Nanoparticles containing synthetic ganglioside GM3 variants as adjuvants in vaccines

Abstract

The present invention describes a method for obtaining nanoparticle adjuvants based on different synthetic ganglioside GM3 variants. Depending on the fine structure of the fatty acids present in the ceramide of the synthetic GM3, adjuvants can be obtained for specifically and exclusively stimulating a humoral or cellular immune response to the associated antigen. In particular, the present invention provides an immunogenic vaccine composition comprising a peptide, polypeptide or protein and the above-mentioned nanoparticles, which are formed by dispersing hydrophobic proteins of the outer membrane complex (OMC) of the bacterium Neisseria meningitidis together with a fully synthetic ganglioside GM3 variant.

Description

Technical Field

[0001] The present invention relates to the fields of immuno-nanotechnology and immuno-oncology, and in particular to therapeutic vaccines for treating individuals with cancer and / or chronic infections caused by oncogenic viruses. In particular, the present invention describes nanoparticle adjuvants specifically for stimulating cellular or humoral immune effectors specifically in these patients, and further provides corresponding vaccine compositions. Prior art

[0002] After decades of failed clinical trial results, the field of cancer therapeutic vaccines has failed to achieve the hope of becoming an effective and low-toxic treatment that is beneficial to patients. The recent clinical success obtained with immune checkpoint inhibitor antibodies (Abs) has encouraged commercial and scientific interests in immuno-oncology, including cancer vaccines. From this point of view, the failure of these therapeutic vaccines may be attributed to factors such as the following: incorrect selection of antigens, the use of relatively ineffective carriers / adjuvants, and the use of the vaccine in a separate form (i.e., not combined with other immunomodulators that allow correction of the negative effects imposed by the tumor microenvironment) (Branca MA et al., (2016) Nat Biotech 34 (10): 1019-24).

[0003] Traditionally, cancer vaccines use proteins classified as autologous tumor-associated antigens in their formulations, which, although abnormally expressed in tumor cells, are also present in normal tissues. The lack of solid evidence for the clinical effectiveness of these vaccines may be partly due to the central tolerance process, through which the elimination of T cells with receptors with high affinity for most of these autoantigens occurs (Tran E. et al., (2017) Nat Immunol 18 (3): 255-62). Therefore, the success of cancer vaccines based on this type of antigen will rely on the use of new specialized adjuvants that can enhance the specific response of effector T cells (which are relatively weak at the beginning) so as to naturally transform tumors into sources of new antigens. This makes it possible to mobilize the powerful effects of multispecific and personalized cytotoxic T lymphocytes (CTLs), which can eradicate malignant lesions.

[0004] Recently, the prior art reflects the changes in the evaluation of antigens for designing successful cancer vaccines, which is to select tumor neoantigens. An attractive source of these neoantigens is derived from the personalized detection of individual tumor mutations. Another more limited source of this type of antigen comes from the sequence of oncogenic viral proteins. Although the advantage of these mutated peptides may be that they are new to the immune system (not present in normal tissues) and therefore more immunogenic, the success of new vaccines designed with these neoantigens will also rely on new adjuvants that can maximize CTL responses (wherein the best availability of antigens is sought by allowing them to be presented by antigen presenting cells (APCs) in the case of correct maturation) and also correct the immunosuppressive effects of the tumor microenvironment. In addition, the identification of true new epitopes is an invalid act in practice. Sequencing studies have identified thousands of somatic mutations in individual tumors and bioinformatics programs have predicted hundreds of peptides that can bind to specific MHC. But when they are isolated and studied by mass spectrometry, the vast majority of these new epitopes do not exist in real tumors, and even worse, only a few can stimulate CTL responses. This means that current methodologies for predicting and validating neoepitopes are far from being routinely used to enable personalized immunotherapy to enter clinical practice (Editorial, (2017) Nat Biotech 35(2):97).

[0005] In all types of vaccines, the choice of a suitable adjuvant is a key element to achieve the desired success. The basic goal of cancer therapeutic vaccines is to induce the activation and proliferation of B lymphocytes, T lymphocytes and innate immune mediators, so that humoral and cellular immune effectors that can recognize and destroy tumor cells appear, thereby leading to an increase in survival rate and quality of life of patients. In this sense, an ideal adjuvant should first optimize the availability of antigens to APCs. Secondly, effective stimulation of these APCs should be obtained so that they express the necessary co-stimulatory signals and secrete specific cytokines and chemokines. Third, TME should be able to be adjusted to neutralize immunosuppressive effects. Today, in practice, there are no adjuvants with all these characteristics.

