., DNA, including cDNA, or RNA, including mRNA) encoding modified interferon-α2c polypeptides having interferon-α2c activity, such as the modified interferon-α2c polypeptides described above. For example, in some embodiments, the present disclosure provides a nucleic acid encoding a gamma-modified interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and has the following amino acid substitutions: L9A, F47A, L117A, F123A, and L128A. In some embodiments, the present disclosure provides a nucleic acid encoding a modified interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and has the following amino acid substitutions: L9A, F47A, L117A, F123A, L128A, I147T, and L157A. In embodiments, the disclosure provides a nucleic acid encoding a modified interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and has the following amino acid substitutions: L9A, F47A, N65A, L66A, L117A, F123A, and L128A. In embodiments, the disclosure provides a nucleic acid encoding a modified interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and has the following amino acid substitutions: L9A, L17A, F47A, N65A, L66A, L117A, F123A, L128A, I147T, and L157A.

[0134] In embodiments, the modified interferon-α2c also includes the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein these added amino acids comprise one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE, or a fragment thereof. In embodiments, the modified interferon-α2c disclosed herein includes the addition of one or more of the amino acid sequence APARSPSTQPWE (SEQ ID NO:26) or a fragment thereof. In embodiments, the modified interferon-α2c includes the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein these added amino acids comprise one or more sequences having at least 70%, 80%, or 90% homology to APARSPSTQPWE (SEQ ID NO:26) or a fragment thereof, wherein the amino acids at positions 5, 7, 9, and 10 of SEQ ID NO:26 are not substituted. In embodiments, the amino acids comprising one or more N- or O-glycosylation sites (e.g., the one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE) or fragments thereof may be added to the N-terminus and / or C-terminus of the modified interferon-α2c polypeptides disclosed herein. In embodiments, the fragments of APARSPSTQPWE are at least 5, at least 6, at least 7, at least 8, at least 9, and / or at least 10 amino acids in length. In the above polypeptide embodiments, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0135] In some embodiments, a nucleic acid of the present disclosure is provided that encodes one or more modified interferon-α2c polypeptides of the present disclosure, for example, but not limited to, a vector or plasmid comprising a nucleic acid (e.g., DNA or RNA) encoding at least one modified interferon-α2c polypeptide. In some embodiments, the present disclosure is directed to a cell comprising a vector or plasmid of the present disclosure.

[0136] [Modified GMOP-Interferon-α2c Polypeptides and Nucleic Acids] In some embodiments, the modified interferon-α2 polypeptides disclosed herein, including those described above, are modified GMOP-interferon-α2c polypeptides that have interferon-α2c activity and have reduced immunogenicity or a reduced tendency to induce an immune response compared to wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In some embodiments, the modified IFN-α2c polypeptides include the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein these added amino acids comprise one or more sequences having at least 60%, 70%, 80%, 90%, or 95% homology to APARSPSTQPWE or a fragment thereof. In some embodiments, the modified IFN-α2c disclosed herein includes the addition of one or more of the amino acid sequence APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof. In some embodiments, the modified IFN-α-2c comprises the addition of amino acids comprising one or more N- or O-glycosylation sites, wherein the added amino acids comprise one or more sequences having at least 70%, 80%, or 90% identity to APARSPSTQPWE (SEQ ID NO: 26) or a fragment thereof, and wherein the amino acids at positions 5, 7, 9, and 10 of SEQ ID NO: 26 are not substituted. In some embodiments, the amino acids comprising one or more N- or O-glycosylation sites (e.g., the one or more sequences having at least 60%, 70%, 80%, 90%, or 95% identity to APARSPSTQPWE or a fragment thereof) may be added to the N-terminus and / or C-terminus of the modified IFN-α2c polypeptide disclosed herein. In some embodiments, the fragment of the sequence APARSPSTQPWE is at least 5, at least 6, at least 7, at least 8, at least 9, and / or at least 10 amino acids in length.

[0137] In embodiments, the modified GMOP-interferon-α2c polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171. In embodiments, the modified GMOP-interferon-α2c polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type interferon-α2c (SEQ ID NO: 23) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. In several embodiments, the modified GMOP-interferon-α2c polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171. In embodiments, the modified GMOP-interferon-α2c polypeptide comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions change the amino acid at said position to alanine, glycine, or threonine. In the above polypeptide embodiments, the modified GMOP-interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0138] In some embodiments, the modified GMOP-interferon-α2c polypeptide with interferon-α2c activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 131, 137, and 142, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2c polypeptide with interferon-α2c activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has the mutations L23A, F61A, L131A, F137A, and L142A. In embodiments of the above polypeptides, the modified GMOP-interferon-α2c polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or the wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In embodiments of the above polypeptides, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0139] In several embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 131, 137, 142, 161, and 171, wherein the substitutions include changing the amino acid at the position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has the mutations L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2c polypeptide has reduced immunogenicity or a tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In some embodiments of the above polypeptides, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0140] In several embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% homology to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 79, 80, 131, 137, and 142, wherein the substitutions include changing the amino acid at said position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% homology to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has the mutations L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2c polypeptide has reduced immunogenicity or a tendency to elicit an immune response compared to the wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In some embodiments of the above polypeptides, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0141] In several embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% homology to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and has one or more amino acid substitutions at any position selected from the set consisting of 23, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitution comprises changing the amino acid at that position to alanine, glycine, or threonine. In some embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity comprises an amino acid sequence at least 60%, 70%, 80%, 90%, or 95% identical to wild-type GMOP-interferon-α2c (SEQ ID NO: 23) and includes the following mutations: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A. In some embodiments of the above polypeptides, the modified GMOP-interferon-α2c polypeptide has reduced immunogenicity or a tendency to elicit a reduced immune response compared to the wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In some embodiments of the above polypeptides, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0142] In embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity is selected from the group consisting of SEQ ID NOs: 35-38. In embodiments, the modified GMOP-interferon-α2c polypeptide having interferon-α2c activity is selected from the group consisting of SEQ ID NOs: 35 and 36. In embodiments, the modified GMOP-interferon-α2c polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In embodiments, the modified GMOP-interferon-α2c polypeptide consists of the amino acid sequence of SEQ ID NO: 38. In embodiments, the modified GMOP-interferon-α2c polypeptide consists of the amino acid sequence of SEQ ID NO:36. In embodiments, the modified GMOP-interferon-α2c polypeptide consists of the amino acid sequence of SEQ ID NO: 37. In the above polypeptide embodiments, the modified interferon-α2c polypeptide has reduced immunogenicity or a reduced tendency to elicit an immune response compared to wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In the above polypeptide embodiments, the modified interferon-α2c polypeptide may be isolated, synthetic, or recombinant.

[0143] In embodiments, modified GMOP-interferon-α2c polypeptides having interferon-α2c activity, such as those described above, have a relative antiviral activity of between 5% and 95% compared to the wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In embodiments, modified GMOP-interferon-α2c polypeptides having interferon-α2c activity, such as those described above, have a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23). In several embodiments, modified GMOP-interferon-α2c polypeptides having interferon-α2c activity, such as the modified GMOP-interferon-α2c polypeptides described above, have a relative antiviral activity of between 20% and 80% compared to wild-type interferon-α2c polypeptide (SEQ ID NO: 24) and / or wild-type GMOP-interferon-α2c (SEQ ID NO: 23).

[0144] In some embodiments, the modified GMOP-interferon-α2c polypeptides disclosed herein with interferon-α2c activity, such as those described above, have a rate of antiproliferative biological activity of between 0% and 50%. In some embodiments, the modified GMOP-interferon-α2c polypeptides with interferon-α2c activity, such as those described above, have a rate of antiproliferative biological activity of less than 10%. In some embodiments, the modified GMOP-interferon-α2c polypeptides with interferon-α2c activity, such as those described above, have a rate of antiproliferative biological activity of less than 5%.

[0145] In embodiments, the modified GMOP-interferon-α2c polypeptides disclosed herein that have interferon-α2c activity, such as the modified GMOP-interferon-α2c polypeptides described above, have an apparent plasma clearance rate (Cl ) of between 5 mL / h and 200 mL / h. app In embodiments, a modified GMOP-interferon-α2c polypeptide having interferon-α2c activity, such as the modified GMOP-interferon-α2c polypeptides described above, has an apparent plasma clearance rate (Cl ) of less than 115 mL / h. app In embodiments, modified GMOP-interferon-α2c polypeptides having interferon-α2c activity, such as the modified GMOP-interferon-α2c polypeptides described above, have an apparent plasma clearance rate (Cl ) of less than 50 mL / h. app )

[0146] In some embodiments, the present disclosure provides polynucleotides or nucleic acids (e.g., DNA, including cDNA, or RNA, including mRNA) encoding modified GMOP-interferon-α2c polypeptides having interferon-α2c activity, such as the modified GMOP-interferon-α2c polypeptides described above. For example, in some embodiments, the present disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 23 and has the following amino acid substitutions: L23A, F61A, L131A, F137A, and L142A. In some embodiments, the present disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 23 and has the following amino acid substitutions: L23A, F61A, L131A, F137A, L142A, I161T, and L171A. In embodiments, the disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 23 and has the following amino acid substitutions: L23A, F61A, N79A, L80A, L131A, F137A, and L142A. In embodiments, the disclosure provides a nucleic acid encoding a modified GMOP-interferon-α2c polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 23 and has the following amino acid substitutions: L23A, L31A, F61A, N79A, L80A, L131A, F137A, L142A, I161T, and L171A.

[0147] In some embodiments, there is provided a nucleic acid of the present invention encoding one or more modified GMOP-interferon-α2c polypeptides of the present disclosure, for example, but not limited to, a vector or plasmid comprising a nucleic acid (e.g., DNA or RNA) encoding at least one modified GMOP-interferon-α2c polypeptide. In some embodiments, the present disclosure is directed to a cell comprising a vector or plasmid of the present disclosure.

[0148] In some embodiments, the modified interferon-α2 polypeptides described herein are conjugated or linked (e.g., fused in-frame, chemically linked, or otherwise conjugated) to a heterologous polypeptide. With reference to one or more modified interferon-α2 polypeptides of the present disclosure, the term "heterologous polypeptide" is intended to mean that one or more modified interferon-α2 polypeptides of the present disclosure are heterologous to or not naturally contained in the heterologous polypeptide. In some embodiments, one or more modified interferon-α2 polypeptides of the present invention may be attached to the C-terminus (with or without a linker, as known in the art) and / or the N-terminus (with or without a linker, as known in the art) of the heterologous polypeptide.

[0149] The present disclosure also provides chimeric or fusion polypeptides (which may, in embodiments, be isolated, synthetic, or recombinant) of which one or more modified interferon-α2 polypeptides disclosed herein are a part. In embodiments, one or more modified interferon-α2 polypeptides of the present disclosure may be conjugated or linked (e.g., fused in frame, chemically linked, or otherwise conjugated) to a small molecule, drug, or drug fragment (e.g., but not limited to, a drug or drug fragment that binds with high affinity to a defined receptor).

[0150] As used herein, two polypeptides (or regions of polypeptides) are substantially homologous or identical if their amino acid sequences share a certain percentage or more of identity, e.g., at least about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, usually at least about 70-75%, more usually at least about 80-85%, more usually about 90% or more, and more usually 95% or more homology or identity. Percent homology can be determined as known in the art. For example, to determine the percent homology or identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the sequence of one polypeptide or nucleic acid molecule to allow for optimal alignment with the other polypeptide or nucleic acid molecule). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid "identity" is equivalent to amino acid "homology"). As known in the art, the percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence homology of polypeptides is usually measured using sequence analysis software.

[0151] In some embodiments, the present disclosure also encompasses polypeptides (e.g., the modified interferon-α2 polypeptides and modified interferon-α2 compositions disclosed herein) that share a lower degree of identity but are sufficiently similar to perform one or more of the same functions performed by the polypeptides encoded by the nucleic acid molecules of the present invention. Similarity is determined by conservative amino acid substitutions. Such substitutions substitute a given amino acid in a polypeptide with another amino acid with similar properties. Conservative substitutions are likely to be phenotypically silent. Typical conservative substitutions include the substitution of one each of the aliphatic amino acids Ala, Val, Leu, Met, and Ile; the exchange of hydroxyl residues Ser and Thr; the exchange of acidic residues Asp and Glu; the exchange of amide residues Asn and Gln; the exchange of basic residues Lys and Arg; and the exchange of aromatic residues Phe, Trp, and Tyr. Guidance regarding which amino acid changes are likely to be phenotypically silent can be found (Bowie JU et al., (1990), Science, 247(4948):130610, which is incorporated herein by reference in its entirety). For example, amino acid sequences with interferon function can be identified by performing a protein-protein BLAST (blastp) search against a non-redundant protein sequence database using the amino acid sequences of these proteins as queries. Searches can be performed with default parameters at the National Center for Biotechnology Information (NCBI) website (http: / / blast.ncbi.nlm.nih.gov).

[0152] Fragments and variants of the disclosed modified IFNα-2 polypeptides and polynucleotides are also encompassed by the present disclosure. "Fragment" is intended to mean a portion of a polypeptide or polynucleotide. Fragments of the polypeptide or nucleotide sequences disclosed herein retain the biological activity of the polypeptides disclosed herein and thus may encode polypeptide fragments that retain interferon-α2 activity (e.g., antiviral biological activity) with reduced immunogenicity compared to wild-type interferon-α2. In embodiments, the present disclosure also encompasses fragments of variants of the polypeptides and polynucleotides described herein.

[0153] In several embodiments, variant polypeptides (e.g., variants of the modified interferon-α2 polypeptides of the present disclosure) can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations, or any combination thereof. Variant polypeptides can be fully functional (e.g., retaining interferon-α2 activity, such as antiviral biological activity) or can lack function in one or more activities. Fully functional variants typically contain only conservative mutations or mutations in non-critical residues or non-critical regions. Functional variants can also contain substitutions of similar amino acids that result in no change or a minor change in function (e.g., reducing immunogenicity and retaining antiviral biological activity). Alternatively, such substitutions can have some positive or negative effect on function. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or deletions in critical residues or critical regions. In several embodiments, the modified interferon-α2 polypeptides disclosed herein may differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations, or any combination thereof, provided that the variants retain biological activity (e.g., IFNα2 activity, such as antiviral activity) and have reduced immunogenicity (compared to wild-type interferon-α2).

[0154] In some embodiments, the fully functional variant of modified interferon-α2 does not contain mutations in one or more critical residues or regions. In some embodiments, the one or more critical residues of modified interferon-α2 that should not be mutated include: residues involved in biological activity, residues in functional hotspots that are highly conserved among various wild-type interferon alleles (e.g., between species), residues involved in binding to the natural receptor of interferon, residues involved in structural interactions that are important for the structural integrity of natural interferon, residues involved in disulfide bonds of natural interferon (e.g., intramolecular disulfide bonds that occur in natural interferon upon proper folding in its natural environment in vivo), and / or residues that are glycosylation sites in natural wild-type interferon (including N-glycosylation sites and O-glycosylation sites).

[0155] In several embodiments, the modified IFNα-2 polypeptides disclosed herein, including fully functional variants of the disclosed modified interferon-α2, do not contain mutations in one or more critical residues or regions, wherein the one or more critical residues or regions are selected from the group consisting of: residues in functional hotspots, residues that are highly conserved among various wild-type interferon alleles (e.g., between species), residues involved in disulfide bonds in native interferons (e.g., intramolecular disulfide bonds that occur in native interferons upon proper folding in their natural environment in vivo), and / or residues that are glycosylation sites in native wild-type interferons (including N-glycosylation sites and O-glycosylation sites).

