Hybrid protein, hybrid nucleotide sequence, nucleic acid molecule, vector for hybrid protein expression, use of hybrid protein or nucleotide sequence, formulation, process for producing a formulation and method for treating lesions and / or cancer caused by human papillomavirus (HPV) infection

Abstract

The present invention describes an approach for improving immunogenic and therapeutic performance in the prevention and / or treatment of lesions and / or cancer caused by or associated with the human papillomavirus (HPV). More specifically, the approach involves a hybrid protein selected from two entities with improved immunogenic and therapeutic potential, use thereof and a formulation comprising same. Also disclosed are nucleotide sequences, a vector for the expression of recombinant proteins, as well as a process for producing a formulation comprising said entity and a therapeutic method. The invention applies to the fields of medicine, pharmacy and biotechnology.

Description

Descriptive Report of Invention Patent HYBRID PROTEIN, HYBRID NUCLEOTIDE SEQUENCE, NUCLEIC ACID MOLECULE, PROTEIN EXPRESSION VECTOR HYBRID, USE OF HYBRID PROTEIN OR NUCLEOTIDE SEQUENCE, FORMULATION, PROCESS FOR PRODUCING A FORMULATION AND METHOD FOR TREATMENT OF LESIONS AND / OR CANCER CAUSED BY HUMAN PAPILLOMAVIRUS (HPV) INFECTION Field of Invention

[0001] The present invention describes an approach to improve immunogenic and therapeutic performance in the prevention and / or treatment of lesions and / or cancer caused by or associated with the human papillomavirus (HPV). More specifically, the approach involves a hybrid protein selected from two entities with improved immunogenic and therapeutic potential, its use, and a formulation comprising said entity. Furthermore, nucleotide sequences, a vector for the expression of recombinant proteins, as well as a process for producing a formulation comprising said entity and a therapeutic method are disclosed. The invention is within the fields of Medicine, Pharmacy, and Biotechnology. Background of the Invention

[0002] Human papillomavirus (HPV) is transmitted primarily through sexual contact and is the most common viral infection of the reproductive tract. Most sexually active women and men will be infected at some point in their lives, and some may experience persistent infections. If left untreated, these infections can progress to pre-cancer and cancer in the anogenital region. HPV is considered the causative agent of cervical cancer, accounting for 95% of cases. A study published by BMC Public Health indicates that for every new case of HPV-induced cervical cancer, there are six cases of pre-cancer. 1 . In addition to the REPLACEMENT SHEET (RULE 26) cervical cancer, HPV is responsible for 90% of anal cancer cases, 75% of vaginal cancer cases, 70% of vulvar cancer cases, 70% of oropharyngeal cancer cases and about 60% of penile cancer cases 2In 2020, there were 852,620 cases of cancer caused by HPV, in both men and women. The study "Changes of new cases from 2020 to 2040" produced by the Global Cancer Observatory indicates a growing trend in the number of cases of these types of cancer, which could reach 1,205,162 by 2040.

[0003] To combat this public health problem, several prophylactic and therapeutic methods are available. For example, prophylactic HPV vaccines based on VLPs (virus-like particles), such as Gardasil and Cervarix, have been developed. However, these approaches do not protect individuals with established infection or those who already have precursor lesions. In these cases, available treatments, such as surgery, radiotherapy, and some chemotherapy, are often invasive and expensive, as well as lacking specificity. Furthermore, cancer recurrence and the development of resistance to conventional treatments are problems faced by many patients. Therefore, there is a need for new antitumor strategies that are safe, effective, and less traumatic for the patient.

[0004] Over the past two decades, therapeutic vaccines capable of teaching the immune system to specifically recognize and eliminate cancer cells have been developed. 34 . In the case of precursor lesions and cancers caused by HPV-16, these vaccines have been based primarily on the expression of a chimeric protein resulting from the genetic fusion of glycoprotein D (gD) of Herpes simplex virus type 1 (HSV-1) with the E7 oncoprotein of HPV-16 5 4 . Furthermore, these studies demonstrate that this technology can be applied in the form of vaccines based on DNA and the purified recombinant protein itself. 3 4 .

[0005] In the construction of these chimeric proteins, the gD component acts as an adjuvant, since when interacting with the HVEM cellular receptor, it promotes REPLACEMENT SHEET (RULE 26) activation of NF-KB and blocks co-inhibitory signals mediated by BTLA and CD160 ligands, resulting in the activation of antigen-specific T lymphocytes 6-10 . Furthermore, gD can act as a molecular vector that directs antigens fused to it to populations of dendritic cells specialized in antigen cross-presentation, important in the generation of antitumor responses. 11 . Finally, contact with the gD protein delays the expression of PD-L1 in dendritic cells, allowing the immunostimulatory function of these cells to last for a longer period.

[0006] However, this technology also has technical limitations, such as poor tumor growth control and unsatisfactory immunogenic activity. Therefore, this technology must continue to evolve. In this sense, changes in the formulations of both therapeutic vaccines mentioned, such as the use of other adjuvants, 11 ' 13 16, association with delivery methods 14 , to other agents 12 15 and chemotherapeutics 17 are some of the improvements already described. The references below outline the prior art mentioned.

[0007] 1. Tao, L. et al. Prevalence and risk factors for cervical neoplasia: A cervical cancer screening program in Beijing. BMC Public Health 14, 1-9 (2014).

[0008] 2. Dong, Z. et al. Human Papillomavirus-Based Immunodiagnosis and Immunotherapy for HPV-Induced Lymphomas. Frontiers in Immunology vol. 11 Preprint at https: / / doi.org / 10.3389 / fimmu.2020.586796 (2021 ).

