Autologous cancer vaccine enriched with HSP110

Abstract

The invention relates to an autologous cancer vaccine and its preparation method. The autologous vaccine according to the present invention is based on hydroxyapatite and / or tricalcium phosphate particles onto which tumor proteins are adsorbed, these tumor proteins being enriched in HSP110.

Description

Title of the invention: Autologous cancer vaccine enriched with HSP110 technical field

[0001] The present invention relates to the treatment of cancer by immunotherapy. The present invention relates particularly to the production of autologous cancer vaccines. Previous technique

[0002] Immunotherapy is a recognized and well-established therapeutic alternative for treating cancer. It encompasses several different therapies, all based on stimulating the patient's immune system to recognize and attack cancer cells.

[0003] Immune system cells are normally capable of monitoring and detecting the presence of abnormalities within the body's cells. However, most tumors develop several mechanisms to evade this monitoring; this results in immune system tolerance towards the tumor. Stimulating immune cells, such as T lymphocytes, so that they specifically recognize tumor cells makes it possible to overcome this tolerance.

[0004] This stimulation can, for example, be achieved by directly exposing (in vitro or in vivo) tumor antigens to macrophages or related cells so that antigen-presenting cells (APCs) stimulate T lymphocytes. This is the concept of a therapeutic cancer vaccine. For in vivo stimulation, the principle is to extract abnormal proteins from the tumor and reintroduce them in a form visible to the immune system.

[0005] Therapeutic cancer vaccines may be based on the use of heat shock proteins (HSPs) such as gp96 or HSP70. These proteins are chaperone molecules and associate with numerous peptides, including tumor-specific antigens for each patient. They thus constitute a molecular fingerprint of the tumor to be eradicated and differ from one patient to another and from one tumor to another. For the same tumor, this fingerprint evolves over time.

[0006] This vaccination strategy requires purifying the vaccine proteins from each tumor against which they are to immunize the patient, and at a specific time. The purification protocol is lengthy, difficult to industrialize, and subject to multiple endotoxin contaminations. It is standard practice to purify HSPs from a tumor homogenate that undergoes a series of centrifugations, precipitations, Con A chromatography, electrophoretic analysis, and Mono Q FPLC chromatography. US patents Nos. 6,447,781, 6,436,404, 6,410,028, 6,383,494, and 6,030,618 describe methods for purifying HSP proteins using SEPHAROSE Con ​​A chromatographic columns.

[0007] More recently, new techniques for the simpler purification of tumor proteins have been developed. Patent application WO2006 / 122914 describes the usefulness of using hydroxyapatite particles (HAP) to purify vaccine proteins from tissue extracts. This application specifically describes a one-step process for preparing tumor antigens in a form recognizable by the immune system, enabling large-scale application by personnel without biochemical qualifications. WO2006 / 122914 further describes how HAP powders can be used as vaccine adjuvants and how hydroxyapatite powder adsorbed with tumor-specific antigens can be used directly as a drug against that tumor.Thus, the same PAH powder can be used both to purify tumor-specific proteins and to stimulate the immune system against them when the powder and proteins are injected together.

[0008] Patent application WO2014 / 184453 further describes the development of a process for producing an improved hydroxyapatite and / or tricalcium phosphate powder. The powder thus obtained is contacted with tumor proteins directly purified from tumor biopsies, thereby marking the cellular identity of the tumor, in order to produce a therapeutic antitumor vaccine that increases the level of innate immunity without additional toxicity or side effects.

[0009] This technique is safe, can be used in conjunction with chemotherapy, and produces no toxic residues. The inventors have demonstrated that T lymphocytes are able to recognize tumor cells after vaccination. The vaccination cycle is very similar to that used for the anti-infective vaccine. The vaccine is injected into the subcutaneous tissue, and several injections are required (one per week for 4 weeks and one per month for 4 months).

[0010] This type of immunotherapy can make the difference between remission and a cure for certain cancers with a poor prognosis. This has been demonstrated, for example, in veterinary medicine, where this vaccination increases the survival rate of dogs compared to chemotherapy alone for a fatal cancer such as high-grade B-cell lymphoma (DLBCL). Excellent results have also been obtained for solid tumors such as bone cancers (osteosarcomas), mast cell tumors, and melanomas.