[0006] As explained by Khong H. et al. (Khong H. et al., (2016) J Immunother Cancer 4:56), among the adjuvant variants that have proven to be most interesting for cancer vaccines are microparticles and nanoparticles, because they can have some desirable properties, especially also acting as vaccine carriers. These particle preparations can be designed so that the accompanying antigen can be effectively directed to specific APCs by adjusting the particle size, stiffness and net charge. Particle vaccines with diameters in the range of 500-2000 nm are preferentially captured by APCs at the injection site and move to lymph nodes (LNs), while particles between 20 and 200 nm are passively drained to LNs, where they are acquired by resident APCs. The microparticles and nanoparticles most commonly used for these purposes are liposomes, proteoliposomes, synthetic polymers and natural polymers. The advantages and limitations of each of these systems have been described in detail in the prior art.

[0007] Of particular relevance to the present invention is the description of a synthetic nanoparticle adjuvant containing ganglioside GM3 in its composition by Xu F. et al. (Xu F. et al., (2016) ACS Nano Vol. 10: 1189-1200). The adjuvant is obtained by coating gold nanoparticles having a diameter of 40 to 80 nm with a lipid membrane into which ganglioside GM3 is inserted. In this way, the principle of glycolipid-receptor specific interactions can be advantageously utilized for orientation, in particular sialyllactose with Siglec1 (CD169) (a marker overexpressed on activated dendritic cells (DCs) that interact with CD4 in LNs). + Although these GM3-coated nanoparticles selectively accumulate in the CD169 cells present in the popliteal LN in vivo + In DC, but there is no evidence of its possibility as an adjuvant in immunization experiments with reference antigens, nor any evidence of efficacy in tumor models. On the other hand, this principle only works in nanoparticles with a diameter range of 40 to 80 nm.

[0008] Molina et al. describe in patent US 7,776,346 a method for preparing an effective immunogen by associating very small sized proteoliposomes (VSSP) with a poorly immunogenic antigen, which is formed by hydrophobic conjugation of the outer membrane protein complex (OMPC) of the bacterium Neisseria meningitidis with the ganglioside GM3, which can be considered to be the closest to the technical solution of the present invention. Rodríguez et al. have described in detail in US 6,149,921 a method for obtaining these VSSPs, in which it is emphasized that the ganglioside GM3 used to obtain these proteoliposomes should be obtained from biological sources, mainly from hybridoma masses generated due to the industrial production of monoclonal antibodies. Similarly, Estévez et al. (Estévez et al., (2000) Vaccine Vol. 18: 90-197) teach that these VSSPs can also be produced by using GM3 obtained starting from canine erythrocytes. It is known in the prior art, as taught, for example, by Lee H et al. (Lee H et al., (2011) Int J Mass Spectr Vol. 305: 138-150), that the ganglioside GM3 obtained from any natural source consists of a variable mixture of various molecular species, wherein the composition of oligosaccharides is fixed but ceramides are variable, with various different types of fatty acids occurring. In addition to the inconvenience of using components of animal or tumor origin to produce pharmaceutical preparations, the use of GM3 of biological origin also moderately hinders the acquisition of VSSPs with the desired highly reproducible characteristics.

[0009] With regard to the present invention, it is reasonable to establish that no previous technical solution or scientific publication describes a VSSP variant obtained by a preparation process using completely synthetic variants of different GM3 with a chemically defined structure, which has the surprising property that its specialized use as an adjuvant for therapeutic vaccines ultimately depends on the molecularly homogeneous ganglioside GM3 used in its acquisition and that, due to said specialization, an adjuvant with advantageous properties relative to VSSP produced with GM3 from biological sources is provided. Thus, the novelty of the present invention is that two new nanoparticle adjuvants of the VSSP type are provided. One of them uses a completely synthetic ganglioside GM3, GM3 (18:0), whose ceramide contains stearic acid, and which is mainly useful for inducing a strong antibody response against the accompanying antigen. The other adjuvant is obtained by using a completely synthetic ganglioside GM3, GM3 (18:1), whose ceramide contains oleic acid, and which is particularly effective in inducing specific effector responses of CTL cells.

[0010] Brief description of the invention

[0011] In one embodiment, the invention relates to adjuvants comprising nanoparticles formed by associating fully synthetic variants of the ganglioside GM3 with hydrophobic proteins of the outer membrane complex of the bacterium Neisseria meningitidis; in particular, these adjuvants have stearic acid or oleic acid in the ceramide of the ganglioside GM3.

[0012] In a second aspect, the present invention relates to a vaccine composition comprising the nanoparticle adjuvant mentioned above, and a peptide, polypeptide or protein as an antigen, and optionally other adjuvants (which may be alumina or an oily adjuvant, but are not limited to these). In particular, the antigens constituting part of these vaccine compositions are the extracellular domains of growth factor receptors or parts thereof, such as HER1, HER2 or HER3 alone or in combination; or the peptide PyrGnRHm1-TT generated starting from the hormone GnRH. The vaccine composition can be used in the preparation of a medicament for treating cancer or chronic viral infection caused by an oncogenic virus.

[0013] In a particular embodiment, the present invention relates to the use of a vaccine composition comprising the nanoparticle adjuvant object of the present invention (alone or in combination) for stimulating an antigen-specific humoral or cellular immune response in a patient.

