A SYNTHETIC PEPTIDE

Abstract

A synthetic peptide having six O-glycosylation sites, which, when bound to a protein, improves pharmacokinetic parameters and is recognized by a monoclonal antibody for detection, quantification, or purification, comprising the sequence: APARSPSPTPTPTPT. Also included is a fusion protein comprising said synthetic peptide, a nucleic acid molecule comprising said peptide and said fusion protein. Furthermore, methods for detecting, quantifying, and purifying the peptide and / or the fusion protein comprising the peptide herein are also described.

Description

The present invention describes a peptide that combines the ability to confer improvements in the biological activity of proteins of therapeutic interest and provides an operational advantage to its production process from mammalian cells. STATE OF THE ART In recent decades, there has been significant development of therapeutic recombinant proteins; however, their low stability and short half-life in circulation pose the greatest obstacles to their clinical applications. Many of these compounds have molecular masses (MM) below 50 kDa, exhibiting high plasma clearance rates. Therefore, strategies have been implemented to prolong their circulation time in order to achieve adequate therapeutic doses. Several mechanisms are involved in the plasma clearance of proteins; however, one of the most frequently used pathways is renal. The physicochemical and structural characteristics of the glomerular filtration barrier are largely responsible for the elimination mechanisms. Thus, the size of a protein, which is its hydrodynamic radius, as well as its physicochemical properties, represent the starting point for improving its plasma half-life. To this end, manipulating a post-translational modification such as glycosylation is a tool capable of conferring changes in the aforementioned characteristics. Thus, glycoengineering, a methodology based on modifying the carbohydrate content and / or structure of proteins, has been proposed as a strategy to address these issues. Glycosylation is the most important post- / co-translational event carried out by eukaryotic cells and affects numerous properties of glycoproteins, including their solubility, pharmacokinetics, bioactivity, secretion, plasma clearance rate, and antigenicity. Thus, glycoengineering focuses on adding oligosaccharide chains to proteins to improve the aforementioned properties (primarily, prolonging their plasma half-life). Previous studies have addressed this topic by applying strategies that add N- or O-glycosylation sites. In particular, O-glycoengineering has been successfully carried out by fusing peptides capable of incorporating O-glycans to proteins of pharmacological interest, such as growth hormone (hGH), erythropoietin (EPO), thyrotropin (TSH), and follicle-stimulating hormone (FSH).Thus, in such proteins, it was possible to increase the half-life in circulation, without affecting the acquisition of their native conformation, secretion, or in vitro bioactivity. The technology of fusing peptides / polypeptides to the N- or C-terminal ends of a protein of interest has been used to improve the therapeutic properties of various molecules. Currently, there are developments involving the fusion of proteins to the following peptides / polypeptides: human albumin {585 amino acids} - polypeptide called XTEN {864 amino acids} various peptides (in the form of highly repeated structures) - CTP peptide (28 amino acids) that carries sites susceptible to O-glycosylation - peptides that carry sites susceptible to N-glycosylation Furthermore, O-glycosylation as a strategy to improve the pharmacokinetic properties of therapeutic proteins has been successfully implemented using peptide fusion methods with known O-glycosylation efficiency. The use of such fusion peptides is due to the fact that generating O-glycosylation sites directly on the protein sequence requires significant modifications (more than the 1-3 amino acids that are conventionally modified to generate a consensus N-glycosylation site). Such modifications would imply an extensive conformational change and, therefore, a change in biological activity. Regarding the use of peptides exhibiting sites of known O-glycosylation efficiency, developments such as the peptide called CTP exist in the literature. This peptide corresponds to the C-tert sequence of 28 amino acids with a high proportion of residues.Ser and Pro, which derives from the human chorionic gonadotropin (hCG) hormone. It exhibits 4 sites where the presence of carbohydrates has been identified, representing a very extensive amino acid structure for only 4 O-glycosylated sites. Patent EP2049144B1 describes polypeptides with an amino acid sequence that includes one or more glycosylation sites, however