[0011] Finally, the international application published under reference WO2019 / 175500 discloses the possibility of producing autologous vaccines from hydroxyapatite and / or tricalcium phosphate powder in contact with tumor proteins contained not in a tumor biopsy, but directly in a serum or plasma sample. This procedure greatly facilitates the production of autologous vaccines prepared from hydroxyapatite and / or tricalcium phosphate powder, and makes it possible to obtain results just as satisfactory as those obtained with vaccines prepared from tumor biopsies.

[0012] Autologous vaccines prepared from hydroxyapatite powder and / or tricalcium phosphate therefore represent a major advance in cancer treatment. Nevertheless, there remains a need for more effective treatments offering improved therapeutic efficacy. Summary

[0013] Quite surprisingly, the present inventors have demonstrated that it is possible to improve the therapeutic properties of autologous vaccines prepared from hydroxyapatite powder and / or tricalcium phosphate, by ensuring that they contain, as tumor proteins, a large proportion of HSP110 heat shock proteins.

[0014] The inventors have indeed demonstrated that autologous vaccines prepared from tumor cell lines not expressing HSP110 resulted in significantly lower levels of apoptosis and tumor regression than those obtained from vaccines enriched in HSP110. A high HSP110 content leads to improved intratumoral lymphocyte infiltration and potentiation of the T-cell response.

[0015] Without wishing to be bound by any particular theory, the present inventors believe that the high HSP110 content of the vaccine according to the invention allows for the binding of a large number of tumor antigens present in the biological sample obtained from the patient. The concentration of an HSP110 vaccine in a biological sample would thus increase the content of tumor antigens, thereby potentiating the effects of the vaccine.

[0016] Also, according to a first aspect, the present invention relates to an anticancer vaccine comprising hydroxyapatite and / or tricalcium phosphate particles and tumor proteins, in which at least 5% by weight of said tumor proteins are HSPHO.

[0017] The present invention also relates to a method for preparing an autologous cancer vaccine, said method comprising the following steps:

[0018] a) extraction of proteins contained in a biological sample obtained from a cancer patient; and

[0019] b) contacting the extracted proteins with hydroxyapatite and / or tricalcium phosphate particles,

[0020] in which said process further includes a step of verifying the presence of HSP110 among the proteins extracted in step a. and / or in the mixture obtained after step b.

[0021] The present invention also relates to the therapeutic use of the vaccine described above for the treatment of cancer. Brief description of the figures Fig. 1

[0022] [Fig. 1] A. Evolution of tumor volume of CT26 cell line in Balb / c mice treated with APAVAC HSP110+, APAVAC shHSPHO, or HA (+SEM) (n=10). B. Evaluation of cell apoptosis by immunofluorescence on tumor sections labeled for caspase 3 (C3C). The ratio of C3C+ cells to the total number of cells is shown (n=5). *p<0.5. Detailed description of the invention

[0023] As indicated above, the present inventors have demonstrated that the presence of the heat shock protein HSP110 significantly enhances the effects of an autologous cancer vaccine composed of hydroxyapatite (HA) particles and / or tricalcium phosphate onto which tumor proteins are adsorbed. The present invention therefore relates to an autologous cancer vaccine enriched with HSP110.

[0024] Also, according to a first aspect, the present invention relates to an anticancer vaccine comprising hydroxyapatite and / or tricalcium phosphate particles and tumor proteins, in which at least 5% by weight of said tumor proteins are HSPHO (heat shock protein 110).

[0025] The concept of "vaccine" is well known to those skilled in the art. This term can generally be defined as a pharmaceutical composition which, once administered to a living organism, stimulates its immune system and develops protective immunity against a specific disease.

[0026] The vaccine according to the present invention is an anti-cancer vaccine, that is to say, in a patient with cancer, it aims to create an immune response against cancer cells.