[0014] In addition, the present invention is directed to a method of treatment for administering to a subject in need thereof a vaccine composition of the present invention, by subcutaneous (SC), intradermal, intramuscular, intratumoral route or by direct application to a mucosa, at a frequency of once every fifteen days for a total of at least five induction doses, and then at monthly maintenance doses for at least six months. The composition may be administered simultaneously, in stages or alternately. DETAILED DESCRIPTION OF THE INVENTION

[0016] Vaccine composition

[0017] The vaccine composition used in the present invention is formed by combining a nanoparticle adjuvant with a peptide, polypeptide or protein as an antigen (particularly the entire extracellular domain of a growth factor receptor or a portion thereof; a hormone associated with a determined stage of tumor progression, such as GnRH, but not limited to these), wherein the nanoparticle adjuvant is formed by dispersing the hydrophobic protein of the OMPC of the bacterium Neisseria meningitidis together with a fully synthetic ganglioside GM3 variant.

[0018] The type of fatty acid comprised by the ceramide of the ganglioside GM3 forming part of the VSSP of the invention will depend on the purpose of the vaccine: where it is desired to favour an antibody response, stearic acid (GM3 18:0) is used, whereas if a CTL response is desired, oleic acid (GM3 18:1) is employed. It is also possible to induce a dual antibody and CTL response in the host against the same antigen by appropriately combining immunizations with these two vaccine formulations. Both VSSPs comprising GM3 (18:0) and VSSPs with GM3 (18:1) can be used alone or as part of an adjuvant in combination with other agents, such as alumina or oily adjuvants, but not limited to the latter.

[0019] The vaccine composition of the present invention using GM3 (18:0) as adjuvant induces higher quality in terms of humoral response, recognition of tumor lines, inhibition of receptor activation in tumor cells and reduction of viability of these cells compared to vaccine formulations using gangliosides obtained from natural sources as adjuvants.

[0020] The vaccine composition of the present invention using GM3 (18:1) as an adjuvant induced higher quality in CTL responses compared to vaccine formulations using gangliosides obtained from natural sources as adjuvants.

[0021] Therapeutic applications and treatment methods

[0022] The present invention provides vaccine compositions specifically designed to induce antibody responses or CTL responses against the growth factor receptor family, which is a particularly useful solution for antigen systems of this purpose in immuno-oncology, where both antibody responses and specific CTL responses are known to be effective but difficult to optimize in a single vaccine formulation and with a single administration regimen.

[0023] It is also an object of the present invention to introduce vaccine formulations specifically designed to generate high titers of neutralizing antibodies against hormones that are relevant at defined stages in tumor progression, particularly GnRH, although not limited to this hormone.

[0024] Another object of the present invention is to provide a therapeutic vaccine composition capable of generating a potent specific antibody response or CTL response in all immunocompromised patients suffering from cancer or chronic viral infection caused by oncogenic viruses.

[0025] The vaccine compositions of the present invention can be introduced into the patient by SC, intradermal, intramuscular, intratumoral routes or by direct application to the mucosa.

[0026] Among the types of cancer that can be treated with the vaccine composition that is the object of the present invention, include cancers of epithelial origin. More particularly, among the examples of these cancers, include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), hepatocellular carcinoma, stomach cancer (including gastrointestinal cancer), pancreatic cancer, head and neck cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, breast cancer, prostate cancer, salivary gland cancer, kidney cancer, prostate cancer, vaginal cancer, thyroid cancer, anal cancer and penile cancer.

[0027] The dosage range of the extracellular domain of the growth factor receptor or part thereof to be used in humans in the vaccine composition of the present invention is 200 μg to 1 mg, preferably 400 μg to 900 μg. The dosage range of GnRH to be used in the vaccine composition of the present invention is 500 μg to 3 mg, preferably 1 mg to 2.5 mg. The VSSP is administered in a range between 100 μg and 1 mg, preferably between 200 μg and 600 μg (depending on the content of OMPC).

[0028] The composition is administered to the subject in the following manner: once every fifteen days for at least a total of five induction doses, and then monthly maintenance doses for at least six months. If it is desired to induce a dual response of antibodies and CTLs against the same antigen in the host, immunization with these two vaccine preparations can be combined simultaneously, in stages or alternately.

[0029] Preparation of Nanoparticle Adjuvant VSSP GM3 (18:0)

[0030] First, in a reactor with stirring, OMPC of Neisseria meningitidis is dispersed in a Tris-HCL buffer solution containing a mixture of sodium deoxycholate (10-40 mM) and sodium dodecyl sulfate (1-10 mM) for a period of 1 to 36 hours. Next, a synthetic ganglioside (GM318:0) of 0.1 to 3 times the mass of the added OMPC is added, and stirring is extended. The detergent is then removed by ultrafiltration or a dialysis system. The remaining solution after ultrafiltration is concentrated to adjust its concentration to the desired dose of 0.1 to 1 mg / ml of OMPC, and sterilized by filtration in a sterile capsule with a pore size of 0.2 μm.

