MODIFIED INTERFERON WITH REDUCED IMMUNOGENICITY

Abstract

This document describes a modified interferon with reduced immunogenicity comprising the substitution of at least 3 amino acids. It further describes a nucleic acid molecule encoding said interferon; a recombinant protein expression cell comprising said nucleic acid molecule encoding said modified interferon; wherein said recombinant protein expression cell comprises a plasmid or vector containing said nucleic acid molecule.

Description

This document describes a modified interferon with reduced immunogenicity comprising the substitution of at least three amino acids. It also describes a nucleic acid molecule encoding said interferon; a recombinant protein expression cell comprising said nucleic acid molecule encoding said modified interferon; wherein said recombinant protein expression cell comprises a plasmid or vector containing said nucleic acid molecule. Ί If it is an Amendment, indicate the No. d< Exp 20150103131 ZW PAIR I am born Transaction: 151.72449 PATENTS Amount: $1228 Date / Time: 09 / 29 / 2015 12:43:38.667 Agent: TERENTINO, FLORENCIA ADRIANA NATIONAL INSTITUTE OF INDUSTRIAL PROPERTY NATIONAL PATENT ADMINISTRATION APPLICATION FOR A PATENT OF INVENTION ARGENTINE REPUBLIC I. APPLICANT(S) UTILITY MODEL APPLICATION Sheet NUMBER OF APPLICANTS NATIONAL UNIVERSITY OF THE LITTORAL CUIT / CUIL / CDI: 30-54667055-0 Enter Name and Surname or Company Name (of one of them, the rest in ANNEX) ID card Individuals: Marital status: Nuptials Spouse's name and surname: ID card Bv. PELLEGRINI 2750 / 3 3 wlietellB™ Location: SANTA FE Postal Code No. 3000 Country of Residence: AR MARTÍNEZ PASSAGE 2626 - SANTA FE - SANTA FE 3000 Legal Address - Street - No. - Location - Province Postal Code Email Address: fterentino@fcjs. uη 1.edu.ar Telephone: 0342-4551211 Ext. 126 II. OBJECT Title of the invention: MODIFIED INTERFERON WITH REDUCED IMMUNOGENICITY Character of the Patent / Utility Model INDEPENDENT Additional to: Patent No. Divisional Application No. Application No. PRIORITY (LAW 17.011) MICROORGANISM DEPOSIT COUNTRY NUMBER DATE DEPOSIT DATE ACCESS NO. TO DEPOSIT L. Γν p ! Name of the Depository Institution -j AdmihíS! Yes. i'!·^ 1-íhCí'jrtLí NTnOL Aü^ UU rutUIUUL' ISSÓN | iNTIh o U yes Address of the Institution Country Depositor's Data Origin of the Biological and Genetic Material Yo: gf Continued on attached sheet: Yo FLORENCIA ADRIANA TERENTINO DECLARES UNDER OATH THAT HE HOLDS THE CHARACTER OF PROXY The mandate is currently in force and the company is registered in [Companies / Register]. COMPANY REPRESENTED BY WHO THAT HIS Registration Details in RPC / IGJ Date: Number Folio Volume: IV. MANDATE ~~ --- Power of Attorney registered with the INPI under number: 78,631 IN THIS ACT, THE FOLLOWING IS AUTHORIZED: (Surname and First Name and ID Number j FLORENCIA ADRIANA TERENTINO, ID No. 34.563.221 For all those purely procedural tasks such as making breakdowns, withdrawing testimonies, certificates, titles, copies and notifications in the file. Responding to hearings, withdrawing applications, making requests (only when the Authorized Party is an Industrial Property Agent) POWER OF ATTORNEY IS ENCLOSED AGENT NO. 2.194 V. PRIOR DISCLOSURE STATEMENT For the purposes of Article 5 of Law 24.481, it is stated that the present invention has been previously disclosed: |NO I (YES / NO) If yes, on what date: | | VI. OBSERVATIONS LEGAL STATUS: The National University of the Littoral was created by National Law No. 10,861. FLORENCIA ADRIANA TERENTINO signs in the capacity of business manager of the National Council for Scientific and Technical Research (CONICET). It is hereby stated that the data provided in this form constitutes a sworn statement; any falsehood contained herein will have the corresponding legal consequences. NOTE: Payment of the corresponding fee must be made at the time of submission or during the first two hours of business on the following business day. Failure to make payment within this period will automatically result in the submission being considered not made and having no effect. Signature of Florence A. Terentino LAWYER Signature of the legally authorized person(s) INTERNAL USE ONLY. THE PRESENTATION CONSISTS OF | | PAGES. CHANGE OF ADDRESS / EMAIL / TELEPHONE: DATE | » Real Address - Street No. Town / City: | | Postal Code No. | Country of Residence: Legal Address - Street - No. - Town / City - Province Postal Code Email Address: | Telephone: CHANGE OF ATTORNEY / AUTHORIZED: DATE New Attorney or Authorized: TRANSFER 0 CHANGE OF BUSINESS TYPE: | iNsrn Exp.'. 20150103131 INPl T,W ”™449 PATENTS Impo-'e: $1228, Date / Time: 09 / 29 / 2015 12:43:38.667 Agent: TERENTINO, FLORENCIA ADRIANA Title of the invention MODIFIED INTERFERON WITH REDUCED IMMUNOGENICITY Nature of the Patent / Utility Model INDEPENDENT National Council for Scientific and Technical Research (CONICET) CUIT / CUIL / CDl: 30-54666038-5 Enter Name and Surname or Company Name DNI Natural Persons: Marital Status: Marriage Spouse's Name and Surname: DNI Rivadavia Avenue 1917 Real Address - Street; No.; Floor; Apt. City: Autonomous City of Buenos Aires Postal Code No. 1033 Country of Residence: AR Date: Number Folio No. Took: Legal Entities: Registration Details in RPC / IGJ Email address: patents@conicet.gov.ar LAWYER Legal representative details Signature of applicant or their legal representative CUIT / CUIL / CDl: Enter Name and Surname or Company Name DNI Natural Persons: Marital Status: Marriage Spouse's Name and Surname: DNI l· Real Address - Street; No.; Floor and Apt. City: Postal Code No. Country of Residence: Legal Entities: Registration Details in RPC / IGJ Date: Number Folio No. Volume: Email Address: Telephone: L 5: f jf Yo Legal representative details Signature of the applicant or their legal representative i Descriptive Report of the Patent of Invention About: Modified interferon with reduced immunogenicity Requested by: 1-National University of the Littoral 2- National Council for Scientific and Technical Research Inventors: Etcheverrigaray, Marina Mufarrege, Eduardo Federico De Groot, Anne Searls Martin, William D. Home: 1-Bv. Pellegrini 2750, Santa Fe, Santa Fe, CP 3000 2- Av. Rivadavia 1917, City of Buenos Aires, CP 1033 For a period of: 20 years QUALIFICATION MODIFIED INTERFERON WITH REDUCED IMMUNOGENICITY TECHNOLOGICAL FIELD The present invention relates to the development of therapeutic molecules of pharmaceutical interest for application to humans. STATE OF THE ART Recombinant proteins for therapeutic use have attracted the interest of numerous biotechnology companies due to their impact on controlling many human diseases, from microbial infections to the treatment of various cancers and arthritis. For this reason, the tireless efforts being made to develop biotherapeutics capable of generating an effective and sustained biological response are not