[0009] 3. Diniz, MO, Lasaro, MO, Ertl, HC & Ferreira, LCS Immune responses and therapeutic antitumor effects of an experimental DNA vaccine encoding human papillomavirus type 16 oncoproteins genetically fused to herpesvirus glycoprotein D. Clin Vaccine Immunol 17, 1576-83 (2010)

[0010] 4. Porchia, B. F. M. M. etal. Purified herpes simplex type 1 glycoprotein D (gD) genetically fused with the type 16 human papillomavirus E7 oncoprotein enhances antigen-specific CD8 + T cell responses and confers protective antitumor immunity. Mol Pharm 8, 2320-2330 (2011 ).

[0011] 5. Lasaro, M. O., Diniz, M. O., Reyes-Sandoval, A., Ertl, H. C. & Ferreira, L. C. S. Anti-tumor DNA vaccines based on the expression of human FOLHA DE SUBSTITUIÇÃO (REGRA 26) papillomavirus-16 E6 / E7 oncoproteins genetically fused with the glycoprotein D from herpes simplex virus-1 . Microbes Infect. 7, 1541-1550 (2005).

[0012] 6. Lasaro, M. O. et al. Targeting of antigen to the herpesvirus entry mediator augments primary adaptive immune responses. Nat Med 14, 205-212 (2008).

[0013] 7. Sciortino, M. T. et al. Involvement of gD / HVEM interaction in NF-kB- dependent inhibition of apoptosis by HSV-1 gD. Biochem. Pharmacol. 76, 1522- 1532 (2008).

[0014] 8. Sciortino, M. T. et al. Involvement of HVEM receptor in activation of nuclear factor kappaB by herpes simplex virus 1 glycoprotein D. Cell. Microbiol. 10, 2297-311 (2008).

[0015] 9. Steinberg, M., Cheung, T. C. & Ware, C. F. The Signaling Networks of the Herpesvirus Entry Mediator (TNFRSR14) in Immune Regulation. Immunol Rev 244, 169-187 (2012).

[0016] 10. Cheung, T. C. et al. Unconventional ligand activation of herpesvirus entry mediator signals cell survival. Proc. Natl. Acad. Sci. U. S. A. 106, 6244-9 (2009).

[0017] 11 . Porchia, B. F. M. M. etal. Herpes simplex virus glycoprotein D targets a specific dendritic cell subset and improves the performance of vaccines to human papillomavirus-associated tumors. Mol. Cancer Ther. 16, 1922-1933 (2017).

[0018] 12. Diniz, M. O., Cariri, F. A. M. O., Aps, L. R. M. M. & Ferreira, L. C. S. Enhanced therapeutic effects conferred by an experimental DNA vaccine targeting human papillomavirus-induced tumors. Hum. Gene Ther. 24, 861-70 (2013).

[0019] 13. Aps, L. R. M. M. et al. Bacillus subtilis spores as adjuvants for DNA vaccines. Vaccine 33, 2328-34 (2015).

[0020] 14. Sales, N. S. etal. In vivo electroporation enhances vaccine-mediated therapeutic control of human papilloma virus, associated tumors by the activation of multifunctional and effector memory CD8+ T cells. Vaccine 35, 7240-7249 FOLHA DE SUBSTITUIÇÃO (REGRA 26) (2017).

[0021] 15. Moreno, ACR et al. The Combined Use of Melatonin and an Indoleamine 2,3-Dioxygenase-1 Inhibitor Enhances Vaccine-Induced Protective Cellular Immunity to HPV16-Associated Tumors. Front. Immunol. 9, 1-14 (2018)

[0022] 16. Pagni, R.L., Silva, O., Rodrigues, K.B., Sales, S. & Moreno, C.R. 2,3-dioxygenase as potential adjuvant targets for tumor immunotherapy. Front. Immunol. 1-15 (2022).

[0023] 17. Porchia, BFMM etal. Active immunization combined with cisplatin confers enhanced therapeutic protection and prevents relapse of HPV-induced tumors at different anatomical sites. Int. J. Biol. Sci. 18, 15-29 (2022).

[0024] In addition to the articles referenced above, in the search for the state of the art in patent literature, the following documents related to the topic were found:

[0025] Brazilian patent PI1003749, held by USP, protects vectors / plasmids harboring the DNA sequence for the expression of the hybrid protein gDE7, which consists of a polypeptide segment of the HPV-16 E7 oncoprotein between two segments corresponding to a modified form of the glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1). Furthermore, the aforementioned DNA and protein sequence are protected, as are their use and pharmaceutical and / or veterinary formulations for the treatment of infectious and / or degenerative diseases, or for the preparation of vaccine adjuvants for other antigens. It is worth noting that the protein protected by the patent is different from the proteins presented in this patent application.

[0026] Brazilian patent BR1 120120049283, held by USP, protects an isolated nucleic acid sequence and an expression vector with codons optimized for the expression of the HPV E7 protein fused to the HSV gD protein in mammalian cells. The document also discloses a synergistic immunogenic composition comprising said expression vector combined with another vector encoding a cytokine for the production of vaccines for the prevention of HPV-induced tumors. Therefore, REPLACEMENT SHEET (RULE 26) This patent differs from the present invention in that it uses the native form of HSV-1 glycoprotein D and requires a combination of vectors to achieve the desired effect.

[0027] Thus, from what can be inferred from the literature researched, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here, in the eyes of the inventors, has novelty and inventive activity compared to the state of the art.

[0028] Therefore, improvements are still needed in the performance of therapeutic vaccines against lesions and / or cancer caused by HPV infection. Summary of the Invention

[0029] The present invention solves the problems of the prior art by providing an approach to improve immunogenic and therapeutic performance in the prevention and / or treatment of lesions and / or cancer caused by or associated with the human papillomavirus (HPV).

