Polypeptide comprising a mutant form of human VEGF-A having rearrangement of disulfide bonds and a composition containing the same

Abstract

The present invention relates to a polypeptide comprising a functional mutant of an isoform of human vascular endothelial growth factor A (VEGF-A) folded by non-natural rearrangement, wherein the second and fourth cystes of the polypeptide chain of the mutant form only intramolecular crosslinks, while the seventh and eighth cystes are only part of the intermolecular bonds. The present invention further comprises an antigenic agent containing at least one of these polypeptides, and a pharmaceutical composition comprising such antigenic agent and a vaccine adjuvant. The antigenic agent according to the present invention is used in the manufacture of drugs, in the promotion of angiogenesis, in the treatment of diseases associated with inflammation and immunosuppression, and in the recovery of the immune system.

Description

Technology Field

[0001] The present invention relates to the fields of biotechnology and human health. The present invention provides a polypeptide comprising a functional mutant of human vascular endothelial growth factor A (VEGF-A) folded into a non-natural arrangement of disulfide bridges. The present invention provides a basis for the production of compositions comprising these polypeptides used as preventive and therapeutic agents for pathologies progressing through angiogenesis, inflammation, and increased immunosuppression. Background Technology

[0002] The VEGF-A system and its receptors are a molecular complex, and their interactions specifically regulate the growth, permeability, plasticity, and motility of endothelial cells, thereby having a positive effect on pathological angiogenesis. Dysregulation of VEGF-A expression and its receptors occurs in both tumor cells and stroma, where they are particularly expressed and regulate endothelial cell function. The paracrine effects of tumor VEGF-A on endothelial cells surrounding tumors and the autocrine effects of this growth factor on tumor cells and stromas expressing their receptors have been described primarily over the past 20 years (Carmeliet, Nat Med, 2003: 9: 653-60, Mashreghi et al., J Cell Physiol, 2018: 233: 2949-65).

[0003] VEGF-A induces proliferation, motility, and tissue growth in endothelial cells, leading to the formation of new blood vessels with arrangement and maturation that depend on the concentration gradient of VEGF-A and its isoforms, as well as the presence of other proangiogenic factors and their receptors (Carmeliet, Nat Med, 2003: 9: 653-60). In addition to this primary function, evidence regarding the expression of type 1 and 2 VEGF-A receptors and co-receptors in myeloid-derived cells indicates that this factor is a major mediator of inflammatory and immunosuppressive processes. For example, VEGF-A is known to interact with type 1 receptors in dendritic cells and reduce maturation via NFkB (Gabrilovich, et al., Nat Med, 1996: 2: 1096-103; Gabrilovich, et al., Blood, 1998: 92: 4150-66). Additionally, VEGF-A binds to a type 2 receptor (VEGFR2) induced in effector T cells as a regulatory mechanism that inhibits interferon-gamma (IFN-γ) secretion (Ziogas, et al., Int J Cancer, 2012: 130: 857-64). VEGF-A is also an essential mediator of the inflammatory process and induces phenotypical changes in macrophages and neutrophils in the microenvironment of lesions, particularly those associated with neoplastic and arthritis phenomena (Voron, et al., Front Oncol, 2014: 4: 70).

[0004] In this regard, the VEGF-A protein family and their receptors are targets of active and passive immunotherapy in diseases that progress through the already described processes. Targeting VEGF-A and its receptors has been validated by the approval of passive immunotherapy with Avastin, Sorafenib, and Sunitinib as a first-line treatment for various tumor types and age-related macular degeneration (AMD) (Wentink, et al., Biochim Biophys Acta, 2015: 1855: 155-71). Active immunotherapy using these growth factors, which imply the induction of autoimmune responses, experiences slower development. Until 2002, only a small number of studies targeting VEGF-VEGFR were reported. Most of these efforts have been devoted to the immunogenicity, anti-angiogenic, and anti-tumor effects of heterologous variants (Wei, et al., Proc Natl Acad Sci USA, 2001: 98: 11545-50), or to studies on proteins with high structural and functional homology to VEGFA (patent application numbers WO 99 / 45018 and WO 00 / 53219). The use of heterologous variants induces high neutralization and specific antibody titers in the absence of cellular response, whereas the use of homologous molecules results in a weak immune response with little evidence of anti-tumor or anti-metastatic effects. One of these strategies has been reflected in clinical practice.

[0005] The use of functional mutants of VEGF-A in active immunization specific to this factor was described in 2002 using naked deoxyribonucleic acid (DNA) or recombinant proteins fused to an immunostimulatory sequence used as an adjuvant (International Patent Application No. PCT / CU03 / 00004).

[0006] Administration of VEGF-A mutants (Arg82, Lys84, His86 → Ala82, Ala84, Ala86; or Arg82, Lys84, His86 → Glu82, Glu84, Glu86) results in the induction of a T-specific antitumor response when using naked DNA-based immunization (Bequet-Romero, et al., Angiogenesis, 2007: 10: 23-34)), or protein-plus adjuvant-based (Morera, et al., Angiogenesis, 2008: 11: 381-93). With protein-based formulations, the induction of VEGF-A specific antibodies that inhibit binding to VEGFR1 and VEGFR2 was achieved (Morera, et al., Angiogenesis, 2008: 11: 381-93; Morera, et al., Vaccine, 2012: 30: 368-77). It was possible to induce antibodies that neutralize this interaction despite mutations introduced into VEGF-A that interfere with protein binding to VEGFR-2. Using this vaccine strategy, relevant antitumor and anti-metastatic effects are demonstrated in murine models of melanoma (MB16F10), lung carcinoma (3LLD122y TC1), breast carcinoma (F3II), and colorectal carcinoma (CT26). In the case of this variant, a direct cellular response was demonstrated in syngeneic tumor cells accompanied by the secretion of IFN-γ after incubation with VEGF-A (Bequet-Romero, et al., Vaccine, 2012: 30: 1790-9). Unlike other strategies, this uses a recombinant antigen produced in bacteria representing VEGF-A isoform 121 in which the cysteine ​​residue involved in VEGF-A dimer formation has not been removed. The use of this strategy in the study species demonstrated the potential presence of a superior immune response (Sanchez Ramirez, et al., BMC Immunol, 2017: 18: 39).

[0007] Using strategies such as changing adjuvants and concentrations, and altering the amount of administered antigen, higher immune responses were achieved with benefits in preclinical models and clinical settings (Morera, et al., Angiogenesis, 2008: 11: 381-93; Gavilondo, et al., Vaccine, 2014: 32: 2241-50; Perez Sanchez, et al., Hum Vaccin Immunother, 2015: 10-203 7; Sanchez Ramirez, et al., BMC Immunol, 2017: 18: 39). These higher immune responses occur without impairment of the physiological parameters of the study species while maintaining the autoregulatory characteristics of the response. Antibody levels obtained under the immunization schedule were significantly lower than those obtained after the injectable administration of therapeutic antibodies. The previously described factors indicate that there remains potential to improve the response to the vaccine without causing adverse effects.

[0008] Therefore, obtaining human VEGF-A variants with increased anti-angiogenic, anti-tumor, anti-metastatic, anti-inflammatory, or immune-restoring effects is still of interest, which enables the improvement of the immune response achieved in individuals immunized with antigen preparations containing such variants. means of solving the problem

[0009] The present invention solves the aforementioned problem by providing a polypeptide comprising a functional mutant of a human VEGF-A isoform folded by a non-natural rearrangement of disulfide crosslinks, wherein the second and fourth cystes form only intramolecular bonds, and the seventh and eighth cystes of the same molecule are detected only as part of intermolecular crosslinks.

[0010] In the case of the present invention, the functional mutant of VEGF-A is a molecule having 95% sequence identity compared to the natural mutant, but differs from the former in binding to VEGFR2, and this fact does not induce signal transduction associated with receptor binding.

[0011] The polypeptides described herein relate to human VEGF proteins, particularly VEGF-A and its isoforms, which have not been previously described. They were primarily produced by introducing changes to the polypeptide purification process defined by SEQ ID NO:2 (Morera, et al., Angiogenesis, 2008: 11: 381-93). Their analysis showed increased stability, immunogenicity, and antitumor effects compared to the original antigen preparation PVM (Examples 1, 2, 3, 4, 5).

[0012] To obtain a polypeptide related to the present invention comprising a functionally mutant form of a human VEGF-A isoform, amino acids involved in VEGFR2 binding were mutated. In one embodiment of the present invention, the polypeptide is VEGF-A 121 , VEGF-A 145 , VEGF-A 165 , VEGF-A 189 , and VEGF-A 206 It features a human VEGF-A isoform selected from the group including. The immunogenicity-increasing cysteine ​​array claimed in the present invention is VEGF-A 121 It is extended into an isoform that shares the standard cysteine ​​structure described for (Fig. 1) and exhibits the same therapeutic effect. Therefore, VEGF-A using the same cloning and expression system 145 , VEGF-A 165 , VEGF-A 189 and VEGF-A 206A sequence corresponding to was inserted. A comparative evaluation between a product that preserves the cysteine ​​knot and a product that does not indicate that the increase in immunogenicity described for PVM and PVM-I can be reproduced in the absence of this standard structure (Example 2).

[0013] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and second cystes, the third and fourth cystes, and the fifth and sixth cystes; and intermolecular bonds are formed between the seventh cysteine ​​of the two polypeptide chains, the eighth cysteine ​​of the two polypeptide chains, and the last cysteine ​​of the two polypeptide chains.

[0014] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and second cystes, the third and fourth cystes, and the fifth and sixth cystes; and intermolecular bonds are formed between the seventh and eighth cystes of two different polypeptide chains and between the last cysteine ​​of two polypeptide chains.

[0015] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and second cystes, the third and fifth cystes, and the fourth and sixth cystes; and intermolecular bonds are formed between the seventh cysteine ​​of the two polypeptide chains, the eighth cysteine ​​of the two polypeptide chains, and the last cysteine ​​of the two polypeptide chains.

[0016] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and second cystes, the third and fifth cystes, and the fourth and sixth cystes; and intermolecular bonds are formed between the seventh and eighth cystes of two different polypeptide chains and between the last cysteine ​​of two polypeptide chains.

[0017] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and second cystes, the third and sixth cystes, and the fourth and fifth cystes; and intermolecular bonds are formed between the seventh cysteine ​​of the two polypeptide chains, the eighth cysteine ​​of the two polypeptide chains, and the last cysteine ​​of the two polypeptide chains.

[0018] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and second cystes, the third and sixth cystes, and the fourth and fifth cystes; and intermolecular bonds are formed between the seventh and eighth cystes of two different polypeptide chains and between the last cysteine ​​of two polypeptide chains.

[0019] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and last cysteine, the second and third cysteine, and the fourth and fifth cysteine; and an intermolecular bond is formed between the seventh cysteine ​​of the two polypeptide chains and between the eighth cysteine ​​of the two polypeptide chains.

[0020] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and last cysteine, the second and third cysteine, and the fourth and fifth cysteine; and an intermolecular bond is formed between the seventh and eighth cysteine ​​of two different polypeptide chains.

[0021] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and last cysteine, the second and fourth cysteine, and the third and fifth cysteine; and intermolecular bonds are formed between the seventh cysteine ​​of the two polypeptide chains and between the eighth cysteine ​​of the two polypeptide chains.

[0022] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and last cysteine, the second and fourth cysteine, and the third and fifth cysteine; and an intermolecular bond is formed between the seventh and eighth cysteine ​​of two different polypeptide chains.

[0023] In one embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and last cysteine, the second and fifth cysteine, and the third and fourth cysteine; and an intermolecular bond is formed between the seventh cysteine ​​of the two polypeptide chains and between the eighth cysteine ​​of the two polypeptide chains.

[0024] In another embodiment, a polypeptide comprising a functional mutant of a human VEGF isoform comprises a rearrangement of intramolecular disulfide crosslinks formed between the first and last cysteine, the second and fifth cysteine, and the third and fourth cysteine; and an intermolecular bond is formed between the seventh and eighth cysteine ​​of two different polypeptide chains.

[0025] In a specific embodiment of the present invention, a polypeptide comprising a functional mutant of a human VEGF-A isoform further comprises an amino-terminal segment that increases its expression in bacteria and a carboxy-terminal segment that facilitates the purification process.

[0026] In a preferred embodiment that does not limit the scope of the invention, a recombinant protein isomer mixture comprising 46 amino acids of the amino-terminal segment of protein P64K from Neisseria meningitidis (SEQ ID NO: 24) is used. In the chimeric protein, the aforementioned amino-terminal segment and the amino-terminal portion of VEGF isoform 121 are separated by 13 amino acids connecting these two segments. Additionally, the protein has a carboxy-terminal 6-histidine sequence useful for antigen generation. The preservation of Cys 110 and 119 within the fusion polypeptide corresponding to Cys 51 and 60 of natural VEGF-A (Fig. 1) is likely to promote the formation of multiple structural isomers along with secondary structural changes of the protein. Only previous studies by Bequet-Romero et al. and Morera et al. have used VEGF-A variants containing such cysteine. Nevertheless, in all of these, intramolecular disulfide crosslinking was detected between cysteine ​​2 and 4 of the polypeptide chain. The presence of these two Cys and the antigen-generating process in which Cys 2 and 4 form only intramolecular bonds constitute a novel approach to the production of proteins for vaccine purposes. Overall, the strategy leads to the exposure of sites that induce an increased immunological response. The former may be due to recognition by antibodies that neutralize VEGF-A binding to type 2 receptors, or the fact that antigen digestion by proteasomes is promoted, allowing new peptides to be presented by antigen-presenting cells.

[0027] A specific embodiment of the present invention is a polypeptide having an amino acid sequence comprising SEQ ID NO: 18 to SEQ ID NO: 23. These have increased immunogenicity compared to polypeptides that preserve the tertiary structure described for VEGF-A isoform 121. Modifications of the tertiary structure explaining the desired superior effect were detected in these polypeptides. Since changes in the secondary structure have previously been described as not being optimized for generating an effective immune response to human VEGF-A, this superior immunogenicity was surprising (Wentink, et al., Proc Natl Acad Sci USA, 2016: 113 : 12532-7).