surprising. Although in most cases these proteins (cytokines, growth factors, and monoclonal antibodies, among others) are biosimilars—that is, molecules virtually identical to those produced by the human body—numerous cases of immune responses developed as a consequence of administering these drugs have been reported. The prevalence of these 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. This undesirable response can produce various effects of varying complexity and severity depending on the circumstances. The most common consequences are hypersensitivity reactions, anaphylaxis, autoimmune diseases, or a decrease in treatment efficacy due to the development of neutralizing antibodies against the drug (ADAs). For these reasons, the agencies responsible for regulating the quality of biosimilars in different parts of the world have developed detailed guidelines that allow for the evaluation of the different characteristics of these products. In particular, prolonged administration of IFN-α2 or IFN-β1 can lead to a breakdown of immunological tolerance to self-antigens in some patients, resulting in the production of anti-IFN antibodies. These antibodies can bind to the IFN molecule in such a way as to produce virtually no effect, or they can alter the pharmacokinetics of the cytokine, or in certain cases neutralize its activity by binding to a region of the protein that prevents interaction with its receptor on the cell surface of target cells. The results of numerous clinical studies have demonstrated that the development of anti-IFN-α antibodies frequently occurs in patients with chronic hepatitis C or neoplastic diseases treated with IFN-α or IFN-β2β. The incidence of anti-IFN-α antibody production is highly variable and has been reported to range from 7% to 60%.A lack of standardization in clinical trials and differences in treatment regimens undoubtedly explain, in part, the apparent variation in the incidence of anti-IFN-α antibodies in patients treated with this therapy. Furthermore, pegylation of the molecule has increased its plasma half-life, allowing for weekly dosing and improved efficacy compared to the native molecule. Pegylation is often incorporated as a strategy to reduce the immunogenicity of recombinant proteins because it exerts spherical hindrance, frequently reducing antigen presentation. However, there is evidence that 8% of patients with chronic hepatitis C who did not respond to therapy with pegylated IFN-α and ribavirin presented neutralizing anti-IFN-α antibodies, while none of the patients who cleared the HCV virus after treatment with IFN-α showed these levels. detectable levels of these antibodies. Furthermore, it has been demonstrated that polyclonal antibodies produced in patients treated with IFN-α2α or IFN-α2β cross-neutralized both proteins, as well as pegylated derivatives of the same and native IFN-α. These results highlight the need to develop IFN-α2β-4N variants that exhibit reduced immunogenicity, modifying as much as possible those amino acids directly involved in the interaction of the immunogenic epitope with MHC class II molecules during antigen presentation and thus avoiding the consequent immune response. Several factors that lead to the generation of neutralizing antibodies have been described. These are mainly divided into two groups: Extrinsic factors: Route of administration, dose, formulation, additives, glycosylation, and contaminants. Contamination of the product with pro-inflammatory agents or nonspecific mitogenic compounds such as LPS (bacterial lipopolysaccharide) and the development of aggregates can generate a critical signal required for antibody induction. Furthermore, proteins that are denatured during formulation may be more immunogenic than their intact counterparts because these products may present novel epitopes recognized by B lymphocytes that might not be present on the original molecule, leading to the stimulation of an immune response.27 In contrast, glycosylation that occurs during product biosynthesis could reduce its immunogenicity due to its ability to mask potential epitopes and their subsequent recognition. Intrinsic factors: The presence of immunogenic epitopes that lead to the formation of antibodies. The initial challenges have been successfully overcome in most cases through a meticulous production process and improvements in the purification stage. Good results have also been achieved through the use of certain excipients that stabilize the biotherapeutic agent, as well as through the introduction of oligosaccharides or polyethylene glycol residues. However, intrinsic factors pose a significant challenge, since B lymphocyte activation contributes to antibody development, even in cases where the therapeutic agent is virtually identical to the autologous protein. B cell activation can be of two types: T cell-independent (T-independent) or T cell-dependent (T-dependent). In the first case, certain structural features of the molecule, such as polymeric repeats, constitute the signals required to stimulate the activation of a specific group of B cells. The antibodies developed as a result of activation are primarily of the low-affinity IgM type. In contrast, T cell-dependent activation relies primarily on the primary protein sequence. In this scenario, the molecule is endocytosed, processed, and the resulting peptides are presented on the surface of antigen-presenting cells (APCs) such as dendritic cells, macrophages, or B lymphocytes, within the context of the Major Histocompatibility Complex Class II. These sequences can, in some cases, be recognized by T helper (Th) cells (via their cell surface receptor, known as the T cell receptor, or TCR) and trigger an immune response that leads to B cell activation and the subsequent production of neutralizing antibodies. The fundamental difference between the two types of responses (T cell-independent and T cell-dependent) lies in the fact that in the latter, the antibodies developed are of the IgG type, exhibit greater affinity, and are generated in a more prolonged and sustained manner. T cell-dependent B cell activation begins when a specific group of B cells interacts with certain protein epitopes through their antigen receptors (IgM / IgD) on the cell surface. This interaction with the antigen induces the receptors to initiate an activation signal (signal 1). This signal promotes the internalization of the complete protein, which is then processed into small peptide epitopes. These epitopes are presented within the MHC Class II groove on the B cell surface. These cells also co-express the CD40 molecule on their surface.When Th lymphocytes interact via their TCR and the CD40 ligand (CD154) with epitope-MHC class II complexes and CD40 (on the surface of the B lymphocyte), they trigger signal 2. This signal activates the B lymphocyte, and the T lymphocytes produce, among other