[0030] In the present invention, a modified protein selected from two hybrid proteins, or their corresponding nucleotide sequences, exhibits improved immunogenic and therapeutic potential. The constructs of the present patent application lack the signal peptide, delete the C-terminal portion of the gD protein comprising the transmembrane region, and add extender sequences to the C- and / or N-terminal region. Furthermore, the hybrid proteins described herein are administered as exogenous antigens, so they are processed and presented in the context of MHC class I and II molecules, generating CD4 and CD8 T lymphocyte responses, in addition to antibodies. Formulations containing these proteins are stable and can be stored at barely refrigerated temperatures, making distribution and storage relatively easy.

[0031] In a first object, the present invention presents a hybrid protein comprising an extender portion linked to the N and / or C terminal region of a modified portion of the HSV-1 glycoprotein D fused to REPLACEMENT SHEET (RULE 26) HPV E7 oncoprotein. Preferably, HPV refers to viral subtypes with oncogenic potential, most preferably HPV-16.

[0032] In a second object, the present invention presents a hybrid nucleotide sequence comprising a coding sequence of an extender portion linked to a coding sequence of the modified HSV-1 glycoprotein D fused to a coding sequence of the HPV E7 protein.

[0033] In a third object, the present invention presents a nucleic acid molecule characterized by the nucleotide sequence of SEQ ID NO: 5, or degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO: 2.

[0034] In a fourth object, the present invention presents a nucleic acid molecule characterized by the nucleotide sequence of SEQ ID NO: 6, or degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO: 3.

[0035] In a fifth object, the present invention presents a vector for the expression of hybrid protein, as defined above, comprising a genetic selection marker and one or more promoters functionally linked to a recombinant nucleotide sequence selected from the group consisting of SEQ ID NO: 5 or SEQ ID NO: 6.

[0036] In a sixth object, the present invention presents the use of the hybrid protein, as defined above, or of the hybrid nucleotide sequence, as previously defined, for the preparation of a medicine for the treatment of lesions and / or cancer caused by human papillomavirus (HPV) infection.

[0037] In a seventh object, the present invention presents a formulation comprising at least one hybrid nucleotide sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 17 and / or SEQ ID NO: 18, and at least one pharmaceutically acceptable excipient.

[0038] In an eighth object, the present invention presents a formulation comprising at least one hybrid protein selected from the group REPLACEMENT SHEET (RULE 26) consisting of SEQ ID NO:2 and / or SEQ ID NO:3, and at least one pharmaceutically acceptable excipient.

[0039] In a ninth object, the present invention presents a process for producing a formulation, comprising a step of mixing the hybrid protein or nucleotide sequence, as defined above, with at least one pharmaceutically acceptable excipient.

[0040] In a tenth object, the present invention presents a method for treating lesions and / or cancer caused by human papillomavirus (HPV) infection comprising the administration of a hybrid protein or a nucleotide sequence, as defined above.

[0041] In an eleventh object, the present invention presents a hybrid protein or a hybrid sequence, as defined above, for use as a medicine.

[0042] In a twelfth object, the present invention presents a hybrid protein or a hybrid sequence, as defined above, for use in the treatment of lesions and / or cancer caused by human papillomavirus (HPV) infection.

[0043] Degenerate sequences of the nucleotide sequences described above, which encode the same product, are also objects of the invention and should be considered as within the scope of the appended claims.

[0044] These and other objects of the invention will be immediately appreciated by those skilled in the art and will be described in detail below. Brief Description of the Figures

[0045] The following figures are presented:

[0046] Figures 1 A, 1 B, and 1 C show the in silico prediction of the secondary structure of the hybrid proteins SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively, using the AlphaFold Protein Structure Database. The gD protein is represented in dark gray; the HPV-16 E7 oncoprotein is represented in intermediate gray; and the extender amino acids are represented in light gray. REPLACEMENT SHEET (RULE 26)

[0047] Figures 2A, 2B and 2C show the analysis of the electrophoretic profile (Figure 2A) and recognition of recombinant proteins by Western Blot (Figures 2B and 2C). 1 - Molecular weight marker; 2 - recombinant protein of SEQ ID No: 3; 3 - recombinant protein of SEQ ID No: 2; 4 - recombinant protein of SEQ ID No: 1; 5 - gD. Run performed in 15% SDS-PAGE and recognition by anti-His (Thermo) and anti-gD (1 D3) antibodies.

[0048] Figures 3A, 3B, 3C, and 3D show the activation capacity of monocyte-derived dendritic cells (MoDCs) mediated by the different recombinant proteins, analyzed by the increase in the expression of surface molecules (Figures 3A and 3B) and cytokine production (Figures 3C and 3D). MoDCs were incubated with the different stimuli for 48 hours. Subsequently, membrane phenotype analysis was performed by flow cytometry and cytokine determination by CBA (Cytometer Bead Array). The data demonstrate the mean of replicates of a representative experiment of a total of three experiments performed with similar results. One-way ANOVA and Bonferroni post-test. **** pc0.0001; ** pc0.01; * p<0.05; ns = not significant.

[0049] Figures 4A, 4B, 4C, 4D, and 4E show the antitumor activity of the different recombinant proteins analyzed through TC-1 cell implantation, which is the most relevant model of HPV-associated tumors in C57BL / 6 mice. One day after tumor implantation, the animals began treatment with two 60 pg doses of each protein subcutaneously, with a 7-day interval between doses. Tumor presence was monitored for at least 60 days, twice weekly. The largest (D) and smallest (d) diameters of the tumors were obtained using a caliper, and the animals were euthanized when one of the diameters reached 15 mm. The graphs demonstrate the results for tumor area, represented by D x d in mm. 2 , from two experiments performed. In the upper right corner of each graph, the number of tumor-free mice / total number of mice is represented.