[0156] In embodiments, amino acid residues believed to be non-essential for the function of the polypeptides disclosed herein, including fully functional variants of the disclosed modified interferon-α2 (e.g., IFNα-2b mutants, IFNα-2a mutants, IFNα-2c mutants, GMOP-IFNα-2b mutants, GMOP-IFNα-2a mutants, and GMOP-IFNα-2c mutants), may be substituted conservatively or non-conservatively, and such amino acid substitutions will not significantly reduce the functional properties of the polypeptide. In embodiments, amino acid residues believed to be essential for the function of the polypeptides disclosed herein, including fully functional variants of the disclosed modified interferon-α2 (e.g., IFNα-2b mutants, IFNα-2a mutants, IFNα-2c mutants, GMOP-IFNα-2b mutants, GMOP-IFNα-2a mutants, and GMOP-IFNα-2c mutants), should not be substituted conservatively or non-conservatively, and should not be conservative, as such amino acid substitutions may significantly reduce the functional properties of the polypeptide. In several embodiments, the IFNα2 polypeptides disclosed herein, including fully functional variants of the disclosed modified interferon-α2 (e.g., IFNα-2b variants, IFNα-2a variants, IFNα-2c variants, GMOP-IFNα-2b variants, GMOP-IFNα-2a variants, and GMOP-IFNα-2c variants), do not contain mutations (which may be either conservative or non-conservative substitutions) in one or more critical residues or regions of WT native human IFN-α2.In several embodiments, the one or more critical residues or regions of WT native human IFN-α2 are selected from the group consisting of: residues involved in biological activity, residues of functional hotspots, residues that are highly conserved among various wild-type interferon alleles (e.g., between species), residues involved in binding of interferon to its native receptor, residues involved in structural interactions that are important for the structural integrity of native interferon, residues involved in disulfide bonds of native interferon (e.g., intramolecular disulfide bonds that occur in native interferon upon proper folding in its natural environment in vivo), and / or residues that are glycosylation sites in native wild-type interferon (including N-glycosylation sites and O-glycosylation sites). In embodiments, the one or more critical residues or regions of WT native hIFN-α2 involved in the biological activity of hIFN-α2 are selected from the group consisting of 22, 26, 27, 30, 31, 33, 34, 36, 68, 79, 85, 120, 121, 122, 124, 129, 131, 132, 144, and 146. Most conservative and non-conservative amino acid substitutions for such amino acid residues are likely to reduce the functional properties of the polypeptide (e.g., IFNα2 activity, including antiviral activity). In embodiments, the one or more critical residues or regions of WT native hIFN-α2 that are functional hotspots are selected from the group consisting of 30, 33, 144, 145, 148, and 149. Most conservative and non-conservative amino acid substitutions for such amino acid residues are likely to reduce the functional properties of the polypeptide (e.g., IFNα2 activity, including antiviral activity). In embodiments, the one or more critical residues or regions of WT native hIFN-α2 that are highly conserved among various wild-type IFN-α2 alleles (e.g., among species) are selected from the group consisting of 91, 122, 150, and 154 (and optionally: 30, 33, 144, 145, 148, and 149). Most conservative and non-conservative amino acid substitutions for such amino acid residues will likely decrease the functional properties of the polypeptide (e.g., IFN-α2 activity, including antiviral activity).In embodiments, the one or more critical residues or regions of WT native hIFN-α2 involved in binding of hIFN-α2 to its native receptor are selected from the group consisting of 5, 6, 12, 13, 15, 16, 19, 20, 22, 26, 27, 30-37, 39-41, 46, 68, 76, 77, 79, 80, 82, 83, 85, 86, 89, 90, 93, 94, 97, 118, 120, 121, 124, 125, 127, 131-136, 144-146, 148, 149, 15, and 153. Most conservative and non-conservative amino acid substitutions of such amino acid residues are likely to reduce functional properties (e.g., IFN-α-2 activity, including antiviral activity). In embodiments, the one or more critical residues or regions of WT native hIFN-α2 involved in structural interactions critical to the structural integrity of hIFN-α2 are selected from the group consisting of 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 91, 114, 115, 118, 121, 122, 125, 132, 150, and 154. Most conservative and non-conservative amino acid substitutions of such amino acid residues are likely to reduce the functional properties of the polypeptide (e.g., IFN-α2 activity, including antiviral activity). In embodiments, the one or more critical residues or regions of WT native hIFN-α2 involved in structural interactions critical to the structural integrity of hIFN-α2 are selected from the group consisting of 36, 41, 42, 91, 122, 129, 150, and 154. Most conservative and non-conservative amino acid substitutions for such amino acid residues are likely to reduce the functional properties of the polypeptide (e.g., IFNα2 activity, including antiviral activity). In embodiments, the one or more critical residues or regions of WT native hIFN-α2 involved in disulfide bonds of native hIFN-α2 (e.g., intramolecular disulfide bonds that occur in hIFN-α2 upon proper folding in its native environment in vivo) are selected from the group consisting of 1, 29, 98, and 138. Most conservative and non-conservative amino acid substitutions for such amino acid residues are likely to reduce the functional properties of the polypeptide (e.g., IFNα-2 activity, including antiviral activity).In embodiments, the one or more critical residues or regions that are sites of glycosylation in native wild-type hIFN-α2 (including N- and O-glycosylation sites) are selected from the group consisting of 106. It is believed that the polypeptides disclosed herein having the described modifications / substitutions will confer the desired activity (e.g., IFNα-2 activity, including antiviral activity). Stated another way, it is believed that the amino acid substitutions described herein will not significantly reduce the functional properties of the polypeptides disclosed herein.

[0157] In embodiments, the modified interferon-α2 (e.g., a fully functional variant of the disclosed modified interferon-α2 (e.g., an IFNα-2b variant, an IFNα-2a variant, an IFNα-2c variant, a GMOP-IFNα-2b variant, a GMOP-IFNα-2a variant, a GMOP-IFNα-2c variant)) does not comprise a mutation (e.g., an amino acid substitution) at one or more amino acids occupying positions selected from the group consisting of 4, 23, 70, and 77 in hIFN-α2. In embodiments, the modified interferon-α2 (e.g., a fully functional variant of the disclosed modified interferon-α2 (e.g., an IFNα-2b variant, an IFNα-2a variant, and an IFNα-2c variant)) does not comprise an amino acid substitution of an Asn residue at one or more amino acids occupying positions selected from the group consisting of 4, 23, 70, and 77 in HIFN-α2. In embodiments, the modified interferon-α2 does not comprise one or more amino acid substitutions at amino acid positions selected from the group consisting of 4, 23, 70, and 77 in hIFN-α2.

[0158] In embodiments, the modified GMOP-interferon-α2 (e.g., fully functional variants of the disclosed modified GMOP-IFNα-2b, GMOP-IFNα-2a, and GMOP-IFNα-2c variants) does not comprise a mutation (e.g., an amino acid substitution) at one or more amino acids occupying positions selected from the group consisting of 18, 37, 84, and 91 of GMOP-hIFN-α2. In embodiments, the modified interferon-α2 (e.g., fully functional variants of the disclosed modified GMOP-IFNα-2b, GMOP-IFNα-2a, and GMOP-IFNα-2c variants) does not comprise an amino acid substitution of an Asn residue at one or more amino acids occupying positions selected from the group consisting of the following positions in GMOP-hIFN-α2:18, 37, 84, 91: In several embodiments, the modified interferon-α2 (e.g., fully functional variants of the disclosed modified GMOP-IFNα-2b variants, GMOP-IFNα-2a variants, and GMOP-IFNα-2c variants) does not contain one or more amino acid substitutions at amino acid positions selected from the group consisting of positions 18, 37, 84, and 91 in GMOP-hIFN-α2.

[0159] In embodiments, the modified interferon-α2 of the present disclosure may include allelic or sequence variants ("variants") or analogs thereof, or may include chemical modifications (e.g., pegylation, glycosylation). In embodiments, the modified interferon-α2 polypeptides described herein are hyperglycosylated. In embodiments, the modified interferon-α2 retains the same functions performed by interferon polypeptides encoded by the nucleic acid molecules of the present disclosure, particularly maintained biological activity and reduced immunogenicity. In embodiments, the modified interferon-α2 can provide high relative antiviral activity. In embodiments, the modified interferon-α2 can result in reduced immunogenicity. In embodiments, the modified interferon-α2 can result in reduced antiproliferative biological activity. In embodiments, the modified interferon-α2 can result in an improved pharmacokinetic profile. In embodiments, the modified interferon-α2 can result in improvements in protein synthesis and purification of the modified interferon-α2.

[0160] The polypeptides of the present disclosure can be modified in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the instantly disclosed polypeptides can be prepared by DNA mutations. Methods for mutagenesis and polynucleotide modification are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; US Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), which is incorporated herein by reference. Conservative substitutions, such as exchanging one amino acid for another with similar properties, may be optimal. Typical conservative substitutions include single substitutions between aliphatic amino acids Ala, Val, Leu, and Ile, exchanges between hydroxyl residues Ser and Thr, exchanges between acidic residues Asp and Glu, exchanges between amide residues Asn and Gln, exchanges between basic residues Lys and Arg, and exchanges between aromatic residues Phe and Tyr. Guidance regarding which amino acid changes are likely to be phenotypically silent is available (Bowie JU et al., (1990), Science, 247 (4948):130610, which is incorporated herein by reference in its entirety). For purposes of this disclosure, polypeptides can include, for example, modified forms of naturally occurring amino acids such as D-stereoisomers, non-naturally occurring amino acids; amino acid analogs; and mimetics.

[0161] It should be clarified that the modifications / substitutions presented by the deimmunized mutants of GMOP-IFNα-2 correspond to amino acids that are not involved in the biological structure or function of the cytokine, i.e., these mutations can be made to any of the following interferon mutants: IFN-α2b, GMOP-IFN-α2b, or any other mutant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2A, IFN-α2c, and GMOP-IFN-α2c).

[0162] Methods for producing the modified interferon-α2 polypeptides of the present disclosure will vary widely, depending on the nature of the various components that make up the molecule. For example, an isolated polypeptide (e.g., an isolated modified interferon-α2 polypeptide) can be purified from cells that naturally express it, purified from cells engineered to express it (recombinant), or synthesized using known protein synthesis methods. Synthesis procedures can be selected to be simple, provide high yields, and result in a highly purified and stable product. For example, the polypeptides disclosed herein can be produced either from the nucleic acids disclosed herein or by using standard molecular biology techniques, such as recombinant technology, mutagenesis, or other means known in the art. The isolated polypeptide can be purified from cells that naturally express it, purified from cells engineered to express it (recombinant), or synthesized using known protein synthesis techniques. In embodiments, the polypeptides disclosed herein are produced by recombinant DNA or RNA technology. In embodiments, the polypeptides disclosed herein can be produced by expression of the recombinant nucleic acids disclosed herein in a suitable host cell. For example, a nucleic acid molecule encoding a polypeptide is cloned into an expression cassette or expression vector, and the expression cassette or expression vector is introduced into a host cell, and the polypeptide is expressed in the host cell.The polypeptide can then be isolated from the cell by an appropriate purification scheme using standard protein purification techniques.Alternatively, the polypeptide can be produced by a combination of ex vivo procedures such as protease digestion and purification.Additionally, the polypeptides disclosed herein can be produced using site-directed mutagenesis techniques or other mutagenesis techniques known in the art (see, e.g., James A. Brannigan and Anthony J. Wilkinson, 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, which are incorporated herein by reference in their entireties).

[0163] In various embodiments, the present disclosure is also directed to methods for synthesizing modified interferon-α2 (e.g., modified IFNα-2b polypeptide, modified IFNα-2a polypeptide, modified IFNα-2c polypeptide, modified GMOP-IFNα-2b polypeptide, modified GMOP-IFNα-2a polypeptide, and modified GMOP-IFNα-2c polypeptide). The modified interferon-α2 polypeptides disclosed herein with improved properties (e.g., reduced immunogenicity) can be generated by genetic modification in one of the various ways described herein. As used herein, the term "modified interferon-α2" can refer to the group of modified interferon-α2s disclosed herein that have intentionally altered amino acid sequences, i.e., "non-wild-type" amino acid sequences, or to microbial organisms (depending on the placement of either term as an adjective) whose genomes have been intentionally modified with respect to (at least) the specific modified interferon-α2 molecules described herein, or both. Such modification can be achieved by recombinant techniques, in which one or more genes are transferred into a target microbial organism from a second, different microbial organism. Recombinant techniques can be achieved using fully synthetic DNA that is introduced into the target microbial organism using conventional methods. Such modification can also be achieved by engineering techniques, in which nucleic acids within the target microbial organism are modified, typically via site-directed mutagenesis, resulting in the conversion of at least one nucleic acid to a different nucleic acid and thus the modification of one or more enzymes. Any of the above methods, as well as combinations of the methods described throughout this application, can also be employed. Thus, it will be understood that the modified interferon-α2 molecules disclosed herein can be produced either in vivo, i.e., by genetically modified microorganisms, or in vitro.

[0164] In some embodiments, the present disclosure provides a method for generating the amino acid substitutions to reduce immunogenicity. The method comprises generating point mutations in the nucleotide sequence of a gene encoding a natural human interferon (e.g., natural IFN-α2, natural GMOP-IFN-α2) by site-directed mutagenesis in the gene. The method comprises the following steps: 1. cloning the gene encoding a natural human interferon (e.g., natural IFN-α2, natural GMOP-IFN-α2) into an appropriate plasmid; 2. using site-directed mutagenesis to generate the mutations necessary to generate the modified interferon-α2 of the present disclosure; and 3. cloning the modified gene from step 2 into an appropriate expression vector. In some embodiments, the expression vector is selected from a group of vectors capable of carrying the gene of the present disclosure and further comprising the elements necessary for expressing the gene of interest in eukaryotic cells.

[0165] In several embodiments, the site-directed mutagenesis technique of the present disclosure involves the use of oligonucleotides specifically designed for this purpose. This technique consists of two steps. In the first step, two separate PCR reactions are performed using oligonucleotides that hybridize to the ends of the fragment cloned into an appropriate vector and an oligonucleotide that hybridizes to an internal region of the gene to be mutated and contains a point mutation corresponding to the amino acid substitution (described herein) that reduces immunogenicity. A reaction mixture is generated in tube a using the reverse external oligonucleotide and the direct oligonucleotide mut a, and another reaction mixture is generated in tube b using the direct external oligonucleotide and the reverse oligonucleotide mut b. The PCR products from both reactions are purified by agarose gel electrophoresis and used as templates for the second step. This second step consists of a second PCR reaction using the direct and reverse external oligonucleotides. The first three cycles are performed without adding primers to allow hybridization and extension of the complete product (fill-in), and then they are added for amplification.

[0166] In some embodiments, to obtain multiple amino acid substitution sites that reduce immunogenicity within the modified interferon-α2 disclosed herein, the modified interferon-α2 is constructed sequentially as follows: first, site-directed mutagenesis is used to generate modified interferon-α2 with amino acid substitution sites, and then the modified interferon-α2 is used as a starting template for generating new amino acid substitution sites.

[0167] In embodiments, the present disclosure is directed to a method for producing modified interferon-α2, comprising the steps of: a) transforming or transfecting a prokaryotic cell with a suitable prokaryotic expression vector containing a gene encoding modified interferon-α2; b) selecting a clone expressing the modified interferon-α2 polypeptide; c) culturing the clone in a suitable culture medium; d) purifying the product; e) in vitro glycosylation of the modified interferon-α2 polypeptide expressed by the clone of step c); and f) purifying the modified interferon-α2. In embodiments, the glycosylation in step e) of the method is hyperglycosylation of the modified interferon-α2 polypeptide.