things, the cytokine IL-4 (in a type 2 Th lymphocyte response) or interferon γ (type 1 Th lymphocytes), leading to the maturation of the immune response. Furthermore, it is important to note that without the participation of T lymphocytes, which provide the second signal, B lymphocytes undergo programmed cell death (apoptosis). For this reason, attenuating a T lymphocyte-mediated immune response has become the focus of attention in the process known as recombinant protein deimmunization for therapeutic purposes. The state of the art provides documents that seek to modify interferons in order to improve their half-life, antiviral or antiproliferative biological activity, and immunogenicity. US patent 7767799 describes a series of variants that aim to improve interaction with the receptor, its antiviral and antiproliferative activity without producing a reduction in immunogenicity. Patent application US2009043076A1 describes an immunogenic interferon beta (IFN-β) with reduced immunogenicity. A study identified three regions with a high probability of exhibiting immunogenicity: R1, spanning amino acids 49 through 72, and R2, spanning amino acids 124 through 145. Based on the identification of these regions, the patent applicants propose various modifications (substitutions) to reduce the immunogenicity of the IFN. The application includes tests verifying the antiproliferative activity of the modified IFN in a B-cell lymphoma cell line (Daudi), confirming its antiproliferative activity. However, the patent application does not describe any in vivo or in vitro assays to demonstrate the reduction in immunogenicity of the modified IFN.This parameter has only been evaluated using an algorithm for detecting potentially immunogenic regions, but the corresponding experimental validation has not been carried out. International application W00034317A2 proposes a method to reduce the immunogenicity of various biologically active molecules, including IFN-α2. Using MPT software (Biovation Aberdeen, UK), they identified several potential regions of IFN-α that can bind to MHC Class II. Five regions or clusters were identified: R1 contains amino acid residues from positions 7 to 40, R2 residues from 45 to 70, R3 residues from 79 to 103, R4 residues from 108 to 132, and R5 residues from 140 to 163. These regions also contain potential epitopes. Based on these regions, they propose modifications to IFN-α's immunogenicity, but they fail to demonstrate this improvement. Furthermore, the regions they propose are not the same as those proposed herein. IFNs that would reduce the lack of trials that invention, because in said patent the residues 15, 27, 65, among others are modified. The present invention solves the technical problem that previous inventions do not solve by providing an interferon with proven antiviral biological activity and reduced immunogenicity. BRIEF DESCRIPTION OF THE FIGURES. Figure 1: Average IC50 values ​​determined for each peptide and the eight HLA alleles analyzed. Mutations that led to an increase in the average IC50 (reduction in antigenicity / immunogenicity) are highlighted in a box at the top of the figure. Figure 2: Western blot assay performed on the culture supernatants of cell lines that produce the different versions of IFN-ot2b-4N. The figure shows that all the cell lines analyzed produce the corresponding IFN variant appropriately. Figure 3: Preliminary antiviral activity assay performed with the culture supernatants of the cell lines that produce the different de-immunized variants of IFN-α2β-4N. As can be seen, the supernatant corresponding to variant 2 did not show antiviral activity. Figure 4: Specific productivity assay of IFN-cc2b4N(VAR1). The different cell clones were analyzed based on their productive capacity. Selected and cryopreserved clones are highlighted in dark gray. In particular, the study continued with clone 10. Figure 5: Specific productivity assay of IFN-α2β4N(VAR3). The different cell clones were analyzed based on their productive capacity. Selected and cryopreserved clones are highlighted in dark gray. In particular, the study continued with clone 9. Figure 6: Polyacrylamide gel electrophoresis under denaturing conditions of the different variants of IFN-α2β. Figure 7: Isoelectric focusing assay showing the glycoform content of the different variants analyzed. The differential migration pattern is mainly due to the sialic acid content in each molecule. Figure 8: Antiviral biological assay of non-glycosylated (NG), O-glycosylated (WT) and hyperglycosylated (4N) interferons. The quantification of the specific activity of each molecule was determined using an international standard (NIBSC). Figure 9: Antiviral biological assay of de-immunized variants of IFN-α2β-4N. The quantification of the specific activity of each molecule was determined through an international standard (NIBSC). Figure 10: Frequency of HLA-DRB1 allotypes in the study cohort (light gray) and in the world population (dark gray). Results of the study of HLA-DRB1 Class II MHC molecule allele typing using Luminex technology and comparison with their frequency in the world population. Figure 11: Comparative analysis of the immunogenicity of the different variants of IFN-ot2b, through the quantification of IFN-γ secreted into the culture medium as a consequence of lymphocyte proliferation. Figure 12: Comparative analysis of the immunogenicity of the different variants of IFN-a2b, through the quantification of IL-4 secreted into the culture medium as a consequence of lymphocyte proliferation. Figure 13: Comparative analysis of the thermal stability of IFN-α2β variants. Incubations were performed at seven different temperatures for 10 minutes. BRIEF DESCRIPTION OF THE INVENTION. The present invention describes a modified interferon with reduced immunogenicity, characterized in that it comprises the substitution of at least three amino acids at positions 9, 18, 47, 117, 123, and 128 with amino acids selected from the group comprising alanine and glycine. Preferably, the modified interferon comprises the substitution of the amino acids at positions 9, 18, and 47. Alternatively, the modified interferon comprises the substitution of the amino acids at positions 9, 47, 117, 123, and 128. The modified interferon of the present invention is selected from the variants comprising: alpha interferon, beta interferon, gamma interferon, non-glycosylated interferon, glycosylated interferon; preferably, the interferon of the present invention is an IFN-α2β-4N. The modified interferon of the present invention comprises the sequence SEQN°1 - IFN-α2β-4N VARI: CDLNQTHSAGSRRTLMLLAQMRNISLFSCLKDRHDFGFPQEEFGNQAQKAETIPVLHEMIQQIF NLFSTNDSSAAWNETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDS1AAVRKYAQRITA YLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE. This interferon maintains its antiviral activity at 85% between 