[0050] Figures 5A and 5B show the antitumor activity of the different REPLACEMENT SHEET (RULE 26) recombinant proteins analyzed by implanting TC-1 cells (10 5 cells), the most relevant model of HPV-associated tumors in C57BL / 6 mice. One day after tumor cell implantation, the animals began treatment with two doses of 60 pg of each protein subcutaneously, with a 7-day interval between doses. The presence of tumors was monitored for at least 60 days, twice weekly. Tumor diameters were obtained using a caliper, and the animals were euthanized when one of the diameters reached 15 mm. The graphs demonstrate survival (Figure 5A) and the frequency of tumor-free mice (Figure 5B), resulting from two combined experiments. Survival curve analysis: Log-rank (Mantel-Cox). **** p < 0.0001; *** p < 0.001 versus PBS group.

[0051] Figures 6A and 6B show the cellular immune response assessed by the ICS assay of mouse peripheral blood cells 7 days after the 2nd dose of treatment. Approximately 1 x10 6 cells were stimulated in vitro with the MHC I-restricted CD8 T cell-specific peptide E749-57 (SEQ ID NO: 7) for 12 hours in the presence of CD28 and Brefeldin, labeled with anti-CD3, -CD8, IFN-y, or TNFa antibodies, and evaluated by flow cytometry. The frequency of CD3 cells + CD8 + IFN-Y + (Figure 6A) and CD3 + CD8 + TNFa + (Figure 6B) represent the proportion of CD8 T lymphocytes that respond specifically against tumor cells in the evaluated period. One-way ANOVA and Bonferroni post test. *** pc0.001 ; ** pc0.01 ; * p<0.05; ns = not significant. Detailed Description of the Invention

[0052] The present invention provides an approach to improve immunogenic and therapeutic performance in the prevention and / or treatment of lesions and / or cancer caused by or associated with human papillomavirus (HPV).

[0053] The hybrid proteins presented here comprise modifications in the sequence of the HPV-16 E7 oncoprotein and / or the HSV-1 glycoprotein D. REPLACEMENT SHEET (RULE 26) These modifications include: a) point mutations (C24G and E26G) in the E7 protein to prevent its interaction with its cellular targets, thus avoiding the oncogenic potential of E7; b) deletion of the C-terminal portion of glycoprotein D to improve the production, purification and affinity of the molecule for the HVEM cellular receptor; c) addition of histidine tags to the N- and / or C-terminal portion; and d) addition of extender amino acids to the N- and / or C-terminal portion to stabilize the sequence and favor the interaction of the protein with the HVEM cellular receptor. Thus, the invention provides control of tumor growth and increased activation capacity of human dendritic cells differentiated from monocytes.

[0054] Furthermore, the nucleotide sequence defined by SEQ ID NO: 6 presents codons optimized for expression in E. coli, increasing production yield by up to 15 times when compared to the expression of the hybrid protein encoded by SEQ ID NO: 5.

[0055] Advantageously, the protein formulations according to the present invention are easily evaluated in vitro for biological activity in various contexts. For example, in immune cells from patients with various HPV-related diseases (precursor lesions or cervical cancer, anal cancer, oropharyngeal cancer, among others).

[0056] The biological activity demonstrated in the examples of the present invention provides evidence that the immune system activation mechanism mediated by these proteins is maintained and indicates therapeutic efficacy in different clinical contexts. This characteristic contributes to personalized patient treatment.

[0057] In a first object, the present invention presents a hybrid protein comprising an extender portion linked to the N and / or C terminal region of a modified portion of the HSV-1 glycoprotein D fused to the HPV E7 oncoprotein. Preferably, HPV refers to viral subtypes with oncogenic potential, more preferably, HPV-16.

[0058] In one embodiment, this hybrid protein comprises a REPLACEMENT SHEET (RULE 26) sequence AAl -AA2-(AA3)n-(AA4)rrAA5-(AA3)n-(AA4)n-(AA6)m-(AA7)p-AA8-(AA9)q- (AAio)r-(AAn)s, where: AAi is an extender moiety with between 10 and 35 amino acids; AA2 is a portion of the HSV-1 glycoprotein D between amino acids 26 to 269 or 21 to 269; AA3 is an apolar amino acid; AA4 is a polar amino acid; AAÕ is a portion of HPV E7 antigen between amino acids 1 and 98, in which, optionally, residues at positions 24 and 26 are replaced by a non-polar amino acid; AAe is a nonpolar amino acid; AA7 is a polar amino acid; AAs is a portion of the HSV-1 glycoprotein D between amino acids 270 to 341 or 268 to 339; AA9 is an apolar amino acid; AA10 is an acidic amino acid; AA11 is a tail containing 6 histidines n, m, p, q, r, s are independently an integer from 0 to 1.

[0059] Nonpolar amino acids consist of alanine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, valine, and methionine.

[0060] Polar amino acids consist of asparagine, glutamine, cysteine, tyrosine, threonine, and serine.

[0061] Acidic amino acids consist of either aspartic acid or glutamic acid.

[0062] In one embodiment, the extender portion is defined by SEQ ID NO: 8 or SEQ ID NO: 13.

[0063] In one embodiment, the hybrid protein is defined by SEQ ID NO:2 or SEQ ID NO:3.