[0168] In embodiments, the present disclosure also provides nucleic acids (e.g., DNA, RNA, vectors, viruses, or hybrids thereof, all of which may be isolated, synthetic, or recombinant) encoding all or part of one or more modified interferon-α2 polypeptides of the present disclosure and / or chimeric or fusion polypeptide compositions of the present disclosure. In embodiments, the nucleic acids further comprise or are contained within expression cassettes, plasmids, and expression vectors, or recombinant viruses; optionally, the nucleic acids, or expression cassettes, plasmids, expression vectors, or recombinant viruses are contained within cells, optionally human or non-human cells, and optionally, the cells are transduced with the nucleic acids, or expression cassettes, plasmids, expression vectors, or recombinant viruses. In embodiments, the cells are transformed, transfected, or otherwise engineered to contain within them, for example, one or more polypeptides of the present disclosure (modified interferon-α2 polypeptides); isolated, synthetic, or recombinant nucleic acids, expression cassettes, plasmids, expression vectors, or recombinant viruses disclosed herein; and / or isolated, synthetic, or recombinant chimeric or fusion polypeptide compositions disclosed herein. In some embodiments, the cells may be mammalian, bacterial, insect, or yeast cells. In some embodiments, the nucleic acid molecules of the present disclosure may be inserted into a vector and used, for example, as an expression vector or a gene therapy vector. Gene therapy vectors can be delivered to a subject, for example, by intravenous injection, local administration (U.S. Pat. No. 5,328,470), or stereotactic injection (Chen SH et al., (1994), Proc Natl Acad Sci USA, 91(8):3054-7, which are incorporated herein by reference in their entireties). Pharmaceutical formulations of gene therapy vectors can include the gene therapy vector in an acceptable diluent or can consist of a slow-release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical formulation can include one or more cells that produce the gene delivery system.Such pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration. In embodiments, the present disclosure is directed to a cell comprising the vector of the present disclosure. In embodiments, the cell may be a mammalian cell, a bacterial cell, an insect cell, or a yeast cell.

[0169] With respect to polynucleotides, "variants" include deletions and / or additions of one or more nucleotides at one or more internal sites within the polynucleotide sequences disclosed herein, and / or substitutions of one or more nucleotides at one or more sites within the polynucleotide sequences disclosed herein. Those skilled in the art will recognize that variants of the polynucleotides of the present invention are constructed so that the open reading frame is maintained. With respect to polynucleotides, conservative variants include sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the present invention. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, such as the polymerase chain reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated using site-directed mutagenesis, that still encode polynucleotides having the desired activity of the present invention (i.e., encoding polypeptides having the desired biological activity described herein, i.e., antipathogenic activity, antifungal activity, antialgal activity, and / or enzymatic activity against chitin and / or polyglucuronic acid). Generally, variants of a particular polynucleotide of the present invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence similarity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.

[0170] Variants of a particular polynucleotide (i.e., a reference polynucleotide) of the present disclosure can also be evaluated by comparing the percent sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. When any given pair of polynucleotides of the present invention is evaluated by comparing the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0171] The polynucleotides provided herein (whether RNA, DNA, expression cassettes, vectors, viruses, or hybrids thereof) encoding all or part of one or more polypeptides of the present disclosure can be isolated from a variety of sources, genetically engineered, amplified, synthetically produced, and / or recombinantly expressed / made. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including, for example, in vitro, bacterial, fungal, mammalian, yeast, insect, or plant cell expression systems. In some embodiments, the polynucleotides provided herein are synthesized in vitro by well-known chemical synthesis techniques (e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066, all of which are incorporated herein by reference in their entireties).Furthermore, techniques for manipulating polynucleotides provided herein, such as subcloning, labeling probes (e.g., random primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization, and the like, are well described in the scientific and patent literature (e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); Current Protocols In Molecular Biology, Ausubel, ed., John Wiley & Sons, Inc., New York (1997); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed., New York (1997). Elsevier, NY (1993), all of which are incorporated herein by reference in their entireties).

[0172] In embodiments, the present disclosure is directed to a characterized cell line comprising a nucleic acid encoding a modified interferon-α2 as disclosed herein. In embodiments, the cell line is suitable for producing a modified interferon-α2 as disclosed herein. In preferred embodiments, the cell line suitable for producing a modified interferon-α2 as disclosed herein is selected from the set of CHO-K1, HEK293, NS0, BHK, Sp2 / 0, CAP, and CAP / T. In embodiments, the present disclosure is also directed to a method for obtaining a eukaryotic cell line, wherein the cell line comprises a gene encoding a modified interferon-α2 as disclosed herein inserted into a suitable expression vector, for producing the modified interferon-α2 as disclosed herein. Preferably, the eukaryotic cell line is a CHO-K1 cell line. In several aspects, the present disclosure is directed to a method for producing a modified interferon-α2 as disclosed herein, the method comprising: a) culturing the transformed or transfected eukaryotic cell line with an expression vector containing a gene encoding a modified interferon-α2 polypeptide as disclosed herein; and b) isolating the expressed and secreted modified interferon-α2 polypeptide from the culture medium.

[0173] In some embodiments, the present disclosure is directed to a method for purifying a modified interferon-α2 polypeptide as disclosed herein. In some embodiments, the step of purifying the modified interferon-α2 polypeptide comprises purification by immunoaffinity chromatography. In some embodiments, the step of purifying the modified interferon-α2 polypeptide comprises purification by immunoaffinity chromatography, wherein the purification by immunoaffinity chromatography comprises the use of an anti-nonglycosylated rhIFN-α2b mAb CA5E6 antibody. In some embodiments, the step of purifying the modified interferon-α2 polypeptide comprises purification by immunoaffinity chromatography, wherein the purification by immunoaffinity chromatography comprises the use of an anti-hGM-CSF monoclonal antibody (designated mAb CC1H7). In some embodiments, the step of purifying the modified interferon-α2 polypeptide further comprises determining the concentration of the purified modified interferon-α2 polypeptide after purification (e.g., by immunoaffinity chromatography). In a preferred embodiment, said determining the concentration of purified modified interferon-α2 polypeptide is determined by spectrophotometric quantitation.

[0174] In several embodiments, the modified interferon-α2 compounds or compositions of the present disclosure (such as one or more modified interferon-α2 polypeptides, polynucleotides, microorganisms expressing one or more polypeptides or polynucleotides, expression cassettes, plasmids, expression vectors, chimeric or fusion polypeptides, recombinant viruses, and / or pharmaceutical compositions of the present disclosure) can be purified to homogeneity or can be partially purified. However, it is understood that preparations in which the modified interferon-α2 compositions are not purified to homogeneity are useful. The important feature is that the preparation permits the desired function of the modified interferon-α2 even in the presence of significant amounts of other components. Thus, the present disclosure encompasses various degrees of purity. In one embodiment, the term "substantially free of cellular material" includes preparations of modified interferon alpha 2 having less than about 30% (by dry weight) other proteins (e.g., contaminating proteins), less than about 20% other proteins, less than about 10% other proteins, less than about 5% other proteins, less than about 4% other proteins, less than about 3% other proteins, less than about 2% other proteins, less than about 1% other proteins, or any value or range therebetween.

[0175] In embodiments, where the modified interferon-α2 compounds or compositions of the disclosure are recombinantly produced, the modified interferon-α2 compositions can also be substantially free of culture medium, e.g., culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the modified interferon-α2 polypeptide, nucleic acid, or chimeric or fusion polypeptide preparation. The phrase "substantially free of chemical precursors or other chemicals" includes preparations of polypeptides, nucleic acids, or chimeric or fusion polypeptides that are separated from chemical precursors or other chemicals involved in the synthesis of modified interferon-α2. The phrase "substantially free of chemical precursors or other chemicals" includes, for example, preparations of modified interferon-α2 polypeptides, nucleic acids, or chimeric or fusion polypeptides having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, less than about 5% chemical precursors or other chemicals, less than about 4% chemical precursors or other chemicals, less than about 3% chemical precursors or other chemicals, less than about 2% chemical precursors or other chemicals, or less than about 1% chemical precursors or other chemicals.

[0176] In several embodiments, the modified interferon-α2 polypeptide compounds or compositions of the present disclosure can be produced by standard recombinant DNA or RNA techniques as known in the art. For example, DNA or RNA fragments encoding different polypeptide sequences can be ligated together in-frame according to conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, polymerase chain reaction (PCR) amplification of nucleic acid fragments can be performed using anchor primers that generate complementary overhangs between two consecutive nucleic acid fragments that can be subsequently annealed and reamplified to generate a chimeric nucleic acid sequence (Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, (2014)). ND, 1992), FM Asubel et al. (eds), Green Publication Associates, New York, NY (Publ), ISBN: 9780471566355, which are incorporated herein by reference in their entireties. Additionally, one or more polypeptides (e.g., modified interferon-α2 polypeptides) of the present disclosure (e.g., one or more modified interferon-α2 polypeptides of the present disclosure comprising, consisting essentially of, or consisting only of one or more sequences consisting of SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, and 27-42) can be inserted into a heterologous polypeptide or into a non-naturally occurring location in a polypeptide through recombinant techniques, synthetic polymerization techniques, mutagenesis, or other standard techniques known in the art. For example, protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques or other mutagenesis techniques known in the art (e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, which are incorporated herein by reference in their entireties).

[0177] Furthermore, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). The nucleic acid molecule encoding the modified interferon-α2 of the present invention can be cloned into such an expression vector so that the fusion moiety is linked in-frame to at least one modified interferon-α2. Such linking of the fusion moiety can be performed, for example, to improve the purification yield of the protein.

[0178] Pharmaceutical Compositions and Formulations In embodiments, one or more modified interferon-α2 polypeptides, chimeric polypeptides, polynucleotides, microorganisms expressing one or more polypeptides or polynucleotides, expression cassettes, plasmids, expression vectors, and / or recombinant viruses (hereinafter, "modified interferon-α2 compounds or compositions of the present disclosure") can be included in a pharmaceutical composition or formulation. In embodiments, the pharmaceutical composition or formulation generally includes a modified interferon-α2 compound or composition of the present disclosure and a pharmaceutically acceptable carrier and / or excipient. In embodiments, the pharmaceutical composition is suitable for administration. The pharmaceutically acceptable carrier and / or excipient will be determined, in part, by the particular composition being administered and the particular method used to administer the composition. Accordingly, suitable formulations of pharmaceutical compositions for administering the modified interferon-α2 compositions disclosed herein are numerous and varied (see, e.g., Remington's Pharmaceutical Sciences, (18 TH (See, e.g., J.D., 1990), Mack Publishing Co., Easton, PA Publ.) In embodiments, pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0179] The terms "pharmaceutically acceptable," "physiologically acceptable," and grammatical variations thereof, referring to compositions, carriers, excipients, and reagents, are used interchangeably to indicate that the material can be administered to or by a subject without producing undesirable physiological effects that would prohibit administration of the composition. For example, a "pharmaceutically acceptable excipient" refers to an excipient that is generally safe, non-toxic, and useful in preparing a desired pharmaceutical composition, including excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous. One of ordinary skill in the art would be able to determine the appropriate timing, sequence, and dosage of administration of the modified interferon-α2 compositions of the present disclosure.

[0180] In some embodiments, preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.Non-aqueous vehicles such as liposomes and fixed oils can also be used.The use of such media and compounds for pharmaceutically active substances is well known in the art.As long as any conventional media or compounds are incompatible with the modified interferon-α2 compounds or compositions of the present disclosure, and as described above, their use in the composition is contemplated.Supplementary active compounds can also be incorporated into the composition.

[0181] In some embodiments, the modified interferon-α2 compound or composition of the present disclosure is formulated to suit its intended route of administration.The modified interferon-α2 compound or composition of the present disclosure can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally; intravaginally; intramuscularly, or as an inhalant.In some embodiments, the modified interferon-α2 compound or composition of the present disclosure can be directly injected into a specific tissue where deposits have accumulated.In other aspects, intramuscular injection or intravenous infusion can be used to administer the modified interferon-α2 compound or composition of the present disclosure.In some methods, the modified interferon-α2 compound or composition of the present disclosure is administered as a sustained-release composition or device, such as, but not limited to, a Medipad® device.

[0182] In some embodiments, the modified interferon-α2 compounds or compositions of the present disclosure can be administered in combination with other agents that are at least partially effective in treating various medical conditions, such as those described herein. For example, the modified interferon-α2 compounds or compositions of the present disclosure can be administered in combination with other agents that stimulate the antiviral activity of the immune system, improve the pharmacokinetic parameters of the composition, enhance and / or complement the natural biological activity of interferon-α2, and / or reduce the immunogenicity of the composition.

[0183] In some embodiments, solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain, but are not limited to, the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial compound such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citric acid, or phosphate, and a compound for adjusting tonicity such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, water, ethanol, DMSO, glycol, propylene glycol, dried skim milk, and the like. The composition may also contain a pH buffering agent and a wetting or emulsifying agent. In embodiments, the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0184] In some embodiments, pharmaceutical compositions or formulations suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers are physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. In some embodiments, modified interferon-α2 formulations may contain aggregates, fragments, degradation products, and post-translational modifications, to the extent that these impurities have reduced immunogenicity and high relative antiviral activity similar to that of pure modified interferon-α2. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, or the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic compounds, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition a compound that delays absorption, for example, aluminum monostearate and gelatin.

[0185] In some embodiments, sterile injectable solutions can be prepared by incorporating the modified interferon-α2 compound or composition of the present disclosure into a suitable solvent in the required amount, optionally with one or a combination of the ingredients listed above, and then sterilizing by filtration.Generally, dispersions are prepared by incorporating a binder into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above.For sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from its solution that has previously been sterile-filtered.In addition, the modified interferon-α2 compound or composition of the present disclosure can be administered in the form of a depot injection or implant preparation, which can be formulated in a way that allows the active ingredient to be released continuously or in a pulsatile manner.

[0186] In embodiments, the solution or suspension of the pharmaceutical composition or formulation is maintained at a pH at which the modified interferon-α2 polypeptide is in its native structural conformation. In embodiments, the pH is maintained at or below pH 10. In embodiments, the pH is maintained at or below pH 7. In embodiments, the pH is maintained between pH 3 and 10. In embodiments, the pH is maintained between pH 4 and 9. In embodiments, the pH is maintained between pH 5 and 8. In embodiments, the pH is maintained between pH 6 and 7.5. In embodiments, a buffer is provided to maintain the pH at a desired level. In embodiments, the buffer is a phosphate buffer. In embodiments, the buffer is an acetate buffer.

[0187] In some embodiments, the solution or suspension of the pharmaceutical composition or formulation contains a surface adsorption inhibitor. In some embodiments, the surface adsorption inhibitor inhibits adsorption of the components of the pharmaceutical composition or formulation to a surface (e.g., a glass or plastic ampoule, syringe, or vial) enclosing the composition or formulation. In a preferred embodiment, the surface adsorption inhibitor inhibits adsorption of one or more modified interferon-α2 polypeptides to a glass surface enclosing the composition or formulation. In some embodiments, the pharmaceutical composition or formulation is enclosed in a borosilicate glass ampoule, syringe, or vial, and the pharmaceutical composition or formulation contains a surface adsorption inhibitor (e.g., a surface adsorption inhibitor that inhibits adsorption of one or more recombinant interferon-α2 polypeptides to a borosilicate glass surface). In some embodiments, the surface adsorption inhibitor is polysorbate 80. In some embodiments, the surface adsorption inhibitor is albumin.

[0188] In some embodiments, the solution or suspension of the pharmaceutical composition or formulation includes a degradation inhibitor. In some embodiments, the degradation inhibitor inhibits the degradation of the modified interferon-α2 polypeptide. In some embodiments, the degradation inhibitor inhibits the oxidative degradation of the modified interferon-α2 polypeptide. In some embodiments, the degradation inhibitor inhibits the oxidative degradation of the modified interferon-α2 polypeptide, wherein the degradation inhibitor is benzyl alcohol.