[0028] In the established purification process for the polypeptide identified as SEQ ID NO:2, structural isomers of VEGF-A having a Cys-nodular structure are produced, which are structural isomers similar to those of the wild-type molecule as shown in Example 1. Unexpectedly, changes in the purification process lead to an abundance of an isomer family exhibiting intramolecular disulfide bonds, including Cys 110 and 119, corresponding to Cys 51 and 60 of VEGF-A, which naturally form intermolecular crosslinks. In the antigen preparation designated herein as PVM-I, other intermolecular bonds including Cys 161, 163, and 175 were observed, which is a fact associated with the formation of oligomeric structures stabilized by disulfide crosslinks. Prior art does not contain information regarding the formation, stabilization, and sequence of the folding described herein. This is related to the fact that almost all studies dealing with the recombinant generation of VEGF-A are conserved in the sequences of Cys residues equivalent to Cys 51 and 60 of natural VEGF-A. When these cystes are conserved in the primary sequence, natural disulfide crosslinking was reproduced using the renaturation process (Pizarro, et al., Protein Expression and Purification, 2010: 72: 184-93). Therefore, the detection of novel non-normal bonds without natural bonds, and their relationship to the increased stability and biological activity of PVM-1 preparations, is an unexpected and surprising discovery of the present invention.

[0029] The present invention is not limited to a specific form for generating structural isomers present in the antigen preparation PVM-I. As shown in Example 7, these isomers can be obtained and isolated by using other procedures. Likewise, in the above example, the equivalent contribution of the identified isomers is presented in terms of increased immunogenicity. Several structural isoforms coexist within the protein fraction with low resistance to trypsin-based digestion, and they exhibit similar functions for generating an increased specific immune response.

[0030] PVM-I formulations consist of a mixture of isomers or specific isomers separated using reverse-phase chromatography capable of efficiently separating 12 isomer variants. Since the administration of the separated isomers regenerates at an immunological level, either of the isomer mixtures is potentially effective, and this effect was observed for formulations containing all of them (Example 7). This may be related to the fact that in all cases, peptide generation by endoproteases such as trypsin is preferred, and trypsin is one of the essential components of the proteasome system responsible for generating peptides to be presented to the immune system within the frame of MHC molecules (Pamer, et al., Annu Rev Immunology 1998: 323-358).

[0031] In addition, the subject of the present invention is an antigen preparation comprising at least a polypeptide of a functional mutant of a VEGF-A isoform folded by non-natural rearrangement of disulfide crosslinks, wherein the second and fourth cystes of the polypeptide chain form only intramolecular bonds, and the seventh and eighth cystes of the polypeptide chain form only intermolecular crosslinks. The antigen preparation comprises at least one pharmaceutically acceptable excipient or diluent. The immunogen dose may be administered in a pharmaceutically acceptable vehicle that is non-toxic and does not exhibit an intrinsic therapeutic effect. Such vehicles include ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins, buffers, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of vegetable fatty acids, water, salts and electrolytes, polyvinylpyrrolidone, cellulose-based materials, and polyethylene glycol. In the present invention, tris chloride buffer is preferentially used as a vehicle for vaccine preparations.

[0032] Since the primary objective of the present invention is a polypeptide having a secondary / tertiary structure significantly different from that found in nature, the present invention is not limited to antigen preparations obtained by the given procedure.

[0033] A series of isomers consisting of polypeptides containing mutations of VEGF-A isoforms can be obtained in a homogeneous composition using other strategies, including purification of structural isomers using reverse-phase high-pressure liquid chromatography (RP-HPLC) or controlled recovery of the original form in the absence of denaturalization agents. As shown in Example 7, structural isoforms can be separated using RP-HPLC, and their independent use at the same concentration yields similar effects in terms of immunogenicity, antitumor, and antimetastatic effects compared to the previously described mixture (PVM-I).

[0034] According to a preliminary study using Freund adjuvants, the PVM-I formulation is an antigenic variant with higher immunogenicity and stability (Example 1). This study was extended to adjuvants and formulations related to treatment scenarios in non-human mammals (Examples 2-5, 9-13) and humans (Examples 14, 15). The use of antigenic variants with higher stability is relevant to productive and commercial situations, while increased immunogenicity provides an excellent therapeutic solution (Examples 2-5, 9-15).

[0035] In a preferred embodiment, the present invention also describes a pharmaceutical composition comprising a functional mutant of human VEGF-A that folds in a non-natural rearrangement of disulfide crosslinks, wherein the second and fourth cystes of the polypeptide sequence form only intramolecular bonds, while the seventh and eighth intermolecular cystes form only intermolecular crosslinks.

[0036] The compositions related to the present invention contain at least pharmaceutically acceptable adjuvants. To increase the immune response, the structural isomers described in the present invention may be combined with previously described adjuvants. These include inorganic salts, immunostimulants such as cytokines, molecular adjuvants (CD40, CD154, constant chain of MHC type I, LFA3), saponins, muramyl dipeptide derivatives, oligonucleotide CpG, lipopolysaccharides, monophosphoryl lipid A and polyphosphases, lipid particles (i.e., Freund adjuvants, MF59, and Montanide), liposomes, nanoparticles, virososomes, ISCOMS, cochelates; microparticle adjuvants, poloxamers, viral and bacterial antigens. Additionally, mucosal adjuvants are included. In certain embodiments, the adjuvant is selected from the group consisting of oil-based adjuvant, aluminum salt, proteoliposome, and proteoliposomes conjugated to gangliosides.

[0037] When an antigen is administered with a pharmaceutically acceptable adjuvant, as a first or second-line treatment with or without other antitumor agents, it can contribute to the reduction and even elimination of the primary tumor and prevent the appearance of new metastases (Examples 2-5, 9-12, 14). In addition, this type of immunotherapy can be used to treat acute and chronic inflammatory processes (asthma, dyspnea, endometriosis, atherosclerosis, tissue edema), infectious diseases (hepatitis, Kaposi's sarcoma), autoimmune diseases (diabetic psoriasis, rheumatoid arthritis), diabetic retinopathy, macular degeneration, neovascular glaucoma, hemangiomas, and angiofibromas (Examples 12, 13, and 15).

[0038] In particular, the present invention describes the administration of vaccine antigens on a weekly schedule when formulated into a group of adjuvants designated as NAcGM3-VSSP. These adjuvants can be obtained by conjugating natural or synthetic forms of ganglioside N-acetyl GM3 into the outer membrane vesicles of *N. meningitidis* (U.S. Patent US6149921; International Application WO201986056A1; Regalado, et al., Organic Process Research & Development 2013: 17: 53-60). Here, these adjuvant variants, also known as VSSP (Very Small Size Proteoliposomes), are referred to as NAcGM3-VSSP. Those containing synthetic N-acetyl GM3 differ among them in the length of the fatty acid added to the ganglioside. In general, using variants containing stearic acid (sNAcGM3-VSSP) or oleic acid (oNAcGM3-VSSP) reproduces the results obtained using natural variants of gangliosides in terms of immunogenicity, antitumor, and antimetastatic effects. Under all these conditions of the formulation, when PVM-1 was used as an antigen compared to PVM (Examples 4-12), superior antitumor, antimetastatic, anti-inflammatory, and immune-restoring effects were obtained.

[0039] Histopathological studies revealed a significant trend in all cases toward a decrease in the number of functional blood vessels, tumor cell density, and the mitotic-to-apoptotic ratio in primary tumors, which was found to be inversely associated with tumor growth. Given that cancer-related mortality is primarily linked to metastasis, the antigen agents were further evaluated in aggressive models of spontaneous and experimental lung metastasis. In all cases, PVM-I-based agents demonstrated a significantly superior anti-metastatic profile compared to that obtained with the vaccine antigen PVM.

[0040] Analysis of metastatic foci in treated animals exhibited characteristics similar to those described for treated primary tumors, with a decrease in vascular density and mitotic / apoptotic balance in parallel with an increase in the number of necrotic foci correlated with the number of metastases found. Interestingly, vaccine-based treatment was able to reduce not only the number of metastases but also their size and proliferative capacity, indicating the transplantation and growth of foci, which is a dual effect of therapeutic intervention on the metastatic process (Example 9).

[0041] It is noteworthy that the antigen preparation of the present invention is administered every two weeks when formulated with aluminum phosphate. When this adjuvant is used, superior antitumor, antimetastatic, anti-inflammatory, and immune recovery effects were obtained when PVM-I was used as the antigen compared to PVM.

[0042] At similar concentrations and across all tested adjuvants, the PVM-I formulation is significantly superior to PVM in terms of the specific antibody response achieved and serum neutralization ability. These effects were observed in the two mouse strains used, demonstrating a wide range of responses achievable with the therapy across various haplotypes. Similarly, for NAcGM3-VSSP-based formulations, PVM-1 is an excellent antigen choice for inducing cellular responses that directly eliminate homologous tumor cells. The novel antigen formulation also demonstrated higher immunogenicity when analyzed in species sharing higher homology to natural VEGF-A, such as human primates.

[0043] For therapeutic application, the vaccine of the present invention is administered to mammals, preferably humans, in pharmaceutically acceptable doses via routes known to those skilled in the art. The described antigen preparation may be administered concurrently with or sequentially with other treatments.

[0044] The administration of the novel antigen agent PVM-I significantly reduces tumor growth of various origins, including melanoma, lung, breast, and colon cancers. These results were obtained from tumor models involved in the transition of other anti-tumor therapies into clinical practice, demonstrating the applicability of this vaccine strategy to the clinical setting of cancer treatment. Since an increase in antigen dosage is associated with an increase in biological effects related to the immune response, the amount of antigen used for each application varies depending on the desired effect.

[0045] The present invention also revealed for the first time the use of a polypeptide comprising a functional mutant of a human VEGF-A isoform folded by the non-natural rearrangement of disulfide crosslinks in the manufacture of drugs for treating diseases associated with increased angiogenesis, inflammation, and immunosuppression. In certain embodiments, the disease to be treated is selected from the group comprising cancer, macular degeneration, diabetes, rheumatoid arthritis, and edema.

[0046] The novel antigen agent PVM-I is useful for active immunotherapy in human cancers. Its use in the treatment of neoplastic diseases replicates the immunological effects described in the preclinical stage. In immunized patients, VEGF-A specific antibodies were detected as well as clones of T lymphocytes that secrete IFN-gamma in response to VEGF-A stimulation. The presence of this immune response was associated with a significant increase in survival. A long-term immune response was observed in the group of patients who achieved a complete response. This is a unique finding for cancer vaccines. Furthermore, it is interesting that therapeutic success is not associated with complete plasmatic VEGF-A ablation. This factor may be related to the absence of side effects similar to those described for other therapeutic alternatives targeting the VEGF / VEGFR2 system in both preclinical and clinical studies. Similarly, by achieving a reduction in circulating VEGF-A and systemic neutralization of VEGF-A's ability to bind to VEGFR2, a new antigen agent administered as a pharmaceutically acceptable adjuvant can be used to treat another group of pathologies associated with excessive angiogenesis.

[0047] The present invention does not limit the use of the antigenic agents disclosed herein to specific diseases and exemplifies how said therapy is effective in such situations. Thus, administering the antigenic agents of the present invention in the presence of aluminum phosphate or sNAcGM3-VSSP rescues the immune system of tumor-bearing animals (Example 11). Antigenic agent PVM-I was also shown to have an increased anti-inflammatory effect compared to the antigenic agent referred to herein as PVM in relation to a collagen-induced arthritis model in DBA / I mice (Example 12). Similarly, antigenic agent PVM-1 had a more significant anti-angiogenic effect in an animal model of corneal injury and in human age-related macular degeneration (Examples 13 and 15). This exemplifies the potential utility of this strategy in the treatment of non-neoplastic diseases. The reduction in corneal vascularization resulting from the administration of VEGF-A to this compartment represents the potential of a strategy for treating age-related macular degeneration and diabetic retinopathy, diseases characterized by increased local levels of VEGF-A, which can be controlled from an immune response that induces active immunization with a preparation containing the polypeptide of the present invention.

[0048] In another aspect, the present invention relates to the use of a pharmaceutical composition comprising an antigenic agent with at least one polypeptide comprising a functional mutant of a human VEGF-A isoform that folds into a non-natural rearrangement of disulfide crosslinks in restoring the immune system. The novel antigenic agent brings about superior recovery of the immune system in addition to exhibiting better immunogenicity. Analysis of suppressor myeloid and regulatory T cells was performed on primary and metastatic lesions in animals treated with the antigenic agent of the present invention. This analysis indicated that the number of these suppressor cells is reduced when the vaccine agent is administered in a schedule with different adjuvants. The function of the suppressor cells and the number of cells at the systemic level are also affected (Example 10).

[0049] The adjuvant effect of the antigen preparation disclosed in the present invention was demonstrated in an experimental model expressing ovalbumin antigen (OVA). When the PVM-1 vaccine antigen was administered sequentially or in combination with OVA in the adjuvant, OVA-specific cellular and humoral immune responses were significantly increased. Furthermore, in relation to class I presenting molecules, the effect of vaccination with PVM-I on the cross-presentation of VEGF-A and OVA was observed (Example 11). Brief explanation of the drawing

[0050] Figure 1a shows the sequence alignment of the amino acid sequences from natural VEGF-A and the amino acid sequences present in the antigen preparation PVM in isoforms 121, 145, 165, 206, and 189. Cysteine, indicated by black C(C), is a cysteine ​​involved in intramolecular cross-linking, and the underlined ( C ) forms intermolecular bonds. Fig. 1b is a schematic diagram showing the disulfide bond arrangement in a natural VEGF-A variant. Figure 2 is a schematic diagram of disulfide bonds detected in antigen preparation PVM-I. Figure 3 shows the evaluation of the presence of standard "cystine nodules" of VEGF-A using natural polyacrylamide gel electrophoresis (in the absence of dithiotheritol or beta-mercaptoethanol). Figure 3a shows the analysis of PVM, CHO-VEGF-A, and VM proteins after trypsin digestion. Figure 3b shows the analysis of proteins in two lots of PVM-1 preparations after trypsin digestion: the PVM-1 preparation (lanes 2-5) and the control (lanes 6-7). Molecular weight markers were tested in the lanes labeled PM1 and PM2. Figure 4 shows the evaluation of humoral and cellular responses in advanced cancer patients immunized with PVM-I administered in NAcGM3-VSSP adjuvant (I) or aluminum phosphate (II). Figure 4a shows the VEGF-A specific IgG titers for immunized patients. Figure 4b shows a study on the ability of immunized patient serum to block VEGF-VEGFR2 interactions. Figure 4c shows the evaluation of VEGF-specific cellular responses measured by INF-gamma ELISPOT. In all cases, the values ​​represent the difference found compared to the values ​​at the start of treatment. Figure 5 shows an analysis of the immune response and effects on survival in patients immunized with the antigen preparation PVM-I. Survival time is expressed as a function of the positive immune response detected for the group that received the antigen from NAcGM3-VSSP adjuvant (I) or aluminum phosphate (II). Specific details for implementing the invention

[0051] (Example)

[0052] Examples 1. PVM and PVM - I Purification and Characterization of Antigen Preparations

[0053] DNA encoding human VEGF-A isoform 121, in which amino acids 82, 84, and 86 are glutamic acid-mutated, was cloned into vector PM238 (Morera, et al., Angiogenesis, 2008: 11: 381-93). The 100% DNA sequence was verified and designated as SEQ ID NO:1. In this genetic construct, the ampicillin resistance gene was interrupted by the kanamycin resistance gene without a change in expression level. The plasmid was transformed into E. coli strain BL21, and the transformant with the highest expression level in a chemically defined medium was selected. This medium was designed to maximize the expression of the recombinant protein in the absence of animal components. The protein was purified according to the method described by Morera et al. (Morera, et al., Angiogenesis, 2008: 11: 381-93). Briefly, the protein was 50 mM NaH2PO4 buffer; Extraction was performed for 16 hours at 300 mM NaCl; 6 M urea; pH 7.8; 4 °C, and purified by nickel affinity chromatography according to the manufacturer's instructions (QIAGEN). The buffer was changed to 10 mM Tris at pH 7.4 in size exclusion chromatography of Matrix G25 (GE Healthcare).