they do not have sites susceptible to identification or purification. Patent application DE102012105416 (Al) relates to a mechanism for achieving adequate glycosylation of a. A peptide already containing a potential N- or O-glycosylation sequence is fused to a donor protein that also contains N- or O-glycosylation sites. After expression of the protein-peptide construct, the desired glycosylation is achieved in the peptide, which is then cleaved from the protein by proteases. In this case, the peptide constitutes the biopharmaceutical of interest and belongs to the group of hematopoietically active peptides, blood pressure-regulating peptides, digestion-regulating peptides, opioid family peptides, tumor-associated glycopeptides, among others. Patent application JP2002000276 describes a sequence of five amino acids: AB-Thr / Ser-C-D where A and B can be any amino acid but at least one of them must be a charged amino acid and C and D can be any amino acid but at least one of them must be a charged amino acid. US patent application 2013266604 (1A) concerns a glycopeptide called GLYCOTAG, which is modified by N-glycosylation. The bacterium C. jejuni is used as the host for its production. European patent EP1342730 (Al) uses the peptide CTP, which contains O-glycosylation sites, in order to increase the plasma half-life of erythropoietin. This peptide corresponds to the 28-amino-acid C-tert sequence with a high proportion of Ser and Pro residues, from which the human chorionic gonadotropin hormone is derived. It exhibits four sites where carbohydrates have been identified, representing a very extensive amino acid structure for only four sites. O-glycosylated. In patent application EP2093235 (A1), extensive point mutations are made in the interferon (IFN) molecule to generate N- or O-glycosylation sites that are present in other IFNs, and mutations are also made in protease cleavage sites. That is, they do not add an amino acid sequence (peptide) to increase the carbohydrate content but rather they mutate the protein sequence in a point manner. The use of shorter peptides simplifies the preparation of fusion proteins, reduces the likelihood of affecting their biological activity, and decreases the risk of immunogenicity resulting from the modification of natural proteins. The peptide described in the present invention constitutes a useful label for detecting, quantifying, and purifying recombinant proteins using a monoclonal antibody. In other words, its use combines the ability to enhance the biological activity of proteins of therapeutic interest with improvements to their production process from mammalian cells. Furthermore, in pharmacokinetic experiments in experimental animals, it was observed that the variant containing the peptide of the present invention exhibited an elimination phase half-life approximately 3 times longer compared to that of the native protein and a decrease in clearance between 3 and 4 times lower than the native protein. Furthermore, the peptide variant of the present invention exhibits an increase in both thermal stability and stability against serum proteases. BRIEF DESCRIPTION OF THE FIGURES Figure 1. SDS-PAGE / western blot using polyclonal anti-hIFN-a2b antibodies for the detection procedure, MMM: molecular mass marker, IFNNG: non-glycosylated IFN produced in E. coli, IFNwt: wild-type IFN produced in CHO cells, GMOPmIFN: IFN fused to the GMOPm peptide and CTPIFN: IFN fused to the CTP peptide. These last two are also produced in CHO cells. Figure 2. Isoelectric focusing / western blot using polyclonal antibodies anti-hIFN-a2b for the detection of IFN isoforms and their fusion derivative to GMOPm (GMOPmIFN). Figure 3. SDS-PAGE / western blot using the antihGM-CSF CC1H7 mAb as the detection antibody. Figure 4. Sandwich ELISA assay using the CC1H7 mAb as the capture antibody and the anti-hIFN-a2b polyclonal antibody as the detection antibody. Diluent I: PBS-BSA 0.1% (w / v)-Tween 0.05% (ν / ν); Diluent II: diluent I containing 1 M NaCl. Figure 5. Pharmacokinetic profile of wild-type IFN and GMOPmIFN in experimental animals. The average plasma biological activity ± SD (n=4) was plotted as a function of the time elapsed since injection. Figure 6. Stability of the native IFN molecule and its GMOPm peptide fusion variant during incubation with human plasma Figure 7. Thermal stability of the native molecule of IFN and its fusion variant to the GMOPm peptide. BRIEF DESCRIPTION OF THE INVENTION The present invention describes a synthetic peptide having 6 O-glycosylation sites, which, when bound to a protein, improves pharmacokinetic parameters and is also recognized by a monoclonal antibody for its detection, quantification, and purification, comprising the sequence: APARSPSPTPTPTPT (SEQ ID N°4). In addition, this peptide has affinity