[0189] In embodiments, the pharmaceutical composition or formulation comprises a sterile powder for extemporaneous preparation of a sterile injectable solution or dispersion, the sterile powder comprising: a dry powder formulation of one or more modified interferon-α2 polypeptides, a bulking agent, and a surface adsorption inhibitor. In embodiments, the bulking agent is glycine. In embodiments, the surface adsorption inhibitor is albumin. In embodiments, the sterile powder further comprises one or more antimicrobial preservatives. In embodiments, the one or more antimicrobial preservatives are selected from the group consisting of m-cresol, benzyl alcohol, and phenol. In embodiments, the sterile powder further comprises dibasic sodium phosphate and monobasic sodium phosphate. In embodiments, the sterile powder is provided as a whole tablet-like solid, pieces, and / or a loose powder. In embodiments, the dry powder formulation of one or more modified interferon-α2 polypeptides is a lyophilized powder. In embodiments, the one or more modified interferon-α2 polypeptides are provided with a desired specific activity. In embodiments, the sterile powder is stored at a low temperature prior to administration to a subject. In embodiments, the sterile powder is stored at a temperature ranging from 2°C to 8°C prior to administration to a subject. In embodiments, prior to administration to a subject, the sterile powder is reconstituted with a diluent to provide a sterile solution. In embodiments, the reconstitution is accomplished by dissolving the sterile powder in a diluent (e.g., by stirring, swirling, inverting, shaking, vortexing, or other means known and understood in the art) to produce a sterile solution. In embodiments, the diluent comprises one or more components selected from the group consisting of sterile water, sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, EDTA, polysorbate 80, and m-cresol. In embodiments, the resuspension is performed in a single-use vial, ampoule, or syringe. In embodiments, the sterile solution provides one or more modified interferon-α2 polypeptides at a desired concentration. In embodiments, the desired concentration of genetically modified interferon-α2 polypeptide is greater than or equal to 1×10 6 ~100×106 In some embodiments, the desired concentration of modified interferon-α2 polypeptide is 10×10 6 ~50×10 6 In some embodiments, the desired concentration of modified interferon-α2 polypeptide is 1×10 IU / mL. 6 ~10×10 6 IU / mL. In embodiments, the desired concentration of modified interferon-α2 polypeptide is reduced for a maintenance dose during maintenance treatment of a condition in a subject. In embodiments, the sterile solution is stored at a low temperature prior to administration to a subject. In embodiments, the sterile solution is stored at a temperature ranging from 2° C. to 8° C. prior to administration to a subject.

[0190] In some embodiments, the pharmaceutical composition or formulation comprises a solution or suspension containing one or more modified interferon-α2 polypeptides and one or more ingredients, wherein the ingredients are selected from the group consisting of sterile water, sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, EDTA, one or more surface adsorption inhibitors (e.g., polysorbate 80), one or more antimicrobial preservatives (e.g., m-cresol), one or more bulking agents, and one or more degradation inhibitors. In some embodiments, the solution or suspension comprises one or more modified interferon-α2 polypeptides, sterile water, sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, EDTA, polysorbate 80, and m-cresol. In some embodiments, the one or more modified interferon-α2 polypeptides are provided with a desired specific activity. In some embodiments, the solution or suspension provides the one or more modified interferon-α2 polypeptides at a desired concentration. In some embodiments, the desired concentration of modified interferon-α2 polypeptide is greater than or equal to 1×10. 6 ~100×10 6 In some embodiments, the desired concentration of modified interferon-α2 polypeptide is 10×10 6 ~50×10 6In some embodiments, the desired concentration of modified interferon-α2 polypeptide is 1×10 IU / mL. 6 ~10×10 6 IU / mL. In embodiments, the desired concentration of modified interferon-α2 polypeptide is reduced for a maintenance dose during maintenance treatment of a condition in a subject. In embodiments, the solution or suspension is stored at a low temperature prior to administration to a subject. In embodiments, the solution or suspension is stored at a temperature ranging from 2° C. to 8° C. prior to administration to a subject.

[0191] In some embodiments, the pharmaceutical composition or formulation solution or suspension comprises one or more modified interferon-α2 polypeptides, a salt, and a buffering agent. In some embodiments, the buffer is provided to maintain the pH at a desired level. In some embodiments, the buffer is a phosphate buffer and the salt is sodium chloride. In some embodiments, the pharmaceutical composition or formulation comprises a sterile powder for extemporaneous preparation of a sterile injectable solution or dispersion, the sterile powder comprising a dry powder formulation of one or more modified interferon-α2 polypeptides. In some embodiments, the sterile powder further comprises one or more ingredients selected from the group consisting of anhydrous dibasic sodium phosphate, monobasic sodium phosphate dihydrate, sucrose, and polysorbate 80. In some embodiments, the sterile powder is provided as a whole tablet-like solid, pieces, and / or a loose powder. In some embodiments, the dry powder formulation of one or more modified interferon-α2 polypeptides is a lyophilized powder. In some embodiments, the one or more modified interferon-α2 polypeptides are provided with a desired specific activity. In some embodiments, the sterile powder is stored at a low temperature prior to administration to a subject. In some embodiments, the sterile powder is stored at a temperature ranging from 2°C to 8°C prior to administration to a subject. In some embodiments, the sterile powder is stored at room temperature prior to resuspension. In embodiments, the sterile powder is stored at a temperature ranging from 15°C to 30°C prior to resuspension. In embodiments, prior to administration to a subject, the sterile powder is reconstituted with a diluent to provide a sterile solution. In embodiments, the reconstitution is accomplished by dissolving the sterile powder in a diluent (e.g., by stirring, swirling, inverting, shaking, vortexing, or other means known and understood in the art) to generate a sterile solution. In embodiments, the diluent consists of sterile water. In embodiments, the resuspension is performed in a single-use vial, ampoule, or syringe. In embodiments, the resuspension is performed in a dual-chamber cartridge, where a first chamber contains the sterile powder and a second chamber contains the diluent, and prior to injection, the components of the two chambers are combined to generate a sterile solution. In embodiments, the dual-chamber cartridge is used to inject the sterile solution into a subject via an injection device that is part of the dual-chamber cartridge. In embodiments, the sterile solution provides the one or more modified interferon-α2 polypeptides at a desired concentration.In embodiments, the desired concentration of the modified interferon-α2 polypeptide is 50 to 500 mcg / mL. In embodiments, the desired concentration of the modified interferon-α2 polypeptide is 100 to 300 mcg / mL. In embodiments, the desired concentration of the modified interferon-α2 polypeptide is 100 to 2000 mcg / mL. In embodiments, the desired concentration of the modified interferon-α2 polypeptide is 400 to 1200 mcg / mL. In embodiments, the sterile solution is stored at a low temperature prior to administration to a subject. In embodiments, the sterile solution is stored at a temperature ranging from 2°C to 8°C prior to administration to a subject.

[0192] In some embodiments, a pharmaceutical composition or formulation of a modified interferon-α2 compound or composition of the present disclosure is co-administered with one or more other pharmaceutical compositions or formulations selected from the group consisting of ribavirin (e.g., REBETOL®), PegIntron®, and INTRON-A®.

[0193] In some embodiments, oral compositions generally contain an inert diluent or an edible carrier and can be enclosed in a gelatin capsule or compressed into a tablet. In some embodiments, for oral therapeutic administration, a binder can be incorporated with an excipient and used in the form of a tablet, a troche, or a capsule. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is orally applied and swished to expectorate or swallowed. Pharmaceutically compatible binding compounds and / or adjuvant materials can be included as part of the composition. In some embodiments, tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; fillers such as starch or lactose, disintegrating compounds such as alginic acid, Primogel or cornstarch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening compounds such as sucrose or saccharin; or flavoring compounds such as peppermint, methyl salicylate or orange flavoring.

[0194] For administration by inhalation, the modified interferon-α2 compounds or compositions of the present disclosure can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0195] In some embodiments, the systemic administration of the modified interferon-α2 compound or composition of the present disclosure can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, the modified interferon-α2 compound or composition of the present disclosure can be formulated into a hard ointment, salve, gel, or cream, and can be applied topically or through transdermal patch technology, as is generally known in the art.

[0196] In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0197] In some embodiments, the modified interferon-α2 compounds or compositions of the present disclosure are prepared with carriers that protect the modified interferon-α2 compositions against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be commercially obtained, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art (U.S. Patent No. 4,522,811, which is incorporated herein by reference in its entirety). In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure can be implanted within or linked to a biopolymer solid support that allows for the sustained release of the modified interferon-α2 composition to the desired site.

[0198] In some embodiments, it is particularly advantageous to formulate oral or parenteral compositions into dosage unit form for ease of administration and uniformity of dosage.Dose unit form as used herein refers to a physically discrete unit suitable as a unit dose for the subject to be treated, and each unit contains a predetermined amount of binding agent calculated to achieve desired therapeutic effect in association with necessary pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined by and directly depends on the inherent properties of binding agent and the specific therapeutic effect to be achieved, and the inherent limitation of the technology of compounding such modified interferon-α2 compound or composition of the present disclosure for the treatment of subject.

[0199] [How to use] The modified interferon-α2 compounds or compositions of the present disclosure (such as one or more modified interferon-α2 polypeptides, polynucleotides, microorganisms expressing one or more polypeptides or polynucleotides, expression cassettes, plasmids, expression vectors, chimeric or fusion polypeptides, recombinant viruses and / or pharmaceutical compositions of the present disclosure) find use in the protection against / treatment of melanoma, melanoma (including malignant melanoma), acute and chronic hepatitis C (including in patients with compensated liver disease), acute and chronic hepatitis B, acute and chronic non-A, non-B hepatitis, Kaposi's sarcoma (including AIDS-associated Kaposi's sarcoma), multiple sclerosis, genital warts, leukemia (including hairy cell leukemia), lymphoma (including follicular lymphoma), condyloma acuminata, and other viral infections (including SARS-CoV-2 infection, ZIKV infection, CHIKV infection, or influenza A infection). In embodiments, the present disclosure provides for the use of a compound or composition of the present disclosure, a modified interferon-α2 compound or composition of the present disclosure, as disclosed herein, for the manufacture of a medicament for the treatment of melanoma, melanoma (including malignant melanoma), acute and chronic hepatitis C (including patients with compensated liver disease), acute and chronic hepatitis B, acute and chronic non-A, non-B hepatitis, Kaposi's sarcoma (including AIDS-associated Kaposi's sarcoma), multiple sclerosis, genital warts, leukemia (including hairy cell leukemia), lymphoma (including follicular lymphoma), condyloma acuminata, other viral infections (including SARS-COV-2 infection, ZIKV infection, CHIKV infection, or influenza A infection), and the like.

[0200] In several aspects, the present disclosure is directed to methods of preventing or treating one or more medical conditions in a subject, comprising administering one or more modified interferon-α2 compounds or compositions of the present disclosure, and the prevention or treatment of medical conditions in a subject by administering one or more modified interferon-α2 compounds or compositions of the present disclosure. Medical conditions include, for example, melanoma, melanoma (including malignant melanoma), acute and chronic hepatitis C (including patients with compensated liver disease), acute and chronic hepatitis B, acute and chronic non-A, non-B hepatitis, Kaposi's sarcoma (including AIDS-associated Kaposi's sarcoma), multiple sclerosis, genital warts, leukemia (including hairy cell leukemia), lymphoma (including follicular lymphoma), condyloma acuminata, and other viral infections (including SARS-CoV-2 infection, ZIKV infection, CHIKV infection, or influenza A infection). In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure can be used in combination with other proteins or compounds used to treat subjects with a medical condition to reduce adverse events or to increase the efficacy of the co-administered compound.

[0201] In certain embodiments, the present disclosure is directed to a method for treating, for example, chronic hepatitis C, comprising administering one or more modified interferon-α2 compounds or compositions of the present disclosure, and preventing or treating chronic hepatitis C in a subject by administering the one or more modified interferon-α2 compounds or compositions of the present disclosure. The modified interferon-α2 compounds or compositions of the present disclosure can be used in combination with other proteins or compounds used to treat subjects with chronic hepatitis C to reduce adverse events or enhance the efficacy of co-administered compounds. In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure (e.g., GMOP-IFN-α2 mutants, IFN-α2 mutants, etc.) lack antiproliferative properties while maintaining antiviral activity, representing an interesting therapeutic alternative for treating chronic hepatitis C. In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure exhibit high relative antiviral activity while reducing immunogenicity in treating chronic hepatitis C.

[0202] In embodiments, the present disclosure is directed to a method for treating, for example, chronic hepatitis B, comprising administering one or more modified interferon-α2 compounds or compositions of the present disclosure, and preventing or treating chronic hepatitis B in a subject by administering one or more modified interferon-α2 compounds or compositions of the present disclosure. The modified interferon-α2 compounds or compositions of the present disclosure can be used together with other proteins or compounds used to treat subjects with chronic hepatitis B to reduce adverse events or enhance the efficacy of co-administered compounds. In embodiments, the modified interferon-α2 compositions of the present disclosure (e.g., GMOP-IFN-α2 mutants, IFN-α2 mutants) lack antiproliferative properties while maintaining antiviral activity, representing an interesting therapeutic alternative for treating chronic hepatitis B. In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure exhibit high relative antiviral activity while reducing immunogenicity in treating chronic hepatitis B.

[0203] Emerging viral infections, such as SARS-CoV-2, ZIKV, CHIKV, and influenza A, have become a global public health concern due to the rapid spread of their etiologic agents to new regions, increasing human infections, and a lack of new treatments and / or effective vaccines. In embodiments, the present disclosure is directed to methods of treating, for example, SARS-CoV-2 infection (and / or related diseases caused by SARS-CoV-2, including COVID-19), ZIKV, CHIKV, or influenza A, including administering one or more modified interferon-α2 compounds or compositions of the present disclosure, and preventing or treating the infection or disease in a subject by administering one or more modified interferon-α2 compounds or compositions of the present disclosure. In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure can be used in combination with other proteins or compounds used to treat a subject with a medical condition to reduce adverse events or enhance the efficacy of co-administered compounds. In embodiments, the modified interferon-α2 compounds or compositions of the present disclosure (e.g., GMOP-IFN-α2 mutants, IFN-α2 mutants) lack antiproliferative properties while maintaining antiviral activity, representing an interesting therapeutic alternative for the treatment of SARS-COV-2 (and / or related diseases caused by SARS-COV-2, including COVID-19), ZIKV, CHIKV, or influenza A. In embodiments, one or more compounds or compositions of the present disclosure, as described above, exhibit high relative antiviral activity with reduced immunogenicity in the treatment of ZIKV, CHIKV, or influenza A.

[0204] In embodiments of the above-described methods, the modified interferon-α2 compound or composition of the present disclosure is co-administered with one or more other pharmaceutical compositions or formulations, hi embodiments, the one or more other pharmaceutical compositions or formulations are selected from the group consisting of ribavirin (e.g., REBETOL®), PegIntron®, and INTRON-A®.

[0205] The methods described herein can be carried out, for example, by utilizing a pre-packaged kit containing at least one pharmaceutical formulation or composition for the treatment and / or prevention of a disease described herein (including melanoma or viral infection and / or related disease), which can be conveniently used, for example, in a clinical setting to treat a subject exhibiting a medical condition or family history described herein. In one embodiment, the kit further includes instructions for use of at least one modified interferon-α2 composition of the present disclosure to treat a subject exhibiting symptoms or a family history of a medical condition described herein. [Example]

[0206] The examples set forth below should not be construed as limiting the scope of the invention in any way. In light of this disclosure, numerous embodiments within the scope of the claims will be apparent to those of skill in the art.