[0054] The generated protein preparations were evaluated by mass spectrometry to confirm their amino acid sequences. ESI-MS and ESI-MS / MS (electrospray ionization mass spectrometry and electrospray ionization serial mass spectrometry, respectively) were obtained using a QTOF-2 orthogonal hybrid configuration spectrometer (Micromass, UK) with a Z-spray electrospray ionization source (NanoESI). The molecular weight of the reduced protein preparation (21,569.13 Da) was separated from it by 13 amino acids that act as a bridge between these polypeptides, which are fused to a fragment of bacterial protein P64K at the amino terminus and contain a histidine tail at the carboxy terminus. Mutations introduced into the human VEGF-A sequence were identified, and glutamic acid was detected instead of the amino acids corresponding to the natural VEGF-A sequence at positions 141, 143, and 145 (arginine, lysine, and histidine, respectively).

[0055] The integrity of the amino terminuses in the samples was confirmed from the peptide mixture obtained from digestion by Glu-C endoproteinase. In the ESI-MS spectrum, the peptide 1 VDKRMALVE 9 A signal corresponding to (double charge, theoretical m / z 530.78) was observed. This peptide was sequenced by ESI-MS / MS, and its sequence was identical to that expected at the amino terminus of the protein. In the ESI-MS spectrum of the reduced intact protein, a signal corresponding to the C-terminal end was detected by increasing the voltage at the mass spectrometer's input cone to induce fragmentation at the source. The analysis results showed that the peptide after sequencing by ESI-MS / MS 177 KPRRGSRAHHHHH 190The carboxyl terminus sequence was confirmed in accordance with (double charge, m / z 875.46). Overall, the primary structure of the polypeptide present in the antigen preparation (SEQ ID NO: 2) could be confirmed through the sequencing results. Hereinafter, the protein preparation defined as SEQ ID NO: 2 is referred to as PVM.

[0056] In the study of fermentation and purification conditions, superior recovery rates were achieved compared to the previously described process (Morera, et al., Angiogenesis, 2008: 11: 381-93) by modifying growth conditions during fermentation, shortening extraction time, adjusting specific protein loading in nickel affinity chromatography, and incorporating a detergent into the washing step. Briefly, the fermentation temperature during the growth phase was changed to 28 °C, and expression was induced by raising the temperature only to 37 °C without chemical inducers. The protein extraction time from 6M urea biomass was also shortened from 16 hours to 2 hours, and a washing step was introduced into nickel affinity chromatography using 0.1% Triton X114. Additionally, the formulations produced from molecular exclusion chromatography were formulated with mannitol (40 mg / mL), sucrose (10 mg / mL), and 10 mM Tris-HCl at pH 7.4. This final protein preparation is referred to as PVM-I, and below it is compared with the PVM preparation obtained in the initial process.

[0057] For both formulations, the total protein concentration was evaluated using the percentage of purity for micro-coomassie and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at dual wavelengths (620 and 450 nm). Immuno-identification was performed by immunoblotting using monoclonal antibodies that recognize different segments present on the antigen. In these studies, the PVM and PVM-I formulations were identical.

[0058] The conformation and stability of these lyophilized protein preparations were analyzed using analytical-scale molecular exclusion chromatography on a Superdex 200 XK 10 / 300 column (GE-Healthcare). A buffer solution of Tris-HCl 10 mM, NaCl 150 mM, pH 7.4 was used as the mobile phase at a flow rate of 0.5 mL / min. The lyophilized protein preparations were suspended in 1 mL of water, and comparative data confirmed that PVM and PVM-I exhibited identical retention profiles in this type of chromatography at the time of reconstitution. In both cases, soluble aggregates with a molecular weight greater than 670 kDa were formed according to the molecular weight standards used. However, the reconstituted solution of PVM-I exhibited distinct characteristics in terms of the stability over time of the added components, indicating changes in structural composition. The reconstituted PVM-I formulation maintains a retention time profile for 30 days at 4°C in molecular exclusion chromatography, whereas the PVM formulation begins to lose stability significantly at 72 hours (Table 1). According to the analysis of the area under the curve on the chromatography chart, the stereochemical change significantly increases from 30 days to 14% of the PVM protein mass at 72 hours when more than 45% of the original stereochemical shape is lost.

[0059] Kinetic evaluation of stereochemical stability of formulations PVM and PVM-I. Retention time (minutes) 15.8 23.1 26.9 Days after formulation / reconstitution The total area under the curve % PVM / 0 days 95.9±1.2 2.3±0.9 1.8±0.9 PVM-I / 0 days 97.3±2.8 2.1±0.9 1.2±0.23 PVM / 3 days 85.2±1.2 0.8±0.9 4.0±0.9 PVM-I / 3 days 96.5±3.4 2.6±0.65 1.7±0.43 PVM / 30 days 52.3±1.2 27.4±0.9 20.3±0.9 PVM-I / 30 days 94.9±3.1 3.1±0.54 2.0±0.18

[0060] main: The average value Expressed as ± standard deviation for 5 experimental replications.

[0061] To evaluate the immunogenicity of PVM and PVM-1 protein preparations, humoral immune responses generated in mice were assessed. Ten animals per group from two mouse strains (C57Bl / 6 and BALB / c) were used, and 100 μg of the protein preparation was administered at weekly intervals at a total volume of 250 μL (protein / adjuvant ratio 1:1 v / v) to the complete Freund adjuvant (SIGMA) as the first single dose and to the incomplete Freund adjuvant (SIGMA) as the second single dose. Serum from the animals was collected one week after the second immunization. The titers of specific antibodies against human VEGF-A present in serum and the ability to neutralize the interaction between VEGF-A and its type 2 receptor were evaluated by ELISA as previously described (Boequet-Romero, et al., Vaccine, 2012: 30: 1790-9). Titer results are expressed as the highest dilution rate at which the presence of antibodies specific to VEGF-A is detected. Neutralization ability is expressed as the percentage of the maximum VEGF-A / VEGFR2 binding that the antibodies present in serum can clear.

[0062] When comparing immunogenicity, the seroconversion rate when immunized with the new antigen preparation (PVM-I) showed a significant increase compared to the previous PVM (PVM), as shown in Table 2, even though the same amount was administered.

[0063] Immunogenicity of formulations PVM and PVM-I VEGF -A specific IgG potency (1 / Dilution) PVM PVM -I t- Student C57Bl / 6 10655 ± 776 18655 ± 334 < 0,0001 BALB / c 21399 ± 2343 26648 ± 2749 0,0002 In VEGFR2 Korea VEGF Inhibition rate of -A binding ( % ) PVM PVM -I t- Student C57Bl / 6 36,7 ± 3,5 51,6 ± 2,3 < 0,0001 BALB / c 55,9 ± 7,7 67,8 ± 8,4 0,004

[0064] main: IgG The station 1: Indicated by dilution. The average value It is expressed as the mean ± standard deviation, and the p-value is for each mouse strain Student It corresponds to the t-test (Student t-test).

[0065] The formation of disulfide crosslinks was studied in both preparations, taking into account that (a) the monomers of these protein preparations have the same primary sequence (SEQ ID NO:2), (b) nine cysteine ​​residues are present, and (c) the immunogenicity, thermal and stereochemical stability of human VEGF-A (constituting 63% of the PVM and PVM-I polypeptide sequences) is associated with the formation of a standard cysteine ​​structure that is widely characterized for wild-type proteins and their family.

[0066] The arrangement of disulfide crosslinks in protein preparations was evaluated in advance through trypsin-based proteolytic studies and analyzed by electrophoresis and molecular exclusion chromatography under non-denaturing conditions on a Superose 12 XK 10 / 300 column using 200 mM Tris-HCl, pH 8.0 as the mobile phase at 0.5 mL / min.

[0067] Since the characteristic configuration of cysteine ​​in natural VEGF-A is known to confer resistance to trypsin digestion (Keck, et al., Arch Biochem Biophys, 1997: 344: 103-13), the present invention compares the digestion of PVM and PVM-I with this enzyme. To this end, the final purification buffer was replaced with 200 mM Tris-HCl pH 8.0, and the proteins were incubated at 37°C for 16 hours in the presence or absence of trypsin at a ratio of 50:1 (protein:trypsin). Subsequently, samples were collected and the efficiency of trypsin digestion was evaluated by non-denaturing natural electrophoresis (Fig. 3). The following samples were used as controls for the correct stereotype of crosslinking in VEGF-A molecules: 1) VM protein: human VEGF-A isoform 121 with R80, K82, and H84 mutations of E produced from the periplasm of Escherichia coli (Gavilondo, et al., Vaccine, 2014: 32: 2241-50), and 2) CHO-VEGF protein: human VEGF-A isoform 121 obtained from transfection of the eukaryotic cell line CHO (Chinese Hamster Ovary) (Sanchez Ramirez, et al., J Immunoassay Immunochem, 2016: 37: 636-58). These VEGF-A variants were purified by a non-denaturing process in the absence of a reducing agent. Figure 3 illustrates how resistance to digestion using trypsin is achieved in preparations from bacterial pericytoplasm, as 90% is recovered from the fraction moving to a lower molecular weight than the undigested protein, which is consistent with the estimated molecular weight and the presence of a standard cysteine ​​structure, also known as a "cysteine ​​nodule," in the dimeric form of mutants with or without the P64K segment (19.902 kDa) (Figure 3a).

[0068] Analysis of the PVM formulation showed a similar phenomenon not observed in the PVM-I formulation, in which peptides corresponding to cysteine ​​nodes did not appear (Fig. 3b). Considering this, peptides generated from the digestion of the PVM-I formulation using trypsin or GluC were analyzed by mass spectrometry as described for PVM, but the samples were not subjected to a reduction process. Table 3 shows the detected disulfide bonds.

[0069] Summary of monoisotopic molecular masses of peptides generated during PVM-I digestion m / z Theoretical value z m / z experimental values detail 1272.93 3 1272.92 There is an intramolecular SS between cysteine ​​85 and 110. 83 SM 114 peptide 993.98 4 993.96 There is an intramolecular SS between cysteine ​​85 and 110. 83 SM 115 peptide 1324.97 3 1324.95 1251.78 4 1251.74 Mutant R 141 Containing 116 CK 160 → E, K 143 → E and H 145 → E, and the two intramolecular SS between cysteine ​​116, 119, 120, and 127 927.44 1236 43 927.441236 from trypsin digestion containing intramolecular disulfide bonds between cysteine ​​85 and 175 ( 83 SK 107 )-SS-( 175 CR 179 ) 733.31 2 733.35 From trypsin digestion containing two intermolecular disulfide bonds between cysteine ​​161, 163, 161, and 163 ( 161 CK 166 )-SS-( 161 CK 166 ) peptide 617.30 2 617.31 from trypsin digestion containing intermolecular disulfide bonds between cysteine ​​175 and 175 of another molecule ( 175 CR 179 )-SS-( 175 CR 179 ) peptide 1036.70 864.1 56 1036.74 864.12 From Glu-C-based digestion containing intramolecular disulfide bonds between cysteine ​​85 and 175 ( 79 VE 103 )-SS-( 174 KH 190 ) peptide 744.32 3 744.31 From Glu-C system digestion containing intramolecular disulfide bonds between cysteine ​​110, 116, and 120 of another molecule 104 YE 123 peptide 824.03 3 824.03 From Glu-C system digestion containing intramolecular disulfide bonds between cysteine ​​161 and 161 104 YE 123 peptide ( 153 ME 162 )-SS-( 153 ME 162 )

[0070] The consistency of the presence of the aforementioned disulfide bonds was analyzed and verified in a total of 8 lots of final products. The identified structures appear in both the active medicinal ingredient and the final formulation. The specific arrangement of disulfide crosslinks found in PVM-1 is significantly different from that described for the wild-type molecule of VEGF-A 121 and other isoforms of this protein. They also differ from the arrangements described for the protein when obtained by recombinant technology in various prokaryotic and eukaryotic expression systems (Keck, et al., Arch Biochem Biophys, 1997: 344: 103-13). A thorough search was performed for intermolecular crosslinks between cysteine ​​110 and 119, corresponding to the second and fourth cystes of natural VEGF-A. Even when these cysteine ​​residues form intramolecular bonds in the wild-type molecule, the corresponding peptide was not detected in trypsin or Glu-C digested PVM-I formulations under any experimental conditions.

[0071] According to the prior art, the most stable structure for VEGF-A corresponds to a structure that preserves the so-called "cysteine ​​nodules." However, studies performed with PVM and PVM-I protein preparations suggest that higher stability under purified conditions is associated with variants lacking the natural structure. Similarly, despite the data from Timmerman et al. (US Patent Application US2012 / 0231000), a comparative analysis of PVM-1 immunogenicity in Freund adjuvants compared to PVM indicates that the former, containing the isomer mixture of Figure 2, exhibits greater immunogenicity.

[0072] This study indicates that changes in the disulfide binding arrangement occur in the PVM-I formulation compared to PVM, resulting in higher stability and reproducibility according to a new protocol for antigen generation. Figure 2 shows 12 variants of crosslinks established in a stable form among the 9 cystes detected in the polypeptide sequence of PVM-I.