for the monoclonal antibody CC1H7. In another aspect of the present invention, a fusion protein is described, characterized in that it comprises a protein with biological activity and said synthetic peptide; where the sequence of said peptide can be repeated at least twice; furthermore, said biologically active protein comprises cytokines, hormones, growth factors, antibodies, blood factors, receptors, neuroactive peptides, among others. Preferably, it comprises the interferon family, colony-stimulating factors, hematopoietic factors, nerve growth factors, growth hormone family, platelet-derived factors, insulin-like growth factors, fibroblast growth factors, vascular endothelial growth factors, hormones, antibodies, blood factors, and receptors for such proteins. More preferably, said biologically active protein is an interferon that forms part of the fusion protein described in SEQ ID No. 5. Furthermore, the present invention comprises the incorporation of a signal peptide sequence into the polypeptide sequence for the export of the I fusion protein to the extracellular medium. Preferably, the signal sequence linked to the synthetic peptide and the biologically active protein comprises SEQ ID No. 6. Said fusion protein comprises a half-life at least 2.5 times longer than that of the biologically active protein in the absence of said synthetic peptide; It maintains at least 80% of its residual antiviral biological activity at a temperature between 50 and 70°C; it retains 100% of its remaining antiviral biological activity after 170 hours of incubation in plasma. Furthermore, the present invention comprises a nucleic acid molecule encoding the synthetic peptide comprising SEQ ID No. 1. It further comprises a nucleic acid encoding the fusion protein comprising a biologically active protein and the synthetic peptide comprising SEQ ID No. 2. The present invention further comprises a DNA molecule encoding the signal peptide linked to the synthetic peptide and the biologically active protein comprising SEQ ID No. 3. It further comprises a plasmid for the expression of said nucleic acid molecules, and a recombinant protein expression cell comprising said nucleic acids. Furthermore, another aspect of the present invention comprises a method for detecting said synthetic peptide comprising recognizing said sequence with a monoclonal antibody having affinity for said sequence; where the monoclonal antibody is CC1H7; where said method comprises an intracellular localization method; where furthermore, said method is for identification and quantification. Specifically, these methods include: Western blot; dotblot. Another aspect of the present invention comprises a method for purifying said fusion protein comprising a biologically active protein and the synthetic peptide of the invention, wherein said purification method comprises at least one immunoaffinity chromatography step. DETAILED DESCRIPTION OF THE INVENTION The present invention comprises the use of a peptide that combines the ability to confer improvements in the biological activity of proteins of therapeutic interest and provides an operational advantage to their production process from mammalian cells. This technology allows the generation of new therapeutic proteins that incorporate O-glycans into their structure by fusing them to a peptide tag of ISamino acids, the main object of the present invention, called GMOPm, whose sequence is the following SEQ ID No. 4: APARSPSPTPTPTPT. This sequence is derived from the N-terminal region of human granulocyte-macrophage colony-stimulating factor or hGM-CSF (APARSPSP) and has 6 potential O-glycosylation sites. The first 7 residues of the GMOP peptide (APARSPS) are part of a tag recently postulated in our laboratory as a new useful tool for detecting, quantifying and purifying recombinant proteins. Furthermore, the first four amino acids (APARs) of this peptide were identified as part of a linear epitope recognized by an anti-hGM-CSF monoclonal antibody (mAb) called CC1H7. The CC1H7 epitope-paratope interaction exhibited the unique characteristic of modifying its affinity with variations in ionic strength, representing an operational advantage for the development of immunochemical techniques such as ELISA, immunoaffinity chromatography, and Western blot, among others. Thus, the developed technology is based on the generation of chimeric proteins resulting from the fusion of a protein of pharmaceutical interest, such as hIFN-α2b, with the peptide GMOPm. The fusion proteins can contain the peptide GMOPm fused to the N-terminus and / or C-terminus of the protein of interest, in different ratios of peptide GMOPm / protein of interest (Pl), namely: GMOPm-Pl; (GMOPm)2-Pl; (GMOPm)3-Pl; (GMOPm)2-Pl-GMOPm; (GMOPm)3-Pl-GMOPm, among other variants. Therefore, the incorporation