[0207] (1) In silico identification of immunogenic sites of therapeutic drugs T cells specifically recognize epitopes presented by antigen-presenting cells (APCs) in the context of MHC (major histocompatibility complex) class II molecules. These T-helper epitopes can be expressed as linear sequences of 7–30 consecutive amino acids that fit into the MHC class II binding groove. Many computer algorithms have been developed and used to detect class II epitopes within protein molecules of various origins (De Groot AS et al., (1997), AIDS Res Hum Retroviruses, 13(7):539–41; Schafer JR et al., (1998), Vaccine, 16(19):1880–4; De Groot AS et al., (2001), Vaccine, 19(31):4385–95; De Groot AS et al., (2003), Vaccine, 21(27–30):4486–504). These "in silico" predictions of T-helper epitopes have been successfully applied to the design of vaccines and deimmunization of therapeutic proteins, namely antibody-based drugs, Fc-fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, artificial protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytic agents (Dimitrov DS, (2012), Methods Mol Biol, 899:1-26). The EpiMatrix® system (EpiVax, Providence, Rhode Island) is a set of prediction algorithms coded into a computer program useful for predicting class I and class II HLA ligands and T cell epitopes. The EpiMatrix® system uses a 20x9 coefficient matrix to model the interactions between specific amino acids (20) and binding positions (9) within HLA molecules. To identify putative T cell epitopes present in a given input protein, the EpiMatrix® system first analyzes the input protein to create a set of 9-mer frames, each of which overlaps the last amino acid by 8. Each frame is then scored for predicted affinity to one or more common alleles of human HLA molecules. Typically, these are DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501 (Mack et al., (2013), Tiss Antig, 81(4):194-203). Briefly, for any 9-mer peptide, specific amino acid codes (one for each of the 20 naturally occurring amino acids) and relative binding positions (1-9) are used to select coefficients from a prediction matrix. Individual coefficients are derived using a proprietary method similar, but not identical, to the pocket profile method originally developed by Sturniolo (Sturniolo T et al., 1999, Nat Biotechnol, 17(6):555-61). Individual coefficients are then summed to generate a raw score. The raw EpiMatrix® scores are then normalized with respect to the distribution of scores obtained from a very large set of randomly generated peptide sequences. The resulting "Z" scores are normally distributed and can be directly compared between alleles.

[0208] EpiMatrix® Peptide Scoring Peptides with an EpiMatrix® "Z" score of 1.64 or higher (approximately the top 5% of any peptide set) were determined to have a significantly higher probability of binding to the predicted MHC molecule and were designated "hits." Peptides with a score of 2.32 or higher (the top 1%) were significantly more likely to bind, with most published T cell epitopes falling within this score range. Previous studies have also demonstrated that EpiMatrix® accurately predicts published MHC ligands and T cell epitopes.

[0209] Identification of T-cell epitope clusters. Candidate T-cell epitopes are not randomly distributed throughout a protein sequence; they tend to cluster together. T-cell epitope clusters range in length from 9 to approximately 30 amino acids and, considering affinity for multiple alleles and multiple frames, contain 4 to 40 binding motifs. After epitope mapping, the result set generated by the EpiMatrix® algorithm was screened for the presence of T-cell epitope clusters and epibars using a proprietary algorithm known as Clusterimer®. Briefly, the EpiMatrix® scores for each analyzed 9-mer peptide were aggregated and compared against a statistically derived threshold. High-scoring 9-mer peptides were then extended one amino acid at a time. The scores of the extended sequences were then re-aggregated and compared to a revised threshold. This process was repeated until the proposed extension no longer improved the cluster's overall score. Highly immunogenic regions, defined as those with a score of 10 or greater (including multiple "hits" against many different HLA DR alleles), were identified as T cell epitope clusters. These regions contain a substantial number of putative T cell epitopes and EpiBars® that demonstrate a high potential for MHC binding and T cell reactivity.

[0210] [Prediction of amino acids involved in HLA binding] The contribution of each amino acid in these regions to HLA binding was assessed using the OptiMatrix tool (part of the EpiVax ISPRI toolkit for deimmunization). OptiMatrix begins by examining the "critical" residues that contribute most to MHC-binding affinity across multiple 9-mer frames and multiple HLA alleles. The program then iteratively replaces all 19 alternative amino acids at any position in the protein sequence (operator-defined input allows the list to be limited to naturally conserved variants) and reanalyzes the predicted immunogenicity of the resulting sequence. To avoid adverse effects on the protein's structure and, therefore, biological activity, a comprehensive literature search for critical residues was performed to identify amino acids that were not candidates for modification.

[0211] Example 1. In silico immunogenicity prediction and deimmunized protein design Binding of peptides to HLA molecules is a critical first step required for T cell responses. Indeed, one of the most important determinants of a protein's immunogenicity is the strength of peptide binding to MHC molecules (Lazarski CA et al., (2005) Immunity. 23: 29-40). To analyze the potential immunogenicity of GMOP-IFN (SEQ ID NO: 10), the complete amino acid sequence was screened using EpiMatrix. This study revealed a high content of T cell epitopes in the protein sequence (Figure 1A). Further analysis using the ClustiMer algorithm allowed the identification of putative 9-mer MHC-binding peptides and their association to cluster regions. A total of six clusters were defined, spanning the following residues of GMOP-IFN (SEQ ID NO: 10): 20-43, 58-72, 70-89, 121-141, 131-154, and 158-179. Five of the six predicted MHC-binding clusters overlapped with previously reported T cell epitopes.

[0212] Next, OptiMatrix was used to identify critical residues that inhibit or reduce MHC II binding affinity. Modifications not identified as critical for biological activity or receptor binding were selected from the OptiMatrix suggested changes. These results were then compared with the MHC binding cluster predictions from ClustiMer. Based on this comparison, 10 sites for modification in GMOP-IFN-α2b (SEQ ID NO: 10) were selected, corresponding to the following positions in the amino acid sequence: 23, 31, 61, 79, 80, 131, 142, 137, 161, and 171. These 10 mutations were introduced in different combinations into the GMOP-IFN-α2b sequence (SEQ ID NO: 10) to generate mutants. Each amino acid was mutated to alanine (except for position 161, where the amino acid was mutated to threonine). All of these mutations were introduced to generate GMOP-IFN-VAR1 (SEQ ID NO: 2), and the effect of the mutations on T cell epitope content is shown in Figure 1B.

[0213] The L9A, F47A, L117A, F123A, and L128A modifications of the hIFN-α2b molecule (SEQ ID NO: 12) were found to be important for binding to specific HLA molecules. Therefore, these modifications (corresponding to positions L23A, F61A, L131A, F137A, and L142A of GMOP-IFN-α2b in SEQ ID NO: 10) were mutated to develop GMOP-IFN-VAR2 (SEQ ID NO: 4). Two additional protein variants, GMOP-IFN-VAR3 (SEQ ID NO: 6) and GMOP-IFN-VAR4 (SEQ ID NO: 8), with seven mutations were also created to reduce the antigenicity of clusters 158-179 and 70-89, respectively. The modifications to produce GMOP-IFN-VAR3 (SEQ ID NO: 6) were as follows: The modifications to produce GMOP-IFN-α2b (SEQ ID NO: 8) were as follows: L23A, F61A, N79A, L80A, L131A, F137A, and L142A. Table 1 summarizes the GMOP-IFN-α2b mutants that were created.

[0214] The immunogenicity score for each variant was calculated using EpiMatrix as described above. As shown in Figure 2, the EpiMatrix immunogenicity global score for each variant is significantly reduced compared to the parent molecule.

[0215] [Table 1] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-α2b correspond to amino acids that are not involved in the biological structure or function of the cytokine, as detailed above. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, the following variants: IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c) with or without one or more GMOP sequences appended.

[0216] (2) Gene expression, and protein production, purification, and characterization (cell culture) Chinese hamster ovary (CHO-K1) cells were cultured in a previously described basal culture medium (Kratje RB, Wagner R, (1992), Biotechnol. Bioeng. 39: 233-242) supplemented with 5% (v / v) fetal calf serum (FCS) (PAA, Argentina). Human embryonic kidney (HEK293T) cells were cultured in DMEM supplemented with 10% (v / v) FCS and 2 mM glutamine. Madine Darby bovine kidney (MDBK) cells were cultured in minimum essential medium (MEM; Gibco, USA) supplemented with 10% (v / v) FCS. Bioassays were performed using MEM supplemented with 2% (v / v) FCS (assay medium). The human Daudi cell line was maintained in RPMI 1640 medium (Gibco) plus 10% (v / v) FCS. All cells were incubated at 37°C in a humidified atmosphere of 5% CO2.

[0217] (Construction of lentiviral vectors and assembly of lentiviral particles) A plasmid (GeneWiz, USA) containing the hIFN-α2b coding sequence was digested with SalI and XbaI enzymes, and the released DNA fragments corresponding to each GMOP-IFN mutant were cloned into a lentiviral plasmid (pLV) (A: Oberbek A. (2011) BiotechnolBioeng 108(3):600-610. B: Chusainow J. (2009) BiotechnolBioeng 102(4):1182-1196). The identity of all constructs was verified by DNA sequencing. Research-grade HIV-based LV particles containing the three hIFN-α2b analog transgenes were produced according to the protocols suggested by Naldini et al. (1996, Science, 272: 263-267) and Dull et al. (1998, J. Virol. 72: 8463-71). Adherent HEK293T cells were cultured in 10-cm plates and simultaneously cotransfected with four plasmids: a packaging plasmid (pMDLg / pRRE) (Dull et al. (1998) J. Virol. 72:8463-71), a Rev expression plasmid (pRSV-Rev) (Naldini et al. (1996) Science 272:263-267), an envelope plasmid expressing VSV-G (pMD2.G) (Dull et al. (1998) J. Virol. 72:8463-71), and the corresponding transfer vectors (pLVs) containing the transgenes. All plasmids were introduced into the cells by liposome-mediated gene transfer using LipofectAMINE 2000 Reagent (Invitrogen, USA) according to the supplier's instructions. The supernatant containing lentiviral particles (LVP) was collected 72 hours after transfection. (lentiviral transduction) 6.0 x 10 per well 4Transduction was performed by incubating 1 ml of LVP-containing supernatant with cells seeded in 6-well plates (Greiner). The medium was replaced with fresh medium 24 hours after transduction. To eliminate remaining wild-type cells, a selection step was initiated 96 hours after transduction by replacing the supernatant with fresh growth medium containing 10 μg ml puromycin (Sigma-Aldrich, USA). The selection medium was replaced every 3–4 days with increasing concentrations of puromycin until the control cells died.

[0218] (Production and purification of GMOP-IFN mutants) The transformed cells were expanded for the production of GMOP-IFN variants, and the productivity of each cell line was assessed by measuring rhIFN-α2b concentration and cell counting. Cells were cultured at 500 cm in growth medium. 2 The cells were grown to confluence in three-neck flasks. The medium was then changed to basal medium (production medium) supplemented with 0.5% (v / v) FCS. Every 48 or 72 hours, the conditioned medium was collected and replaced with fresh production medium. The harvest was clarified by centrifugation and stored at -20°C. Protein was purified by immunoaffinity chromatography using the anti-nonglycosylated rhIFN-α2b mAb CA5E6 (which has been shown to effectively bind various IFN variants) coupled to CNBr-activated Sepharose 4B (GE Healthcare) as previously described (Ceario N et al. (2008) Biochimie 90:437-449). The concentration of the purified GMOP-IFN variants was determined by spectrophotometric quantification.

[0219] (rhIFN-α sandwich ELISA) GMOP-IFN mutant yields from culture supernatants were quantified by a specific sandwich ELISA assay described by Ceaglio et al. (2008, Biochimie. 90: 437-449) based on the capture of IFN-α2b (and its different versions) by the monoclonal antibody (mAb) CA5E6 immobilized on a polystyrene plate and subsequent recognition by immunoglobulins (Igs) present in rabbit anti-IFN-α2b polyclonal serum (C7).

[0220] In a 96-well flat-bottom polystyrene plate (Greiner), 1 g.ml of mAb CA5E6 diluted in Na2CO3 / NaHCO3 50 mM pH 9.6 solution was added. -1 100 liters of (100 ng / well) was sensitized (sensitization solution), which was then incubated at 37°C for 1 hour and at 4°C overnight.

[0221] Nonspecific interaction sites were blocked using 200 L of bovine serum albumin (BSA, Sigma) 1% (P / V) in PBS (blocking solution) per well, which was then incubated at 37°C for 1 hour.

[0222] The first incubation consisted of bacterial ifn-2b standard (Gema Biotech, Argentina) at 10 ng / ml -1 to 0.078ng.ml -1 A 1:2 serial dilution of 100 μl of IFN-2b was performed from the sample to be analyzed. For this purpose, a 0.1% BSA (P / V) solution in PBS supplemented with 0.05% (V / V) Tween 20 was used (dilution solution). The samples were serially diluted in medium to allow comparison with the standard within the linear range of the curve. The incubation was carried out for 1 hour at 37°C. To assess possible nonspecific binding of the reagent, a check was carried out without adding IFN-2b (negative control). To do this, IFN was replaced by 100 μl of dilution solution during this step.

[0223] The second incubation was performed by adding 100 μl of rabbit serum C7 anti-IFN-2b diluted 1:1,000 in diluent, and incubated at 37°C for 1 hour.

[0224] The third incubation was performed by adding 100 μL of rabbit anti-immunoglobulin goat antibody conjugated with the enzyme peroxidase (DAKO, Denmark) at a 1:2,000 dilution in diluent, followed by incubation at 37°C for 1 hour.

[0225] The revealing reaction consisted of a substrate of H2O2 (0.015 vol) diluted in 50 mM citric acid / sodium phosphate solution, pH 5.3 (revealing solution), and 0.5 mg / ml o-phenylenediamine chromogen (OPD, Sigma). -1 The enzyme reaction was revealed by adding 100 μL of the solution per well and incubating for 15 minutes at room temperature in the dark. The enzyme catalyzed the reduction of the substrate and simultaneously oxidized the color-developing reagent, resulting in color development. The reaction was stopped by adding 50 μL of H2O42N, and the color was read at 492 nm using a microtiter plate reader (Labsystems Multiskan MCC / 340, Finland).

[0226] Quantitation was performed by plotting absorbance values ​​based on the concentration of IFN-2b used as the standard and the dilution rate of the samples, both of which were displayed on a logarithmic scale. Sample concentrations were determined using a parallel linearity test (D: Milano, F. (2001) Bachelor's Thesis in Biotechnology. Design and Validation of Bioassays for the In Vitro Biological Evaluation of Drugs. Faculty of Biochemistry and Biological Sciences, UNL, Santa Fe, Argentina.). (SDS-PAGE and Western blotting) SDS-PAGE analysis was performed according to standard methods using a 15% (w / v) polyacrylamide resolving gel and a 5% (w / v) stacking gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (BioRad). Blots were blocked with 5% (w / v) nonfat milk in Tris-buffered saline (TBS) for 1 hour and probed with rabbit anti-rhIFN-α2b polyclonal antibody. After 1 hour, the blots were incubated with the same peroxidase label as described for ELISA. Immunoreactive bands were visualized using an ECL® Chemiluminescent Western Blotting Analysis System (GE Healthcare). Washing between steps was performed with TBS containing 0.05% (v / v) Tween 20 (TBS-T). Dilutions were prepared in TBS-T containing 0.5% (w / v) nonfat milk.

[0227] Example 2. GMOP-IFN Deimmunized Mutants: Production and Purification The GMOP-IFN mutants were synthesized, cloned into third-generation lentiviral vectors, and expressed in CHO cells. After selecting the cells with puromycin (300 μg / ml), culture supernatants from stable cell lines were preliminarily screened for rhIFN-α2b production and biological potency by sandwich ELISA and antiviral assay, respectively.