[0064] In particular, the hybrid protein of SEQ ID NO: 2 is formed by: i) an extender amino acid sequence and a histidine tail (SEQ ID NO: 8), followed by ii) a first segment of the HSV-1 glycoprotein D REPLACEMENT SHEET (RULE 26) corresponding to amino acids 21 to 269 (SEQ ID NO: 9) linked to the sequence of iii) HPV-16 E7 antigen corresponding to amino acids 1 to 98 of the viral protein (SEQ ID NO: 10), which in turn is linked to iv) the second segment of HSV-1 glycoprotein D corresponding to amino acids 268 to 339 (SEQ ID NO: 11) followed by a v) second extender amino acid sequence comprising a histidine tail (SEQ ID NO: 12).

[0065] In particular, the hybrid protein of SEQ ID NO: 3 is formed by: i) an extender amino acid sequence and a histidine tail (SEQ ID NO: 13), followed by ii) a first segment of HSV-1 glycoprotein D corresponding to amino acids 26 to 269 (SEQ ID NO: 14) linked to the sequence of iii) HPV-16 E7 antigen corresponding to amino acids 1 to 98 of the viral protein, in which the cysteine ​​and glutamic acid at positions 24 and 26, respectively, have been replaced by glycine (SEQ ID NO: 15); in turn, this sequence is linked to iv) the second segment of HSV-1 glycoprotein D corresponding to amino acids 270 to 341 (SEQ ID NO: 16). Between portions ii)-iii) and iii)-iv) there are amino acids LQ and LQGT, respectively.

[0066] In a second object, the present invention presents a hybrid nucleotide sequence comprising a coding sequence of an extender portion linked to a coding sequence of a modified HSV-1 glycoprotein D fused to a coding sequence of the HPV E7 protein.

[0067] In one embodiment, the hybrid nucleotide sequence is defined by SEQ ID NO: 19, wherein: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is thymine; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR. REPLACEMENT SHEET (RULE 26)

[0068] In one embodiment, such a hybrid nucleotide sequence is a DNA nucleotide sequence defined by SEQ ID NO: 5.

[0069] In one embodiment, the hybrid nucleotide sequence is defined by SEQ ID NO: 23, wherein: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is uracil; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR.

[0070] In one embodiment, this hybrid nucleotide sequence is an RNA nucleotide sequence defined by SEQ ID NO: 17.

[0071] In another embodiment, the hybrid nucleotide sequence is defined by SEQ ID NO: 20, wherein: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is thymine; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR.

[0072] In one embodiment, this hybrid nucleotide sequence is a DNA nucleotide sequence defined by SEQ ID NO: 6.

[0073] In another embodiment, the hybrid nucleotide sequence is defined by SEQ ID NO: 24, wherein: R is A or G; Y is C or T; REPLACEMENT SHEET (RULE 26) H is A, C or T; N is A, C, G or T; T is uracil; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR.

[0074] In one embodiment, this hybrid nucleotide sequence is an RNA nucleotide sequence defined by SEQ ID NO: 18.

[0075] In a third object, the present invention presents a nucleic acid molecule characterized by the nucleotide sequence of SEQ ID NO: 5, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO: 2.

[0076] In a fourth object, the present invention presents a nucleic acid molecule characterized by the nucleotide sequence of SEQ ID NO: 6, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO: 3.

[0077] In a fifth object, the present invention presents a vector for the expression of a hybrid protein, as defined above, comprising a genetic selection marker and one or more promoters functionally linked to a recombinant nucleotide sequence selected from the group consisting of SEQ ID NO: 5 or SEQ ID NO: 6.

[0078] In a sixth object, the present invention presents the use of the hybrid protein, as defined above, or of the hybrid nucleotide sequence, as previously defined, for the preparation of a medicine for the treatment of lesions and / or cancer caused by human papillomavirus (HPV) infection.

[0079] In one embodiment, the hybrid protein or nucleotide sequence, as defined above, is used in the preparation of a medicament for the treatment of lesions and / or cancer caused by HPV-16 infection.

[0080] In one embodiment, the medicine is in the form of a vaccine REPLACEMENT SHEET (RULE 26) therapy.

[0081] In a seventh object, the present invention presents a formulation comprising at least one hybrid nucleotide sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 17 and / or SEQ ID NO: 18, and at least one pharmaceutically acceptable excipient.

[0082] In an eighth object, the present invention presents a formulation comprising at least one hybrid protein selected from the group consisting of SEQ ID NO:2 and / or SEQ ID NO:3, and at least one pharmaceutically acceptable excipient.

[0083] In one embodiment, the concentration of the hybrid proteins in the formulations is from 1 pg to 70 pg. Preferably, from 20 pg to 70 pg. More preferably, from 40 pg to 70 pg. Even more preferably, 60 pg.

[0084] In one embodiment, the formulation prepared for administration via the nasal, intramuscular, subcutaneous, or intradermal route. Preferably, via the subcutaneous route.

[0085] In a ninth object, the present invention presents a process for producing a formulation, comprising a step of mixing the hybrid protein or the nucleotide sequence, as defined above, with at least one pharmaceutically acceptable excipient.

[0086] In a tenth object, the present invention presents a method for treating lesions and / or cancer caused by human papillomavirus (HPV) infection comprising the administration of a hybrid protein or a nucleotide sequence, as defined above.

[0087] In an eleventh object, the present invention presents a hybrid protein or a hybrid sequence, as defined above, for use as a medicine.

[0088] In a twelfth object, the present invention presents a hybrid protein or a hybrid sequence, as defined above, for use in the treatment of lesions and / or cancer caused by human papillomavirus (HPV) infection. REPLACEMENT SHEET (RULE 26)

[0089] Degenerate sequences of the nucleotide sequences described above, which encode the same product, are also objects of the invention or, at least, considered equivalent means to those disclosed in the present patent application.