[0027] In the context of the present invention, "cancer" can be any cancer for which immunotherapy may be indicated. Cancer can particularly be chosen from a non-exhaustive list including melanomas, carcinomas or adenocarcinomas (which develop from epithelial cells and / or glandular cells), and sarcomas (which develop from tissue cells). connective or muscular), tumors of the central nervous system, tumors of the hematopoietic system such as leukemias and lymphomas. We can also include various cancers of infectious origin, the most representative of which are cervical cancer, primary liver cancer, and gastric cancers.

[0028] In a particular embodiment, the cancer is chosen from the group consisting of osteosarcomas, B or T lymphomas, breast tumors, melanomas, hemangiosarcomas, mastocytomas, fibrosarcomas, brain or central nervous system tumors, schwanomas, mesotheliomas, seminomas, teratomas and blastomas.

[0029] In a preferred embodiment, the cancer is chosen from a glioblastoma, a sarcoma (such as osteosarcoma or fibrosarcoma), a melanoma, a carcinoma or an adenocarcinoma.

[0030] In a particular embodiment, the cancer is disseminated cancer, i.e. presenting metastases (patients classified in the NxMx category according to the TNM classification).

[0031] An “autologous” vaccine refers to a vaccine in which the tumor proteins / antigens are obtained from the patient intended to be vaccinated.

[0032] The "patient" or "subject" is a mammal. It can be human or animal, such as a dog, a horse or a cat.

[0033] The concept of "tumor proteins / antigens" is well known to those skilled in the art. These are proteins and / or molecules specifically expressed by tumor cells and which can be recognized by T and B lymphocytes.

[0034] In the context of the present invention, some of the tumor proteins contained in the vaccine are HSP110. “HSP110” (for heat shock protein 110), also known as HSP105 or HSPH1, is a protein belonging to the family of large heat shock proteins. It is encoded by the hsphl gene. Like all heat shock proteins, HSP110 acts as a chaperone and is produced in response to cellular stress. HSP110 is well known to those skilled in the art (see, for example, Gibouin, Vincent Cabaud. Functional characterization of the first HSPHO inhibitors. Diss. Université Bourgogne Franche-Comté, 2023; Berthenet, Kevin. The HSP110 protein: role in tumor development and on the immunogenicity of colorectal cancer. Diss. Université de Bourgogne, 2015). On average, HSP110 represents 0.5 to 1% by weight of total cytosolic proteins after activation by heat shock (Easton, Douglas P., Yoshiyuki Kaneko, & John R. Subjeck.Cell stress & chaperones 5.4 (2000): 276). The vaccine according to the invention is enriched in HSP110 proteins, meaning that the proportion of HSP110 proteins relative to total proteins is significantly higher than that measured in vivo. Thus, within the framework of the present invention, at least 5%, particularly. at least 10%, more particularly at least 15%, preferably at least 20%, even more preferably at least 30% by weight of the tumor proteins contained in the vaccine are HSPHO.

[0035] Knowing the molecular characteristics (size, sequence, structure, etc.) of HSPl 10, a person skilled in the art is perfectly capable of determining its content in a given medium, and particularly the percentage of HSPl 10 present in a protein mixture. Several methods for doing so are available and commonly used. Such techniques include, for example, ELIS A, protein transfer (Western blot), mass spectrometry, fluorimetric and / or colorimetric methods, or methods based on absorbance measurement. All these methods are well documented and easily performed.

[0036] "Hydroxyapatite" is a mineral belonging to the calcium phosphate family, The formula is CaiO(PO4)6(OH)2, and the crystal structure is hexagonal. Hydroxyapatite is the hydroxylated member of the apatite group. The ions in the crystal lattice are substitutable by other ions of similar charge and size. There are also tunnels in the lattice that can accommodate small molecules such as certain amino acids. These characteristics give this mineral very specific adsorption properties.