[0228] The proteins were purified by a single-step immunoaffinity chromatography using CNBr-activated Sepharose with a monoclonal antibody (CA5E6) as a ligand. The protein variants contained in the supernatant were loaded onto the matrix at no more than 40% of the theoretical capacity. No loss of cytokine was observed during the flow-through or washing steps. Protein concentrations were measured spectrophotometrically at a wavelength of λ = 280 nm (Figure 8).

[0229] Protein purity was analyzed by SDS-PAGE followed by Coomassie blue staining (Figure 3). All protein preparations showed a similar mobility shift in SDS-PAGE. However, non-glycosylated rhIFN-α2b was also detected in all samples, reflecting the presence of a less efficiently occupied O-glycosylation consensus sequence. Densitometry profiles of GMOP-IFN-VAR2 and GMOP-IFN-VAR3 showed purities of over 94% with bovine serum albumin (BSA) as the primary contaminant. In contrast, the purity levels of GMOP-IFN-VAR1 and GMOP-IFN-VAR4 proteins were approximately 80%, likely due to lower protein binding to CA5E6 mAb.

[0230] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-2b correspond to amino acids that are not involved in the biological structure or function of the cytokine. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, variants such as IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c) with or without the addition of one or more GMOP sequences.

[0231] (3) In vitro activity measurement method (Antiviral assay) Antiviral biological titration assays for interferons quantitate the inhibitory activity of these cytokines on viral spread or replication (Familletti G et al., (1981), Methods Enzymology 78: 387-394). The simplest and most convenient procedure is to measure the ability of interferon to protect susceptible cells from the cytopathic effects of lytic viruses over a range of cytokine concentrations.

[0232] The biological antiviral activity of rhIFN-α2b was determined by its ability to inhibit the cytopathic effects caused by vesicular stomatitis virus (VSV) on MDBK cells (Familletti PC et al., (1981), Methods Enzymol. 78: 387-394; Rubinstein S et al., (1981 J. Virol. 37: 755-8). To evaluate the effect of modifications on the antiviral activity of GMOP-IFN mutants, MDBK cells were seeded (2.5 × 10 per well) in culture microtiter plates in growth medium [MEM supplemented with 10% SFB (V / V)]. 4 cells) and incubated overnight at 37°C.

[0233] After removing the culture supernatant, 20 μg / ml of rhIFN-a2b (WHO international standard (NIBSC95 / 566)) was added. 1 From 0.16Uml- 1 Serial 1:2 dilutions of rhIFN-α2b or test samples of each GMOP-IFN mutant were added to the assay medium. The plates were then incubated at 37°C for 6 hours. After removing the supernatant, the monolayers were infected with 1.6 PFU of VSV virus per cell. Viral replication was allowed to proceed until clear cytopathic activity was observed in control wells (no rhIFN-α2b). The medium was discarded, and cells were fixed and simultaneously stained with a solution of 0.75% (w / v) crystal violet in 40% (v / v) methanol (Merck). After 15 minutes at 37°C, the plates were washed with distilled water to remove the dye, and the fixed dye was solubilized in 20% (v / v) acetic acid. The plates were read at λ = 540 nm using a microtiter plate reader with the ability to homogenize the plates before reading. The signal intensity for each dilution was reported as the average absorbance measured in five wells.

[0234] The absorbance data were plotted on a logarithmic scale as a function of the activity of IFN-α2b (standard) and the dilution of the sample, and the biological activity (AB) of each molecule was calculated by comparing it with the standard using a parallel line test. From these results, the quotient of the AB value and the molecule concentration in the sample was calculated to determine the specific biological activity (ABE) of each protein.

[0235] Finally, the relative antiviral activity values ​​in percentage were determined by forming the quotient of the ABE of the IFN-α2b-WT molecule (180 ± 50 IU / ng) and the ABE of each of the GMOP-IFN-α2b mutants.

[0236] Antiproliferative Assay To measure the ability of rhIFN-α2b to inhibit cell proliferation, an in vitro bioassay was performed using Daudi cells (Nederman T et al., (1990), Biologicals. 18: 29-34). Serial 1:2 dilutions of rhIFN-α2b WHO International Standard from 50 U / ml to 0.02 U / ml, or 1:2 dilutions of test samples of each GMOP-IFN mutant, were placed in the wells of a microtiter plate. Prewashed Daudi cells (5 × 10 per well) were then added. 3 The plates were incubated at 37°C for 96 hours. Cell proliferation was assessed using the CellTiter 96™ AQueous Non-Radioactive Cell Proliferation Assay (Promega) with two reagents: MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] at a concentration of 2 mg / ml; -1 and PMS [phenazine methosulfate] concentration 0.92 mg.ml -1 ) was used. WHO international standard: Escherichia coli-produced rhIFN-2b (NIBSC95 / 566) was used.

[0237] In a sterile 96-well flat-bottom plate, rhIFN-a2b standard in RPMI medium (growth medium) supplemented with 10% SFB (V / V) was added at an activity concentration of 50 UI.ml -1 to 0.39UI.ml -1 50 μl of each of the 1:2 serial dilutions was placed per well. Each protein was analyzed under the same conditions, with appropriate initial dilutions to allow comparison with the standard within the linear range of the dose-response curve.

[0238] The Daudi cell line was cultured during the growth stage. 5 cellml -1 A suspension of 50 μl was prepared, 50 μl of which was added to each well, and the mixture was incubated in a stove at 37° C. for 96 hours.

[0239] To demonstrate the assay, 20 μl of a colorimetric reagent prepared by mixing 2 mL of MTS solution with 100 μL of PMS solution per plate was added per well. The plate was incubated at 37°C for 5 hours. This colorimetric method measures cell proliferation by highlighting the presence of dehydrogenase enzymes found in metabolically active cells, whose activity is directly related to the number of viable cells present in culture. The dehydrogenase enzymes catalyze the bioreduction of MTS to a soluble (blue) formazan colorimetric reagent that absorbs at 490 nm. PMS acts as an electron donor in the redox reaction. The amount of product produced is directly proportional to the number of metabolically active cells in the crop. The absorbance of the colorimetric reagent was measured at 492 nm using a microplate reader against a plate background reading of 690 nm. This assay was reproduced in triplicate.

[0240] Absorbance values ​​were plotted against the corresponding standard activity data and sample dilution values ​​on a logarithmic scale. The antiproliferative bioactivity value of each novel molecule was calculated using the standard using the parallel line comparison method.

[0241] Finally, specific antiproliferative bioactivity values ​​were determined by forming a ratio between volumetric activity and protein concentration.

[0242] Example 3A. GMOP-IFN-VAR2 and GMOP-IFN-VAR3 exhibited high residual antiviral activity and null antiproliferative properties. A deimmunization strategy was used, aiming to modify the most immunogenic amino acids without altering residues directly involved in antiviral activity. The effect of these modifications on the biological activity of the cytokine was evaluated by in vitro antiviral activity assays. As recommended by the European Pharmacopoeia, MDBK cells were used as targets for viral infection with VSV virus. The relative antiviral activity of GMOP-IFN-α2b mutants relative to GMOP-IFN-α2b (190 ± 50 UI / ml) was determined by their ability to inhibit the cytopathic effect caused by vesicular stomatitis virus on MDBK cells and normalized to the activity of GMOP-IFN-α2b (Figures 9, 10, and 11). Preliminary antiviral activity tests were performed using cell line culture supernatants from the production strains of each GMOP-IFN-α2b mutant. All supernatants exhibited antiviral activity, although at different levels (Figure 9).

[0243] The relative antiviral activity of the GMOP-IFN-α2b mutants to that of GMOP-IFN-α2b (190 ± 50 UI / ml) was then determined using purified GMOP-IFN-α2b and the GMOP-IFN-α2b mutants as described above (Figures 10 and 11). A significant decrease in residual antiviral activity was observed for GMOP-IFN-VAR1 and GMOP-IFN-VAR4 (0.06% and 0.17%, respectively) (Figure 10). As a result, both proteins were discarded from further study. On the other hand, as shown in Table 2, GMOP-IFN-VAR2 and GMOP-IFN-VAR3 retained most of their original antiviral activity (72% and 35%, respectively) (Figure 11). This reflects the fact that a partial reduction in IFN-receptor interaction was still evident despite the selection of immunogenic residues limited to those not directly involved in biological activity.

[0244] [Table 2] During antiviral therapy with rhIFN-α, one of the most common side effects is a decrease in neutrophil counts, i.e., neutropenia, which is frequently associated with dose adjustment or early discontinuation (Saleh MI and Hindi NN, (2018), Naunyn. Schmiedebergs. Pharmacol. 391: 953-963). Therefore, to further characterize the antiproliferative activity of GMOP-IFN-VAR2 and GMOP-IFN-VAR3, we used an in vitro bioassay to measure their ability to inhibit cell proliferation in Daudi cells. Despite not altering residues directly involved in the protein's biological activity, a significant reduction in their specific antiproliferative activity was observed for both protein variants. As shown in Table 3, both GMOP-IFN-VAR2 and GMOP-IFN-VAR3 exhibited less than 1% of their original antiproliferative potency (0.5 ± 0.2 Ung- for GMOP-IFN-VAR2). 1 , and 0.4 ± 0.1 Ung- for GMOP-IFN-VAR3 1 ) Taken together, these results indicate that the same cellular receptors are responsible for the biological activity of both hIFN-α2b, indicating a greater sensitivity of IFN antiproliferative activity to changes in cytokine structure. These results are highly positive, given that high antiproliferative activity is generally associated with undesirable side effects of IFN-2b therapy, such as neutropenia, which creates susceptibility to serious infections (bacterial, viral, fungal, etc.).

[0245] [Table 3] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-2b correspond to amino acids that are not involved in the biological structure or function of the cytokine. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, variants of: IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c), with or without the addition of one or more GMOP sequences.

[0246] Example 3B. Comparative Residual Antiviral and Antiproliferative Properties of IFN-α Variants Compared to Other Interferons The effects of various modifications on the biological activity of the IFN-α2b cytokine were evaluated by an in vitro antiviral activity assay. MDBK cells were used as targets for viral infection with VSV, as recommended by the European Pharmacopoeia. The relative antiviral activity of hyperglycosylated GMOP-IFN variants 1 to 4 (SEQ ID NOs: 2, 4, 6, and 8, respectively) to GMOP-IFN-α2b (190 ± 50 UI / ml) was determined by their ability to inhibit the cytopathic effect caused by vesicular stomatitis virus on MDBK cells and normalized to the activity of GMOP-IFN-α2b.

[0247] Several other purified IFN-α2b mutants were also generated to compare their biological activity with the hyperglycosylated GMOP-IFN mutant. These mutants include PEGylated IFN-α2b, non-glycosylated IFN-α2b, non-glycosylated GMOP-IFN mutants 1-4 (SEQ ID NOs: 2, 4, 6, and 8, respectively), and 4N-IFN. 4N-IFN is a hyperglycosylated IFN-α2b mutant in which mutations were introduced into native hIFN-α2b by substituting Asn to provide a consensus N-glycosylation site consisting of an Asn-Xaa-Ser / Thr tripeptide, where X can be any residue except proline. 4N contains mutations to Asn at positions 4, 23, 70, and 77 of hIFN-α2b.

[0248] Hyperglycosylated GMOP-IFN-VAR1 and hyperglycosylated GMOP-IFN-VAR4 are predicted to have reduced residual antiviral activity. Furthermore, PEGylated IFN-α2b, nonglycosylated IFN-α2b, nonglycosylated GMOP-IFN variants 1-4, and 4N-IFN are predicted to have reduced residual antiviral activity. Hyperglycosylated GMOP-IFN-VAR2 and hyperglycosylated GMOP-IFN-VAR3, on the other hand, are predicted to retain or retain most of their original antiviral activity. This reflects the fact that a partial reduction in IFN-receptor interaction is still evident, despite the limited selection of immunogenic residues not directly involved in biological activity.

[0249] During antiviral therapy with rhIFN-α, one of the most common side effects is a decrease in neutrophil counts, or neutropenia, which is frequently associated with dose adjustment or early discontinuation (Saleh MI and Hindi NN, (2018), Naunyn. Schmiedebergs. Pharmacol. 391: 953-963). Therefore, to further compare the antiproliferative activity of hyperglycosylated GMOP-IFN-VAR2 and hyperglycosylated GMOP-IFN-VAR3 with PEGylated IFN-α2b, nonglycosylated IFN-α2b, nonglycosylated GMOP-IFN variants 1-4, and 4N-IFN, in vitro bioassays were used to measure their ability to inhibit Daudi cell proliferation. Despite not altering residues directly involved in the protein's biological activity, hyperglycosylated GMOP-IFN-VAR2 and hyperglycosylated GMOP-IFN-VAR3 exhibited significantly reduced specific antiproliferative activity. Conversely, we do not expect a significant decrease in specific antiproliferative activity for PEGylated IFN-α2b, nonglycosylated IFN-α2b, nonglycosylated GMOP-IFN variants 1-4, and 4N-IFN. Taken together, these results suggest that IFN antiproliferative activity is more sensitive to changes in cytokine structure, given that the same cellular receptor is responsible for the biological activity of both hIFN-α2b variants. These results are highly encouraging, given that high antiproliferative activity is generally associated with undesirable side effects of IFN-2b therapy, such as neutropenia, which can lead to susceptibility to serious infections (bacterial, viral, fungal, etc.).

[0250] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-2b correspond to amino acids that are not involved in the biological structure or function of the cytokine. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, variants of IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c) with or without the addition of one or more GMOP sequences.

[0251] (4) Physicochemical characterization (Isoelectric focusing) Isoelectric focusing (IEF) was performed on a 1 mm thick 8% (w / v) polyacrylamide gel containing 7 M urea. To define the pH range, 30% (w / v) ampholyte (5-7) and 70% (w / v) ampholyte (2-4) (Pharmalyte, GE Healthcare) were mixed. The gel was prefocused for 30 minutes at 10 W, 2000 V, and 100 mA. Then, 5–20 μl of sample was applied 1 cm from the cathode and electrophoresed for 90 minutes under the same conditions as the prefocusing step. Components separated by IEF were detected by Coomassie blue staining.

[0252] [Evaluation of suitable O-glycosylation sites in GMOP-IFN and its mutants] Because there are no known consensus recognition sequences for O-glycosyltransferases, neural network prediction of mucin-type GalNAc O-glycosylation sites was performed using NetOGlyc 3.1 Server software (Julenius K et al., (2005), Glycobiology. 15: 153-164).

[0253] Example 4. GMOP-IFN deimmunized mutants exhibited distinctive electrophoretic profiles. To further characterize the charge-based heterogeneity of each protein variant, we performed isoelectric focusing (IEF) assays. For wild-type IFN, rhIFN-α2b, produced in CHO-K1 cells, four electrophoretic bands were observed, representing isoforms with O-glycan structures that differed in the content of sialic acid attached to the native Thr106 O-glycosylation site.

[0254] The high content of glycan structures attached to O-glycosylation sites in GMOP-IFN was evidenced by the presence of approximately seven isoforms located in the most acidic region of the gel. Interestingly, both deimmunized variants exhibited distinct electrophoretic profiles compared to the original molecules. A total of 11 electrophoretic bands were detected for both proteins, three of which were located in the most acidic end of the gel. Furthermore, GMOP-IFN-VAR3 exhibited a lower content of the most basic isoform (Figure 4). These results are consistent with the analysis of mucin-type GalNAc O-glycosylation sites using NetOGlyc 3.1 server software. This algorithm predicted the occurrence of five O-glycosylation sites in GMOP-IFN and six in GMOP-IFN-VAR2 and GMOP-IFN-VAR3.

[0255] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-2b correspond to amino acids that are not involved in the biological structure or function of the cytokine. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, variants of: IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c), with or without the addition of one or more GMOP sequences.