65° and 75°C. Furthermore, this interferon generates a positive IFN-γ response rate of up to 39% and a positive IL-4 response rate of up to 4%. Alternatively, the modified interferon of the present invention comprises SEQN°3 - IFN-γ2b-4N VAR3: CDLNQTHSAGSRRTLMLAAQMRNISLFSCLKDRHDFGFPQEEFGNQAQKAETIPVLHEMIQQ IFNLFSTNDSSAAWNETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQ RITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE. Where said Interferon maintains all its antiviral activity between 65° and 75°C. Furthermore, this interferon generates a positive IFN-γ response rate of up to 27% and a positive IL-4 response rate of up to 8%. In another aspect of the present invention, a nucleic acid molecule encoding for said modified interferon is described. In another aspect of the present invention, a plasmid and vector comprising said modified interferon are described. In another aspect of the present invention, a recombinant protein expression cell is described comprising said nucleic acid molecule encoding said modified interferon; wherein said recombinant protein expression cell comprises a plasmid or vector containing said nucleic acid molecule. DETAILED DESCRIPTION OF THE INVENTION. The present invention focuses on the development of variants of a glycosylated version of interferon-a-2b (IFNa2b-4N) that exhibit reduced immunogenicity with respect to the original molecule and the variants currently available on the market. The modified interferon of the present invention refers to an interferon whose amino acid sequence has been modified; where said modification comprises the substitution of at least 3 of any of the amino acids at positions 9, 18, 47, 117, 123, 128 by amino acids selected from the set comprising: Alanine and Glycine. A previous in silico analysis revealed that IFN-α2β is potentially immunogenic. The same study conducted with IFN-α2β-4N showed that this variant was even more immunogenic than its unmodified counterpart. Further investigation identified the regions of the protein responsible for this undesirable effect. Subsequently, the mutations to be introduced into the cytokine sequence were determined in order to decrease its immunogenicity without eliminating its biological activity. Using in vitro techniques, the modifications that effectively produce an impact on immunogenicity were identified, and possible variants of IFN-a2b-4N containing them were designed in different combinations. These proteins were produced and purified in animal cells, and their biological activity and immunogenicity were evaluated using ex vivo assays. These experiments revealed not only the high immunogenicity of IFN-α2b-4N and commercial versions of IFN-α2b but also markedly reduced immunogenicity in two of the three variants developed. Considering the undesirable effects associated with the administration of IFN-α2β, autoimmune neutralizing antibodies, such as the generation of or even the development of alternative diseases, these variants are extremely promising for the treatment of viral infections in patients. Using a suite of immunoinformatics programs, it was possible to predict the regions (or peptides) responsible for the potential immunogenicity of the cytokine (Figure 1). These regions are assigned a score related to the probability that the sequence will trigger an immune response. Furthermore, some of the programs used allow for the identification of the amino acids that contribute most significantly to the immunogenicity of the peptide containing them. Based on this information, sequential mutations were performed on the primary protein sequence to reduce the score of each peptide. In this way, not only were the immunogenic peptides within IFN-α2β-4N defined (original peptides or ORG), but modifications that allowed reducing their immunogenicity (modified peptides or MOD) were also determined. To confirm the experimental predictions, the analyzed peptides were generated by chemical synthesis and evaluated based on their ability to bind purified class II human MHC molecules. This allowed for the identification of the residues actually involved in the cytokine's immunogenicity. Figure 2 illustrates the mutations that reduced the immunogenicity of each analyzed peptide. Based on the identification of those mutations that led to a significant reduction in the immunogenicity of the cytokine, three new variants of IFNcc2b-4N were produced that retained the glycosylation sites and at the same time contained different combinations of the modifications that would lead to lower immunogenicity. Example 1: Identification of the immunogenic regions of the IFN-α2β-4N molecule (epitopes) Several computer algorithms exist that can make such predictions, differing primarily in their ability to accurately predict overall immunogenicity and epitopes within the protein. Therefore, choosing the right software for this task is critical. For this reason, the immunoinformatics analysis stage was performed using the toolset developed by EpiVax Inc., a company based in Providence, Rhode Island, USA. Island) and is a leader in the analysis of the immunogenicity of proteins for therapeutic purposes, with a long track record and worldwide recognition. The first step involved identifying the specific regions of the molecule responsible for this characteristic, that is, the immunogenic epitopes. These small sequences can be recognized by more than one type of MHC Class II molecule (promiscuous epitopes) and therefore elicit an immune response in a larger portion of the world's population. Overall, the result of this search yields a variety of 9 amino acid sequences, where some of these residues directly contact the groove formed by the molecules that make up the MHC Class II and the rest are involved in binding to the T lymphocyte receptor (TCR). Example 2: Grouping of epitopes into clusters or peptides (original peptides) Once the highly immunogenic and promiscuous epitopes were identified, the next step was to group them into peptides of between 15 and 25 amino acids, so as to perform the experimental validation tests with a smaller number of sequences that include one or more epitopes. Example 3: Identification of the most relevant amino acids in the immunogenicity of each peptide and replacement by residues that allow reducing its immunogenic potential and at the same time do not produce a disruption of the structure of the protein as a whole (modified peptides). The final stage of the predictive process involved determining the individual contribution of each amino acid to the overall immunogenicity of each peptide. To achieve this, An iterative replacement of each residue with the remaining 19 was performed, and the resulting immunogenicity was evaluated in each case. At this stage, the possibility of making conservative or natural changes to the molecule was analyzed using a stack of sequences