[0073] Examples 2. NAcGM3 - In VSSP Administered antigen preparation PVM and PVM Comparison of immunogenicity of -I

[0074] Mouse strains C56BL / 6 and BALB / c, which differed in their ability to generate humoral or cellular immune responses to the antigen test challenge, were used. In both cases, 100 µg of the antigen preparation was administered per single dose to a) 100 µg of NAcGM3-VSSP containing natural gangliosides, b) 100 µg of sNAcGM3-VSSP incorporating stearic acid and gangliosides, or c) 100 µg of oNAcGM3-VSSP incorporating oleic acid and gangliosides. Ten animals per group were immunized for eight weeks according to a weekly schedule. Serum from the animals was collected one week, and up to the third and eighth weeks after each immunization. The antibody titer specific to human VEGF-A and the ability to neutralize the interaction between VEGF-A and its type 2 receptor were evaluated by ELISA as described (Bequet-Romero, et al., Vaccine, 2012: 30: 1790-9).

[0075] Specific IgG titers increased with increasing immunization cycles and reached a maximum one week after the eighth immunization. The results corresponding to this experimental point are shown in Table 4. As can be seen, for both strains, significantly superior results were obtained compared to the PVM-1 antigen preparation in terms of specific titers and inhibition of VEGF-A binding to the receptor after the eighth immunization. A comparative analysis of the three variants of the adjuvant NAcGM3-VSSP indicates that the vaccine preparations are equally immunogenic.

[0076] Immunogenicity evaluation results for PVM and PVM-I antigen preparations VEGF -A specific IgG potency ( 1: Dilution ) NAc-GM3 VSSP PVM PVM -I t- Student C57Bl / 6 6500 ± 221 8592 ± 631 < 0.0001 BALB / c 29785 ± 3662 38870 ± 5574 < 0.0001 sNAc-GM3 VSSP C57Bl / 6 5900 ± 634 7664 ± 547 < 0.0001 BALB / c 25879 ± 2669 35990 ± 2899 < 0.0001 oNAc-GM3 VSSP C57Bl / 6 6205 ± 554 8120 ± 421 < 0.0001 BALB / c 30879 ± 4100 37555 ± 3552 < 0.0001 In VEGFR2 Korea VEGF Inhibition rate of -A binding ( % ) NAc-GM3 VSSP PVM PVM-I t-Student C57Bl / 6 25.8 ± 4.5 39.9 ± 2.2 < 0.0001 BALB / c 62.2 ± 5.9 79.4 ± 3.1 < 0.0001 sNAc-GM3 VSSP C57Bl / 6 21.4 ± 2.3 37.3 ± 3.5 < 0.0001 BALB / c 60.2 ± 1.9 77.3 ± 3.6 < 0.0001 oNAc-GM3 VSSP C57Bl / 6 27.5 ± 1.9 38.5 ± 3.3 < 0.0001 BALB / c 60.2 ± 1.9 77.3 ± 3.5 < 0.0001

[0077] main: Student The p-value corresponding to the t-test is displayed.

[0078] Examples 3. Antigen preparations administered to aluminum phosphate PVM and PVM Comparison of immunogenicity of -I

[0079] Mouse strains C56BL / 6 and BALB / c were used. 100 micrograms of the antigenic agent were administered to 0.7 mg equivalents of Al3+ (aluminum phosphate) per single dose. Ten animals per group were immunized a total of four times at a biweekly frequency. Serum from the animals was collected one week after the second and fourth immunizations. Human VEGF-A specific antibody titers and their ability to neutralize the interaction between VEGF-A and VEGFR2 were evaluated by ELISA as described (Bequet-Romero, et al., Vaccine, 2012: 30: 1790-9).

[0080] An increase in titer was observed as the number of single doses administered increased for the two antigen types and two mouse strains. The results for serum samples obtained after the fourth immunization are shown in Table 5. For both strains, significantly superior results were obtained for the PVM-1 antigen preparation after the fourth immunization in terms of specific antibody titer and inhibition of VEGF-A binding to the receptor.

[0081] Analysis of immunogenicity results for PVM and PVM-I VEGF -A specific IgG potency ( 1: Dilution ) PVM PVM -I Student -t C57Bl / 6 15560 ± 781 29576 ± 891 0.001 BALB / c 53000 ± 1799 70660 ± 3469 < 0.0001 In VEGFR2 Korea VEGF Inhibition rate of -A binding ( % ) PVM PVM -I Student -t C57Bl / 6 39.7 ± 3.8 55.3 ± 5.6 0.001 BALB / c 65.3 ± 6.5 76.5 ± 4.9 0.006

[0082] main: Student The p-value corresponding to the t-test is displayed.

[0083] Examples 4 . NAcGM3 - In VSSP Administered PVM or PVM -I antigen By immunizing with a preparation Evaluation of induced cellular response.

[0084] Cellular responses were analyzed as described by Bequet-Romero et al. (Bequet-Romero, et al., Angiogenesis, 2007: 10: 23-34; Morera, et al., Vaccine, 2012: 30: 368-77). In this case, responses were evaluated only in relation to the adjuvant NAcGM3-VSSP. Animals (n = 10 per group) were immunized in the manner described for this adjuvant in Example 2, sacrificed one week after the last immunization, and cells isolated from mouse spleens were co-incubated with synonymic tumor cells labeled with the CFSE (carboxyfluorescein succinimidyl ester) fluorophore at a ratio of 100:1 (effector cells: labeled tumor cells). Cells isolated from mouse spleen were co-incubated with CFSE (carboxyfluorescein succinimidyl ester) fluorescence at a ratio of 100:1 (effecter cells: labeled tumor cells).

[0085] For this study, melanoma cell line B16F10, EL4 lymphoma and lung carcinoma 3LL-D122, which are cognates of the C56BL / 6 strain, and colon carcinoma line CT26, breast F3II, and kidney RENCA, which are cognates of the BALB / c strain, were selected. Analysis of surviving cells by flow cytometry (Space ML-PARTEC) after co-incubation revealed that in both mouse strains, administration of the PVM-1 agent resulted in a direct cellular response of greater intensity, even when similar single doses of the two antigen agents were administered. Table 6 shows the results corresponding to the percentage of tumor cells that died when replaced by lymphocytes of immunized animals, and represents the average value of cells corresponding to treatment with the adjuvants as a 100% survival rate. In two mouse strains (C57Bl / 6 and BALB / c), administration of the antigenic agent PVM-I (corresponding to group IV in all cases) was superior to the use of PVM agents in terms of inducing an effective cellular response.

[0086] Evaluation of cell-mediated cytotoxicity of PVM and PVM-I administration in NAcGM3-VSSP against mouse strains BALB / c and C57Bl / 6 Treatment group (I) No treatment (II) NAcGM3-VSSP PVM (III) NAcGM3-VSSP PVM -I (IV) NAcGM3-VSSP Brothers Strain C57Bl / 6 B16F10 1.498 ± 6.080 a 27.14 ± 3.034 b 45.52 ± 11.92 c 0.8686 ± 6.24 a EL-4 8.440 ± 2.532 a 37.38 ± 1.909 b 49.65 ± 5.658 c 10.05 ± 6.560 a 3LL -D122 5.978 ± 7.115 a 23.80 ± 7.287 b 41.34 ± 2.160 c 0.1457 ± 6.552 a strain BALB / c CT26 4.891 ± 6.341 a 24.03 ±7.184 b 47.33 ± 6.384 c 8.482± 4.804 a F3II 9.588 ± 6.578 a 40.97 ± 9.524 b 69.65 ± 3.678 c 11.97 ± 5.824 a RENCA 1.051 ± 2.996 a 34.73 ± 10.52 b 55.96 ± 7.523 c 0.8838 ±3.230 a

[0087] main: Dunnet Statistical comparison using a post-test: Different characters indicate a significant difference (p<0.05), and are displayed in ascending order according to the mean value.

[0088] Examples 5 For the treatment of solid tumors in mice PVM and PVM - Efficacy of I-based immunization

[0089] In all cases, groups of 12 mice were immunized. The results of immunization using sNAcGM3-VSSP or aluminum phosphate are described for two tumor models. When the adjuvant sNAcGM3-VSSP was used, animals were immunized by subcutaneous inoculation for 8 weeks with a total weekly dose of 200 μL containing 100 μg of the corresponding antigen and 100 μg of the adjuvant. For aluminum phosphate, animals were immunized 4 times on a bi-weekly schedule. Each time, 100 μg of the antigen was administered in the form of aluminum phosphate in a total volume of 200 μL containing 0.7 mg equivalent of Al3+.

[0090] The melanoma model B16F10 was evaluated in mouse strain C57BL / 6. Three days after 4th immunization (in the presence of sNAcGM3-VSSP) or 2nd immunization (in the presence of aluminum phosphate), a total of 20,000 cells were subcutaneously inoculated into mice in 100 μL of DMEM culture medium. Table 7 shows the weight of primary tumors surgically removed from animals euthanized 25 days after tumor test infection.

[0091] Comparative evaluation of antigen preparations PVM and PVM-1 in sNAcGM3-VSSP and aluminum phosphate administration in subcutaneous melanoma model B16F10 therapy Tumor weight (g) VEGF -A / VEGFR2 Binding inhibition Cellular response ( % dissolution) No treatment 2.523 ± 0.3705 a 2.4 ± 2.1 c 5 ± 2.3 c sNAcGM3-VSSP PVM 1.748 ± 0.6057 b 47.3 ± 3.5 b 40 ± 7.1 b sNAcGM3-VSSP PVM-1 1.199 ± 0.3434 c 75.8 ± 4.5 a 62 ± 8.4 a sNAcGM3-VSSP excipient 2.933 ± 0.2133 a 4.1 ± 2.1 c 12 ± 3.8 c Aluminum Phosphate PVM 1.822 ± 0.5987 b 59.2 ± 1.9 b 35 ± 6.5 b Aluminum Phosphate PVM-1 1.266 ± 0.6532 c 80.2 ± 1.9 a 55 ± 5.4 a Aluminum phosphate excipient 2.941 ± 0.5778 a 2.2 ± 1.3 c 8 ± 3.8 c

[0092] main: Dunnet Statistical comparison using post-hoc testing: Different characters indicate a significant difference (p<0.05) and On the average value Displayed in ascending order accordingly

[0093] As observed in Table 7, treatment of animals with the antigenic agents in both adjuvants resulted in a significant reduction in tumor growth up to 25 days after the tumor test infection, which was attributed to the use of the new antigenic agent (PVM-I), exhibiting more significant inhibition. This antitumor response correlated with the presence of an immune response specific to VEGF-A in both humoral and cellular forms (p<0.05; Pearson test).

[0094] Similarly, the antitumor effect was evaluated in mouse strain BALB / c using the same immune system. Test tumor infection was also performed 3 days after 4th immunization (in the presence of NAcGM3-VSSP) or 2nd immunization (in the presence of aluminum phosphate) with 25,000 cells of CT26 synonyms for colorectal carcinoma using this strain. A comparative analysis of tumor growth 30 days after test infection showed a superior antitumor effect due to immunization with antigen preparation PVM-1 compared to the use of PVM preparations (Table 8).

[0095] Comparative evaluation of antigen preparations PVM and PVM-1 in NAcGM3-VSSP and aluminum phosphate administration in colon cancer model CT26 therapy volume Weight (g) VEGF -A / VEGFR2 Binding inhibition Cellular response ( % dissolution) No treatment 2.523 ± 0.3705 a 2.4 ± 2.1 c 5 ± 2.3 c sNAcGM3-VSSP PVM 1.748 ± 0.6057 b 47.3 ± 3.5 b 40 ± 7.1 b sNAcGM3-VSSP PVM-1 1.199 ± 0.3434 c 75.8 ± 4.5 a 62 ± 8.4 a sNAcGM3-VSSP excipient 2.933 ± 0.2133 a 4.1 ± 2.1 c 12 ± 3.8 c Aluminum Phosphate PVM 1.822 ± 0.5987 b 59.2 ± 1.9 b 35 ± 6.5 b Aluminum Phosphate PVM-1 1.266 ± 0.6532 c 80.2 ± 1.9 a 55 ± 5.4 a Aluminum phosphate excipient 2.941 ± 0.5778 a 2.2 ± 1.3 c 8 ± 3.8 c

[0096] main: Dunnet Statistical comparison using post-hoc testing: Different characters indicate a significant difference (p<0.05) and On the average value Shown in ascending order accordingly.

[0097] Similar to previous studies, these results correlated with the presence of humoral and cellular immune responses specific to VEGF-A, which were superior when using antigen agent PVM-I (p<0.05; Pearson test). In both studies, histological analysis of serial sections of extracted tumors demonstrated the anti-angiogenic, pro-apoptotic, and anti-proliferative effects of vaccination, which appeared significantly more favorable with the use of antigen agent PVM-I (p<0.05, Dunnet test in all analyses).

[0098] Examples 6. VEGF Based on isoforms 145, 165, 189, and 206 from -A polypeptide variant's Manufacturing and characterization

[0099] As shown by the alignment in Figure 1, since all VEGF-A isoforms share the cysteine ​​residue present in isoform 121, strategies used to increase immunogenicity may be valid for the entire protein family. To verify this hypothesis, isoforms 145, 165, 189, and 206 were cloned from the mRNA (messenger ribonucleic acid) of HeLa tumor cells. Automated sequencing was performed using the CCDS database updated in June 2018 (see 12 / 23 / 2019) https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi release 22 The sequence was confirmed based on the content reported in ).

[0100] Simply put, the strategy described in Example 1 was used because all isoforms of VEGF-A can be obtained in a single amplification reaction and can be perfectly separated due to their different sizes. In all cases, the mutations described in Example 1 were introduced to exclude the induction of VEGFR2 activation from the administration of the antigen preparation. The amino acid sequences corresponding to the proteins that produced the protein variants PVM145, PVM165, PVM189, and PVM206 were named SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0101] As described, after cloning the protein variant in vector pM238, the protein was obtained according to the following two processes: a) the process described by Morera et al. (Morera, et al., Angiogenesis, 2008: 11: 381-93) and b) the process described in Example 1. The resistance to trypsin digestion of the two purified variants was compared for each isoform. Analysis of the digestion products was performed by molecular exclusion chromatography. The results showed that, as described for PVM-I, when variants of the VEGF-A isoform are obtained according to the second purification strategy (PVM145-I, PVM165-I, PVM189-I, PVM206-I), resistance to trypsin digestion is observed at less than 5% of the total protein mass. This resistance was reflected in the presence of a signal corresponding to a dimer of "cysteine ​​nodules," which is obtained only when the disulfide crosslinks are set according to the arrangement existing in nature (signal between 23 and 25 minutes) (Table 9). Therefore, when a protein preparation is obtained according to the process described in Example 1, it is digested by more than 95% when incubated with trypsin, compared to the corresponding product produced by the initial process described by Morera et al., as shown in Table 9. The peptide, which is described in detail in Table 3 (Example 1) and confirms the absence of standard cysteine ​​nodules, was also confirmed by ESI-MS / S for the digestation of antigenic preparations PVM145-I, PVM165-I, PVM189-I, and PVM206-I with trypsin, and was not detected in preparations PVM145, PVM165, PVM189, and PVM206.