[0037] The hydroxyapatite particles according to the present invention are obtained via the same process as that described in application WO2014 / 184453. This document describes, in particular, a process for producing calcium phosphate hydroxyapatite particles, CaIO(PO4)6(OH)2, consisting of a slow precipitation at high temperature, obtained by the double decomposition of a calcium salt and a phosphorus salt in a basic medium. The reaction takes place at a constant temperature in a large reaction volume and is followed by a maturation phase and a washing phase with water. The precipitate thus obtained undergoes a further transformation step: solid / solution separation. This last step is carried out either by filtration, oven drying and crushing, or by spray drying using a fluidized bed.Regardless of the solid / solution separation technique chosen, the powder will undergo two transformation steps specific to the application of the invention: a particle size selection step by dry sieving to retain only the particle size range of interest, less than 25 µm or between 25 and 45 µm, followed by a final step during which the powder will be sintered at an optimal temperature to ensure melting of the grains, resulting in very specific powder surface conditions (preferably greater than or equal to 30 m² / g). These last two steps can be reversed: selection then sintering, or sintering then selection. "Sintering" is a process consisting of heating a powder. without bringing it to the point of melting. In the context of the present invention, sintering is, for example, carried out at a temperature between 400°C and 600°C. The resulting PAH can be in powder form and undergo one or more washings.

[0038] The vaccine according to the present invention comprises this hydroxyapatite and / or tricalcium phosphate powder loaded with tumor antigens. The present invention also relates to a method for preparing such a vaccine.

[0039] In this context, the present invention relates to a method for obtaining an autologous cancer vaccine, said method comprising the following steps:

[0040] a. extraction of proteins contained in a biological sample obtained from a patient with cancer;

[0041] b. contacting the extracted proteins with hydroxyapatite and / or tricalcium phosphate particles,

[0042] wherein said process further comprises a step of verifying the presence of HSP110 among the proteins extracted in step a. and / or in the mixture obtained after step b.

[0043] The “biological sample” can be any sample from which Tumor antigens can be extracted. These samples can be liquids, tissues, cell samples, organs, biopsies, etc.

[0044] According to a particular embodiment, the biological sample is a tumor sample, in particular a tumor biopsy. A tumor sample is typically obtained by taking a cell sample in vivo, directly from the patient's tumor. Preferably, the sample is taken by puncture from the tumor, for example, from a biopsy of a patient with cancer. It may also consist of tumor samples comprising tumor cells from the patient to be treated, previously collected, such as homogenates, lyophilized products, dialysates, or a centrifuged pellet.

[0045] According to another embodiment, the biological sample may also be a blood sample such as a whole blood sample, a serum sample, or a plasma sample. A serum or plasma sample is typically obtained from a whole blood sample taken from the patient to be treated. The blood is subjected to centrifugation to separate the heavier components from the supernatant. This supernatant constitutes the plasma if it is anticoagulated blood or the serum if the blood has coagulated naturally.

[0046] In the context of the present invention, the proteins contained in the biological sample are extracted by any method known and commonly used by those skilled in the art. Typically, the mass of the tumor proteins / antigens extracted from the sample is between 60 kDa and 170 kDa.

[0047] When the sample is a tumor sample, the tumor (cytoplasmic) proteins can be extracted as follows:

[0048] - possibly freezing of tumor tissue;

[0049] - crushing of tumor tissue,

[0050] - solubilization or suspension of cytoplasmic tumor antigens in a solution (for example a NaHCO3 solution);

[0051] -centrifugation,

[0052] - separation of the pellet and the supernatant.

[0053] A person skilled in the art is also familiar with a large number of techniques for extracting proteins from a serum or plasma sample. Typically, tumor proteins / antigens are extracted by precipitating the serum or plasma sample in a saline solution; the resulting mixture is then centrifuged, and the pellet obtained represents the extracted proteins.