of the GMOPm peptide integrates functional and operational advantages into the production process of recombinant proteins of therapeutic interest, since it provides improvements in their pharmacokinetic properties and, therefore, in their biological potency in vivo, while also facilitating the immunochemical procedures related to their quantification, quality determination, and purification. The developed technology, based on the use of the GMOPm peptide, confers therapeutically relevant proteins with properties inherent to increased glycosylation. Specifically, it promotes an increase in molecular mass and charge, which leads to a decrease in plasma clearance and thus contributes to increased biological activity in vivo. It also provides technological improvements by facilitating the development of certain stages in the production process of the proteins of interest. These improvements are based on the property of the GMOPm peptide to be detected by the mAb CC1H7 due to the presence of the linear and N-terminal epitope APARSPS. In this way, the GMOPm peptide provides an effective method for detecting fusion proteins by Western Blot assays (Example 6 - Fig. 3), quantifying these proteins by ELISA assays (Example 6 - Fig. 4) and purify them using an immunoaffinity procedure. The latter benefits from the particularity of the epitope APARSPS and the CC1H7 paratope modify the resulting strength of their interactions in the presence of high or low ionic strength (this behavior is evidenced in the ELISA test shown in example 6 - Fig. 4). EXAMPLES EXAMPLE 1: DESIGN OF IFN VARIANTS To evaluate the proposed technology, hIFN-α2b was used as a model molecule. IFNs are cytokines with important therapeutic applications based on their antiviral, antiproliferative, and immunomodulatory activities. These allow for the treatment of various diseases, including hepatitis B and C, and certain cancers such as melanoma, Kaposi's sarcoma, chronic myeloid lymphoma, and angioblastoma. However, their application as a therapeutic agent is limited due to their short half-life in circulation, resulting from rapid renal elimination, strong binding to specific receptors, and / or proteolytic degradation in the blood. This leads to prolonged treatments, requiring the administration of high and frequent doses, with the potential for adverse effects and the generation of an immune response in patients. For this reason, the application of the glycoengineering strategy based on the use of the GMOPm peptide proved attractive to improve the pharmacokinetic properties of this cytokine. In this way, a variant of hIFN-cf2b was designed by fusing the peptide GMOPm to the N-terminal end of said cytokine, obtaining a new molecule called GMOPmIFN that comprises the amino acid sequence SEQ ID N°5, which has 7 potential O-glycosylation sites (6 due to the addition of the peptide sequence GMOPm and 1 naturally present in the hIFN-cf2b molecule). The synthesis of the chimeric DNA, in such a way construction (which also contains the signal peptide derived from the hGM-CSF molecule to allow the secretion of the fusion protein into the extracellular environment) It was requested from the GeneArt company and comprises the sequence SEQ ID N°3 (Lifé Technologies-Invitrogen). EXAMPLE 2: CONSTRUCTION OF THE EXPRESSION VECTOR AND CHO CELL TRANSDUCTION The synthesis products were cloned into the pLV-PLK lentiviral vector. Lentiviral particles containing the transfer vector were assembled and used to transduce CHO-K1 cells, thereby generating stable GMOPmIFN-producing cell lines. The produced molecules were characterized in terms of their degree of glycosylation, functionality, and ability to interact with the previously mentioned anti-hGM-CSF CC1H7 mAb. EXAMPLE 3: EVALUATION OF MOLECULAR MASS BY SDS-PAGE TESTS FOLLOWED BY WESTERN BLOT Using SDS-PAGE / internal blot assays with anti-HIFN-β2b polyclonal antibodies for the detection procedure, it was observed that the variant construct GMOPmIFN was successful in achieving an increase in the molecular mass of hIFN-a2b compared to the non-glycosylated IFN produced in bacteria (IFN NG) and Unmodified IFN produced in CHO-K1 cells, which has a natural O-glycosylation site at amino acid position Thrl06 (IFNwt), was used. This allowed for the successful incorporation of the GMOPm peptide and the subsequent expression of O-type carbohydrates in the new variant (Fig. 1). The culture supernatant containing the GMOPm variant showed a higher molecular weight compared to the IFN produced in bacteria and the naturally O-glycosylated IFN produced in CHO cells, as a consequence of the greater degree of glycosylation provided by the incorporation of the GMOPm peptide into the cytokine. For comparison, an IFN variant tagged with the CTP peptide at its end was also incorporated