[0102] In Freund adjuvants, a comparative analysis of the immunogenicity of these variants was performed as described in Example 1. This study showed superior neutralization of VEGF-A binding to VEGFR2 by serum from animals treated with variants with low trypsin digestion resistance (PVM145-I, PVM165-I, PVM189-I, PVM206-I) compared to variants PVM145, PVM165, PVM189, and PVM206 (Table 9).

[0103] [Table 9] Evaluation of protein recovery rate at 24.8 minutes after trypsin digestion of antigen variants. Analysis of inhibition of VEGF-A binding to VEGFR2 by serum of animals immunized with these variants.

[0104]

[0105] These proteins were used in BALB / c animals test-infected with 10,000 CT26 cells via the subcutaneous route, following an immunization and administration schedule similar to that described in Example 5. The results indicated that 28 days after tumor inoculation, the use of antigen variants in combination with the adjuvant sNAcGM3-VSSP induced superior antitumor effects for variants PVM145-I, PVM165-I, PVM189-I, and PVM206-I compared to formulations PVM145, PVM165, PVM189, and PVM206 (Table 10).

[0106] [Table 10] Comparative analysis of observed antitumor effects using polypeptide variants based on VEGF-A isoforms 145, 165, 189, and 206

[0107]

[0108] Note: Multiple comparison ANOVA Test and Dunnet Results of post-test (different letters indicate a significant difference, p<0.05).

[0109] Regardless of the VEGF-A isoform used, variants that do not exhibit resistance to trypsin digestion were observed to have the highest immunogenicity and antitumor effects.

[0110] Examples 7 Trypsin sensitive variant's using oxidation and selection PVM -I preparation

[0111] Due to the formation of multiple disulfide bonds, multiple conformations coexist in the PVM-1 protein preparation. These can be obtained and separated by a process different from that already described in Example 1, which includes controlled denaturation and denaturation steps in the presence of an oxidizing agent. For the smaller isoform of VEGF-A expressed in E. coli (SEQ ID NO: 2), inclusion bodies were dissolved in 6 M guanidium chloride, and the protein was purified by affinity for nickel in the presence of this denaturing agent according to the manufacturer's recommendations (QIAGEN). The eluted product was then separated by a linear gradient between buffers in reverse-phase chromatography (RP) on a preparative C18 column: (A) trifluoroacetic acid (TFA) in water (0.088% v / v) and (B) 0.084% TFA, 90% acetonitrile in water (v / v). In the fraction from which the protein of interest was eluted, resistance to trypsin digestion was not observed as expected, which is consistent with the denaturation and reduction status of the protein preparation. The protein was then subjected to an oxidative non-denaturation process in Tris-HCl pH 8.6 at 22°C for 20 hours, regardless of the presence of guanidium chloride and β-mercaptoethanol (0.2 mM). After changing the buffer to 10 mM Tris-HCl pH 7.4, the formation of disulfide crosslinks was evaluated by size exclusion chromatography on G25 Sepharose. The formation of "cysteine ​​nodules" was analyzed by digestion with trypsin for 16 hours in 200 mM Tris-HCl buffer, pH 8.0, and a 50:1 ratio (protein:trypsin), as described in Example 1.According to this, re-naturalized proteins in the absence of guanidium chloride showed 90% resistance to digestive degradation (PVM-IA), whereas re-naturalization in the presence of a denaturant resulted in a differential percentage of resistance to digestive degradation, which decreased as the molar concentration of guanidium chloride increased.

[0112] Using 6M guanidium chloride, the largest number of fractions were obtained that were not resistant to trypsin digestion, and all of these were designated as PVM-IO. These fractions were separated by RP-HPLC chromatography on a C18 pre-column. A total of 15 fractions were separated and numbered according to the order of elution in RP chromatography (F1 to F15, Table 11).

[0113] After exchanging the buffer with 10 mM Tris pH 7.4; in a study using BALB / c mice, PVM-IA, PVM-IO formulations, and fractions of the latter were compared to PVM and PVM-I. Protein administration was evaluated using a standard regimen of eight administrations at one-week intervals at a ratio of 200 µg of protein formulation to 100 µg of sNAcGM3-VSSP. Table 11 shows the results of a comparative analysis regarding specific IgG titers against murine VEGF-A, binding inhibition against VEGFR2, induction of cytotoxic cellular responses in both lymph nodes and spleen, and the effects of immunization on subcutaneous CT26 tumor growth.

[0114] [Table 11] Comparative analysis of fractions obtained after oxidative recovery of PVM-I

[0115]

[0116] Note: Multiple comparisons ANOVA Test and Dunnet Post-hoc test results (different letters indicate a significant difference p<0.05).

[0117] Table 11 demonstrates the superior efficacy of the PVM-I formulation compared to the PVM formulation, supporting the results presented in Example 1 regarding the Freund adjuvant. This study explains the antitumor effects directly related to the immune effects previously described and the induction of effective humoral and cellular responses. Analysis of the regenerated protein formulation (PVM-IA) without a reducing agent revealed that its immunogenicity was 7 to 10 times lower and its antitumor effect was lower than that of PVM-I and PVM. However, when the regenerated formulation was used in the presence of 6M guanidium chloride (PVM-IO), results were obtained that were not significantly different from those described for PVM-I. Furthermore, evaluation of the fractions of the PVM-IO formulation indicated that at least 12 of them (F4–F15) had equivalent effects in generating antitumor responses as well as inducing specific humoral and cellular immune responses.

[0118] As described in Example 1, analysis of these fractions by developed trypsin digestion and mass spectrometry accompanied by Edman degradation (Marti, T., Rosselet, SJ, Titani, K. and Walsh, KA (1987) Biochemistry 26, 8099-8109) demonstrated that in fractions F4 to F15, cysteine ​​2 and 4 of the standard structure of cysteine ​​nodes form intramolecular crosslinks, whereas in fractions F1 to F3, intermolecular conjugations of C2-C2, C4-C4, C2-C4, C2-C9, and C4-C9 are detected.

[0119] The sequence of bindings involving cysteine ​​in fractions F4 to F15 below corresponds to the polypeptides identified in the sequences listed as SEQ ID NO: 18 to SEQ ID NO: 23, as described in detail below:

[0120] - F4 corresponds to SEQ ID NO: 18, where the seventh cysteine ​​(position 161) of the two polypeptide chains, the eighth cysteine ​​(position 163) of the two polypeptide chains, and the last cysteine ​​(position 175) of the two polypeptide chains form intermolecular disulfide crosslinks.

[0121] - F5 corresponds to SEQ ID NO: 18, where the seventh cysteine ​​(position 161) of the polypeptide chain, the eighth cysteine ​​(position 163) of the other polypeptide chain, and the last cysteine ​​of the two polypeptide chains form an intermolecular disulfide crosslink.

[0122] - F6 corresponds to SEQ ID NO: 19, where the seventh cysteine ​​(position 161) of the two polypeptide chains, the eighth cysteine ​​(position 163) of the two polypeptide chains, and the last cysteine ​​(position 175) of the two polypeptide chains form intermolecular disulfide crosslinks.

[0123] - F7 corresponds to SEQ ID NO: 19, where the 7th cysteine ​​(position 161) of the polypeptide chain, the 8th cysteine ​​(position 163) of the other polypeptide chain, and the last cysteine ​​of the two polypeptide chains form an intermolecular disulfide crosslink.

[0124] - F8 corresponds to SEQ ID NO: 20, where the seventh cysteine ​​(position 161) of the two polypeptide chains, the eighth cysteine ​​(position 163) of the two polypeptide chains, and the last cysteine ​​(position 175) of the two polypeptide chains form intermolecular disulfide crosslinks.

[0125] - F9 corresponds to SEQ ID NO: 20, where the seventh cysteine ​​(position 161) of the polypeptide chain, the eighth cysteine ​​(position 163) of the other polypeptide chain, and the last cysteine ​​of the two polypeptide chains form an intermolecular disulfide crosslink.

[0126] - F10 corresponds to SEQ ID NO: 21, where the 7th cysteine ​​(position 161) of the two polypeptide chains and the 8th cysteine ​​(position 163) of the two polypeptide chains form an intermolecular disulfide crosslink.

[0127] - F11 corresponds to SEQ ID NO: 21, where the seventh cysteine ​​(position 161) of the polypeptide chain and the eighth cysteine ​​(position 163) of the other polypeptide chain are linked to form an intermolecular disulfide crosslink.

[0128] - F12 corresponds to SEQ ID NO: 22, where the 7th cysteine ​​(position 161) of the two polypeptide chains and the 8th cysteine ​​(position 163) of the two polypeptide chains are connected to form an intermolecular disulfide crosslink.

[0129] - F13 corresponds to SEQ ID NO: 22, where the seventh cysteine ​​(position 161) of one polypeptide chain and the eighth cysteine ​​(position 163) of another polypeptide chain are linked to form an intermolecular disulfide crosslink.

[0130] - F14 corresponds to SEQ ID NO: 23, where the 7th cysteine ​​(position 161) of the two polypeptide chains and the 8th cysteine ​​(position 163) of the two polypeptide chains are connected to form an intermolecular disulfide crosslink.

[0131] - F15 corresponds to SEQ ID NO: 23, where the seventh cysteine ​​(position 161) of one polypeptide chain and the eighth cysteine ​​(position 163) of another polypeptide chain are linked to form an intermolecular disulfide crosslink.

[0132] Examples 8 Analysis of the Relationship between Cysteine ​​2 and 4 in the Generation of Immunogenic Structures

[0133] The results described in Examples 1, 9, and 10 indicate that a common feature of the more immunogenic structure of PVM-1 is the fact that the intramolecular linkage of VEGF-A involves cysteine ​​2 and 4 (Cys 110 and Cys 119 PVM-I) in the primary sequence. Because these residues avoid protein dimer aggregation, they appear to be mutated at a high frequency in studies reviewed to obtain VEGF-A (Jiang, et al., Biochemistry, 2010: 49: 6550-6; Wentink, et al., Proc Natl Acad Sci USA, 2016: 113: 12532-7). In their natural conformations, these are amino acids that form interchain crosslinks to produce VEGF-A dimers. Previous studies by Bequet-Romero et al. and Morera et al. have used primary sequence variants of VEGF-A containing these cystes, but none of them, when observed, show the formation of intrachain disulfide bonds between cysteine ​​2 and 4 in the sequence. The presence of these amino acids in the sequence used to generate the antigen, and the process by which they are obtained such that the mentioned cystes form only intrachain bonds, constitutes a novel approach for obtaining proteins for vaccination purposes. To better characterize the relevance of these residues to the immunogenicity of the antigen preparation, the present invention proceeded to obtain a variant (by site-specific mutagenesis) containing the following change from cysteine ​​to alanine in the primary sequence:

[0134] a) PVM-I Ala110: Cys110 by Ala110

[0135] b) PVM-I Ala119 : Cys119 by Ala119

[0136] c) PVM-I Ala110 ,119 : Cys110 for Ala110 and Cys119 for Ala119.

[0137] To this end, mutations were introduced via nested polymerase chain reaction (PCR) as described, using the oligonucleotide pairs listed in Table 12 (Bequet-Romero, et al., Angiogenesis, 2007: 10: 23-34). Briefly, PCR 1 and 2 were performed, and a plasmid containing the sequence encoding SEQ ID NO: 2 was taken as a template. A mixture for PCR MasterMix (Qiagen) and the oligonucleotides listed in Table 12 were used. The DNA amplified in each reaction was separated from the reaction template onto an agarose gel and purified. The two resulting DNA strands were used as templates for PCR 3 for each molecule listed in Table 12. 25 cycles of PCR were performed, and the resulting DNA was separated from an agarose gel and digested with the enzymes NheI and BamHI (Promega) according to the manufacturer's instructions. The digested DNA was cloned into vector pM238 as described (Morera, et al., Angiogenesis, 2008: 11: 381-93). Autosequencing of the generated DNA confirmed the introduced mutations. The DNA encoding these mutated proteins was transformed into strain BL21(DE3), and polypeptides were produced according to the process described in Example 1 for PVM-I. Polypeptides corresponding to sequences SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 were used in formulation PVM-I Ala110 , PVM-IAla119 , and PVM-I Ala110 ,119 generated.

[0138] Oligonucleotides used in the PCR reaction to obtain the PVM-1 mutant. PCR 1 for Oligonucleotide PCR 2 dragons Oligonucleotide PCR 3 dragons Oligonucleotide PVM - I Ala110 SEQ ID NO: 13, SEQ ID NO: 7 SEQ ID NO: 8, SEQ ID NO: 14 SEQ ID NO: 13, SEQ ID NO: 14 PVM - I Ala119 SEQ ID NO: 13, SEQ ID NO: 9 SEQ ID NO: 10, SEQ ID NO: 14 SEQ ID NO: 13, SEQ ID NO: 14 PVM - I Ala110 ,119 SEQ ID NO: 13, SEQ ID NO: 11 SEQ ID NO: 12, SEQ ID NO: 14 SEQ ID NO: 13, SEQ ID NO: 14

[0139] In a study using BALB / c mice, preparations containing proteins with mutations in cysteine ​​110 and 119 were compared with PVM and PVM-I, where their administration was evaluated as eight doses administered at one-week intervals at a ratio of 200 μg of antigen preparation to 100 μg of adjuvant sNAcGM3-VSSP. The comparative study was conducted in a subcutaneous CT26 colon cancer model with 10 animals per group. Table 13 shows the superior immunogenicity of the PVM-I preparation compared to PVM, which supports the results presented in Example 1. Analysis of the protein preparations with mutations in cysteine ​​110 and 119 indicates that they have approximately 2 times lower immunogenicity than PVM-1 and even PVM, resulting in a lower antitumor effect.