[0054] In the context of the present invention, the extracted proteins and / or the proteins adsorbed onto the hydroxyapatite and / or tricalcium phosphate particles are analyzed to verify that they contain HSPl 10. This step can therefore be carried out between step a and step b, and consist of verifying that the proteins extracted in step a do indeed contain HSPl 10, but it can also be carried out after step b and consist of verifying that the proteins contained in the mixture after step b (i.e., after adsorption onto the hydroxyapatite and / or tricalcium phosphate particles) do indeed contain HSPl 10. This verification can be carried out between steps a and b only, after step b only, but also between steps a and b and after step b. According to a particular embodiment, the step of verifying the presence of HSPi 10 is carried out after step b.That is, after contacting the extracted proteins with hydroxyapatite and / or tricalcium phosphate particles. This analysis can be performed by any method known to those skilled in the art, for example, by subjecting the proteins adsorbed onto the particles to SDS-PAGE electrophoresis. As indicated above, those skilled in the art have several methods at their disposal for detecting and quantifying the presence of HSPL10 in a given medium.

[0055] According to a particular embodiment, the step of verifying the presence of HSP110 among the proteins extracted from the biological sample and / or among the proteins adsorbed onto the hydroxyapatite and / or tricalcium phosphate particles consists of verifying that the HSP110 protein is indeed present in proportions representing at least 5% by weight of the total proteins (i.e., either of the proteins extracted in step a, or of the proteins adsorbed onto the hydroxyapatite and / or tricalcium phosphate particles after step b). The process according to the invention may further include a step in which the proteins extracted in step a or the Proteins adsorbed onto hydroxyapatite and / or tricalcium phosphate particles in step b are enriched in HSP110 to contain at least 5% by weight of HSPHO. This means increasing the HSP110 content until it represents at least 5% by weight of the total proteins extracted in step a and / or the proteins adsorbed onto the hydroxyapatite and / or tricalcium phosphate particles. This can be achieved by specifically selecting and purifying the HSP110 protein from a biological sample obtained from the patient (the sample being as defined above), and / or by adding this purified HSP110 to the total extracted proteins and / or the mixture obtained in step b to increase the HSP110 concentration.

[0056] In a particular embodiment, all the proteins / antigens extracted from the biological sample, including the HSP110 protein, are used and contacted with hydroxyapatite and / or tricalcium phosphate particles to prepare the vaccine according to the present invention. That is to say, the entire pellet of extracted proteins / antigens is contacted with the hydroxyapatite particles.

[0057] Typically, in order to produce the vaccine based on hydroxyapatite particles and / or tricalcium phosphate according to the invention, tumor proteins / antigens (including HSP110) are brought into contact with hydroxyapatite particles by passing said tumor proteins / antigens over a column of hydroxyapatite particles, such as a chromatography column. The tumor antigens may optionally be brought into contact in solution and then washed by centrifugation. The tumor proteins / antigens are then adsorbed onto the surface of the hydroxyapatite particles. The hydroxyapatite particles may particularly be in powder form.

[0058] When a chromatography column is used, it can, for example, be put under pressure.

[0059] Once the hydroxyapatite particles are loaded with tumor proteins / antigens, they are suspended in an injection liquid and injected into the patient from whom the biological sample was obtained.

[0060] Typically, the injection liquid includes an organic injection-facilitating agent such as carboxymethylcellulose.

[0061] Typically, the autologous vaccine prepared within the framework of the present invention is in the form of a suspension.

[0062] The autologous vaccine according to the present invention therefore comprises HA and / or tricalcium phosphate particles having adsorbed the patient-specific tumor antigens (obtained from a biological sample of said patient), including HSP110, and having been resuspended.

[0063] This vaccine is then preferably administered by injection, for example by subcutaneous or intradermal injection. The vaccine may be administered orally or by any other means allowing transmucosal vaccination.

[0064] In this context, the present invention also relates to the use of the autologous vaccine described above in therapy, and particularly in the treatment of cancer in the patient from whom the tumor proteins / antigens were obtained. The invention therefore relates to the autologous vaccine as described herein for its use in the treatment of cancer.

[0065] Another aspect further relates to a method of treating a patient suffering from cancer, said method comprising the following steps:

[0066] i) extraction of proteins contained in a biological sample obtained from a patient with cancer;

[0067] ii) suspension in an injection liquid of the extracted proteins with hydroxyapatite and / or tricalcium phosphate particles; and

[0068] iii) injection of the mixture obtained in step ii) into the patient from whom the serum or plasma sample used in step i) was obtained

[0069] wherein said method further includes checking for the presence of HSP110 among the proteins extracted in step i) and / or among the proteins adsorbed on the hydroxyapatite and / or tricalcium phosphate particles at the end of step ii).