[0031] Preparation of Nanoparticle Adjuvant VSSP GM3 (18:1)

[0032] First, in a reactor with stirring, OMPC of Neisseria meningitidis is dispersed in a Tris-HCL buffer solution containing a mixture of sodium deoxycholate (10-40 mM) and sodium dodecyl sulfate (1-10 mM) for a period of 1 to 36 hours. Next, a synthetic ganglioside (GM318:1) of 0.1 to 3 times the mass of the added OMPC is added, and stirring is extended. The detergent is then removed by ultrafiltration or a dialysis system. The remaining solution after ultrafiltration is concentrated to adjust its concentration to the desired dose of 0.1 to 1 mg / ml of OMPC, and sterilized by filtration in a sterile capsule with a pore size of 0.2 μm.

[0033] Preparation of vaccine compositions of HER1, HER1+HER2 and HER3 with VSSP GM3 (18:0)

[0034] The preferred vaccine composition of the present invention, which comprises HER1 or HER1+HER2 or HER3 as a growth factor receptor and VSSP GM3 (18:0) as an adjuvant and aims to induce a strong specific humoral immune response, can be prepared in the following manner: the contents of a separate vial of the antigen HER1 or HER1+HER2 or HER3 as a solution or a lyophilisate are separately mixed with VSSP GM3 (18:0) stored at 4 to 20° C. at the patient's bedside for 10-30 minutes before injection, so that the final ratio of the mass of the antigen to the mass of VSSP GM3 (18:0) (depending on the content of OMPC) is in the range of 300 μg to 2 mg.

[0035] Preparation of vaccine compositions of HER1, HER1+HER2 and HER3 with VSSP GM3 (18:1)

[0036] The preferred vaccine composition of the present invention, which comprises HER1 or HER1+HER2 or HER3 as a growth factor receptor and VSSP GM3 (18:1) as an adjuvant and aims to induce a strong specific cellular immune response, can be prepared in the following manner: the contents of a separate vial of the antigen HER1 or HER1+HER2 or HER3 as a solution or a lyophilisate are separately mixed with VSSP GM3 (18:1) stored at 4 to 20° C. at the patient's bedside for 10-30 minutes before injection, so that the final ratio of the mass of the antigen to the mass of VSSP GM3 (18:1) (depending on the content of OMPC) is in the range of 300 μg to 2 mg.

[0037] Preparation of vaccine composition of GnRHm1-TT VSSP GM3 (18:0)

[0038] The peptide GnRHm1-TT (EHWSYPLRPG) was produced by replacing the amino acid L-glycine in the sixth position normally occupied by it in the sequence of native GnRH (EHWSYGLRPG) with L-proline during the biosynthesis process. To complete its construction, the epitope QYIKANSKFIGITEL of tetanus toxoid (Junco JA et al., (2007) Vaccine 25:8460-68) was added during a suitable synthesis, following the solid phase method (Hougten et al., (1986) Biotecniques 4:522-6); for the purposes of the present invention, from now on, the peptide will be referred to as PyrGnRHm1-TT.

[0039] The preferred vaccine composition of the present invention, which comprises PyrGnRHm1-TT and VSSP GM3 (18:0) as an adjuvant and aims to induce a strong specific humoral immune response, can be prepared in the following manner: the contents of a separate vial of the antigen PyrGnRHm1-TT as a solution or a lyophilisate are separately mixed with VSSP GM3 (18:0) stored at 4 to 20° C. at the patient's bedside for 10-30 minutes before injection, so that the final ratio of the mass of the antigen to the mass of VSSP GM3 (18:0) (depending on the content of OMPC) is in the range of 600 μg to 4 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 CD11b in the spleen of mice bearing MCA203 tumors treated with VSSP nanoparticles containing various GM3 molecular species + GR1 + The accumulation of cells was measured by flow cytometry.

[0042] Figure 2 Antigen-specific CTL responses induced in vivo in mice immunized with OVA / VSSP GM3(18:1), OVA / VSSP GM3(18:0) and OVA / native VSSP were measured by flow cytometry.

[0043] Figure 3 . Anti-tumor effect in the EG7 model treated with OVA and vaccines containing VSSP adjuvant nanoparticles of various GM3 molecular species. The figure shows the individual values ​​of tumor volume for each group and their averages at day 20 of the experiment. The dotted line indicates the value below which the tumor is considered not to have progressed. The frequency of animals with no tumor progression due to treatment in each group is shown on the right.

[0044] Figure 4 . Titers of IgG antibodies induced by HER3 vaccine preparations adjuvanted with VSSP GM3 (18:0), measured by ELISA.