[0090] Definitions of:

[0091] Nucleotide sequence: refers to the sequence of basic units (nucleotides) that form deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules. In DNA, the nucleotide is composed of a phosphate group, a deoxyribose, and a nitrogenous base selected from adenine (A), thymine (T), cytosine (C), or guanine (G). In RNA, the nucleotide is composed of a phosphate group, a ribose, and a nitrogenous base selected from adenine (A), uracil (T), cytosine (C), or guanine (G). Furthermore, these sequences are degenerate; that is, more than one type of codon can define a single amino acid, so different sequences can produce the same protein.In this context, a serine can be specified by AGY (which includes AGC and AGT) or TCN (which includes TCA, TCC, TCG, and TCT); a leucine can be specified by TTR (which includes TTA and TTG) or CTN (which includes CTA, CTC, CTG, and CTT); an arginine can be determined by CGN (which includes CGA, CGC, CGG, and CGT) or AGR (which includes AGA and AGG).

[0092] Formulation: refers to the combination of at least one active ingredient with at least one excipient. Formulations can be in solid form, such as a powder for reconstitution, or in liquid form, such as solutions and emulsions. Preferably, the formulation is a sterile vaccine that is administered via the nasal, intramuscular, intradermal, or subcutaneous routes.

[0093] Excipient: refers to any component of a formulation other than the active ingredient or compound. These excipients aid in the adequate delivery of the ingredient and in enhancing its effect to achieve the desired purpose. Non-limiting examples that may be used in the context of this patent application include: carriers, such as PBS; adjuvants, such as aluminum salts, saponin, and squalene-based oil-in-water emulsions. REPLACEMENT SHEET (RULE 26) (for example: mf59®), among others.

[0094] The extender sequences inserted into the N-terminal region of the constructs of this application contribute to the stability of the molecules and the exposure of the binding region to the HVEM receptor (see SEQ IDs NOS: 8 and 13). This binding to the HVEM receptor, through gD, triggers the activation of various cell types of the immune system. Thus, the hybrid proteins of this patent application defined by SEQ ID NO: 2 and SEQ ID NO: 3 and their corresponding nucleotide sequences, both DNA and messenger RNA, can be used in the preparation of a drug, preferably in the form of a vaccine with enhanced therapeutic and immunogenic potential, for the treatment of lesions and / or cancer caused by HPV infection. Examples

[0095] The examples shown here are intended only to exemplify one of the numerous ways of carrying out the invention, without, however, limiting its scope.

[0096] Example 1 - Cloning of recombinant proteins

[0097] The nucleotide sequences of the recombinant proteins were used for cloning into an expression vector in Escherichia coli. SEQ ID NO: 4 in the pET-30a vector and SEQ ID NO: 5 and SEQ ID NO: 6 in the pET-28a vector. Then, the E. coli strains BL21(DE3) star (for expression of SEQ ID NO: 1) and BL21(DE3) (for expression of SEQ ID NO: 2 and 3) were transformed with the recombinant plasmids. A single colony was inoculated into Luria Bertani (LB) medium containing the antibiotic kanamycin and the culture was incubated at 37°C at 200 rpm. The strains were individually inoculated into 1 L of LB medium containing kanamycin and grown at 37°C and 200 rpm. Protein expression was induced with 0.5 mM Isopropyl beta-D-1-thiogalactopyranoside (IPTG) at 37 °C, 200 rpm for 4 h when the optical density at 600 nm (DQ600) reached approximately 1.2. Bacterial pellets were harvested by centrifugation and resuspended with lysis buffer followed by sonication. The precipitate after REPLACEMENT SHEET (RULE 26) centrifugation was dissolved using a denaturing agent. The target proteins were obtained by two-step purification using inclusion body wash and a Superdex 200 column (SEQ ID No: 1) or a HisTrap HP 5ml affinity column (SEQ ID No: 2 and SEQ ID No: 3). The target proteins were then sterilized by a 0.22 μm filter before being stored in aliquots. The final concentration of the purified products was determined by quantification on SDS-PAGE using a standard protein with known concentration (Table 1).

[0098] Table 1 - Final production yield of hybrid proteins in prokaryotic cells (E. coli), after upstream and downstream processes from laboratory-scale shaker culture. Quantification was performed on SDS-PAGE using a standard protein with known concentration.

[0099] Example 2 - Determination of purity, molecular weight and characterization of recombinant proteins.

[0100] To determine the purity, molecular weight, and characterization of the recombinant proteins, SDS-PAGE (Figure 2A) and Western blot (Figures 2B and 2C) assays were performed. The results confirmed the approximate molecular weight of 50 kDa for the three proteins, with SEQ ID No. 3 demonstrating a higher degree of purity and the absence of "contaminating" bands. The Western blot results also confirm the presence of histidine in the formulations (Figure 2B), where a more intense band can be observed for the protein SEQ ID No. 2, which, unlike the others, has two histidine tails. The recombinant protein SEQ ID No. 1 showed lower recognition of the histidine tail, consistent with the challenges faced during purification by affinity chromatography. REPLACEMENT SHEET (RULE 26) nickel. All proteins showed recognition of gD (Figure 2C), revealed with an anti-gD monoclonal antibody (1 D-3; recognizes the region of interaction with the HVEM cellular receptor) by the chemiluminescence technique.

[0101] Example 3 - In silico prediction of secondary structure

[0102] Amino acid sequences were used to perform in silico secondary structure prediction using the AlphaFold artificial intelligence program and its database, designed as a machine learning system. The results for structures related to sequences SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3 are shown in Figures 1A, 1B, and 1C, respectively. The images were edited so that the portion corresponding to glycoprotein D was dark gray, the portion corresponding to the HPV-16 E7 oncoprotein was intermediate gray, and the extender amino acids were light gray. In the predictions of the hybrid proteins of SEQ ID No. 2 and SEQ ID No. 3, better structure stabilization can be observed, with the formation of alpha helices and beta-pleated sheets.Furthermore, the conformation of these two hybrid proteins favors greater exposure of the E7 protein (in intermediate gray), which may reflect better epitope presentation and, consequently, increased immune response.