[0256] (5) Immunogenicity evaluation [Preparation of human PBMCs and HLA-DR typing] All blood extraction and handling procedures were approved by the Universidad Nacional del Litoral Research Ethics Committee (Santa Fe, AR). Blood samples were collected by venipuncture from healthy donors aged 18 to 60 years after obtaining informed consent.

[0257] PBMCs were isolated by Ficoll-Paque® PLUS (GE Healthcare Biosciences, SE) density gradient separation according to the manufacturer's instructions, and the buffy coat was collected and washed twice with PBS. PBMCs were collected at a concentration of 1-3 × 10 7 The blood was frozen and stored in liquid nitrogen at a concentration of 1000 cells / ml. An aliquot of blood was previously separated, and HLA-DR allotypes were determined using Luminex Sequencing Technology (PRICAI, Buenos Aires, AR). Typing results were compared with the HLA-DR frequencies in the global population published in the Allele Frequency Net Database (The Royal Liverpool and Broadgreen University Hospitals, NHS Trust website: www.allelefrequencies.net).

[0258] [Ex vivo T cell assay] Ex vivo T cell assays were performed with modifications of Jaber and Baker ((2007), J. Pharm. Biomed. Anal. 43: 1256-1261). Monocytes were isolated from PBMCs of each donor blood sample by differential adherence to plastic (Elkord E et al., (2005), Immunology. 114: 204-12). Adherent cells were retained for differentiation, while nonadherent cells were collected and cryopreserved for further use. To induce the immature phenotype of monocyte-derived DCs, monocytes were incubated for 6 days in growth medium containing 1000 U / ml each of human IL-4 (Millipore, USA) and granulocyte-macrophage colony-stimulating factor (GM-CSF, GemaBiotech, AR), with a medium change on day 3. On day 6, immature dendritic cells were collected, counted, and incubated with rhIFN-α2b mutants or non-antigen (medium or vehicle). The test antigens included in this study were GMOP-IFN and its deimmunized variants. After overnight incubation, DCs were washed to remove foreign antigens and resuspended in growth medium containing recombinant human tumor necrosis factor α (rhTNF, ProsPec, USA), GM-CSF, and IL-4 for 4 days to induce DC maturation. Ag-pulsed DCs were then incubated with autologous cells for 48 hours in medium containing 2 ng / ml human IL-2 (Thermo, USA). Supernatants were collected and assayed for IFN-γ and IL-4 quantification by sandwich ELISA. A negative control (medium or vehicle) and a positive control (phytohemagglutinin, Sigma-Aldrich, USA) were also included.

[0259] [IFN-γ sandwich ELISA] 96-well plates were coated with 100 μl of primary hIFN-γ mAb (clone NIB42, BD, USA) at a concentration of 2 μg / ml and incubated at 37°C for 1 h and then at 4°C overnight. After blocking with 1% (w / v) BSA in phosphate-buffered saline (PBS) at 37°C for 1 h, culture supernatants were added and incubated at 37°C for 2 h. Serial 1:2 dilutions of rhIFN-γ (BD, USA) from 1 ng / ml were also added. Next, 500 ng / ml- 1 Biotinylated hIFN-γ mAb (clone 4S.B3, BD, USA) was added to the plate at 100 μl / well and incubated at 37°C for 1 hour. The plate was then incubated with horseradish peroxidase (HRP)-conjugated streptavidin (RPN4401-AMDEX, USA) diluted 1:5000. After 1 hour, the plate was incubated with substrate solution (0.5 mg / ml o-phenylenediamine in 50 mM phosphate citrate buffer, 0.015% (v / v) H2O2). The reaction was stopped by adding 2N H2SO4, and the absorbance was measured at 492 nm using a microtiter plate reader L (Labsystems Multiskan MCC / 340, Finland). Between each step, plates were washed with PBS containing 0.05% (v / v) Tween 20 (PBS-T). Dilutions were prepared in PBS-T containing 0.1% (w / v) BSA. Assays were performed in triplicate. The stimulation index (SI) was defined as the ratio of cytokine concentrations from protein-loaded samples divided by cytokine concentrations from vehicle-treated samples.

[0260] [Statistical analysis] Differences between treatments were assessed by one-way analysis of variance (ANOVA). If the ANOVA revealed significant differences (p<0.05), a post-hoc Tukey's multiple comparison test was applied.

[0261] [Example 5. Immunogenicity analysis] Ex vivo human PBMC assays are based on measuring immune cell activation after exposure to therapeutic candidates. This allows for the analysis of antigen-specific activation of T cells and the determination of the therapeutic's ability to induce an immune response. These samples contain several relevant immune cells, such as T lymphocytes, as well as antigen-presenting cells (e.g., monocytes, dendritic cells, and B cells). If an immune response results from exposure to a therapeutic, it can be measured by quantifying specific cytokines secreted by activated, potent T cells, such as IFN-γ, IL-4, IL-6, and TNF-α. This provides a suitable experimental platform for assessing the risk associated with immunogenic T cell epitopes potentially present in therapeutic proteins.

[0262] (donor sample) Human immune cell-based assays have been widely used to assess the immunogenicity risk of proteins (Jaber A and Baker M, (2007), J. Pharm. Biomed. Anal. 43: 1256-1261; Mazor R et al., (2012), Proc. Natl. Acad. Sci. USA 109:E3597-603; Lamberth K, et al., (2017), Sci. Transl. Med. 9: 1-12). It is now clear that these experiments are more reliable when performed in donor pools with diverse HLA genotypes and representative of HLA occurrence in the global population. In this study, blood was collected from 20 healthy donors aged 18 to 60 years. A blood aliquot was collected from each donor, and HLA-DR allotypes were determined using Luminex Sequencing Technology. Briefly, this technique involves amplifying an extract of the DRB1 gene by PCR (polymerase chain reaction) and hybridizing it with a specific probe attached to polystyrene spheres labeled with orochrome. These spheres are read by the Luminex team, which detects the PCR product when it hybridizes to the trace attached to the sphere. The HLA-DRB1 alleles expressed by donors show high heterogeneity and are shown in Table 4.

[0263] [Table 4]

[0264] [T cell activation reaction] The antiproliferative effect of endogenous hIFN-α2b on T cells limits its direct incubation with PBMC samples. To circumvent this issue, an alternative protocol was adapted, including a preliminary step for the generation of monocyte-derived DCs (moDCs). Immature DCs were pulsed with different GMOP-IFN mutants at high concentrations for a short incubation time, followed by washing. During this step, immature DCs can endocytose and process antigens. Upon maturation, DCs can present GMOP-IFN-derived peptides bound to MHC class II on their cell surface, where they can stimulate T cell responses. Blood samples were obtained from healthy donors and selected to include the major HLA-DR allotypes expressed in the global population. This allows for the detection of hIFN-α2b-specific T cell responses restricted to specific HLA-DR allotypes. Ex vivo T cell assays and IFN-γ sandwich ELISA were performed to assess the concentrations of IFN-γ and IL-4, as described above. The concentrations of these cytokines in the culture supernatants of samples incubated with the protein under analysis were compared with those of a negative control (dendritic cells incubated with PBS or vehicle and exposed to lymphocytes). Finally, the stimulation rate was calculated from the ratio of IFN-γ levels in the samples to the negative control. From this, the proportion of donors with reduced IFN-γ levels in the supernatants was assessed for each mutant relative to the original GMOP-IFN-2b, with a p value of ≤ 0.05 considered significant between-sample differences.

[0265] As shown in Figure 5, nearly all donors responded to the GMOP-IFN protein, as judged by increased IFN-γ production, compared to the negative control. This result is in good agreement with computational predictions. Also consistent with findings using the EpiMatrix algorithm, a significant reduction in immunogenicity was observed for both GMOP-IFN deimmunized variants, as evidenced by a reduced rate of IFN-γ response in 63% of donors for GMOP-IFN-VAR2 and 42% of donors for GMOP-IFN-VAR3.

[0266] However, when IL-4 secretion (Th2 profile) was analyzed, no measurable levels of the cytokine were detected, although cells incubated with the lectin showed clear T cell activation.

[0267] [HLA-DR restriction for antigen presentation] To confirm that antigen presentation is mediated by HLA-DR molecules, GMOP-IFN-pulsed DCs from three responsive donors were treated with anti-DR antibodies (at two concentrations) before incubation with autologous T cells. Lower T cell activation was observed when DCs were pretreated with anti-DR antibodies, as judged by a decrease in the IFN-γ stimulation index (SI) (Figure 6). Furthermore, this effect became even more pronounced with increasing amounts of antibody, indicating that HLA-DR molecules play an essential role in IFN-induced peptide presentation and subsequent T cell activation.

[0268] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-2b correspond to amino acids that are not involved in the biological structure or function of the cytokine. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, variants of: IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c), with or without the addition of one or more GMOP sequences.

[0269] (6) Pharmacokinetic profile Measuring the biological activity of biopharmaceuticals and assessing their pharmacokinetics can provide valuable information by allowing specific quantification of the active protein fraction in collected biological fluids.

[0270] Example 6A. Comparative Pharmacokinetic Profiles of IFN-α Variants in Rats To assess the impact of the deimmunizing mutations introduced into the sequence of GMOP-IFN-α2b on the protein properties in vivo, the pharmacokinetic parameters of GMOP-IFN-α2b and its deimmunized mutants were analyzed.

[0271] Two-month-old female Wistar rats (Center for Biological Experiments and Bioterio, FCV-UNL) weighing an average of 200 g were housed in a vivarium at 24°C under a 12-h light / dark photoperiod with free access to water and food. Rats were divided into batches of eight and subcutaneously inoculated with a single dose (same mass units) of GMOP-IFN-α2b, GMOP-IFN-α2b(VAR2), or GMOP-IFN-α2b(VAR3). The presence of IFN-α in rat plasma samples was monitored by collecting blood samples at different times postinjection to assess residual antiviral bioactivity. Samples were centrifuged at 100 × g for 10 min at room temperature, and plasma was separated and stored at -20°C for further analysis. Plasma protein concentrations were then plotted against time (Figure 7).

[0272] The behavior of the protein examined after subcutaneous inoculation showed absorption and excretion processes that could be assumed to be first-order processes. Therefore, to describe the behavior of the cytokine, we worked to create a mathematical model in which both the overall absorption rate and the excretion rate could be treated as first-order processes. In this way, we fitted the experimental data to a curve from which we could calculate the constants that characterized it, and ultimately determined the pharmacokinetic parameters shown in Table 5.

[0273] The data were analyzed using a one-compartment model assuming first-order absorption and excretion kinetics. The pharmacokinetic parameters considered here were the maximum plasma protein concentration (C max ), the time required to reach the maximum plasma protein concentration (T max ), and the terminal half-life (t 1 / 2), apparent plasma clearance (Cl ), which is the drug clearance rate (without considering the bioavailability of the drug in the rat body). app Differences between treatment groups were evaluated by ANOVA (p≦0.05) and Tukey's test.

[0274] As shown in Table 5, all IFN-α2 variants exhibited similar absorption and distribution phases, with no significant differences between them. The time at which each protein analog achieved maximum biological activity in plasma (T max ) showed no significant difference, and the initial distribution phase of the cytokine was significantly higher than the maximum T recorded in the wild-type cytokine. value It was shown that the same would be true above (0.6±0.3h).

[0275] Regarding the excretion phase, there was no significant difference between the IFN-α2 mutants. 1 / 2 No difference was detected between the two, all of which were much higher than those described for IFN-2b-WT (0.9 ± 0.2 h).

[0276] However, GMOP-IFN-α2b-VAR3 showed a significantly lower plasma clearance rate (Cl ) compared with GMOP-IFN-α2b. app ) was observed. These differences in proteins may be related to the diversity of glycosidic structures attached to the proteins, as evidenced by isoelectric point assays (Figure 4).

[0277] These results indicate that the improved pharmacokinetic properties obtained by conjugating the glycan-rich peptide to the IFN-α2b molecule are maintained in the deimmunized variant. Furthermore, a further improvement in plasma clearance rate was detected for GMOP-IFN-α2b-VAR3.

[0278] [Table 5]

[0279] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-2b correspond to amino acids that are not involved in the biological structure or function of the cytokine. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, variants of: IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c), with or without the addition of one or more GMOP sequences.

[0280] Example 6B. Comparative Pharmacokinetic Profiles of IFN-α Variants in Rats Compared to Other Interferons To assess the impact of deimmunizing mutations introduced into the GMOP-IFN-α2b sequence on in vivo protein properties, the pharmacokinetic parameters of hyperglycosylated GMOP-IFN-α2b and its hyperglycosylated deimmunized variants are analyzed. Pharmacokinetic parameters for PEGylated IFN-α2b, nonglycosylated IFN-α2b, nonglycosylated GMOP-IFN variants 1-4 (SEQ ID NOs: 2, 4, 6, and 8, respectively), nonglycosylated GMOP-IFN-α2b, and IFN-α4 are also analyzed.

[0281] Two-month-old female Wistar rats (Center for Biological Experiments and Bioterio, FCV-UNL) weighing an average of 200 g were housed in a vivarium at 24°C under a 12-hour light / dark photoperiod with free access to water and food. The rats were divided into batches of eight and subcutaneously inoculated with a single dose (equal mass units) of hyperglycosylated GMOP-IFN-α2b, hyperglycosylated GMOP-IFN-α2b (VAR2), hyperglycosylated GMOP-IFN-α2b (VAR3), PEGylated IFN-α2b, nonglycosylated IFN-α2b, nonglycosylated GMOP-IFN variants 1–4, nonglycosylated GMOP-IFN-α2b, and 4N-IFN. The presence of IFN-α in rat plasma samples was monitored by collecting blood samples at different times postinjection to assess residual antiviral bioactivity. The samples are centrifuged at 100 x g for 10 minutes at room temperature, the plasma separated and stored at -20°C for further analysis. Plasma protein concentrations are then plotted against time.

[0282] Quantification of proteins in plasma is performed by assessing their biological activity. Using the data obtained, the biological activity of each sample is plotted against the time elapsed since molecule inoculation. The behavior of proteins examined after subcutaneous inoculation shows absorption and excretion processes that can be assumed to be first-order processes. Therefore, to describe the behavior of cytokines, a mathematical model is constructed in which the overall absorption and excretion rates can both be treated as first-order processes. In this way, the experimental data are fitted to a curve from which the characterizing constants can be calculated, ultimately determining the pharmacokinetic parameters.

[0283] The data were analyzed using a one-compartment model assuming first-order absorption and excretion kinetics. The pharmacokinetic parameters considered here were the maximum plasma protein concentration (C max ), the time required to reach the maximum plasma protein concentration (T max ), and the terminal half-life (t 1 / 2), apparent plasma clearance (Cl ), which is the drug clearance rate (without considering drug bioavailability in the rat body). app Differences between treatments were assessed by ANOVA (p≦0.05) followed by Tukey's test.

[0284] Hyperglycosylated GMOP-IFN-α2b (VAR2) and hyperglycosylated GMOP-IFN-α2b (VAR3) showed similar absorption and distribution phases, with no significant difference between them. The time at which each protein analog attained maximum biological activity in plasma (T max ) was not significantly different, and the initial distribution phase of the cytokine was significantly shorter than the maximum T recorded in the cytokine wild type. value Conversely, PEGylated IFN-α2b, non-glycosylated IFN-α2b, non-glycosylated GMOP-IFN variants 1-4, non-glycosylated GMOP-IFN-α2b, and 4N-IFN showed reduced maximum T value It is expected to show

[0285] Regarding the excretion phase, hyperglycosylated GMOP-IFN-α2b (VAR2) and hyperglycosylated GMOP-IFN-α2b (VAR3) showed no significant differences. 1 / 2 No significant differences were observed between the IFN-α2b and IFN-α2b, all of which are significantly higher than those described for IFN-2b-WT. Conversely, PEGylated IFN-α2b, nonglycosylated IFN-α2b, nonglycosylated GMOP-IFN variants 1–4, nonglycosylated GMOP-IFN-α2b, and 4N-IFN showed significantly lower t values ​​than those described for IFN-2b-WT. 1 / 2 It is expected to show

[0286] However, GMOP-IFN-α2b-VAR3 showed a significantly lower plasma clearance rate (Cl ) compared with GMOP-IFN-α2b. app) has been observed. The difference between these proteins is thought to be related to the diversity of the sugar chain structures attached to the proteins. On the other hand, PEGylated IFN-α2b, non-glycosylated IFN-α2b, non-glycosylated GMOP-IFN variants 1-4, non-glycosylated GMOP-IFN-α2b, and 4N-IFN showed a high plasma clearance rate (Cl app ) is expected.