homologous to the one of interest, in order to avoid potential structural and / or functional disruptions. This stage was complemented by an exhaustive literature search that allowed for the prior identification of the essential residues from the perspective of the cytokine's structure and function. Once the most suitable modifications to the original peptides were established, they were sequentially introduced, thus generating the corresponding modified peptides. Once the sequences of the original (ORG) and modified (MOD) peptides were obtained, the next step was to experimentally validate the immunoinformatics predictions. The starting point was obtaining the peptides through chemical synthesis. Example 4: In vitro assays to evaluate the binding affinity of synthetic peptides to purified human Class II MHC molecules The first experimental validation assay consisted of evaluating the in vitro interaction capacity of the synthetic peptides (original and modified) with the different types of MHC Class II molecules (alleles). In general, the most frequently analyzed alleles are DRBl*0101, DRBl*0301, DRBl*0401, DRB1*O7O1, DRBl*0801, DRBl*1101, DRB1*13O1, and DRB1*15O1, as they represent more than 90% of the allelic diversity in the human population. The protocol suggested by Moise et al., 2011, was used. This assay is based on the ability of each peptide analyzed to compete for binding to the MHC pocket against a reference peptide, usually conjugated to a detection molecule, for example, biotin. These complexes are then transferred to a multiwell plate where a human MHC-binding antibody has been pre-adsorbed. After successive washes, detection is performed by incubation with streptavidin conjugated to a molecule capable of emitting a quantifiable signal (for example, europium), and the binding affinity of the peptide of interest to the MHC can be estimated by measuring the fluorescence intensity. In this way, peptides with a high binding affinity for MHC molecules will produce a weaker signal compared to those that bind weakly to this complex. Typically, five different concentrations of each peptide are tested, and the information Once obtained, a graph is created that allows the determination of a parameter known as IC50, which is defined as the concentration of the peptide needed to produce inhibition of 50% fluorescence. This technique is particularly useful for determining the effect of mutations introduced into the analyzed peptides by directly comparing the IC50 values ​​of the original and modified versions of each peptide. An increase in IC50 indicates a reduction in the binding affinity between the peptide and the analyzed HLA molecule, that is, a decrease in its antigenicity / immunogenicity. Table 1 summarizes the results obtained from the in vitro assays (IC50) and the immunogenicity scores yielded by the EpiMatrix immuno-informatics algorithm developed by EpiVax, Inc. 101 *0301 N / W 1.49 N / N *0401 N / N / U * 01 N / N / N 1 1.56 N / N / N 1101 N / N .48 N / N *1301 N / N / N 1 N / N / N / N / N1 1.27 1.32 1.44 1.19 N / ONE / ONE / ONE / ONE / ONE / ONE / ONE / ONE / ONE / ONE / U 0.93 1.19 1.26 1 6 1.14 1.17 1.17 1.04 (MOV N / ONE / ONE / ONE / ONE / ONE / ONE / ONE / ONE) (M0D1) N / ONE / ONE / ONE / ONE / ONE / U 1.59 1.60 N / ONE / ONE / ONE / ONE / U 34.60 N / ONE / ONE / ONE / ONE / ONE / U 1. 1.24 N / ONE / ONE / ONE / ONE / ONE / U ( N / ONE / ONE / ONE / ONE / ONE / ONE / U scores thrown by Table 1: Comparative analysis EpiMatrix and the IC50s calculated from the binding assays to 8 HLA alleles for the 14 analyzed peptides. N / B: Not joined. Finally, the calculation of the average of the IC50s was performed in such a way as to consider the global-level effect of the introduced mutations on the interaction with the eight alleles HLA-DRB1 analyzed. Figure 1 illustrates the impact of the different mutations analyzed on the binding between the original and modified IFN-α2b-4N peptides. An increase in the average IC50 is interpreted as a reduction in the affinity of the binding between the HLA-DRB1 molecule and the peptide in question (antigenicity). Of the proposed modifications, 2(MOD), 4-5(MOD), and 5(MOD) showed a positive impact from the point of view of the de-immunization process. Therefore, based on the results obtained, it was decided to produce three variants of IFN16 in animal cells. a2b-4N. Variant 1 (VARI) with 5 mutations at positions 9, 47, 117, 123, 128; variant 2 (VAR2) with 8 mutations at positions 9, 18, 47, 60, 64, 117, 123, 128; and variant 3 (VAR3) with 3 mutations at positions 9, 18 and 47, whose amino acid sequences are: SEQN°1 - IFN-a2b-4N VARI: cdlnqthsAgsrrtlmllaqmrnislfsclkdrhdfgfpqeefgnqAqkaetipvlhemiqqi fnlfstndssaawnetlldkfytelyqqlndleacviqgvgvtetplmkedsiAavrkyAqri tAylkekkysspcawevvraeimrsfslqeslqeslqeslqen SEQN°2 - IFN-a2b-4N VAR2: cdlnqthsAgsrrtlmlAaqmrnislfsclkdrhdfgfpqeefgnqAqkaetipvlhemAqi AnlfstndssaawnetlldkfytelyqqlndleacviqgvgvtetplmkedsiAavrkyAqri tAylkekkyspcawevvraeimrsfslstnlqeslrske SEQN°3 - IFN-a2b-4N VAR3: cdlnqthsAgsrrtlmlAaqmrnislfsclkdrhdfgfpqeefgnqAqkaetipvlhemiqqi fnlfstndssaawnetlldkfytelyqqlndleacviqgvgvtetplmkedsilavrkyfqrit lylkekkyspcawevvraeimrsfslqeslqeslqr The original sequence of the protein is: SEQN°4 - IFN-oí2b-4N: CDLNQTHSLGSRRTLMLQMRNISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIF NLFSTNDSAAWNETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITL YLKEKKYSPCAWEVVRAEIMRSFSSLSTNLQESLRSKE It should be clarified that the modifications / substitutions present in the de-immunized variants of IFN-α2b-4N correspond to those amino acids not involved (according to the literature) in the structure or biological function of the cytokine, nor in the N-glycosylation sites. In other words, these mutations can occur in any of the following interferon variants: IFN-α2b, IFN-α2b-4N, or any other variant of IFN-α2. Example 5: Production of the different de-immunized variants of XFN-a2b-4N in CHO-K1 animal cells. The nucleotide sequences of the different de-immunized IFN-α2β-4N variants were artificially synthesized by Gene-art (Life Technologies). They were digested with appropriate restriction enzymes and cloned into a lentiviral vector that allows strong constitutive expression in eukaryotic cells (A: Oberbek A. (2011) Biotechnol Bioeng 108(3):600-610., B: Chusainow J. (2009) Biotechnol Bioeng 102(4):1182-1196). Example 6: Determination of the presence of the different variants of de-immunized IFN-ot2b-4N by polyacrylamide gel electrophoresis followed by Western blott. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the method of Laemmli (1970). For this purpose, samples were resuspended in a solution of 0.05 M Tris-HCl, 2% (w / v) SDS, 10% (v / v) glycerol, and 5% (v / v) β-mercaptoethanol (β-ME), pH 6.8 (sample preparation solution). The samples were incubated for 3 min at 100°C and then directly plated onto the stacking gel, which contained a 5% (w / v) monomer / branching agent (acrylamide / bisacrylamide) polymerized with a 15% (w / v) acrylamide / bisacrylamide. The gel concentration of separation The run was performed at constant voltage (200V) until the run front reached 0.5 cm from the lower edge of the separating gel. The Mini-Protean 3 vertical modular electrophoresis system (Bio-Rad) was used. Following electrophoresis, the proteins were transferred to a PVDF membrane (BioRad) using a standard submerged protein transfer protocol (C: Hames, BD (1990) One-dimensional polyacrylamide gel electrophoresis. In: Gel electrophoresis of proteins. A practical approach. Hames, BD, Rickwood, D. (eds.) Oxford University Press, Inc., New York, USA. Chapter 1, pp. 1-147). A solution of 25 mM Tris, 192 mM glycine, and 20% (v / v) methanol, pH 8.3, was used as the transfer solution. The transfer was carried out at a constant current intensity of 180 mA for 1 h. The success of the assay was evaluated by detecting the presence of proteins with a 0.25% (w / v) Ponceau Red (Sigma) solution in 15% (v / v) glacial acetic acid and 40% (v / v) methanol. The dye was removed by successive washes with TBS solution (50 mM Tris-HCl, 150 mM NaCl, pH 7.5), and finally, the immunochemical reaction detailed below was carried out on the membranes: 1. Blocking: Nonspecific binding sites were blocked by incubating PVDF membranes with 5% (w / v) skimmed milk solution in TBS for 1 h at room temperature. 2. First incubation: The different membranes were incubated with a polyclonal anti-IFNa2b antibody solution prepared in 5% (w / v) skimmed milk solution in TBS (Western blot diluent). Incubation was carried out for 1 h at room temperature with shaking. 3. Second incubation: The membranes were incubated with rabbit anti-murine immunoglobulin antibodies conjugated with the enzyme peroxidase (Dako, USA). The solutions were prepared in Western blot diluent solution, incubating the membranes for 1 h at room temperature. 4. Washing: After each stage, 3 successive washes were performed by immersing the PVDF membranes in TBS-Tween 20 0.05% (V / V) solution for 5 min. with agitation. 5. Revealed: The analysis was performed using chemiluminescence, employing the commercial ECL™ Western blotting analysis system kit (GE Healthcare) according to the manufacturer's instructions. 500 µA of reagent was added to the membranes, and after a few minutes of incubation, the excess was removed. The membranes were then placed on a nylon support, sealing the ends to prevent contact between the substrate and the developing film. The membranes were exposed to Kodak Biomax-Xar photographic film (Kodak, USA) for varying durations to achieve maximum resolution with good contrast between the chemiluminescent signal and the background. Exposure of the membranes to the photographic film was carried out in a darkroom equipped with a 15 W red lamp. The photographic film was developed manually using the conventional method of fixing (lith graphic fixer, AGFA, Argentina) and developing (GBX developer and enhancer, AGFA, Argentina). The order of sowing and the results obtained by sowing the culture supernatants of the clones that over-express the analyzed variants are shown in Figure 2. As can be seen, the cell lines produce each variant of IFN-α2β-4N appropriately. However, in preliminary antiviral activity assays (see protocol below), the supernatants from the cell line producing IFN-α2β-4N - VAR2 (SEQN°2) showed no evidence of antiviral biological activity (see Figure 3) and were therefore not included in subsequent assays. This result is not surprising considering that this protein had the highest number of mutations in its sequence (n=8), which likely led to a significant change in its structure and, consequently, its biological function. For this reason, this variant was not considered in subsequent assays. Example 7: Cell cloning of the producer lines of the de-immunized variants of IFN-α2β-4N and evaluation of the producer clones Once the cell lines producing the de-immunized variants of IFN-α2β-4N were obtained and their culture supernatants analyzed, each line was cloned using the limit dilution method. The clones were evaluated for their productive capacity using the sandwich ELISA technique described below. This assay is based on the capture of IFN-α2b-4N (or its variants) by the monoclonal antibody (mAb) CA5E6 immobilized on polystyrene plates and its subsequent recognition by immunoglobulins (Igs) present in a rabbit polyclonal anti-IFNα2b serum (C7). The protocol is described below: Sensitization: Flat-bottom polystyrene plates with 96 wells (Greiner) were sensitized with 100 μA of the mAb CA5E6 1 pg.ml'1 (100 ng / well) diluted in 50 mM Na2CO3 / NaHCO3 solution, pH 9.6 (sensitizing solution). They were incubated for 1 h. °C and overnight at 4 °C. Blocking: Blocking of nonspecific interaction sites was performed with 200 μA per well of a 1% (w / v) bovine serum albumin (BSA, Sigma) solution in PBS (blocking solution). Incubated for 1 h at 37 °C. First incubation: 100 μA of dilutions were added Successive 1:2 dilutions of the bacterial IFN-γ2b standard (Gema Biotech, Argentina) were performed, from 10 ng / ml to 0.078 ng / ml, and of the samples to be analyzed. A 0.1% (w / v) BSA solution in PBS with the addition of 0.05% (v / v) Tween 20 (diluent) was used. The samples were tested by performing serial dilutions to the medium in order to compare them with the standard within the linearity range of the curve. The samples were incubated for 1 h at 37 °C. A control without the addition of IFN-α2β was performed to evaluate the possible nonspecific binding of the reagents (negative control). For this purpose, during this stage the IFN was replaced with 100 ml of diluent solution. Second incubation: 100 pl of rabbit C7 anti-IFN-α2b serum diluted 1:1,000 with diluent solution was added. It was incubated for 1 h at 37 °C. Third incubation: 100 pl of goat anti-rabbit immunoglobulin antibody conjugated with the enzyme peroxidase (DAKO, Denmark) were added at a 1:2,000 dilution in diluent solution. It was incubated for 1 h at 37 °C. Development reaction: Development was performed by enzymatic reaction using 0.015 volume H2O2 diluted in 50 mM sodium citrate / phosphate solution, pH 5.3 (developing solution), with the addition of the chromogen o-phenylenediamine (OPD, Sigma) at a concentration of 0.5 mg / ml. 100 µl of this solution were placed in each well, and after 15 min of incubation in the dark at room temperature, color development was observed as the enzyme catalyzed the reduction of the substrate with the simultaneous oxidation of the chromogen. The reaction was stopped by adding 50 µl of 2N H2SO4, and the color reading was taken at λ = 492 nm using a microtiter plate reader (Labsystems Multiskan MCC / 340, Finland). Quantification: Absorbance values ​​were plotted as a