[0070] The patient may receive one or more injections, doses of vaccine. A dose generally comprises between 30 and 50 mg of hydroxyapatite and / or tricalcium phosphate and between 1000 and 2000 pg of protein.

[0071] Preferably, the patient may receive several injections spaced out over time, for example, several days, weeks, or months. In a preferred embodiment, the injections are separated by one week during the first month and then by one month for four months.

[0072] Each injection can ideally be prepared from a new sample of tumor proteins from the patient in case of signs of vaccine escape or ineffectiveness, particularly when there is an increase in the level of tumor markers.

[0073] The autologous vaccine according to the present invention can be used in combination with another anticancer treatment such as radiotherapy, chemotherapy or another immunotherapeutic agent.

[0074] The present invention is presented in more detail in the examples below. These examples are provided for illustrative purposes. Examples

[0075] MATERIALS AND METHOD

[0076] Cell line culture

[0077] The CRC CT26 cell line was purchased from the American Type Culture Collection (ATCC). The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. The CT26 cells were engineered to express the hsphl-targeting shRNA (TRCN0000275617) using the Sigma-Aldrich Mission pLKO.l hPGK-Puro-CMV-tGFP plasmids and were cultured in RPMI containing puromycin (ThermoFisher Scientific, 2.5 pg / mL), 10% SBF, penicillin 100 U / mL, streptomycin 100 pg / mL, and amphotericin B 0.25 pg / mL. The cells were maintained at 37 °C and 5% CO2.

[0078] Production of the (syngeneic) vaccine

[0079] CT26 WT / shHSPl 10 cells (5 x 10⁵) were injected subcutaneously into the right flank of female BALB / c mice (Charles River Laboratories). The mice were euthanized when the tumors reached 500 mm³, and the tumors were collected for subsequent production of AP AVAC according to the standard manufacturing protocol for the treatment in community veterinary practice.

[0080] Western Blot

[0081] The proteins were separated in an 8-10% SDS polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The primary antibodies were incubated as follows: HSP-110 (1 / 1000) and β-Actin (1 / 1000). The secondary antibodies were incubated with the appropriate antibodies (1 / 10000) coupled to horseradish peroxidase (HRP).

[0082] Adaptation of the vaccination schedule

[0083] CT26 cells (5 x 10⁵) were injected subcutaneously into the right flank of female BALB / c mice. The vaccination schedule consisted of subcutaneous administration of AP AVAC or HA (vehicle) three times a week during the first week and once a week during the second week. The mice were euthanized one week after the completion of the vaccination schedule, and immunohistochemistry (IHC) and flow cytometry analyses were performed.

[0084] Immunohistochemical / immunofluorescence analysis

[0085] Histological studies were performed on 5 µm tumor sections. The tissues were deparaffinized, and antigen recovery was carried out using citrate buffer for staining cleaved caspase 3 (C3C) and EDTA buffer for staining CD4 and CD8. Endogenous peroxidases were inhibited with 3% hydrogen peroxide (H2O2) solution. The tissues were fixed with 3% BSA and incubated for 1 hour with the following dilutions: CD4 (1 / 500), CD8 (1 / 500), Casp3 (1 / 200). Horseradish peroxidase (HRP)-coupled secondary antibodies were selected based on the species used for the primary antibodies.

[0086] Epitope development was performed using the NovaRed kit (Vector Labs), and the nuclei were counterstained with Harris hematoxylin. Finally, the slides were mounted with Coverquick 3000 organic mounting medium (VWR). Images were captured using the Axioscope upright optical microscope (Cari Zeiss GmbH) equipped with the Gryphax Naos Progrès Jenoptik camera. Five images per sample were acquired randomly, and image analysis was performed using ImageJ software.