[0045] Figure 5a Titers of specific antibodies against HER1 and HER2 induced by HER1+HER2 bivalent vaccine preparations adjuvanted with VSSP GM3(18:0) compared with those adjuvanted with native VSSP, measured by ELISA.

[0046] Figure 5b Titers of specific antibodies against the subdomains of ECD-HER2 induced by HER1+HER2 bivalent vaccine preparations adjuvanted with VSSP GM3(18:0) compared to those adjuvanted with native VSSP, measured by ELISA.

[0047] Figure 6 Recognition of HER1+ / HER2+ tumor lines by antibodies induced by HER1+HER2 bivalent vaccine preparations adjuvanted with VSSP GM3(18:0) compared to those adjuvanted with native VSSP, measured by flow cytometry.

[0048] Figure 7 Effect of antibodies induced by HER1+HER2 bivalent vaccine preparations adjuvanted with VSSP GM3(18:0) on the viability of the H125 HER1+ / HER2+ tumor line compared to that adjuvanted with native VSSP, measured by MTT colorimetric assay.

[0049] Figure 8 Recognition of the MDA-MB468 (HER1+) tumor line by antibodies induced by HER1 vaccine preparations adjuvanted with VSSP GM3 (18:0) compared to those adjuvanted with native VSSP, measured by flow cytometry.

[0050] Fig. 9 Inhibition of HER1 activation by antibodies induced by HER1 vaccine preparations adjuvanted with VSSP GM3(18:0) compared to those adjuvanted with native VSSP, measured by Western blotting.

[0051] Fig.10 Effect of immunization with the peptide PyrGnRHm1-TT, with and without VSSP GM3(18:0), on the weight of the prostate gland in Copenhagen rats.

[0052] Fig.11Evaluation of tumor growth rate in Copenhagen rats implanted with the Dunning R3327-H tumor line. Example

[0053] Example 1. Administration of VSSP GM3 (18:1) does not increase the number of CD11b+GR1+ cells in the spleen in mice bearing MCA203 tumors

[0054] On day 0, 1 × 10 6 Four groups of female C57BL / 6 mice (3 animals / group) were inoculated with MCA203 cells by SC route. Then, mice in three of these groups were injected with native VSSP, VSSP GM3 (18:1) and VSSP GM3 (18:0) (200 μg of OMPC) by SC route on days 11, 12 and 18, leaving the fourth group as an untreated control, to which phosphate buffer was administered. On day 22, the animals were sacrificed and the spleens removed were processed individually for determination of CD11b by flow cytometry. + Gr1 + The number of cells. Figure 1 ), such as in animals treated with VSSP GM3 (18:1), the mean number of these splenocytes was not increased relative to the mean number induced by the tumor in control animals. + Gr1 + The mean value of the number of cells was significantly increased in the spleen of animals injected with native VSSP and VSSP GM3(18:0) (same letters, p>0.05; different letters, p<0.05, ANOVA and Tukey test).

[0055] Example 2. Vaccination with OVA / VSSP GM3 (18:1) induces up to three times more CD8 + T cell-specific cytotoxicity

[0056] In vivo antigen-specific CTL responses were evaluated in female C57BL / 6 mice (3 animals / group) immunized with three different preparations of the antigen OVA using nanoparticles of natural VSSP, VSSP GM3 (18: 1) or VSSP GM3 (18: 0) as adjuvants. The vaccine (200 μg of OMPC) was administered by SC route on days 0, 1 and 7. In parallel, splenocytes were obtained from naive animals and differentially labeled with CFSE (5 minutes at 37°C). After pulsing with the peptide SIINFEKL (1 μM; 90 minutes at 37°C and 5% CO2), highly labeled (5 μM) cells were used as target cells, while lowly labeled (0.33 μM) cells were used as controls without peptide loading. After the last immunization, washing was performed to remove free peptides, and the two types of splenocytes were mixed in equal proportions and injected into vaccinated animals. After 16 hours, the inguinal lymph nodes of the vaccinated mice were removed and all events corresponding to the two fluorescence intensities were measured by flow cytometry. The percentage of specific lysis was calculated according to the following formula: 100-[(CFSE 高 / CFSE 低 )x 100]. Figure 2 As shown in , the vaccine OVA / VSSP GM3 (18:1) induced a specific lysis close to 60%, which is a value almost three times higher than that achieved with the vaccines OVA / native VSSP and OVA / VSSP GM3 (18:0).