function of the concentrations of IFN-α2β used as a standard and the sample dilutions, both on a logarithmic scale. The concentration of the samples was determined using the parallel lines assay (D: Milano, F. (2001) Undergraduate 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.). Thus, clone 10 and clone 9 from the CHO-K1 cell lines producing IFNOí2b-4N (VARI) and IFN-a2b-4N (VAR3), respectively, were selected to continue the study (see figures 4 and 5). Example 8: Purification of de-immunized variants of IFNa2b-4N by immunoaffinity chromatography Immunoaffinity chromatography can be divided into two stages: A) Preparation of a matrix containing the immobilized antibody. B) Binding of the antigen to the antibody matrix and elution of the same. For the purifications, the ÁKTA Basic and AKTA purifier liquid chromatographs (GE Healthcare) were used, equipped with a pump system (P-900), an automatic fraction collector (FRAC-900), an absorbance detection unit (UV-900) and a pH and conductivity detection unit (PH / C-900). The purification of the de-immunized variants of IFNa2b-4N obtained by culturing the corresponding clones under adherence conditions was carried out using the CNBR-Sepharose-mAbCA5E6 affinity matrix packed on an XK 16 / 20 column. 6 ml of previously unused resin was used for each variant to be purified. Before beginning the purification protocol, the sample was conditioned to ensure the appropriate interaction between the cytokine and the mAb. This involved adding Triton X-100 to the culture supernatant to reach a final concentration of 0.3% (v / v) and then adjusting the pH to 7.5 by adding dilute HCl or NaOH, as needed. Finally, the sample was filtered using a vacuum filtration system with 0.45 µm membranes (Sartorius Stedim, Germany) and a 1000 mL capacity (Nalgene). 500 and acetate cellulose filter holders The protocol developed for purification by immunoaffinity chromatography is described below: Equilibrium: The matrix was equilibrated with 5 VC of equilibration solution (Tris 25 mM; Triton X-100 0.3% (V / V) ; pH 7.5) at a flow rate of 1 mi.min-1. Seeding: A sample volume conditioned to a linear flow of 1 mi.min-1 was seen. The flowthrough was collected in fractions for subsequent analysis. Wash 1: The matrix was washed with 5 VC (Column Volumes) of a solution of Tris 25 mM, pH 7.5; NaCl 0.5 M; Triton X-100 0.2% (V / V). Wash 2: The matrix was washed with 12 VC of a 0.15 M NaCl solution. Each wash was collected in fractions for subsequent analysis. The flow rate applied during the washing stages was equal to that used during the equilibration phase. Elution: The different de-immunized variants of IFNa2b-4N retained were eluted by lowering the pH. For this purpose, 11 cubic centimeters (VC) of a 100 mM glycine solution, pH 2, were used, operating at the same flow rate used during the equilibration step. The fractions were collected and neutralized with 1 M Tris solution, pH 8. All fractions were stored at -20 °C for further analysis. Example 9: Concentration and diafiltration of purified proteins The fractions corresponding to the elution peak of each purification process were mixed, concentrated, and diafiltered against PBS. For this purpose, a 10 kDa Amicon® ultrafiltration membrane was used. Once diafiltered, the purified samples were quantified by spectrophotometric reading at λ=280 nm using an absorptivity coefficient for hIFN-a2b of 0.924 mi .mg’1.cm”1. The results obtained were confirmed by HPLC-C4. The purity of the samples was greater than 98%. Example 10: SDS-PAGE of de-immunized variants of IFNot2b-4N (immunoaffinity purified samples, IA) The different de-immunized versions of IFN-α2β-4N that were produced in animal cells (CHO-Kl) and purified by immunoaffinity chromatography were seeded on a polyacrylamide gel under denaturing conditions in the order shown in Figure 6. As can be seen in Figure 6, the bands corresponding to the de-immunized variants migrate with a greater electrophoretic delay due to their higher content of highly glycosylated isoforms. This effect is particularly pronounced in the IFN-α2β-4N - VAR3 version (lane 6). Example 11: Isoelectric focusing. Although each purified protein has a unique primary sequence, a population of proteins that differ in their glycosylation pattern (glycoproteins, glycoforms, or isoforms) can generally be found within the sample. The main difference between the observed bands is due to the sialic acid content, which gives each molecule a particular electrical charge. The analysis of the isoforms of the IFN-α2β4N variants was performed by isoelectric focusing, using a GE Healthcare system consisting of an electrophoresis tank (Multiphor II), a cooling bath (Multitemp III) and a voltage source (EPS3500XL). The isoelectric focusing technique was performed on 1 mm thick gels with an acrylamide / bisacrylamide concentration of 8% (w / v) and the addition of 7 M urea. The pH range was obtained by mixing 20% ​​(v / v) ampholytes with a pH of 2.5–5 and 80% (v / v) ampholytes with a pH of 3–10. Once the electrophoretic support was prepared, the ampholytes were pre-focused for 1 h at 10 W, 2000 V, and 100 mA to generate the pH gradient. Subsequently, 10 µA of each sample were seeded. Focusing was performed for 40 min under the same conditions as the pre-focusing step. The components thus separated were detected by staining with Coomassie Brilliant Blue. As shown in Figure 7, IFN-α2b-4N-VARI exhibits a glycoform pattern similar to that of the original protein (IFN-α2b-4N), although with a lower content of IFN-α2b-WT (O-glycosylated IFN-α2b). However, IFN-α2b-4N-VAR3 presents a markedly different isoform pattern, characterized by the presence of highly sialized proteins. It is worth noting that the sialic acid content not only contributes to the overall charge of the molecule but also confers a longer half-life, as it restricts binding to hepatic asialo-glycoprotein receptors that have specificity for terminal galactose or N-acetylgalactosamine residues (E: MJ Koury. Trends Biotechnol. 21 (11) (2003) 462-464). Furthermore, carbohydrates can also play an important role in protecting against extracellular proteolytic enzymes by masking cleavage sites and thus reducing their elimination from the system. circulatory (F: N. Jenkins, EMA EnzymeMicrob. Technol. 