[0087] Analysis by flow cytometry

[0088] CT26 tumors were subjected to mechanical dissociation and enzymatic digestion using the Tumor Dissociation Kit mouse and the MACS Dissociator soft (Miltenyi Biotec) in accordance with the manufacturer's instructions.

[0089] The single-cell suspension was then washed twice with RPML1640, resuspended in RPMI supplemented with 10% FBS and 1% PSA, and stimulated in vitro with Cell Stimulation Cocktail (eBioscience) and Protein Transport Inhibitor (PTI, eBioscience) for 3 hours at 37°C. The cells were then harvested and stained with antibodies against CD45, CD4, CD8, PD-1, LAG-3, and Tim-3 before being fixed and permeabilized. The permeabilized cells were then stained for Gzb, IL-2, TNF, and IFN-γ. All data were collected on an AURORA flow cytometer and analyzed using FlowJo software;

[0090] RESULTS

[0091] Determination of the specific role of HSP 110

[0092] To study the role of Hspl 10 in the mode of action of AP AVAC (an autologous vaccine prepared from hydroxyapatite powder as described in the published application under reference WO2014 / 184453), vaccines lacking Hspl 10 were produced. To this end, CT26WT or HSP110 knockdown cells were implanted into BALB / c mice, thereby producing CT26 tumors with and without HSP110 expression.

[0093] These tumors were subsequently used to develop the "APAVAC HSP110+" and "APAVAC shHSPl 10" vaccines. The absence of HSP110 in the HSP110 knockdown tumors and in APAVAC shHSPl 10 was confirmed by western blot analysis.

[0094] To study the impact of HSP110 on the antitumor effect of APAVAC, Balb / c mice were xenografted with CT26 cells. After tumor detection, the vaccination program as described above was implemented. Tumor-bearing mice were treated with APAVAC HSP110+, APAVAC shHSPl 10, or HA (control consisting of hydroxyapatite powder alone, without tumor proteins), and tumor size was monitored by caliper measurements every 2 days.

[0095] The results revealed that AP AV AC HSP110+ resulted in a significant reduction in tumor growth compared to HA, thus confirming the antitumor effects of AP AVAC. Importantly, mice treated with AP AVAC shHSPl 10 exhibited increased tumor progression compared to those treated with AP AVAC HSP110+ (Fig. IA). This observation was confirmed by analyzing tumor cell apoptosis.

[0096] We confirmed this observation by analyzing the apoptosis of tumor cells, assessed by staining of cleaved caspase-3 in tumor slides, showing that AP AVAC HSP110+ induced higher levels of apoptosis in tumor cells than AP AVAC shHSPl 10 and HA (Fig. IB).

[0097] Role of HSPs in APAVAC-mediated T-lymphocyte infiltration in the tumor microenvironment

[0098] It has previously been shown that APAVAC-type vaccines induce increased infiltration of CD4+ and CD8+ T cells into the tumor. To investigate the impact of HSP110 on this effect of APAVAC, an immunohistochemical analysis of CD4+ and CD8+ T cell infiltration in mouse tumor slides was performed. The results revealed a notable increase in the presence of CD4+ and CD8+ T cells in tumors of mice treated with APAVAC HSP110+, compared to the levels observed in mice treated with HA. However, this effect is significantly reduced in mice treated with APAVAC shHSP10. These results indicate the involvement of HSP110 in T cell infiltration into the tumor microenvironment.

[0099] Effect of APAVAC and role of HSP110 in T cell functionality in the tumor microenvironment

[0100] To evaluate the functionality of tumor-infiltrating T cells after vaccination, tumor-infiltrating lymphocytes (TILs) were isolated from CT26 tumor-bearing mice subjected to various treatments for flow cytometry analysis. T cell activation was assessed by analyzing the percentage of CD4+ T cells expressing the immune control markers PD-1 and LAG-3, as well as the percentage of CD8+ T cells expressing PD-1 and TIM-3. T cell functionality was determined by analyzing the intracellular expression of riFN-γ, Granzyme B, TNF-α, and IL-2.