into the electrophoretic run. N-terminal of the cytokine. This tag exhibits 8 potential O-glycosylation sites, 4 of which are confirmed to be occupied by carbohydrates in the natural protein from which it is derived (hCG). Lanes 4 and 5 of Fig. 1 show the GMOPmIFN and CTPIFN variants, respectively. The latter showed a higher molecular mass, presumably due to its larger peptide size (28 aa). CTP peptide vs. GMOPm peptide (15 aa). Regarding the contribution of glycosylation to molecular size, it is only known that the CTP peptide exhibits 8 potential glycosylation sites while GMOPm has 6. If all sites were occupied, glycosylation would likely be a contributing factor to the increase in the molecular mass of CTPIFN relative to GMOPmIFN. However, it is necessary to consider results shown in subsequent examples that illustrate a similar decrease in the plasma clearance rate of the GMOPmIFN molecule compared to the decrease observed for CTPIFN, both studied with respect to the molecule of IFNwt. Regarding the above, it appears that the presence of carbohydrates and their negative charge, rather than the larger amino acid size of the CTP peptide, are more important for achieving a similar decrease in the plasma clearance rate of GMOPmIFN and CTPIFN molecules. It follows that the glycosidic charge of the GMOPm peptide is greater than that of the CTP peptide, and therefore, the greater molecular mass observed in the molecule. CTPIFN is mainly due to the larger peptide size of CTP. In conclusion, the use of the GMOPm peptide would represent an advantage in the search for molecules that increase the carbohydrate content / macromolecular size ratio, due to its property of conferring a high proportion of carbohydrates in a smaller peptide size, for example, compared to the CTP peptide. Thus, by fusing two consecutive GMOP sequences, it would be possible to achieve the same amino acid size as a CTP molecule but with a carbohydrate content two or more times greater, which would translate into a substantial improvement in the pharmacokinetic properties and in vivo biological activity of the molecule to which the aforementioned O-glycosylated peptides were incorporated. EXAMPLE 4: EVALUATION OF THE ISOFORM PROFILE THROUGH ISOELECTROFOCUSING TESTS FOLLOWED BY WESTERN BLOT The isoform profile obtained from isoelectric focusing assays and subsequent western blot allowed the incorporation of new carbohydrates into the hIFN-oí2b molecule by fusing it to the GMOPm peptide. These carbohydrates provide a higher content of sialic acid and, consequently, would decrease its isoelectric point, which was observed as a displacement of the bands towards the more acidic zone of the gel (Fig. 2). EXAMPLE 5: DETERMINATION OF BIOLOGICAL ACTIVITY SPECIFIC ANTIVIRAL AND ANTIPROLIFERATIVE Furthermore, the functionality of the chimeric protein was analyzed by studying its biological activity in vitro in cell cultures, both in relation to its ability to inhibit viral replication (antiviral activity) and to inhibit cell proliferation (antiproliferative activity). As shown in the table I, the incorporation of the GMOPm peptide at the N-terminal end of the 'hIFN-a2b' determined a decrease in the specific biological activity in vitro of the chimeric protein with respect to IFNwt, preserving 53% of the antiviral ABE, and 43% in the case of the antiproliferative ABE. However, this should not be considered an impediment when evaluating this molecule as a therapeutic agent, since there is ample evidence demonstrating the error involved in relying on in vitro assays to predict the in vivo biological activity and therapeutic effects of a molecule. In vitro bioassays do not take into account the pharmacokinetic differences between compounds, nor the differences in body distribution or metabolism. Table I. Determination of the in vitro antiviral and antiproliferative ABE of the IFNwt molecule and its derivative GMOPmIFN present in culture supernatants. Molecule ABE Antiviral (Ul / ng) ABE Antiproliferative (Ul / ng) IFNwt 431.9 + 142.6 150.5 ± 14.6 GMOPmIFN 228.5 ± 96.5 64.2 ± 2.8 EXAMPLE 6: EVALUATION OF INTERACTION ABILITY OF THE EPITOPE APARSPS WITH THE mAbCClH7 Finally, SDS-PAGE / western blot assays, using the anti-hGM-CSF mAb CC1H7 for the detection procedure, allowed us to demonstrate the immunochemical recognition of those molecules containing the peptide APARSPS such as the natural protein that gave rise to the antibody: hGM-CSF, a peptide fusion protein APARSPS: APARSPS-IFN, and the protein carrying the peptide of the present invention: GMOPmIFN. The assay also showed the specificity of the recognition because no reactivity was observed with the IFNwt molecule (Fig. 3). Furthermore, the solid-phase immobilized CC1H7 mAb was able to capture the GMOPmIFN variant in solution in a sandwich