[0103] Example 4 - Validation of the immunomodulatory role

[0104] To validate the immunomodulatory role of the proteins, they were evaluated for their ability to activate human dendritic cells differentiated from monocytes (MoDCs). For this step, blood samples from three healthy donors were collected by apheresis. Monocytes were separated on a Ficoll gradient, enriched by adherence at 37°C for 2 hours, and cultured for 5 days with the cytokines GM-CSF and IL-4 in AIM V medium (ThermoFisher). No. 5 eday the stimuli were added to the MoDCs. After 48 hours of exposure, the MoDCs were analyzed for their membrane phenotype and functional state. To determine the membrane phenotype, the cells were labeled with human antibodies conjugated to specific fluorophores (CD1 1 c-PerCPCy5.5, HLA-DR-APCCy7, CD80-BV605 and CD83-PE) REPLACEMENT SHEET (RULE 26) and evaluated by flow cytometry in the LSR Fortessa equipment (BD Biosciences) using FlowJo software (Tree Star) to analyze the percentage of CD1 1 c+ HLA-DR+ cells that express co-stimulatory molecules (Figures 3A and 3B). The functional status was evaluated by quantifying cytokines secreted in the culture supernatant 48 h after stimulation (Figures 3C and 3D) using the CBA Human Th1 / Th2 / Th17 kit (BD Biosciences, catalog 560485).

[0105] LPS-activated MoDCs were considered as positive controls, and non-activated MoDCs (iDCs) were considered negative controls. MoDCs were considered activated with the recombinant protein when the expression of CD80 and CD83 was increased compared to iDC. The results show that only the recombinant proteins of SEQ ID NO: 2 and SEQ ID NO: 3 were able to induce the upregulation of CD80 and CD83 molecules in all individuals, with concomitant secretion of the pro-inflammatory cytokines TNF-alpha and IL-6.

[0106] The recombinant proteins were also evaluated for their antitumor effect against HPV-16-induced tumors in mice. In the subcutaneous tumor model, 1 x10 5TC-1 cells were injected into the right flank of the animals, followed by the administration of one of the recombinant proteins, also subcutaneously, in the left flank, one day later. A second dose of protein was administered at an interval of 7 days. Tumor growth results were obtained by measuring the smallest and largest diameters to calculate the tumor area (Figures 4A-4E) in untreated animals (control) and those treated with 60 pg of recombinant protein per dose. Furthermore, a group of animals treated with equimolar amounts of the gD (SEQ ID NO: 21) and E7 (SEQ ID NO: 22) proteins co-administered was included to evaluate the impact of the gene fusion strategy on the recombinant proteins.

[0107] The data show that, in general, the treatment of animals with recombinant proteins led to a benefit in controlling the growth of REPLACEMENT SHEET (RULE 26) tumors (Figures 4A-4E). The results also demonstrate that the antitumor effect is only obtained when there is a genetic fusion of gD with the target protein, in this case, HPV-16 E7, since the treatment involving the co-administration of the purified proteins obtained alone was unable to control tumor growth in any of the treated animals, as well as in the untreated control group. Regarding the different proteins, it was possible to observe that those of SEQ ID NO: 1 and SEQ ID NO: 2 induced partial antitumor control, with 8 of 11 animals and 6 of 10 treated animals showing protection, respectively. On the other hand, SEQ ID NO: 3 was able to lead to complete tumor control in all treated animals, generating the best therapeutic protection results compared to the other proteins.

[0108] For survival analysis, the maximum tumor diameter of 15 mm was determined at the time of euthanization. Thus, the data reveal that the groups treated with the protein of SEQ ID NO: 1 or SEQ ID NO: 2 showed an increase in survival compared to untreated controls or those treated with co-administration of gD and E7, reaching 70% (Figure 5A). In contrast, the group treated with the protein of SEQ ID NO: 3 showed 100% survival and 100% tumor-free animals (Figures 5A and 5B) at the end of the 60-day monitoring period.

[0109] Furthermore, as a way to complement the results of tumor growth control, an evaluation of the specific cellular response was performed by analyzing the frequency of CD8 T lymphocytes. +systemic specific antibodies, in turn characterized by the intracellular production of IFN-gamma and TNF-alpha. The analysis was performed using individual blood samples collected seven days after the second dose and the percentage of CD3 T cells + CD8 + IFN-Y and CD3 + CD8 + TNF-a of each animal was calculated by subtracting the frequency of stimulated cells from the frequency of non-stimulated cells with the E7-specific peptide (SEQ ID NO: 7) (Figures 6A and 6B).

[0110] The results reveal an increase in the systemic response of T lymphocytes REPLACEMENT SHEET (RULE 26) CD8 + E7-specific IFN-gamma producers mainly in the groups treated with proteins of SEQ ID NO: 1 and SEQ ID NO: 3, where SEQ ID NO: 3 showed greater homogeneity in the data of frequency of CD3 cells + CD8 + IFN-Y + and CD3 + CD8 + TNFa +in relation to SEQ ID No: 1. Furthermore, the cellular response results corroborate the data obtained from antitumor control for the group treated with co-administered (non-fused) gD and E7 proteins, which did not show an increase in circulating effector cells under the conditions tested.

[0111] Therefore, overall, these results demonstrate the surprising immunogenic and therapeutic potential of the proteins of SEQ ID NO: 2 and SEQ ID NO: 3 and the benefit of these optimizations mainly related to the control of tumor growth in mice and the activation capacity of human MoDCs.