[0057] Example 3. The use of VSSP GM3 (18:1) adjuvant nanoparticles in cancer therapeutic vaccines has a greater anti-tumor effect than the use of natural VSSP

[0058] On day 0, 3 × 10 5 Three groups of female C57BL / 6 mice (10 animals / group) were inoculated by SC route with EG.7 (a variant of EL4 thymoma genetically modified to express OVA as a tumor neoantigen) cells. Then, three consecutive SC immunizations were performed on days 4, 5 and 11 with two different OVA vaccine preparations adjuvanted with natural VSSP (50 μg / dose) and a second preparation adjuvanted with VSSP GM3 (18:1) (200 μg / dose). The mice in the third group were injected with phosphate buffered saline at the same frequency and used as controls for the experiment. Starting from 7 days after tumor inoculation, tumor volume (TV) was measured twice a week. Animals were sacrificed when tumors showed necrosis or when tumor size exceeded 17 mm in diameter. On day 20 ( Figure 3), we see the advantage of therapeutic vaccine OVA / VSSP GM3 (18:1) over formulation OVA / natural VSSP (average TV: VSSPGM3 (18:1), 560 mm 3 ; Natural VSSP, 740mm 3 ; Control, 1640mm 3 )(p=0.037, ANOVA and Tukey test). More importantly, it was seen that in the group treated with the preparation OVA / VSSP GM3 (18:1), 50% of the animals did not show tumor progression in a challenge model such as EG7 (100% of the animals in the control group had progression). In animals vaccinated with OVA / natural VSSP, only 20% achieved protection from progression (p=0.012, chi-square test).

[0059] Example 4. Induction of high titers of specific antibodies against HER3 by the vaccine preparation HER3 / VSSP GM3 (18:0)

[0060] The mice (n=5) of BALB / c strain were immunized by SC route with a vaccine preparation containing 200 μg ECD-HER3 mixed with 200 μg VSSP GM3 (18:0). The immunization was carried out on days 0, 14, 28 and 42. Blood was drawn on day 35 (1st blood draw) and day 56 (2nd blood draw) to process serum, and the titer of specific antibodies against ECD-HER3 was determined by ELISA. For this reason, the plate was coated with 10 μg / mL of ECD-HER3 and incubated at 37°C. After the corresponding blocking, serum dilutions (1 / 100, 1 / 1000, 1 / 5000, 1 / 10000) were added. The reaction was visualized by using a conjugate (Sigma) of anti-mouse IgG antibody / peroxidase and the corresponding substrate of the enzyme.

[0061] Immunized mice developed specific antibodies of the IgG isotype, which reached titers up to 1 / 5000 ( Figure 4 ). This result demonstrates the potential of VSSP GM3(18:0) to activate the humoral branch of the immune system against very poorly immunogenic antigens, such as autologous tumor antigens.

[0062] Example 5. HER1+HER2 bivalent vaccine preparation adjuvanted with VSSP GM3 (18:0) induces higher titers of specific IgG antibodies compared to that adjuvanted with native VSSP

[0063] Mice (n=5) of the BALB / c strain were immunized with a vaccine preparation comprising a mixture of 100 μg ECD-HER1 and 100 μg ECD-HER2 adjuvanted with 200 μg natural VSSP (group 1) or 200 μg VSSP GM3 (18:0) (group 2). The immunization was performed by SC route on days 0, 14 and 28. Blood was drawn on day 35 to process serum. The titer of specific antibodies against the ECD of HER1 and HER2 was determined by ELISA technology. For this purpose, the plate was coated with 5 μg / mL of ECD-HER1 or 5 μg / mL of ECD-HER2 and incubated at 37°C. After the corresponding blocking, serum dilutions (1 / 100, 1 / 1000, 1 / 10000, 1 / 50000, 1 / 100000) were added. The reaction was visualized by using a conjugate of anti-murine IgG / alkaline phosphatase (Sigma) and the corresponding substrate of the enzyme. Pre-immune serum was used as a negative control.

[0064] All immunized mice developed specific antibodies of the IgG isotype. The titer of specific antibodies for ECD-HER1 was higher in Group 2, where the adjuvant used was VSSP GM3 (18:0), relative to the titer of antibodies developed in Group 1, where the adjuvant was native VSSP ( Figure 5a ). On the other hand, although no significant differences were obtained between the titers of antibodies induced in groups 1 and 2 with regard to specific antibodies against ECD-HER2, the titers in group 2, in which the vaccine preparation used VSSP GM3 (18:0) as an adjuvant, had an increasing trend. In view of the trend observed in the recognition of ECD-HER2, titration of polyclonal antibodies (PcAb) purified from immune sera against subdomains of ECD-HER2 expressed on filamentous phage (subdomains I, II, III and IV) was performed. To this end, ELISA plates were coated with 10 μg / mL of PcAb and incubated at 37°C. Then, phage samples expressing different subdomains of HER2 were added, and the colorimetric signal was visualized with an anti-phage antibody conjugated to peroxidase (GE-Healthcare). Figure 5b As can be seen, PcAbs from animals in which VSSP GM3 (18:0) was used as an adjuvant had higher reactivity against subdomains I, III, and IV of ECD-HER2 than antibodies from animals in which native VSSP was used as an adjuvant.