ELISA assay, in which polyclonal anti-hIFN-a2b antibodies were used for the detection reaction (Fig. 4). Additionally, the sample exhibited greater interaction with the CC1H7 capture mAb when diluted in 1 M NaCl solution (diluent IIj) compared to the routine assay diluent. ELISA: PBS-BSA 0.1% (w / v)-Tween 0.05% (v / v) (diluent I). This result confirms the peculiarity that the epitope-paratope CC1H7 interaction modifies its affinity with variations in ionic strength. These results together confirm that the presence of the APAR epitope in the chimeric protein GMOPmIFN allows it to be captured and / or detected in liquid and / or solid phase by the mAb CC1H7, which is an operational advantage, especially for those proteins of interest for which there are no specific antibodies that allow their identification in complex matrices such as culture supernatants. EXAMPLE 7: PRODUCTION AND PURIFICATION OF GMOPmIFN A STARTING FROM CULTURE SUPERNATIVES The GMOPmIFN fusion protein was produced by culturing recombinant CHO-Kl cell lines under adherent conditions and subsequently purified by immunoaffinity chromatography to an anti-hIFN-a2b monoclonal antibody (mAb CA5E6). A high degree of purity (greater than 80%) was achieved, which is suitable for further characterization assays. EXAMPLE 8: EVALUATION OF THE PHARMACOKINETICS OF WILD-TYPE IFN AND GMOPmIFN IN EXPERIMENTAL ANIMALS USING THE SUBCUTANEOUS ROUTE. COMPARISON WITH VALUATIONS PHARMACOKINETICS OF THE CTPIFN MOLECULE. The importance of carbohydrates on various biological properties of glycoproteins has been extensively documented. Oligosaccharides have a marked effect on the solubility, stability, bioactivity, antigenicity, and pharmacokinetics of glycoproteins. A critical property that determines the efficacy of a therapeutic protein is its plasma clearance. Adequate control of drug concentration in plasma over time can lead to greater efficacy and a reduction in unwanted side effects (Marshall et al., 2003). In order to evaluate the influence of the glycosidic portion on the pharmacokinetic parameters of the O-glycosylated variant GMOPmIFN, in vivo experiments were performed using experimental animals. Evaluating the pharmacokinetics of a biopharmaceutical by determining its biological activity provides valuable information, as it allows for the quantification of only the fraction of the protein that is active in the sampled biological fluid. For this reason, it was decided to perform the pharmacokinetic analysis of IFNwt and its glycosylated variant in rats using the same dose, defined in reporter biological activity units of the purified samples. For this experiment, two batches of eight female Wistar rats each, two months old and with an average weight of 200 g, were used. Each batch was divided into two groups of four animals and injected subcutaneously with a dose (250 μA) of 1.105 U per animal of each purified variant (IFN wild type and GMOPmIFN). Blood samples were collected at different time points post-injection, so as to obtain n=4 for each time point, with blood extractions alternating between group 1 (t= 30 min, 2, 4, 8, and 24 h) and group 2 (t=1, 3, 6, 10, and 48 h). The residual biological activity in the plasmas obtained by centrifugation was determined using a reporter gene assay, and the average values ​​for each molecule were plotted as a function of time (Fig. 5). The behavior of the studied proteins after subcutaneous inoculation followed a first-order absorption model with rapid body distribution; therefore, absorption and elimination were assumed to be first-order processes. Thus, considering the equations governing the one-compartment model, the corresponding pharmacokinetic parameters were calculated (Table II) and compared using ANOVA, with p < 0.05 considered statistically significant. Table II. Pharmacokinetic parameters of rhIFN-a2b variants after subcutaneous injection in rats. Variant Tmax Ctníx tt / 2eüm AUC CLapp hlFN-a2b (h) (Ul.mi1) (h) (Ul.li.mr1) (ml.h1) Wild type 0.82 ± 0.41 185.1 ± 133.1 0.88 ± 0.20 309.8 ±48.1 328.3 ± 47.3 GMOPmIFN 1.25 ± 0.40 182.6 ± 28.7 2.57 ± 0.42 1323.3 ± 250.4 77.6 ± 15.8 The parameters were calculated for each animal and are expressed as the average value ± SD (n=4). As can be seen in Table II, the maximum plasma biological activity of wild-type IFN was reached earlier than that of GMOPmIFN (p=0.02). This result indicates an extension of the initial distribution phase of the variant conjugated to the GMOPm peptide, which could be explained by the presence of bound carbohydrates, causing a slower absorption rate from the injection site into the circulation. However, no significant differences were observed between the maximum plasma activities (Cmax) achieved by both molecules. Furthermore, the elimination phase half-life was 2.9 times longer