[0140] [Table 13] Antigen preparations PVM, PVM-I, PVM-IAla110, PVM-I Ala119 and PVM-I Ala110,119 Comparative analysis of

[0141]

[0142] Note: Multiple comparisons ANOVA Test and Dunnet Post-hoc test results (different letters indicate a significant difference p<0.05).

[0143] Examples 9 Antigen agents in the treatment of lung metastases in mice PVM and PVM -I's preclinical Comparative evaluation of uses

[0144] Metastasis is a fundamental cause of cancer death of all origin. Among these, metastasis located in the lungs most frequently leads to patient death due to respiratory complications, replacement of lung tissue by the tumor, and the accumulation of pleural fluid. Lung metastasis constitutes one of the most resistant situations to anti-angiogenic interventions based on a single administration of drugs that eliminate ligands or signaling inhibitors delivering VEGF-A receptors. In this regard, integrating cellular branches of the immune response into the response arsenal for vaccine immunization can play a decisive role.

[0145] To evaluate the efficacy of the vaccine formulations under analysis, a group of 12 mice was immunized with these formulations for the treatment of lung metastases in mice, and tumor test infections were performed according to the requirements of all models. The results of immunization using sNAcGM3-VSSP as an adjuvant are described below for three metastasis models. Animals were immunized by subcutaneous inoculation for 8 weeks at a weekly dose rate of 200 μL total volume containing 100 μg of the corresponding antigen and 100 μg of the adjuvant.

[0146] For the study in mouse strain C57Bl / 6, 250,000 cells of 3LL metastatic lung carcinoma, clone D122, were inoculated onto the paw pads on day 3 after 4th immunization. The primary tumor was surgically removed 20 days after transplantation, and the animals were sacrificed 15 days later to count and characterize lung metastases. Analysis of metastatic load was evaluated by measuring lung weight and the number of macrometastases. Mitotic index studies were performed for each animal by measuring the number of mitotic events per field in 10 fields per cut. Similarly, apoptosis values ​​were quantified per field, and the mitotic-to-apoptotic ratio was calculated.

[0147] Table 14 shows a comparison of lung weights in treated animals. A differential effect was observed between the two antigens favoring PVM-I, an antigenic agent rich in a series of structural isomers completely lacking the disulfide bonds present in natural VEGF-A. Histopathological characterization of metastatic lesions using hematoxylin-eosin staining and analysis of serial sections demonstrated that a specific immune response to VEGF-A also led to a significant reduction in the mitotic / apoptotic balance observed in metastatic lesions.

[0148] Effects of Antigen Agents PVM and PVM-I in a Model of Spontaneous Lung Metastasis of Subcutaneous Tumor 3LL-D12 therapy Lung weight (g) Number of massive transitions Mitosis / Apoptosis ratio No treatment 0.6562 ± 0.336 a 7.727 ± 6.574 a 2.533 ± 0.9415 a PVM in sNAcGM3-VSSP 0.3975 ± 0.11 b 4.273 ± 4.027 b 1.52 ± 0.5993 b PVM-I in sNAcGM3-VSSP 0.279 ± 0.039 c 1.333 ± 1.614 c 0.8731 ± 0.1439 c Excipients in sNAcGM3-VSSP 0.498 ± 0.167 a 9.818 ± 5.382 a 2.881 ± 0.7743 a

[0149] Note: Multiple comparisons ANOVA Test and Dunnet Post-hoc test results (different letters indicate a significant difference p<0.05).

[0150] In the mouse strain BALB / c, the establishment of experimental lung metastases was evaluated following intravenous inoculation with 20,000 CT26 carcinoma cells. Test infection was performed via the retro-orbital plexus 3 days after the fourth inoculation. Animals were sacrificed 30 days after the tumor test infection, and lung tumor load, morphology, cell density, angiogenesis, and mitotic / apoptotic balance were evaluated in histopathological analysis of serial sections of lung tissue. As shown in Table 15, the number of metastases and the area they occupy in the analyzed sections (10 per animal) are reduced by immunization with PVM. The most significant effect was observed when immunized with the PVM-I agent (p < 0.05; Dunnet's post-hoc test). Interestingly, the very high mitotic index (number of mitosis per field) for metastatic lung lesions induced by this model was reduced approximately fourfold when the vaccine agent was administered. As in the previous analysis, the best effect was observed in the group treated with the antigen variant PVM-I.

[0151] Effects of antigen preparations PVM and PVM-I administration in an experimental lung metastasis model following intravenous administration of CT26 colon cancer. therapy Number of massive transitions Mitosis / Apoptosis ratio No treatment 16.22 ± 8.913 c 5.567 ± 1.197 a PVM in sNAcGM3-VSSP 7.778 ± 6.797 b 3.244 ± 1.638 b PVM-I in sNAcGM3-VSSP 1.889 ± 2.977 a 1.8 ± 0.6325 c Excipients in sNAcGM3-VSSP 22.22 ± 10.4 c 6.422 ± 2.524 a

[0152] Note: Multiple comparisons ANOVA Test and Dunnet Post-hoc test results (different letters indicate a significant difference p<0.05).

[0153] In BALB / c mouse strains, a model of F3II metastatic breast cancer that spontaneously metastasizes to the lungs was also evaluated. In this case, a biweekly schedule with an additional group of aluminum phosphate adjuvants was used. Three days after the fourth immunization (in the presence of sNAcGM3-VSSP) or the second immunization (in the presence of aluminum phosphate), a total of 200,000 cells were subcutaneously inoculated into 100 μL of DMEM culture medium.

[0154] Table 16 shows the weight analysis of primary tumors 28 days after tumor transplantation. When animals were treated with antigen variants, as well as adjuvants and administration regimens, tumor growth was significantly reduced; however, it was antigen agent PVM-1 that demonstrated more substantial inhibition (p < 0.05, Dunnet’s post-hoc test). The same table shows the results regarding the number of large lung metastases. As can be seen, the reduction in tumor growth translates into a significant reduction in the number of transplanted metastases. Furthermore, in this study, a decrease in the mitotic / apoptotic balance and an increase in the number of necrotic lesions associated with metastatic lesions were observed. All of these effects were detected more significantly in the group treated with antigen agent PVM-1.

[0155] Effects of administering antigen preparations PVM and PVM-I as two adjuvants in an F3II metastatic breast cancer model. Tumor growth Number of transitions therapy n=10 n=12 No treatment 3.375 ± 0.3701 a 18 ± 3.643 a PVM in sNAcGM3-VSSP 2.231 ± 0.5614 b 7.25 ± 3.306 b PVM-I in sNAcGM3-VSSP 1.579 ± 0.4538 c 2.417 ± 1.621 c Excipients in sNAcGM3-VSSP 3.502 ± 0.4347 a 21.08 ± 4.907 a PVM in aluminum phosphate 2.387 ± 0.5571 b 9.167 ± 3.614 b PVM-1 in aluminum phosphate 1.691 ± 0.3536 c 3.25 ± 2.094 c Excipients in aluminum phosphate 3.508 ± 0.5316 a 21.50 ± 4.075 a

[0156] Note: Multiple comparisons ANOVA Test and Bonferroni Results of post-test (different letters indicate a significant difference p<0.05).

[0157] Overall, the results indicated that the antigen preparation PVM-1 was more effective in reducing the metastatic potential of primary tumors and the transplantability of metastatic cells in the lungs. Furthermore, the superior efficacy of vaccination with the antigen preparation PVM-1 was observed in changes to the metastatic phenotype, with reduced mitotic / apoptotic balance, a higher incidence of necrosis, and cellular lesions exhibiting a significant reduction in angiogenesis.

[0158] Examples 10 Antigen agents for tumor-induced immunosuppression PVM and PVM Effects of immunization using -I

[0159] A group of 15 animals of the BALB / c strain was used, and they were immunized by subcutaneous inoculation with the following variants:

[0160] 1. PVM during sNAcGM3-VSSP (immunization 8 times a week)

[0161] 2. PVM-I in sNAcGM3-VSSP (immunization 8 times a week)

[0162] 3. sNAcGM3-VSSP (Immunization 8 times a week)

[0163] 4. PVM in aluminum phosphate (immunization 4 times every two weeks)

[0164] 5. PVM-I in aluminum phosphate (immunization 4 times every two weeks)

[0165] 6. Aluminum phosphate (immunization 4 times every two weeks)

[0166] In all cases, immunization was performed by subcutaneous inoculation with a total volume of 200 μL. Three days after the last immunization, five animals per group were randomly selected and euthanized to analyze their immune status and that of the experimental controls (groups 3 and 6). This evaluation was performed by flow cytometry of whole blood samples, splenocytes, lymph nodes, and bone marrow.

[0167] The remaining animals in each group were subcutaneously infected with 20,000 CT26 colon cancer cells on their right flanks. On days 14 and 21 after tumor cell injection, five mice per group were euthanized for evaluation, and in this case, analysis of dissociated tumor tissue was added.

[0168] No toxic events at the macroscopic level were proven in any animal, and histopathological analysis showed no damage in any organs analyzed 7 days after the last immunization. Immunological evaluation consists of (1) analysis of serum concentrations of murine VEGF-A; (2) evaluation of T lymphocyte concentrations in whole blood, lymph nodes draining the primary tumor, and the tumor itself; and (3) study of IFN-gamma secretion by intratumoral lymphocytes when exposed to a representative antigen of mutated VEGF-A.

[0169] In untreated animals, murine VEGF-A levels increased with the time of exposure to the tumor, corresponding to an increase in tumor size. In the group immunized with antigen variants, a significant decrease in VEGF-A levels was observed (p<0.001 ANOVA, Dunnet's post-hoc test), which persisted even 30 days after tumor test infection (Table 17). A similar phenomenon was observed for tumor compartments.

[0170] Analysis of mouse VEGF-A concentration in serum and tumor lesions of immune animals. group therapy In serum VEGF -A ( pg / mL) volume middle VEGF -A ( pg / mL) 14th 21st 14th 21st I PVM in sNAcGM3-VSSP 75. 5 ± 26 b 110.2 ± 15 b 137 ± 34 b 250.2 ± 38 b II PVM-I in sNAcGM3-VSSP 52.2 5 ±23 c 62.5 ± 12 c 88.7 ± 27 c 103.1 ± 11 c III sNAcGM3-VSSP 116.5 ± 50 a 183.4 ± 73 a 304.4 ± 31 a 516.1 ± 20 a IV PVM in aluminum phosphate 85.1 ± 16 b 108.7 ± 18 b 161.2 ± 37 b 293 ± 30 b V PVM-I in aluminum phosphate 49.5 ± 19 c 76.3 ± 21 c 103.4 ± 24 c 140.9 ± 20 c VI aluminum phosphate 120.3 ± 35 a 207.6 ± 62 a 420.1 ± 63 a 643.4 ± 87 a VII No treatment 106.2 ± 50 a 195.15 ±73 a 387.4 ± 43 a 601.2 ± 92 a VIII No treatment / no tumors 30 ± 5 27 ± 11

[0171] Note: Multiple comparisons ANOVA Test and Bonferroni Post-test results (different characters indicate significant differences).

[0172] As reported by Gabrilovich et al., the immune system status of sacrificed animals was analyzed at each moment through studies on the ratio of cell populations present in the lymph nodes and the tumors themselves (Gabrilovich D et al. Blood 1998, 92: 4150). For this study, monoclonal antibodies against CD3, CD4, and CD8 labeled with fluorescein isothiocyanate, phycoerythrin, and phycoerythrin-Cy7 were used, which allowed for the visualization of cell populations using a flow cytometer (Sysmex Partec). The results obtained are shown in Table 18.

[0173] Summary of analysis results for the cell population of interest. WB group CD3 CD4 CD8 I PVM in sNAcGM3-VSSP 3260 ± 125 2341 ± 140 789 ± 63 II PVM-I in sNAcGM3_VSSP 3890 ± 446 2652 ± 236 897 ± 115 III sNAcGM3-VSSP 1267± 181 1267± 181 212 ± 58 IV PVM in aluminum phosphate 2950 ± 185 2950 ± 185 690 ± 94 V PVM-I in aluminum phosphate 3575 ± 283 3575 ± 283 795 ± 106 VI aluminum phosphate 1320 ± 230 1320 ± 230 198 ± 63 DLN group CD3 CD4 CD8 I PVM in sNAcGM3-VSSP 123663 ± 3548 78898 ± 2689 32354 ± 1779 II PVM-I in sNAcGM3_VSSP 166621 ± 6652 90003 ± 3223 45000 ± 2600 III sNAcGM3-VSSP 90114 ± 1052 58454 ± 1290 24241 ± 2003 IV PVM in aluminum phosphate 114423 ± 2848 68878 ± 3672 30245 ± 2158 V PVM-I in aluminum phosphate 156771 ± 5542 81123 ± 3117 43222 ± 3485 VI aluminum phosphate 80333 ± 1242 60343 ± 1450 19541 ± 1253 T group CD3 CD4 CD8 % CD8 PD1 - I PVM in sNAcGM3-VSSP 4542 ±145 2525 ± 253 3400 ± 350 75 ± 5.1 II PVM-I in sNAcGM3_VSSP 6995 ± 230 2855 ± 125 3985 ± 180 92 ± 3.1 III sNAcGM3-VSSP 2538 ± 93 1240 ± 52 1418 ± 277 50 ± 4.5 IV PVM in aluminum phosphate 3542 ± 152 1525 ± 98 2400 ± 84 63 ± 4.2 V PVM-I in aluminum phosphate 6583 ± 302 2514 ± 96 3408 ± 102 72 ± 2.4 VI aluminum phosphate 2597 ± 212 1025 ± 98 1610 ± 201 42 ± 3.7

[0174] main: whole blood ( WB ), draining lymph nodes ( DLN ) and subcutaneous tumor (T).

[0175] Analysis of the lymphocyte population in animals 14 days after tumor test infection showed an increase in CD3, CD4, and CD8-positive cell fractions in three analyzed compartments, which is directly associated with the administration of the vaccine formulation. With the use of the antigen variant PVM-I, these cells of the immune system are achieved at significantly higher concentrations (p<0.05 Bonferroni post-hoc test). Furthermore, a study on PD1 markers of CD8 T lymphocytes infiltrating subcutaneous tumors showed that their increase was associated with a negative PD1 phenotype in animals treated with the antigen, and appeared at a higher rate in individuals administered the PVM-1 antigen (Table 18). The increase in CD4 and CD8 cell infiltration was positively and significantly associated with a decrease in tumor mass (r = 0.7147, p < 0.002).

