Autologous cancer vaccine

Abstract

The present invention relates to an autologous cancer vaccine and a method for producing the autologous cancer vaccine, comprising the following steps: a) extracting proteins from a serum or plasma sample obtained from a cancer patient; and b) contacting the proteins extracted in step a) with hydroxyapatite and / or tricalcium phosphate particles.

Description

[Technical Field]

[0001] The present invention relates to the treatment of cancer by immunotherapy, in particular to the production of autologous cancer vaccines. [Background technology]

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

[0003] Cells of the immune system are normally able to monitor and detect the presence of abnormalities within the body's cells. However, most tumors have evolved several mechanisms to evade this surveillance, resulting in immune system tolerance of the tumor. This tolerance can be enhanced by stimulating immune cells, such as T lymphocytes, to specifically recognize tumor cells.

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

[0005] Therapeutic cancer vaccines can be based on the use of heat shock proteins (HSPs), such as gp96 and HSP70. These proteins are chaperone molecules that bind to various peptides, including antigens specific to each patient's tumor. Thus, they constitute the molecular fingerprint of the tumor to be eradicated, which differs from patient to patient and from tumor to tumor. For the same tumor, this fingerprint evolves over time.

[0006] This vaccination strategy requires the purification of the vaccinating protein from each tumor against which the patient needs to be immunized, at a given time. The purification protocol is lengthy, difficult to industrialize, and subject to repeated contamination by endotoxins. Traditionally, HSPs have been purified from disrupted tumor material by a series of centrifugation, precipitation, Con A chromatography, electrophoretic analysis, and Mono QFPLC chromatography. U.S. Patent Nos. 6,447,781, 6,436,404, 6,410,028, 6,383,494, and 6,030,618 describe methods for purifying HSP proteins, including the use of a Con A chaperone chromatography column.

[0007] Patent application WO2006 / 122914 describes the usefulness of using hydroxyapatite particles (HAP) to purify vaccination proteins from tissue extracts. This application more specifically describes a one-step method for producing tumor antigens in a form recognizable by the immune system, which can be used on a large scale by staff who are not qualified biochemists. WO2006 / 122914 also discloses that HAP powder and other calcium salts can be used as vaccination adjuvants, and that hydroxyapatite powder adsorbed with tumor-specific tumor antigens can be used as a drug against the tumor. Thus, the same HAP powder can be used both to purify tumor-specific proteins and to stimulate the immune system against the proteins when the powder and protein are injected together.

[0008] The development of an improved method for producing hydroxyapatite and / or tricalcium phosphate powders is also described in patent application WO 2014 / 184553. The powders thus obtained are contacted with tumor proteins purified directly from tumor biopsies, thereby marking the cellular identity of the tumor, in order to generate therapeutic antitumor vaccines that increase the level of innate immunity without further toxicity or secondary effects.

[0009] This technique is safe, can be combined with chemotherapy, and does not produce toxic residues. The inventors demonstrated that T lymphocytes can recognize tumor cells after vaccination. The vaccination cycle is very similar to that used for anti-infectious disease vaccines. The vaccine is injected into the subcutaneous tissue and requires several injections (once a week for 4 weeks, once a month for 4 months).

[0010] This type of immunotherapy can make the difference between remission and complete recovery in some cancers with a poor prognosis. This has been demonstrated, for example, in veterinary medicine, where vaccination increases dog survival rates compared to chemotherapy alone for deadly cancers such as DLBCL. Excellent results have also been obtained for solid tumors such as bone cancer (osteosarcoma), mast cell tumors, and melanoma.

[0011] However, the production of these vaccines from biopsies has several drawbacks that significantly impair their industrial use, the first of which is the need to biopsy the tumor in an area that is not necrotic and that is representative of the overall tumor antigen level and metastatic potential from that tumor.

[0012] Furthermore, biopsies are not always possible, especially in individuals with multiple debilitating conditions that place them at high anesthetic risk, where the biopsy itself carries a risk of fatality or serious sequelae. This risk is particularly significant in tumors such as pancreatic adenocarcinoma and difficult-to-reach brain tumors, as well as in high-grade prostate tumors, due to the risk of dissemination.

[0013] Furthermore, from a regulatory perspective, managing biopsies is a difficult and complex issue, requiring the establishment of tissue banks, adherence to strict regulations, monitoring the cold chain, and ensuring complex logistics.

[0014] Therefore, the cost of biopsy and subsequent management in tissue banks can easily be higher than the cost of the treatment itself.

[0015] It would therefore be highly advantageous and beneficial to be able to develop cancer vaccines that can be produced without relying on tumor biopsies, while maintaining efficacy comparable to tumor-derived vaccines. Summary of the Invention [Problem to be solved by the invention]

[0016] BRIEF DESCRIPTION OF THE INVENTION Surprisingly, the inventors have succeeded in developing a cancer vaccine that provides results comparable to or better than those obtained with the vaccine described in application WO2014 / 184553 (based on proteins and HAP particles derived from tumor biopsies), but which does not use tumor biopsies.