[0065] Example 6. Greater recognition of HER1+ / HER2+ tumor lines by antibodies induced by HER1+HER2 bivalent vaccine preparations adjuvanted with VSSP GM3 (18:0) compared to adjuvanted with native VSSP

[0066] Mice of the BALB / c strain (n=5) were immunized by the SC route with a vaccine preparation comprising a mixture of 100 μg ECD-HER1 and 100 μg ECD-HER2 adjuvanted with 200 μg native VSSP or with 200 μg VSSP GM3 (18:0). The immunization was carried out on days 0, 14 and 28, and the recognition of tumor lines expressing HER1 and HER2 was evaluated using the serum corresponding to day 35. The lines used were: vaginal epithelial carcinoma A431 (ATCC-CRL 1555), non-small cell lung cancer H125 (ATCC-CRC 5801) and breast cancer SKBR3 (ATCC-HTB 30). The measurements were performed by flow cytometry. For this purpose, 10 of each cell line were blocked with 2% fetal calf serum in phosphate-buffered saline solution. 5 Cells were then incubated with a 1 / 200 dilution of the serum mixture from each treatment group. The binding of specific antibodies to the receptors HER1 and HER2 in tumor cells was visualized by using a conjugate of anti-mouse IgG antibody / fluorescein isothiocyanate (FITC) (Sigma) and by acquiring at least 5000 cells in a flow cytometer. As a negative control, a mixture of pre-immune sera was used for each group evaluated. The sera induced by the vaccine preparation adjuvanted with VSSP GM3 (18:0) recognized the cells of the evaluated tumor lines with higher intensity ( Figure 6 ).

[0067] Example 7. Antibodies induced by a HER1+HER2 bivalent vaccine preparation adjuvanted with VSSP GM3 (18:0) cause a greater reduction in viability of the H125 tumor line than when adjuvanted with native VSSP

[0068] 10 5Cells of the H125 line were incubated for 72 hours with a 1 / 20 diluted serum mixture from BALB / c mice, which were immunized every fifteen days with three doses of a bivalent vaccine preparation of a mixture of 100 μg ECD-HER1 and 100 μg ECD-HER2 adjuvanted with VSSP GM3 (18:0) or natural VSSP. As a negative control, a mixture of pre-immune sera at the same dilution as the immune sera was used. As a positive control, 10 μM of the tyrosine kinase inhibitor AG1478 was used. The effect of immune sera on cell viability was determined by MTT colorimetric assay, and the absorbance was read at 540 nm and 630 nm. Cell viability was determined by the following formula:

[0069]

[0070] Figure 7 It was shown that serum induced by vaccine preparations adjuvanted with VSSP GM3(18:0) significantly reduced cell viability relative to vaccine preparations adjuvanted with native VSSP.

[0071] Example 8. Greater recognition of HER1+ tumor lines by antibodies induced by HER1 vaccine preparations adjuvanted with VSSP GM3 (18:0) compared to those adjuvanted with native VSSP

[0072] Mice of the C57BL / 6 strain (n=5) were immunized by the SC route with a vaccine preparation containing 200 μg of ECD-HER1 adjuvanted with 400 μg of native VSSP or with 400 μg of VSSP GM3 (18:0). The immunization was performed on days 0, 14, 28 and 42, and the recognition of the MDA-MB468 breast cancer line (ATCC-HTB 132) with a high expression of HER1 in its membrane was evaluated using the serum corresponding to day 56. The measurements were performed by flow cytometry. For this purpose, 10% of the cell line was blocked with 2% fetal bovine serum in phosphate buffered saline solution. 5 Cells were then incubated with a 1 / 100 dilution of the serum mixture from each treatment group. The binding of the specific antibody to the receptor HER1 in tumor cells was visualized by using a conjugate of anti-mouse IgG antibody / FITC (Sigma) and by acquiring at least 5000 cells in a flow cytometer. As a negative control, a mixture of pre-immune sera was used for each group evaluated. The sera induced by the vaccine preparation adjuvanted with VSSP GM3 (18:0) recognized the cells of the evaluated tumor lines with higher intensity ( Figure 8 ).

[0073] Example 9. Antibodies induced by HER1 vaccine preparations adjuvanted with VSSP GM3 (18:0) inhibit HER1 activation to a greater extent than those adjuvanted with native VSSP

[0074] Mice of the C57BL / 6 strain were used, divided into four groups (n=5) and immunized by SC route with the following vaccine preparations:

[0075] Group 1: 200 μg ECD-HER1 adjuvanted with 200 μg native VSSP;

[0076] Group 2: 200 μg ECD-HER1 adjuvanted with 200 μg VSSP GM3 (18:0);

[0077] Group 3: 200 μg ECD-HER1 adjuvanted with 400 μg native VSSP;

[0078] Group 4: 200 μg ECD-HER1 adjuvanted with 400 μg VSSP GM3 (18:0).