for the variant GMOPmIFN, compared to that of the native protein. While the plasma activity of wild-type IFN could not be determined after 8 h post-injection, its highly O-glycosylated analog showed detectable biological activity values ​​after 48 h from administration. The addition of new glycosylation-susceptible sites to the native molecule, and their subsequent occupation, resulted in a 4.2-fold reduction in CLapp for the GMOPmIFN variant compared to wild-type IFN (p<0.0001), which is consistent with the increase in the area under the concentration-time curve (AUC). These results demonstrate that the addition of carbohydrates to the rhIFN-β2b molecule improves its pharmacokinetic properties. The reduction in clearance of the variant GMOPmIFN, obtained through glycoengineering by fusing it to a novel peptide containing O-glycosylation sites, could be explained by various mechanisms. One of these involves a decrease in glomerular filtration due to changes in the protein's charge and molecular size (Gooche et al., 1992; Sheffield, 2001). Another mechanism involves decreased receptor affinity, primarily for proteins whose main elimination mechanism is receptor-mediated endocytosis. Specifically, the produced variant contains carbohydrates that increase its molecular size, modify its charge, and decrease its affinity for the receptor, allowing it to maintain a stable plasma concentration for a longer period. Oligosaccharides can also play a crucial role in protecting glycoproteins from extracellular proteolytic enzymes by masking cleavage sites (Jenkins and Curling, 1994). Similarly, pharmacokinetic studies in rats demonstrated that the IFN variant CTPIFN exhibited an apparent clearance similar to GMOPmIFN. It is important to note that although CTPIFN had a higher molecular mass than GMOPmIFN (Fig. 1), the incorporation of the GMOPm peptide into IFN resulted in a plasma clearance rate similar to that observed after the incorporation of the CTP peptide into the cytokine. This is likely due to a higher proportion of glycans in the GMOPmIFN molecule compared to CTPIFN, which may be responsible for greater protection against the action of plasma proteases or for the decrease in glomerular filtration rate, primarily due to their greater negative charge. Furthermore, the incorporation of a smaller glycopeptide to achieve a similar pharmacokinetic effect is highly advantageous, as it implies a decrease in...the probability of structural instability and risk of immunogenicity. Thus, considering that GMOPmIFN exhibits a lower plasma clearance rate due to the greater degree of glycosylation provided by the additional O-glycosylation sites compared to the IFNwt molecule, it is reasonable to consider using the modified peptide to construct variants with multiple GMOPm peptides fused to the N-terminus and / or C-terminus of the protein of interest in varying proportions. This could potentially lead to an even greater improvement in pharmacokinetic parameters. EXAMPLE 9: EVALUATION OF THE STABILITY IN HUMAN PLASMA OF IFN AND ITS DERIVATIVE CONJUGATED WITH THE GMOPm PEPTIDE Many therapeutic peptides and proteins are vulnerable to attack by proteolytic enzymes present in blood plasma, requiring the administration of high doses to achieve the desired pharmacological effect. rhIFN-β2b can be administered subcutaneously, intravenously, or intramuscularly, resulting in different pharmacokinetic profiles. Regardless of the route of administration, the cytokine is rapidly inactivated by body fluids and tissues and eliminated from the plasma within a few hours of administration. One of the elimination mechanisms of rhIFN-β2b is proteolysis and inactivation by serum proteases (Shetcher et al., 2001; Peleg-Shulman et al., 2004). The presence of O-glycans in IFN molecules conjugated with the GMOPm peptide could confer resistance to proteolytic attack, constituting one of the reasons why this molecule exhibits a longer plasma half-life in experimental animals compared to its non-glycosylated homologue. The stability of the extensively glycosylated IFN molecule (GMOPmIFN) in human plasma at 37 °C was analyzed and compared to that of the rhIFN-β2b wild type. For this purpose, an equivalent mass of each was diluted in human plasma and incubated for varying time intervals at 37 °C. The assay was performed in triplicate. The percentage of remaining antiviral biological activity after treatment was plotted as a function of the time elapsed since the beginning of incubation, which lasted for 7 days (Fig. 6). Under the tested conditions, which attempted to mimic a physiological environment, the antiviral biological activity of the rhIFN-α2b wild type showed a marked decline during the course of the experiment, exhibiting a half-life (ti / 2, defined as the time required to reach 50% of