[0287] These results indicate that the improved pharmacokinetics obtained by conjugating glycan-rich peptides to the IFN-α2b molecule can be maintained in the deimmunized variants. Furthermore, a further improvement in plasma clearance rate was detected for GMOP-IFN-α2b-VAR3. Conversely, the improved pharmacokinetic properties of the deimmunized variants were not expected for PEGylated IFN-α2b, nonglycosylated IFN-α2b, nonglycosylated GMOP-IFN variants 1–4, nonglycosylated GMOP-IFN-α2b, and 4N-IFN.

[0288] It should be clarified that the modifications / substitutions exhibited by the deimmunized variants of GMOP-IFN-2b correspond to amino acids that are not involved in the biological structure or function of the cytokine. That is, this experiment and these mutations can be performed on any of the modified IFN-α2 polypeptides (or related modified IFN-α2 compounds and compositions) as disclosed herein, including, for example, variants of: IFN-α2b, GMOP-IFN-α2b, or any other variant of IFN-α2 (including IFN-α2a, GMOP-IFN-α2a, IFN-α2c, and GMOP-IFN-α2c), with or without the addition of one or more GMOP sequences.

[0289] [Equivalent product] While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. Further, this application is intended to cover any variations, uses, or adaptations of the invention including such departures from the present disclosure as come within known or customary practice in the art to which the disclosure pertains and fall within the scope of the appended claims. The present invention includes the following embodiments. [Aspect 1] A modified interferon-α2 polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 12, and further comprising one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. [Aspect 2] A modified interferon-α2 polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 12, and further comprising at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. Aspect 3 A modified interferon-α2 polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 12, and further comprising one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. Aspect 4 A modified interferon-α2 polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 12 and comprising at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions comprise changing the amino acid at said position to alanine, glycine, or threonine. Aspect 5 5. The modified interferon-α2 polypeptide according to any one of embodiments 1 to 4, comprising the mutations L9A, F47A, L117A, F123A, and L128A. Aspect 6 6. The modified interferon-α2 polypeptide of embodiment 5, further comprising the mutations I147T and L157A. Aspect 7 The modified interferon-α2 polypeptide of embodiment 5, further comprising the mutations N65A and L66A. Aspect 8 8. The modified interferon-α2 polypeptide of embodiment 7, further comprising the mutations L17A, I147T, and L157A. Aspect 9 9. The modified interferon-α2 polypeptide of any one of embodiments 1 to 8, wherein the polypeptide has reduced immunogenicity compared to the wild-type interferon-α2b polypeptide of SEQ ID NO:12. Aspect 10 10. The modified interferon-α2 polypeptide of any one of embodiments 1 to 9, wherein the polypeptide has a relative antiviral activity of 5% to 95% compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. Aspect 11 11. The modified interferon-α2 polypeptide of any one of embodiments 1 to 10, wherein the polypeptide has a relative antiviral activity of 10% to 90% compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. Aspect 12 12. The modified interferon-α2 polypeptide of any one of embodiments 1 to 11, wherein the polypeptide has a relative antiviral activity of between 20% and 80% compared to the wild-type interferon-α2b polypeptide of SEQ ID NO: 12. Aspect 13 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 10, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 14 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 10, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 15 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 10, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 16 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 10, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 17 17. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 13 to 16, comprising the mutations L23A, F61A, L131A, F137A, and L142A. Aspect 18 20. The modified GMOP-interferon-α2 polypeptide of embodiment 17, further comprising the mutations I161T and L171A. Aspect 19 20. The modified GMOP-interferon-α2 polypeptide of embodiment 17, further comprising the mutations N79A and L80A. Aspect 20 20. The modified GMOP-interferon-α2 polypeptide of embodiment 19, further comprising the mutations L31A, I161T, and L171A. Aspect 21 21. The modified GMOP-interferon-α2 polypeptide of any one of aspects 13 to 20, wherein the polypeptide has reduced immunogenicity compared to the wild-type GMOP-interferon-α2b polypeptide of SEQ ID NO:10. Aspect 22 22. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 13 to 21, wherein the polypeptide has a relative antiviral activity of 5% to 95% compared to the wild-type GMOP-interferon-α2b polypeptide of SEQ ID NO: 10. Aspect 23 23. The modified GMOP-interferon-α2 polypeptide of any one of aspects 13 to 22, wherein the polypeptide has a relative antiviral activity of between 10% and 90% compared to the wild-type GMOP-interferon-α2b polypeptide of SEQ ID NO: 10. Aspect 24 24. The modified GMOP-interferon-α2 polypeptide of any one of aspects 13 to 23, wherein the polypeptide has a relative antiviral activity of 20% to 80% compared to the wild-type GMOP-interferon-α2b polypeptide of SEQ ID NO: 10. Aspect 25 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 22, and comprising one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitution involves changing the amino acid at said position to alanine, glycine, or threonine. Aspect 26 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 22, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acids at said positions to alanine, glycine, or threonine. Aspect 27 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 22, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 28 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 22, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acids at the positions to alanine, glycine, or threonine. Aspect 29 29. The modified interferon-α2 polypeptide of any one of embodiments 25 to 28, comprising the mutations L9A, F47A, L117A, F123A, and L128A. Aspect 30 30. The modified interferon-α2 polypeptide of embodiment 29, further comprising the mutations I147T and L157A. Aspect 31 30. The modified interferon-α2 polypeptide of embodiment 29, further comprising the mutations N65A and L66A. Aspect 32 32. The modified interferon-α2 polypeptide of embodiment 31, further comprising the mutations L17A, I147T, and L157A. Aspect 33 33. The modified interferon-α2 polypeptide of any one of aspects 25 to 32, wherein the polypeptide has reduced immunogenicity compared to the wild-type interferon-α2a polypeptide of SEQ ID NO:22. Aspect 34 34. The modified interferon-α2 polypeptide of any one of aspects 25 to 33, wherein the polypeptide has a relative antiviral activity of between 5% and 95% compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. Aspect 35 35. The modified interferon-α2 polypeptide of any one of embodiments 25 to 34, wherein the polypeptide has a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. Aspect 36 36. The modified interferon-α2 polypeptide of any one of aspects 25 to 35, wherein the polypeptide has a relative antiviral activity of between 20% and 80% compared to the wild-type interferon-α2a polypeptide of SEQ ID NO: 22. Aspect 37 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 21, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 38 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 21, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions comprise changing the amino acids at the positions to alanine, glycine, or threonine. Aspect 39 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 21, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 40 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 21, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 41 41. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 37 to 40, comprising the mutations L23A, F61A, L131A, F137A, and L142A. Aspect 42 42. The modified GMOP-interferon-α2 polypeptide of embodiment 41, further comprising the mutations I161T and L171A. Aspect 43 42. The modified GMOP-interferon-α2 polypeptide of embodiment 41, further comprising the mutations N79A and L80A. Aspect 44 44. The modified GMOP-interferon-α2 polypeptide of embodiment 43, further comprising the mutations L31A, I161T, and L171A. Aspect 45 45. The modified GMOP-interferon-α2 polypeptide of any one of aspects 37 to 44, wherein the polypeptide has reduced immunogenicity compared to the wild-type GMOP-interferon-α2a polypeptide of SEQ ID NO:21. Aspect 46 46. ​​The modified GMOP-interferon-α2 polypeptide of any one of embodiments 37 to 45, wherein the polypeptide has a relative antiviral activity of between 5% and 95% compared to the wild-type GMOP-interferon-α2a polypeptide of SEQ ID NO: 21. Aspect 47 47. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 37 to 46, wherein the polypeptide has a relative antiviral activity of 10% to 90% compared to the wild-type GMOP-interferon-α2a polypeptide of SEQ ID NO: 21. Aspect 48 48. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 37 to 47, wherein the polypeptide has a relative antiviral activity of 20% to 80% compared to the wild-type GMOP-interferon-α2a polypeptide of SEQ ID NO: 21. Aspect 49 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 24, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 50 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 24, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acids at said positions to alanine, glycine, or threonine. Aspect 51 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 24, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 52 A modified interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 24, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157, wherein the substitutions involve changing the amino acids at said positions to alanine, glycine, or threonine. Aspect 53 53. The modified interferon-α2 polypeptide of any one of embodiments 49 to 52, comprising the mutations L9A, F47A, L117A, F123A, and L128A. Aspect 54 54. The modified interferon-α2 polypeptide of embodiment 53, further comprising the mutations I147T and L157A. Aspect 55 54. The modified interferon-α2 polypeptide of embodiment 53, further comprising the mutations N65A and L66A. Aspect 56 56. The modified interferon-α2 polypeptide of embodiment 55, further comprising the mutations L17A, I147T, and L157A. Aspect 57 57. The modified interferon-α2 polypeptide of any one of aspects 49 to 56, wherein the polypeptide has reduced immunogenicity compared to the wild-type interferon-α2c polypeptide of SEQ ID NO:24. Aspect 58 58. The modified interferon-α2 polypeptide of any one of embodiments 49 to 57, wherein the polypeptide has a relative antiviral activity of 5% to 95% compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. Aspect 59 59. The modified interferon-α2 polypeptide of any one of aspects 49 to 58, wherein the polypeptide has a relative antiviral activity of between 10% and 90% compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. Aspect 60 60. The modified interferon-α2 polypeptide of any one of aspects 49 to 59, wherein the polypeptide has a relative antiviral activity of between 20% and 80% compared to the wild-type interferon-α2c polypeptide of SEQ ID NO: 24. Aspect 61 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 23, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 62 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 23, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions comprise changing the amino acids at the positions to alanine, glycine, or threonine. Aspect 63 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 23, wherein the polypeptide comprises one or more amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 64 A modified GMOP-interferon-α2 polypeptide having interferon-α2 activity, comprising an amino acid sequence having at least 90% homology to SEQ ID NO: 23, wherein the polypeptide comprises at least five amino acid substitutions at any position selected from the set consisting of 23, 31, 61, 79, 80, 131, 137, 142, 161, and 171, wherein the substitutions involve changing the amino acid at said position to alanine, glycine, or threonine. Aspect 65 65. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 61 to 64, comprising the mutations L23A, F61A, L131A, F137A, and L142A. Aspect 66 66. The modified GMOP-interferon-α2 polypeptide of embodiment 65, further comprising the mutations I161T and L171A. Aspect 67 66. The modified GMOP-interferon-α2 polypeptide of embodiment 65, further comprising the mutations N79A and L80A. Aspect 68 68. The modified GMOP-interferon-α2 polypeptide of embodiment 67, further comprising the mutations L31A, I161T, and L171A. Aspect 69 69. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 61 to 68, wherein the polypeptide has reduced immunogenicity compared to the wild-type GMOP-interferon-α2c polypeptide of SEQ ID NO:23. Aspect 70 70. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 61 to 69, wherein the polypeptide has a relative antiviral activity of 5% to 95% compared to the wild-type GMOP-interferon-α2c polypeptide of SEQ ID NO: 23. Aspect 71 71. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 61 to 70, wherein the polypeptide has a relative antiviral activity of 10% to 90% compared to the wild-type GMOP-interferon-α2c polypeptide of SEQ ID NO: 23. Aspect 72 72. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 61 to 71, wherein the polypeptide has a relative antiviral activity of 20% to 80% compared to the wild-type GMOP-interferon-α2c polypeptide of SEQ ID NO: 23. Aspect 73 73. The modified GMOP-interferon-α2 polypeptide of any one of embodiments 1 to 72, wherein the polypeptide has a percent antiproliferative biological activity of less than 20%. Aspect 74 74. The modified interferon-α2 polypeptide of any one of embodiments 1 to 73, wherein the polypeptide has a percent antiproliferative biological activity of less than 10%. Aspect 75 75. The modified interferon-α2 polypeptide of any one of embodiments 1 to 74, wherein the polypeptide has a percent antiproliferative biological activity of less than 5%. Aspect 76 76. The modified interferon-α2 polypeptide of any one of embodiments 1 to 75, wherein the polypeptide has an apparent plasma clearance rate (Clapp) of less than 115 mL / h. Aspect 77 77. The modified interferon-α2 polypeptide of any one of embodiments 1 to 76, wherein the polypeptide has an apparent plasma clearance rate (Clapp) of less than 50 mL / h. Aspect 78 78. The modified interferon-α2 polypeptide according to any one of embodiments 1 to 77, wherein the polypeptide is hyperglycosylated. Aspect 79 79. A nucleic acid encoding one or more of the modified interferon-α2 polypeptides according to any one of embodiments 1 to 78. Aspect 80 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO: 1. Aspect 81 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO: 3. Aspect 82 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO:5. Aspect 83 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO: 7. Aspect 84 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO: 13. Aspect 85 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO: 15. Aspect 86 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO: 17. Aspect 87 80. The nucleic acid of embodiment 79, comprising the nucleic acid sequence of SEQ ID NO: 19. Aspect 88 A plasmid comprising one or more nucleic acid sequences of a nucleic acid according to any one of aspects 79 to 87. Aspect 89 79. A plasmid encoding one or more of the modified interferon-α2 polypeptides according to any one of embodiments 1 to 78. Aspect 90 A ve...

Claims

1. A modified interferon-α2 polypeptide having interferon-α2 activity, the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:

6.

2. 2. The modified interferon-α2 polypeptide of claim 1, wherein the polypeptide has an apparent plasma clearance rate (Clapp) of less than 115 mL / h.

3. A nucleic acid encoding the modified interferon-α2 polypeptide of claim 1 or 2.

4. A vector encoding a modified interferon-α2 polypeptide according to claim 1 or 2 and / or comprising a nucleic acid according to claim 3.

5. A cell line encoding a modified interferon-α2 polypeptide according to claim 1 or 2, and / or comprising a nucleic acid according to claim 3, and / or comprising a vector according to claim 4.

6. A pharmaceutical composition comprising a modified interferon-α2 polypeptide according to claim 1 or 2, and / or a nucleic acid according to claim 3, and / or a vector according to claim 4.

7. A therapeutic agent for treating one or more diseases in a subject, comprising the modified interferon-α2 polypeptide of claim 1 or 2, or a composition comprising the modified interferon-α2 polypeptide of claim 1 or 2.

8. 10. A therapeutic agent comprising the modified interferon-α2 polypeptide of claim 1 or 2, or a composition comprising the modified interferon-α2 polypeptide of claim 1 or 2, for use in treating one or more diseases in a subject, wherein the diseases include melanoma, acute and chronic hepatitis C, acute and chronic hepatitis B, acute and chronic non-A, non-B hepatitis, Kaposi's sarcoma, multiple sclerosis, genital warts, leukemia, lymphoma, condyloma acuminata, SARS-COV-2 infection, ZIKV infection, CHIKV infection, and influenza A infection.

9. 3. A method for purifying modified interferon-α2 polypeptide according to claim 1 or 2, comprising the step of purifying the genetically modified interferon-α2 polypeptide by immunoaffinity chromatography, wherein the purification by immunoaffinity chromatography comprises the use of anti-nonglycosylated rhIFN-α2b mAb CA5E6 antibody, or anti-hGM-CSF monoclonal antibody, or both.

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