16 (1994) 354e364). Example 12: Determination of antiviral biological activity. Interferon antiviral bioassays quantify the inhibitory activity of these cytokines on viral propagation or replication (G: Familletti, PC; Rubinstein, S.; Petska, S. (1981) A convenient and rapid cytopathic effect inhibition assay for interferon. Methods Enzymology 78: 387-394). The simplest and most convenient procedure involves measuring the interferon's ability to protect susceptible cells from the cytopathic effect of a Utic virus across a range of cytokine concentrations. A wide variety of cell line / virus systems exist; in this study, the MDBK cell line / vesicular stomatitis virus (VSV) system was tested. To perform the assay, the World Health Organization (WHO) international standard for IFN-γ2b produced in E. coli obtained from the National Institute for Biological Standards and Control (NIBSC 95 / 566) was used, following the methodology detailed below. The MDBK cell line was cultured in growth medium [MEM supplemented with 10% (V / V) FBS]. When the culture was in the exponential growth phase, the cells were harvested, counted, and resuspended to obtain a cell density of 2.5 x 10⁵ cells / ml to establish a monolayer culture. 100 µA of this cell suspension was seeded into a sterile 96-well culture plate and incubated for 24 h at 37 °C. The culture supernatant was removed by inversion, drained, and 100 μA were placed per well of successive dilutions. 1:2 of the IFN-c<2b standard in MEM medium supplemented with 2% (V / V) FBS (assay medium), from an activity concentration of 20 Ul.ml'1 to 0.156 Ul.ml'1, incubating for 6 h at 37 °C. The supernatant was removed by inversion, drained, and 100 μA of an appropriate dilution of the virus (VSV) was placed in the assay medium, incubating for 18 hours until the maximum cytolytic effect was observed in cells not treated with IFN-α2β. To perform the assay, the culture supernatant was removed by inversion, drained, and 50 µL of a 1% (w / v) crystal violet (Anedra, Argentina) and 40% (v / v) methanol (Merck) solution was added to each well. The cells were incubated for 15 min at 37 °C. During this stage, cells not used by the virus, and therefore remaining adhered to the substrate, were fixed and stained. The stain was then removed, and the cells were washed with distilled water until the absence of stain was verified in the washes. To dissolve the fixed stain, 250 µL of 20% (v / v) acetic acid (Merck) was added. The resulting color was read at λ = 540 nm using a microtiter plate reader, which allows for plate homogenization prior to reading. The absorbance data were plotted against the corresponding activity values ​​of IFN-α2β (standard) and sample dilutions on a logarithmic scale, and the biological activity (BA) values ​​were calculated for each molecule by comparison with the standard using the parallel line assay. Finally, the specific biological activity (SBA) value was determined by calculating the ratio between the AB and the concentration of the molecules in the corresponding samples. Thus, the results of specific antiviral activity for the analyzed interferon variants were as follows: Activity Antiviral Protein IFN-α2β-NG IFN-β2b-WT IFN-O(2b-4N IFN-α2β-4N(VARI) IFN-O(2b-4N (VAR3) Specific (Ul / mg) 253.933.7131 16.600.025 180,626,554 ± 4,019,522 50,833,006 ± 627,682 14,420,356 ± 1,002,108 8,576,214 ± 853,076 Protein IFN-β2b-4N Specific Antiviral Activity (µl / mg) 50,833,006 ± 627,682 IFN-a2b-4N(VARI) 14,420,356 ± 1,002,108 IFN-0í2b-4N (VAR3) 8,576,214 ± 853,076 Relative Antiviral Activity (%) 100.0 28.4 16, 9 Table 2: Comparative assay of the specific antiviral biological activity of the de-immunized versions of IFN-a2b-4N. As can be seen in Table 2, the deimmunized variants of IFN-γ2β-4N exhibit lower specific antiviral activity compared to the original protein. This result is not surprising because the mutations introduced into the protein produced a glycoform pattern enriched in highly glycosylated species (see Figure 7). While this effect is very beneficial in terms of increasing the half-life and stability of the cytokine in serum, it is also often associated with a reduction in receptor-mediated biological activity, such as antiviral and antiproliferative activity (H: Ceaglio N., Etcheverrigaray M., Kratje R., Oggero M. (2008) Biochimie 90 (3):437-49). Example 13: Lympho-proliferation assays from mononuclear cells incubated with different versions of IFN. One of the most commonly used preclinical assays involves evaluating the ex vivo response of immune system cells to a given therapeutic agent. If an immune response occurs as a result of this exposure, it can be measured by quantifying certain cytokines secreted by activated helper T lymphocytes. Generally, the cytokines evaluated are IFN-γ and IL-4, which are representative of, respectively. a Thl and Th2 type response, The initial stage of the trial involves obtaining peripheral blood samples from at least 20 healthy donors, whose HLA-DRB1 genotype is characterized to determine the allele frequency of the cohort and compare it with that of the world population. HLA-DRB1 (Class II) major histocompatibility complex gene typing was performed by PRICAI (First Argentine Center for Immunogenetics) using Luminex technology. Briefly, this technique involves PCR (Polymerase Chain Reaction) amplification of exon 2 of the DRB1 gene, followed by hybridization with specific probes attached to polystyrene beads labeled with fluorochromes. These beads are read by the Luminex instrument, and the PCR product is detected if it hybridizes to the oligos attached to the beads. In this way, a very good correlation was found between the analyzed population and the global population (see Figure 10). This is important because the results obtained can be extrapolated to a larger population, regardless of its genotype. In this study, the following were analyzed: The following versions of IFN: IFN-α2b-NG, IFN-α2b-WT, IFN-α2b-4N, IFN-α2b-4N-VAR1, IFN-α2b-4N-VAR3 and IFN-γ2b-NG (PEG). The latter is a biosimilar of the commercial version PEGINTRON® (Schering-Plough) and consists of a pegylated variant of non-glycosylated interferon (IFN-γ2b-NG). Polyethylene glycol (PEG) not only increases the protein's half-life but also protects it against proteolytic degradation (J: Bukowski, R., Ernstoff, MS, Gore, ME, Nemunaitis, JJ, Amato, R., Gupta, SK, Tendler, CL, 2002. Pegylated interferon alfa-2b treatment for patients with solid tumors: a phase I / II study. J. Clin. Oncol. 20 (18), 3841-3849. / / K: Wang, Y., Youngster, S., Grace, M., Bausch, J., Bordens, R., Wyss, D.F., 2002. Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications. Adv. Drug Deliv. Rev. 54, 547-570 / / L: Bailón, P., Palleroni, A., Schaffer, CA, Spence, CL, Fung, W., Porter, JE, Ehrlich, GK, Pan, W., Xu, Z. , Modi, MW, Farid, A., Berthold, W., 2001. Rational design of a potent, long-lasting form of interference: a 40 kDa branched polyethyleneglycolconjugated inferíeron-_-2a for the treatment of hepatitis C. Bioconjugate Chem. 12, 195-202.). With these lessons we proceeded to carry out the lymphoproliferation tests that will continue in detail.

Claims

1. A modified and isolated human interferon α with reduced immunogenicity, characterized in that it comprises the substitution of between 3 and 5 amino acids at positions 9, 18, 47, 117, 123, and 128 by amino acids selected from the group comprising: Alanine and Glycine. Eighteen claims follow.