[0101] The results showed that treatment with APAVAC HSP110+ induces significantly higher levels of CD4+ T cells co-expressing PD-1 and LAG-3, as well as CD8+ T cells co-expressing PD-1 and TIM-3, compared with treatment with HA.

[0102] Despite the common association of these markers with a state of exhaustion, CD8+ and CD4+ T cells from mice treated with APAVAC HSP110+ presented a more efficient phenotype. Specifically, CD4+ T cells exhibited significantly higher levels of intracellular IFN-γ, IL-2, and TNF-α compared to tumors from HA-treated mice. Furthermore, CD8+ T cells displayed significantly higher levels of IFN-γ and TNF-α. Consistent with these findings, similar trends were observed in the expression of IL-2 and Granzyme B.

[0103] Interestingly, after the removal of HSP110 from APAVAC (APAVAC shHSPHO), mice showed a marked decrease in lymphocyte activation and functionality, to levels comparable to those in the HA-treated group. This is indicated by a reduced percentage of CD4+ co-expressing PDI and LAG3 and CD8+ co-expressing PDI and TIM3, as well as a decrease in intracellular cytokine expression compared to the values ​​observed in the APAVAC HSP110+ treated group. These data suggest that APAVAC elicits a T-cell response against the tumor, with HSP110 playing a crucial role in mediating this effect.

[0104] CONCLUSION

[0105] The development of a murine model to validate the mode of action of the autologous cancer vaccine, AP AVAC, has revealed its promising potential as an immunotherapeutic approach. AP AVAC HSP110+ demonstrates its ability to elicit a robust antitumor immune response, as evidenced by the increased infiltration of CD4+ and CD8+ T cells into the tumor microenvironment.

[0106] The functionality of these infiltrated T cells is demonstrated by the expression of immune checkpoints, the production of pro-inflammatory cytokines and the release of granzyme B. In addition, the efficacy of the immune response induced by AP AVAC HSP110+ is demonstrated in vivo, where treated mice show a significant reduction in tumor growth and an increase in apoptosis, and ex vivo where tumor cells co-cultured with splenocytes from mice treated with APAVAC HSP110+ show a higher mortality rate.

[0107] The study of the role of HSP110, using an HSP110-depleted APAVAC, highlights its crucial importance in achieving a therapeutic response, affecting both tumor progression and T-lymphocyte infiltration into the tumor microenvironment. This underscores the potential of HSP110 as a key contributor to boosting the therapeutic efficacy of APAVAC.

Claims

Demands

1. Cancer vaccine comprising hydroxyapatite and / or tricalcium phosphate particles and tumor proteins, wherein at least 5% by weight of said tumor proteins are HSPi 10 (heat shock protein 110).

2. A process for preparing an autologous cancer vaccine, said process comprising the following steps: a. extraction of proteins from a biological sample obtained from a cancer patient; b. contacting the extracted proteins with hydroxyapatite and / or tricalcium phosphate particles, wherein said process further comprises a step of verifying the presence of HSP110 among the proteins extracted in step a. and / or in the mixture obtained after step b.

3. A method according to claim 2, wherein the verification step consists of verifying that at least 5% by weight of the proteins extracted in step a. and / or of the proteins contained in the mixture obtained after step b. are of HSPI 10.

4. A process according to claim 2 or 3, wherein said process further comprises a step in which the proteins extracted in step a. and / or the proteins contained in the mixture obtained after step b. are enriched in HSP110 so as to contain at least 5% by weight of HSP110.

5. A method according to any one of claims 2 to 4, wherein said biological sample is a tumor sample.

6. A method according to any one of claims 2 to 4, wherein said biological sample is a serum or plasma sample.

7. A method according to any one of claims 2 to 6, wherein said patient is a human, a dog, a horse or a cat.

8. A method according to any one of claims 2 to 7, wherein the cancer is selected from melanomas, carcinomas, adenocarcinomas, sarcomas, tumors of the central nervous system, leukemias, lymphomas and cancers of infectious origin. 16

9. Autologous vaccine obtained according to the process of any one of claims 2 to 8.

10. Vaccine claim 1 or 9 for its use in the treatment of cancer.

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