[0176] Tumor leukocytes were isolated by a positive selection process in a column using magnetic beads coated with antibodies specific to CD45 (Miltenyi), and IFN-gamma secretion in response to the addition of mutated VEGF-A in the culture medium was evaluated using an ELISPOT-type system (MABTECH). An increase in the secretion of this cytokine was observed only in animals vaccinated with the antigen vaccine, and was higher in the group administered the PVM-1 antigen for both adjuvants (p < 0.05, Bonferroni post-hoc test) (Table 19). This demonstrated not only increased cell infiltration but also that these infiltrating leukocytes possessed higher activity associated with the administration of the vaccine formulation.

[0177] Analysis of tumor CD45+ infiltrates by IFN-gamma ELISPOT. group IFN -Number of gamma secretor clones / 10 6 CD45 + cell I PVM in sNAcGM3-VSSP 120 ± 34 c II PVM-I in sNAcGM3_VSSP 352 ± 130 a III sNAcGM3-VSSP 42 ± 14 d IV PVM in aluminum phosphate 105 ± 23 c V PVM-I in aluminum phosphate 203 ± 102 b VI aluminum phosphate 32 ± 12 d

[0178] Examples 11 . Regarding the immunogenicity generated for another antigen test infection PVM -1 Evaluation of the adjuvant effect of immunization.

[0179] Given the potential for immune recovery, we investigated whether immunity with PVM-I could induce auxiliary effects through increased humoral and cellular responses specific to VEGF-A as well as unrelated antigens. This potential effect was investigated by evaluating the degree of specific immunity to antigens present in the tumor and antigens administered with the vaccine.

[0180] The ovarian tumor model ID8-OVA was used for tumor test infections in C57Bl / 6 animals and the parental line ID8 in an in vitro lysis study. Animals (n=10 per group) were administered 2.5 million tumor test cells intraperitoneally, and the immunization schedule began 3 days later as follows:

[0181] a) PVM-I (200μg), sNAcGM3-VSSP (100μg): Administered subcutaneously weekly for 8 weeks,

[0182] b) OVA (1 mg), sNAcGM3-VSSP (100 μg): Administer subcutaneously weekly for 2 weeks. sNAcGM3-VSSP (100 μg): Administer subcutaneously weekly for the remaining 6 weeks.

[0183] c) PVM-I (200μg), sNAcGM3-VSSP (100μg), OVA (1mg): administered subcutaneously weekly for 2 weeks, and PVM-I (200μg), sNAcGM3-VSSP (100μg): administered subcutaneously weekly for the remaining 6 weeks.

[0184] d) sNAcGM3-VSSP (100μg): Administered subcutaneously weekly for 8 weeks,

[0185] e) OVA (1 mg): Administer subcutaneously weekly for 2 weeks. sNAcGM3-VSSP (100 μg): Administer subcutaneously weekly for the remaining 6 weeks.

[0186] Animals were euthanized 1 week after the 8th immunization and 60 days after tumor transplantation. In this tumor model, animal body weight is correlated with tumor growth. Table 20 shows the observed synergistically increased antitumor effect by administering OVA in the presence of PVM-1 in sNAcGM3-VSSP.

[0187] Analysis of humoral responses to VEGF-A and OVA was performed using an ELISA-type system on collected serum as described in Examples 1, 2, and 3. Specific cellular responses were analyzed using tumors for specific cytolysis studies by in vitro co-incubation of splenocytes with ID8 and ID8-OVA tumor cell lines (described in Example 4). Additionally, specific stimuli (peptide OVA) for 72 hours 257-264 , or VEGF KDR -The secretion of IFN-gamma in the supernatant of splenocytes isolated from immunized animals in response to the addition of ) was investigated. In the latter case, an ELISA type assay was used for IFN-gamma detection according to the manufacturer's instructions (Biolegend). The results are summarized in Tables 20 and 21.

[0188] Animal body weight and IgG titers specific to VEGF-A and OVA antigens at the end of the study. group Weight (g) VEGF -A about potency Regarding the OVA potency PVM -I / NAcGM3 - VSSP 24,90 ± 0,7 b 10580 ± 2056 a 3800 ± 152 c OVA / NAcGM3 - VSSP 25,76 ± 1,4 b 135 ± 22 b 10080 ± 1202 b PVM-I / NAcGM3-VSSP / OVA 23,37 ± 1,7 c 10320 ± 1842 a 20955 ± 1242 a NAcGM3 - VSSP 27,83 ± 2,5 a 200 ± 76 b 140 ± 24 d OVA 29,13 ± 3,8 a 180 ± 54 b 200 ± 52 d

[0189] Note: Multiple comparisons ANOVA Test and Dunnet Post-hoc test results (different letters indicate a significant difference p<0.05).

[0190] Comparative analysis of cell response results. group IFN - Gamma VEGF KDR - IFN -gamma OVA 257-264 CTL -ID8 ( % ) CTL -ID8-OVA (%) PVM -I / NAcGM3 - VSSP 1650 ± 98 a 1200 ± 54 c 35 ± 3 a 33 ± 5.3 b OVA / NAcGM3 - VSSP 10 ± 25 b 3045 ± 39 b 5 ± 2 b 28± 6.1 b PVM-I / NAcGM3-VSSP / OVA 1700 ± 120 a 3800 ± 152 a 35 ± 3 a 79 ± 4.1 a NAcGM3 - VSSP 90 ± 45 b 59 ± 26 d 5 ± 2.1 b 6 ± 1.5 c OVA 25 ± 23 b 100 ± 44 d 6.5 ± 3 b 15 ± 3.4 c

[0191] Note: Percentages indicate the lysis of ID8 or ID8-OVA cells at the final point of the study. Multiple comparisons ANOVA Test and Dunnet Results of post-test (different letters indicate a significant difference p<0.05).

[0192] Humoral (Table 20) and cellular (Table 21) responses are induced for both antigens when administered in conjunction with sNAcGM3-VSSP. Concurrent administration of OVA with immunotherapy containing PVM-I significantly increases the specific response to OVA in animals administered with OVA as an immunogen, as well as in animals administered with PVM-I alone. This fact indicates the potential of a vaccine strategy using this antigen preparation for inducing amplified responses to tumor-expressed antigens that are not found within the vaccine preparation itself. In the group treated with both antigens, humoral responses, IFN-gamma secretion, and ID8-OVA cell lysis suggest that there is a synergy inducing superior effects as well as an additive effect. These factors indicate the potential to use this strategy to increase immune responses to other tumor-associated antigens.

[0193] Examples 12 . From 2 supplements PVM or PVM - In a collagen-induced arthritis model by immunization with -I In vivo Protection test.

[0194] A group of 20 DBA / 1 mice (H-2q haplotype) were immunized, which are susceptible to collagen-induced arthritis. The animals were administered investigational antigen preparations (PVM or PVM-I) containing aluminum phosphate or sNAcGM3-VSSP adjuvants. For sNAcGM3-VSSP, a dosage regimen of 100 μg of antigen and 100 μg of adjuvant was used, administered eight times at weekly intervals; for aluminum phosphate, 0.7 mg of Al was administered every two weeks. 3 + 100 μg of antigen was administered in four doses. The treatment groups were defined as follows:

[0195] I. PVM in sNAcGM3-VSSP

[0196] II. PVM-I in sNAcGM3-VSSP

[0197] III. sNAcGM3-VSSP

[0198] IV. PVM in Aluminum Phosphate

[0199] V. PVM-I in aluminum phosphate

[0200] VI. Aluminum Phosphate.

[0201] Three days after 4th immunization with NAcGM3-VSSP or 2nd immunization with aluminum phosphate, induction of autoimmune arthritis was initiated by immunization with chicken collagen II (Sigma) according to a previously described model (Campbell IK et al Eur. J. Immunol. 30: 1568, 2000). This immunization was repeated on day 26 to complete the induction of the autoimmune response. Signs of erythema (1), inflammation (2), or joint stiffness (3) were evaluated daily according to an arthritis index with a score of 0 to 3 for each bridge, with a maximum value of 12. Mice began to show clinical symptoms of arthritis on day 23 after induction and reached a peak incidence on day 50. Table 22 shows the analysis of arthritis incidence in animals of different experimental groups. On days 40 and 55, it was observed that the incidence of arthritis was significantly reduced in the vaccinated groups (I, II, IV, V) compared to the control groups III and VI, who received a placebo of the adjuvant.

[0202] Incidence of arthritis at two evaluation points group Up to 40 days Incidence rate Up to 55 days Incidence rate I 20 / 7 (35%) 20 / 9 (45%) II 20 / 4 (20%) 20 / 6 (30%) III 20 / 12 (60%) 20 / 16 (80%) IV 20 / 8 (40%) 20 / 12 (60%) V 20 / 6 (30%) 20 / 8 (40%) VI 20 / 13 (65%) 20 / 15 (75%)

[0203] Examples 13. In non-human primates PVM and PVM Evaluation of the immunogenicity of -I and analysis of the immunization effect on experimental laser-induced choroidal neovascularization.

[0204] The ability of antigenic preparations PVM and PVM-I to induce relevant immune responses in more autologous situations was evaluated in non-human primates (Chlorocebus aethiops sabaeus). Four animals were used per group. 400 µg of antigenic preparation PVM or PVM-I was compared with 200 µg of NAcGM3-VSSP in the aluminum phosphate form or 0.7 mg of Al. 3 +The treatments were administered as follows. The administration schedule was eight doses per week for NAcGM3-VSSP and four doses every other week for aluminum phosphate. VEGF-A-specific IgG antibody titers and the neutralization of its binding to type 2 receptors were evaluated for eight weekly immunizations (NAcGM3-VSSP) or four weekly immunizations (aluminum phosphate). The results are shown in Table 23. As can be seen, both antigen preparations induced specific titers for VEGF-A, and the titers induced by PVM-1 were higher in both adjuvants. These titers, in turn, correlated with an increase in serum neutralizing capacity.

[0205] Analysis of humoral responses in non-human primates. Treatment group animal PVM in aluminum phosphate PVM-I in aluminum phosphate NAcGM3 - VSSP PVM NAcGM3 - VSSP PVM-I 1 17830.9 54315.64 2776.882 5591.977 2 24673.76 45220.69 2585.966 5126.05 3 25177.13 32738.94 2729.897 6105.441 4 10060.54 25681.82 2804.34 4878.99 average 19436 39489 2724 5426 standard deviation 7092 12766 97 541 Mann-Whitney p = 0.0286 p = 0.0286

[0206] Note: Humans who used two supplement choices VEGF -Specific to A potency Displayed.

[0207] To assess the extent to which activation of the cellular response was achieved in parallel with the induction of the humoral response, a conventional delayed-type hypersensitivity (DTH) test was performed as described (Morera, et al., Vaccine, 2010: 28 : 3453-61). At 48 hours, hardness was measured vertically twice using a digital vernier caliper. The area of ​​the lesion was calculated, and the geometric mean was reported. Erythema or inflammation was not considered part of this response. A diameter of 0.5 mm was considered the limit of the detectable response. Results were expressed under the following conditions: (++) > 5 mm 2 ; (+) = 0.5 to 4.99 mm 2 ; (-) = Reaction not detectable. The results of this scoring are shown in Table 24. Furthermore, 6 mm 2Punches (6 per animal) were formed at the sensitization site and analyzed by hematoxylin / eosin staining. At least two sections were analyzed from each biopsy to determine the characteristics of the infiltrates regarding the presence of monocytes, neutrophils, or eosinophils.

[0208] Intradermal injection of the antigen was well tolerated without side effects such as blisters or ulcers. Monkeys in all groups treated with the vaccine responded to human VEGF-A in different combinations with adjuvants. No response was reported in any of the immunization groups at the control site where saline was injected. Histopathological evaluation confirmed the presence of a potent DTH-type response to VEGF-A inoculation. Biopsies obtained from the human VEGF-A injection site were consistent with a DTH scene accompanied by abundant infiltration of macrophages and lymphocytes.

[0209] Score of DTH response in non-human primates immunized with PVM or PVM-I in the presence of two adjuvants. experimental group Test infection strain solution hVEGF-A121 1. n 400 µg PVM in NAcGM3-VSSP - (++) 2. 400 µg PVM-I in NAcGM3-VSSP - (++) 3. Plesev in NAcGM3-VSSP - - 4. 400 µg PVM in aluminum phosphate - (++) 5. 400 µg of aluminum phosphate PVM-I - (++) 6. Placebo in aluminum phosphate - -

[0210] Note: Six sites were analyzed per animal. - indicates an undetectable reaction.

[0211] Peripheral blood mononuclear cells (PBMCs) from immunized animals were isolated by a Ficoll gradient, as previously reported, and analyzed for specific cytolysis of synonyms previously incubated with VM and labeled with CFSE. Table 25 shows the results corresponding to the viability analysis of "charged" and CFSE-labeled cells by flow cytometry. At least three replicate analyses per animal indicate that the vaccine formulation induces specific cellular immunity against the cytotoxic antigen within one week after the last immunization, corresponding to the induction period. As can be seen, in both adjuvants, the effect of the vaccine was significantly greater when using the PVM-1 antigen (P < 0.05; Dunnet test).

[0212] [Table 25] Direct cell lysis of VEGF-A "charged" autologous PBMCs isolated from monkeys immunized with PVM or PVM-1 in two adjuvants.

[0213]

[0214] Note: Cytotoxicity is, " Uncharged "The one who is not" PBMC and By comparison, Parenchyma Evaluated using analysis, with CFSE Displayed as a percentage of the tagged group reduction.

[0215] The prevention of experimental laser-induced choroidal neovascularization in non-human primates was also evaluated in these animals. The model reported by Krzystolik et al. was used (Krzystolik MG, et al. 2006. Acta Ophthalmol, 120: 338-346). Four monkeys were used per experimental group. Animals were anesthetized for all procedures by intramuscular injection of ketamine hydrochloride, acepromazine maleate, and atropine sulfate. Proparacaine hydrochloride local anesthesia was also used. One week after the final immunization, choroidal neovascularization (CNV) was induced in the macula by argon laser imaging. The extent of the lesions was detected and measured using photography and fluorescence angiography, and the characteristics of the lesions were evaluated. The development of CNV lesions was assessed before and after laser treatment, as well as at 15, 20, and 29 days. The lesions were analyzed by experts outside the experimental design using the following scales: Grade 1, no hyperfluorescence; Grade 2, hyperfluorescence without effusion; Grade 3, early or mid-stage hyperfluorescence and late leakage; and Grade 4, very bright early or mid-stage transport hyperfluorescence with late leakage extending beyond the edge of the laser spot. The animals were observed daily to evaluate their clinical condition, including ocular abnormalities.