[0112] Those skilled in the art will appreciate the knowledge presented herein and will be able to reproduce the invention in the presented embodiments and in other variants and alternatives, covered by the scope of the following claims. REPLACEMENT SHEET (RULE 26)

Claims

Claims 1. Hybrid protein characterized by comprising an extender portion linked to the N and / or C terminal region of a modified portion of the HSV- glycoprotein D. 1 fused to the HPV E7 oncoprotein.

2. Hybrid protein according to claim 1, characterized in that it comprises a sequence AAi-AA2-(AA3)n-(AA4)n-AA5-(AA3)n-(AA4)n-(AA6)m-(AA7)p-AA8-(AAg)q-(AAio)r-(AAn)s, where: AAi is an extender moiety with between 10 and 35 amino acids; AA2 is a portion of the HSV-1 glycoprotein D between amino acids 26 to 269 or 21 to 269; AA3 is an apolar amino acid; AA4 is a polar amino acid; AAÕ is the HPV-16 E7 antigen between amino acids 1 and 98, in which, optionally, residues at positions 24 and 26 are replaced by a non-polar amino acid; AAe is a nonpolar amino acid; AA7 is a polar amino acid; AAs is a portion of the HSV-1 glycoprotein D between amino acids 270 to 341 or 268 to 339; AAg is a nonpolar amino acid; AA10 is an acidic amino acid; AA11 is a tail containing 6 histidines n, m, p, q, r, s are independently an integer from 0 to 1.

3. Hybrid protein according to claim 1 or 2 characterized in that the extender portion is defined by SEQ ID NO: 8 or SEQ ID NO:

13.

4. Hybrid protein according to one of claims 1 to 3, characterized in that it is defined by SEQ ID NO:2 or SEQ ID NO:

3.

5. Hybrid nucleotide sequence characterized by comprising a coding sequence of an extender portion linked to a sequence encoding the modified HSV-1 glycoprotein D fused to a coding sequence for the HPV E7 protein.

6. Hybrid nucleotide sequence according to claim 5, characterized in that it is defined by SEQ ID NO: 19, where: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is thymine; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR.

7. Hybrid nucleotide sequence according to claim 6, characterized in that it is a DNA nucleotide sequence defined by SEQ ID NO:

5.

8. Hybrid nucleotide sequence according to claim 5, characterized in that it is defined by SEQ ID NO: 23, where: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is uracil; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR.

9. Hybrid nucleotide sequence according to claim 8, characterized in that it is an RNA nucleotide sequence defined by SEQ ID NO:

17.

10. Hybrid nucleotide sequence according to claim 5, characterized in that it is defined by SEQ ID NO: 20, where: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is thymine; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR.

11. Hybrid nucleotide sequence according to claim 10, characterized in that it is a DNA nucleotide sequence defined by SEQ ID NO:

6.

12. Hybrid nucleotide sequence according to claim 5, characterized in that it is defined by SEQ ID NO: 24, where: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is uracil; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR.

13. Hybrid nucleotide sequence according to claim 12, characterized in that it is an RNA nucleotide sequence defined by SEQ ID NO:

18.

14. Nucleic acid molecule characterized by the nucleotide sequence of SEQ ID NO: 5, or degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

2.

15. Nucleic acid molecule characterized by the nucleotide sequence of SEQ ID NO: 6, or degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

3.

16. Vector for the expression of hybrid protein, as defined in one of claims 1 to 4, characterized in that it comprises a genetic selection marker and one or more promoters functionally linked to a recombinant nucleotide sequence selected from the group consisting of SEQ ID NO: 5 or SEQ ID NO:

6.

17. Use of the hybrid protein, as defined in one of claims 1 to 4, or of the hybrid nucleotide sequence, as defined in one of claims 5 to 15, characterized in that it is to prepare a medicament for treating lesions and / or cancer caused by human papillomavirus (HPV) infection.

18. Use of the hybrid protein or nucleotide sequence according to claim 17, characterized in that the HPV is HPV-16.

19. Use of the hybrid protein or nucleotide sequence according to one of claims 17 and 18, characterized in that the medicament is in the form of a therapeutic vaccine.

20. Formulation characterized by comprising at least one hybrid nucleotide sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 17 and / or SEQ ID NO: 18, and at least one pharmaceutically acceptable excipient.

21. Formulation characterized by comprising at least one hybrid protein selected from the group consisting of SEQ ID NO: 2 and / or SEQ ID NO: 3, and at least one pharmaceutically acceptable excipient.

22. Formulation according to claim 21, characterized in that the hybrid protein is at a concentration of 1 pg to 70 pg.

23. Formulation according to one of claims 20 to 22, characterized in that it is presented in the form of administration via the nasal, intramuscular, subcutaneous or intradermal route.

24. Process for producing a formulation characterized by comprising a step of mixing the hybrid protein, as defined in one of claims 1 to 4, or the nucleotide sequence, as defined in one of claims 5 to 15, with at least one pharmaceutically acceptable excipient.

25. Method for treating lesions and / or cancer caused by human papillomavirus (HPV) infection, characterized by comprising the administration of a hybrid protein, as defined in one of claims 1 to 4, or of a nucleotide sequence, as defined in one of claims 5 to 15.

26. Hybrid protein, as defined in one of claims 1 to 4, or a nucleotide sequence, as defined in one of claims 5 to 15, characterized in that it is for use as a medicine.

27. Hybrid protein, as defined in one of claims 1 to 4, or a nucleotide sequence, as defined in one of claims 5 to 15, characterized in that it is for use in the treatment of lesions and / or cancer caused by human papillomavirus (HPV) infection.

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