the initial biological activity) of 123 h, calculated by interpolation on the trend line of the experimental results. Unlike the previous molecule, which retained 40% of its initial biological activity at the end of the experiment, the variant carrying the GMOPm peptide retained between 90% and 100% of its initial activity. Consequently, the unmodified molecule was gradually inactivated, while the remaining one maintained virtually intact biological activity, confirming that the higher O-glycosidic content confers greater resistance to inactivation by plasma proteases at physiological temperature.Such post-translational modification could mask the wandering sites, creating a spherical impediment to the development of enzymatic activity. This characteristic would positively influence the in vivo pharmacokinetics of the hyperglycosylated molecule, contributing to the delayed plasma elimination of the cytokine mediated by the presence of carbohydrates. EXAMPLE 10: EVALUATION OF THE THERMAL STABILITY OF IFN and its derivative GMOPmIFN In the pharmaceutical industry, temperature is one of the most important factors affecting protein stability, and although there is no general mechanism to describe this effect, the higher the temperature, the lower the stability of the polypeptide. The thermal behavior of the molecule was studied IFNwt and its extensively O-glycosylated variant (GMOPmIFN) were tested by incubating each at different temperatures (ranging from 20 to 95 °C) for 10 minutes. Subsequently, residual antiviral biological activity was evaluated (Fig. 7). Both proteins retained intact antiviral biological activity after 10 minutes of incubation at 55 °C. However, a gradual loss of biological activity was observed in both molecules starting at 65 °C as the incubation temperature increased. At this temperature, IFNwt retained only 36% of its initial biological activity, while the GMOPmIFN variant exhibited greater stability, retaining 80% of its activity. Subsequently, the temperatures that produced a 50% decrease in the biological activity of each molecule (Tm) were calculated, obtaining values ​​of 63.4 and 70.1 °C for the IFNwt and GMOPmIFN molecules, respectively.Thus, it is possible to conclude that there is an increase in thermal stability as a consequence of the increased glycosidic content. Previous work related to the chemical glycosylation of proteins such as α-chymotrypsin and β-lactoglobulin {Solá and Griebenow, 2009; Shental-Bechor and. Levy (2008) demonstrated an increase in thermal stability dependent on both glycan content and molecular size. It has been postulated that the increase in the degree of glycosylation stabilizes the native state of the protein by increasing internal non-covalent forces, which generates greater rigidity in its structure. This study demonstrates that glycoengineering via fusion to the GMOPm peptide is a highly attractive option for improving the pharmacokinetic properties of proteins with short circulating half-lives. Furthermore, the strategy employed in this work could offer other advantages, considering that sugars not only play a significant role in determining plasma half-life but also affect other glycoprotein properties, potentially improving their therapeutic efficacy by increasing their stability and solubility or decreasing their immunogenicity. It is important to highlight that the use of peptides for the generation of...O-glycosylation sites avoid the extensive modification that would be required to achieve this objective directly on the protein of interest, which could result in extensive conformational changes leading to a drastic decrease in biological activity and the risk of exacerbating immunogenicity instead of reducing it. Furthermore, this strategy is projected as a universal method that could be applied to any protein of interest without requiring extensive characterization of the amino acids to be modified in the sequence of each native protein to avoid conformational changes and / or loss of biological activity. Following this objective, it is highlighted that the peptides described in the present invention constitute a useful label for detecting, quantifying, and purifying recombinant proteins using a monoclonal antibody. That is, the use of these peptides could increase the biological activity of proteins of therapeutic interest, improving their pharmacokinetics and stability, resulting in less frequent administration and, consequently, a reduction in undesirable side effects. In turn, these peptides would provide advantages in the development of the biopharmaceutical production process of interest from mammalian cells.

Claims

1. An isolated synthetic peptide characterized in that it comprises the sequence SEQ ID No. 4 and comprises 6 sites for O-glycosylation. 29 claims follow.