[0079] The immunization was performed at the 0th, 14th, 28th and 42nd days, and the serum corresponding to the 56th day was used to evaluate the ability of the antibodies produced to inhibit the phosphorylation of HER1 in the presence of EGF. For this reason, the cells of the H292 lung cancer system (CRL 1848) were incubated for 2 hours with a serum mixture of 1 / 100 dilution. Then, the cells were stimulated with 100ng / mL of EGF for 10 minutes to induce the activation of HER1. The effect of the serum on the phosphorylation of HER1 was measured by Western blotting, wherein specific antibodies were used to detect phosphorylated HER1 and beta-actin. As a control for the activation of HER1, cells treated with EGF were used. As a negative control for the inhibition of HER1, a mixture of pre-immune serum with a dilution of 1 / 100 was used, and as a positive control for inhibition, 10 μM of the tyrosine kinase inhibitor AG1478 was used. The obtained photographs were used to perform optical density determination, which enables data to be standardized.

[0080] The sera of the group immunized with the vaccine preparation using VSSP GM3 (18:0) as adjuvant inhibited the activation of the receptor HER1 in terms of phosphorylation more strongly than the sera of the group using native VSSP as adjuvant, both at a dose of 200 μg and at a dose of 400 μg, with the inhibition being more pronounced at a dose of 400 μg ( Fig. 9 ). This result demonstrates the superiority of the vaccine preparation comprising VSSP GM3 (18:0) as adjuvant in terms of the quality of the induced humoral immune response.

[0081] Example 10. Effect of the vaccine formulation PyrGnRHm1-TT / VSSP GM3 (18:0) on the prostate in Copenhagen rats

[0082] Male Copenhagen rats aged 8-12 weeks were used. The animals were divided into 3 groups with 10 animals in each group.

[0083] Group 1: Placebo (Montanide ISA 51VG / VSSP GM3 18:0);

[0084] Group 2: immunized with PyrGnRHm1-TT / Montanide ISA 51VG;

[0085] Group 3: immunized with PyrGnRHm1-TT / Montanide ISA 51VG / VSSP GM3 18:0.

[0086] To prepare the immunogen, the peptide PyrGnRHm1-TT was resuspended in distilled water and VSSP to a final concentration of 750 μg of peptide and 120 μg of VSSP / 250 μL. The mixture was then formulated with Montanide ISA 51VG in a ratio of 50:50 (v / v). Animals received 4 immunizations at a frequency of once every fifteen days.

[0087] Immunization with the peptide PyrGnRHm1-TT emulsified in Montanide ISA 51VG produced a significant reduction in prostate size compared to placebo (p<0.05). However, the difference was much greater when the peptide PyrGnRHm1-TT was emulsified with VSSP (p<0.01) ( Fig.10 ).

[0088] Example 11. Induction of anti-tumor response in the Dunning R3327-H model by using the peptide PyrGnRHm1-TT

[0089] Adult Copenhagen rats were implanted SC with tumor fragments (2×2×2 mm) of the Dunning R3327-H murine tumor model in the distal region of the right hind limb. The animals were divided into four groups that received different treatments:

[0090] Group 1: Placebo: Montanide ISA 51VG / VSSP GM3 18:0;

[0091] Group 2: castrated;

[0092] Group 3: immunized with peptide PyrGnRHm1-TT / Montanide ISA 51VG;

[0093] Group 4: Immunized with peptide PyrGnRHm1–TT / Montanide ISA 51VG / VSSP GM318:0.

[0094] When the tumors reached a diameter of approximately 10 mm, immunizations were started for each group according to the corresponding treatment. A total of 7 immunizations were performed, once every 15 days.

[0095] exist Fig.11 In the placebo group (group 1), a dramatic tumor growth was observed, which was different from the castration group and the immunization group (groups 2, 3 and 4), in which a significant inhibition of tumor growth was seen (p < 0.01). This difference became more significant in the variant PyrGnRHm1-TT emulsified with VSSP GM3 18:0 (p = 0.005).

Claims

1. An adjuvant comprising nanoparticles formed by dispersing hydrophobic proteins of the outer membrane complex of the bacterium Neisseria meningitidis together with a fully synthetic variant of the ganglioside GM3, wherein the fatty acid present in the ceramide of the ganglioside GM3 is stearic acid (18:0) or oleic acid (18:1).

2. A vaccine composition comprising the adjuvant of claim 1, and an antigen selected from the group consisting of: - HER1, HER2 or HER3, alone or in combination; and -peptide PyrGnRHm1-TT.

3. The vaccine composition of claim 2, comprising an additional adjuvant selected from the group consisting of: -aluminium oxide, and -Oily adjuvant.

4. Use of a vaccine composition according to any one of claims 2 to 3 for the preparation of a medicament for the treatment of cancers expressing HER1, HER2, HER3 and / or the peptide PyrGnRHm1-TT.

5. Use of the vaccine composition according to claim 2 in the preparation of a medicament for stimulating an antigen-specific humoral immune response, wherein the fatty acid present in the ceramide of the ganglioside GM3 is stearic acid (18:0).

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