[0216] Of the four grades assigned to laser treatment, Grade 4 corresponds to a clinically severe leakage condition. The lesion is considered to reflect the presence of new choroidal vessels that have grown beyond the laser treatment area or leaked so intensely that fluorescence spread noticeably beyond the vessels. In the placebo group, the mean number of Grade 4 lesions ranged from 45.4% to 50.2% of the laser treatment area. The mean percentage of Grade 4 lesions in the control group was similar to that reported by other authors using this CNV animal model. In contrast, all groups treated with the vaccine showed a significant reduction or the complete absence of Grade 4 lesions, regardless of the adjuvant used. Table 26 shows the percentage distribution of all degrees of damage for the treatment groups at day 29. It is interesting that a significant reduction in higher-grade lesions was obtained with the use of the vaccine PVM-I (ANOVA, Bonferroni post-hoc test, p < 0.05).

[0217] [Table 26] Analysis of the degree of choroidal neovascularization.

[0218]

[0219] Examples 14. Antigen preparations PVM Treatment of human solid tumors by administration of a vaccine composed of -I and an adjuvant.

[0220] This study aims to evaluate the potential of novel antigen agents that induce relevant specific immune responses in relation to human tumor pathology. Patients with advanced-stage solid tumors who had no other treatment options and had not received other treatment for at least 4 weeks prior to the start of administration were selected. Antigen agent PVM-I was administered as a single dose of 800 μg: (a) in the presence of sNAcGM3-VSSP, in a weekly inoculation schedule for 8 weeks, followed by monthly administration, or (b) aluminum phosphate, administered as a four-dose schedule at a bi-weekly frequency, followed by monthly administration.

[0221] Humoral and cellular immune responses were evaluated in serum and PBMCs collected in both cases: before the start of the immunization schedule and one week after the last immunization. Two baseline parameters were evaluated in serum: specific antibody titer against VEGF-A and the ability to neutralize binding to the VEGFR2 receptor. These parameters were determined using two types of ELISA systems (Morera, et al., Vaccine, 2012: 30: 368-77; Sanchez Ramirez, et al., J Immunoassay Immunochem, 2016: 37: 636-58). A titer of 1:500 or higher and a neutralization rate of 10% or higher were defined as positive results.

[0222] Cellular response was evaluated in PBMC samples by IFN-gamma ELISPOT as described (Gavilondo, et al., Vaccine, 2014: 32: 2241-50). Low responders were defined as patients with between 29 and 39 clones positive for IFN-gamma secretion per million CD3-positive cells, intermediate responders as patients with 40 to 79 signal numbers, and high responders as those showing 80 or more signals.

[0223] When PVM-1 was administered with both adjuvants, a specific immune response to VEGF-A was established in terms of the ability to neutralize the interaction between specific antibodies (Fig. 4a) and growth factors and VEGFR2 (Fig. 4b). This response was observed in 70% of patients treated with either of the adjuvants used. Similarly, a significant increase in the responsiveness of PBMCs to stimulation using a variant of human VEG-A (VM) was observed (Fig. 4c). These results indicated that the use of the novel antigenic variant PVM-1 in humans induces a specific immune response to human VEGF-A.

[0224] Figure 5 summarizes the total number of patients and the number of patients who tested positive in all trials for each adjuvant. When using pre-immunization values ​​as a control, results were stratified by considering the number of trials with much higher outcomes. In the graph, each patient is also assigned the number of months survived after the start of vaccine administration (analysis at 24 months). This makes it possible to correlate the extent to which the specific immune response contributes to increased survival in the absence of other tumor-specific therapies. A significant increase in survival was observed in patients with a higher number of positive responses (p < 0.05; Kaplan Meyer).

[0225] Examples 15 Antigenic agents in the treatment of human age-related macular degeneration PVM Clinical uses of -I

[0226] This study aimed to evaluate the potential of the antigen agent PVM-I to induce a specific immune response associated with human age-related macular degeneration (AMD). Patients received bevacizumab via the intravitreal route once a month for three consecutive months at a rate of 125 μg per 50 μL. During the first three months after diagnosis, the administration of this drug or other anti-angiogenic agents, such as aflibercept, ranibizumab, and ramucirumab, is established as the traditional treatment for the initial management of this disease. Following this administration, patients systematically undergo visual acuity testing and phenotypic changes in retinal membrane structure, and depending on these changes, new intravitreal injections of anti-angiogenic agents are indicated or not indicated. The success of treatment is primarily determined by a reduction in the need for intravitreal administration.

[0227] This regimen was administered concurrently with or without the antigen preparation PVM-1 at a single dose of 400 μg: (Group 2) once-weekly for 8 weeks in the presence of sNAcGM3-VSSP, followed by re-administration once a month, or (Group 3) four single-dose regimens every other week in aluminum phosphate, followed by re-administration once a month. Group 1 received only the standard regimen consisting of monthly doses for three consecutive months. After the first three doses of bevacizumab, patients were re-administered only if the lesion had worsened compared to previous ophthalmic analyses. This worsening was defined as vision loss, increased retinal thickness, the presence of intraretinal fluid, or an increase in subretinal fluid (greater than 200 μm or a value observed at the previous visit).

[0228] In the indicated adjuvants and regimens, the administration of the antigen agent significantly reduced the number of intravitreal injections of bevacizumab required to maintain or improve the patient's visual acuity and macular integrity. Table 27 summarizes the number of bevacizumab injections per patient required after the third dose and the specific antibody titers against VEGF-A that induce immunization. The use of immunotherapy with the antigen agent PVM-1 significantly reduces the number of intravitreal injections of bevacizumab administered to the patient compared to the existing regimen (Group 1) (p<0.05 in both cases, Dunnet trial). In both cases, a significant correlation was observed between the reduction in the number of intravitreal administrations and the levels of specific antibodies against human VEGF-A in the patients' serum one week after the fourth immunization for Group 2 (Pearson r=-0.8788, p= 0.0008) and one week after the eighth immunization for Group 3 (Pearson r=-0.7894, p=0.0066).

[0229] Patient evaluation in the study Group 1 Group 2 Group 3 Patient Code A Patient Code A B Patient Code A B FP01 2 FP03 2 5560 FP07 0 15987 FP02 8 FP04 3 4875 FP09 0 24920 FP05 5 FP08 4 2871 FP10 1 3852 FP06 2 FP14 1 3952 FP11 6 1360 FP12 7 FP15 0 10250 FP17 6 1350 FP13 7 FP16 2 5023 FP19 4 2800 FP18 7 FP20 5 995 FP23 2 4600 FP22 8 FP21 4 1885 FP25 4 2750 FP24 6 FP26 2 5587 FP28 5 1900 FP30 5 FP27 6 1190 FP29 5 1530

[0230] A: Necessary In the vitreous humor Number of injections, B: Generated after antigen administration VEGF -A Specific antibody titer, Group 1: Conventional therapy, Group 2: 400 in aluminum phosphate μg of PVM -I, group 3: 400 μg of PVM-1 in sNAcGM3-VSSP.

[0231] Approximately 30% of the patients included in the study had active extrafoveal polyps. This is a poor prognostic factor for patient progression in response to conventional intravitreal administration therapy. This is because the polyp lesions originate in deeper layers of the retina. Interestingly, an analysis of patients with choroidal polypoidal neovascularization showed a significant reduction in the number of active lesions one year after the start of treatment, while the effect was observed in only 20% (1 in 5) of the patients in the group that received conventional therapy (Table 28).

[0232] Evaluation of extrafollicular polyp lesions in study patients. Patient Code Treatment group active Yongjeong number / Number at the start of treatment active Yongjeong's Total number of treatments per year PF12 I 4 / 4 6 / 6 PF18 I 3 / 3 4 / 3 PF24 I 2 / 2 3 / 2 PF20 II 3 / 3 0 / 0 PF21 II 4 / 2 0 / 0 PF27 II 3 / 2 1 / 1 PF11 III 3 / 3 1 / 0 PF17 III 3 / 2 1 / 0 PF28 III 4 / 2 2 / 1 PF29 III 2 / 2 0 / 0

Claims

Claim 1 A polypeptide comprising a mutant of human vascular endothelial growth factor A (VEGF-A) isoform 121 having the amino acid sequence of SEQ ID NO. 2, wherein the mutant is characterized in that all amino acids corresponding to the 82nd arginine, 84th lysine, and 86th histidine of the wild-type VEGF-A isoform 121 are substituted with glutamic acid so as not to be able to bind to the receptor of human vascular endothelial growth factor type 2 (VEGFR2), folds into a non-natural arrangement of disulfide crosslinks, and the 2nd and 4th cystes in the chain of the polypeptide are found only as intramolecular crosslinks, and the 7th and 8th cystes are found only as forming intermolecular bonds. Claim 2 delete Claim 3 delete Claim 4 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a second cysteine, a third cysteine ​​is connected to a fourth cysteine, and a fifth cysteine ​​is connected to a sixth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO. 18 forming a disulfide crosslink, wherein the seventh cystes of the two polypeptide chains are connected, the eighth cystes of the two polypeptide chains are connected, and the last cystes of the two polypeptide chains are connected. Claim 5 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a second cysteine, a third cysteine ​​is connected to a fourth cysteine, and a fifth cysteine ​​is connected to a sixth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO. 18 forming a disulfide crosslink, wherein a seventh cysteine ​​of one polypeptide chain of the two polypeptide chains is connected to an eighth cysteine ​​of the other polypeptide chain, and the last cystes of the two polypeptide chains are connected. Claim 6 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a second cysteine, a third cysteine ​​is connected to a fifth cysteine, and a fourth cysteine ​​is connected to a sixth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO. 19 forming a disulfide crosslink, wherein the seventh cystes of the two polypeptide chains are connected, the eighth cystes of the two polypeptide chains are connected, and the last cystes of the two polypeptide chains are connected. Claim 7 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein the first cysteine ​​is connected to the second cysteine, the third cysteine ​​is connected to the fifth cysteine, and the fourth cysteine ​​is connected to the sixth cysteine; and the intermolecular bond is characterized in that two polypeptide chains having SEQ ID NO. 19 form a disulfide crosslink, wherein the seventh cysteine ​​of one polypeptide chain of the two polypeptide chains is connected to the eighth cysteine ​​of the other polypeptide chain, and the last cystes of the two polypeptide chains are connected. Claim 8 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a second cysteine, a third cysteine ​​is connected to a sixth cysteine, and a fourth cysteine ​​is connected to a fifth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO 20 forming a disulfide crosslink, wherein the seventh cystes of the two polypeptide chains are connected, the eighth cystes of the two polypeptide chains are connected, and the last cystes of the two polypeptide chains are connected. Claim 9 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a second cysteine, a third cysteine ​​is connected to a sixth cysteine, and a fourth cysteine ​​is connected to a fifth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO 20 forming a disulfide crosslink, wherein a seventh cysteine ​​of one polypeptide chain of the two polypeptide chains is connected to an eighth cysteine ​​of the other polypeptide chain, and the last cystes of the two polypeptide chains are connected. Claim 10 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a last cysteine, a second cysteine ​​is connected to a third cysteine, and a fourth cysteine ​​is connected to a fifth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO 21 forming a disulfide crosslink, wherein the seventh cystes of the two polypeptide chains are connected and the eighth cystes of the two polypeptide chains are connected. Claim 11 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a last cysteine, a second cysteine ​​is connected to a third cysteine, and a fourth cysteine ​​is connected to a fifth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO. 21 forming a disulfide crosslink, wherein a seventh cysteine ​​of one polypeptide chain and an eighth cysteine ​​of the other polypeptide chain are connected. Claim 12 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a last cysteine, a second cysteine ​​is connected to a fourth cysteine, and a third cysteine ​​is connected to a fifth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO 22 forming a disulfide crosslink, wherein the seventh cystes of the two polypeptide chains are connected and the eighth cystes of the two polypeptide chains are connected. Claim 13 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein 1 cysteine ​​is connected to the last cysteine, 2 cysteine ​​is connected to the 4th cysteine, and 3 cysteine ​​is connected to the 5th cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO. 22 forming a disulfide crosslink, wherein the 7th cysteine ​​of one polypeptide chain and the 8th cysteine ​​of the other polypeptide chain are connected. Claim 14 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a last cysteine, a second cysteine ​​is connected to a fifth cysteine, and a third cysteine ​​is connected to a fourth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO 23 forming a disulfide crosslink, wherein the seventh cystes of the two polypeptide chains are connected and the eighth cystes of the two polypeptide chains are connected. Claim 15 In claim 1, the intramolecular crosslink comprises a rearrangement of intramolecular disulfide crosslinks, wherein a first cysteine ​​is connected to a last cysteine, a second cysteine ​​is connected to a fifth cysteine, and a third cysteine ​​is connected to a fourth cysteine; and the intermolecular bond is formed by two polypeptide chains having SEQ ID NO. 23 forming a disulfide crosslink, wherein a seventh cysteine ​​of one polypeptide chain and an eighth cysteine ​​of the other polypeptide chain are connected. Claim 16 A polypeptide according to any one of claims 4 to 15, comprising an amino-terminal segment that increases the expression of the polypeptide in bacteria and a carboxy-terminal segment that facilitates purification, having the amino acid sequence of SEQ ID NO.

24. Claim 17 delete Claim 18 delete Claim 19 An antigen preparation for the treatment of a disease selected from the group consisting of melanoma, lymphoma, lung cancer, colorectal cancer, breast cancer, renal cancer and uterine cancer, comprising the polypeptide of claim 16 and a pharmaceutically acceptable excipient or diluent. Claim 20 A pharmaceutical composition for the treatment of a disease selected from the group consisting of melanoma, lymphoma, lung cancer, colorectal cancer, breast cancer, kidney cancer and uterine cancer, comprising the antigen preparation of claim 19 and a pharmaceutically acceptable vaccine adjuvant. Claim 21 A pharmaceutical composition according to claim 20, wherein the vaccine adjuvant is selected from the group consisting of oil adjuvants, aluminum salts, proteoliposomes, and proteoliposomes conjugated to gangliosides. Claim 22 delete Claim 23 delete Claim 24 delete

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