[0017] The present inventors have realised that it may indeed be possible to develop such a vaccine by extracting tumor proteins directly from the blood, particularly serum, of individuals suffering from cancer. [Means for solving the problem]

[0018] In this regard, the present invention relates to a method for producing an autologous cancer vaccine, said method comprising the following steps: a) extracting proteins from a serum or plasma sample obtained from a cancer patient; and b) contacting the proteins extracted in step a) with particles of hydroxyapatite and / or tricalcium phosphate Includes:

[0019] The present invention also relates to an autovaccine obtained from the above-mentioned production method.

[0020] Finally, the present invention also relates to an autologous vaccine obtained from the above-described production method for use in therapy, in particular in the treatment of cancer in the patient from whom the serum and / or plasma sample was obtained (autologous vaccine). [Brief explanation of the drawings]

[0021] [Figure 1] SDS-page of proteins isolated from the surface of vaccine powders produced from various tumors. From left to right: breast adenocarcinoma, diffuse lymphoma, and osteosarcoma. The results are qualitatively and quantitatively different. Scale in kDa. [Figure 2] SDS-page of proteins from serum-prepared vaccines. From left to right: melanoma (first two columns), tongue cancer (next two columns), and hemangiosarcoma (next two columns). Scale in kDa. There are numerous minor contaminants, with the quantitatively major bands between 160 and 110 kDa. This is highly reproducible. [Figure 3] Labeling of glioblastoma sections with anti-human IgG, showing the presence of anti-tumor cell antibodies after vaccination, as indicated by peroxidase labeling (brown deposits). DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention The inventors have demonstrated that it is possible to develop an effective cancer vaccine based on hydroxyapatite particles presenting tumor antigens, where said tumor antigens are obtained from a patient's plasma or serum sample, rather than from a tumor biopsy as is the case in the prior art.

[0023] Thus, in a first aspect, the present invention relates to a method for producing a cancer vaccine, in particular an autologous cancer vaccine, said production method comprising the following steps: a) extracting proteins from a serum or plasma sample obtained from a cancer patient; and b) contacting the proteins extracted in step a) with particles of hydroxyapatite and / or tricalcium phosphate Includes:

[0024] An "autologous" vaccine refers to a vaccine in which the tumor proteins / antigens are derived from the patient who is intended to be vaccinated.

[0025] A "patient" or "subject" is a human or an animal, eg, a mammal, particularly a dog, horse, or cat.

[0026] Serum or plasma samples are typically obtained from blood samples taken from the patient to be treated. The blood is centrifuged to separate the heaviest components from the supernatant, which constitutes plasma if it contains anticoagulated blood, or serum if the blood has naturally clotted.

[0027] The concept of "tumor proteins / antigens" is well known to those skilled in the art: these are proteins and / or molecules that are specifically expressed by tumor cells and can be recognized by T and B lymphocytes.

[0028] According to the present invention, tumor proteins / antigens are extracted directly from serum or plasma samples obtained from patients to be treated. Typically, the mass of tumor proteins / antigens extracted from serum or plasma samples is between 60 kDa and 130 kDa, particularly between 70 kDa and 110 kDa.

[0029] Those skilled in the art are aware of numerous techniques for extracting proteins from serum or plasma samples. Typically, tumor proteins / antigens are extracted by precipitating the serum sample with saline, the resulting mixture is then centrifuged, and the resulting pellet represents the extracted proteins.

[0030] In one particular embodiment, all proteins / antigens extracted from the plasma / serum are used and contacted to prepare the vaccine according to the invention, i.e. the entire "pellet" of proteins / antigens extracted from the plasma / serum is contacted with the hydroxyapatite particles.

[0031] "Hydroxyapatite" has the formula Ca 10 It is a mineral of the calcium phosphate family (PO4)6(OH)2, and its crystal structure has a hexagonal unit cell. Hydroxyapatite is a hydroxylated member of the apatite group. Ions in the crystal unit cell can be replaced by others with similar charge and size, and the unit cell also contains tunnels that can accommodate small molecules, such as certain amino acids. These unique features give this mineral very specific adsorption properties.

[0032] The hydroxyapatite particles according to the invention are obtained in the same way as described in application WO 2014 / 184553, the contents of which are incorporated herein by reference. This document in particular describes calcium phosphate hydroxyapatite Ca obtained by metathesis of calcium and phosphorus salts in a basic medium, including slow precipitation at high temperatures. 10 A method for producing (PO4)6(OH)2 particles is described. The reaction is carried out in a large reaction volume at a constant temperature, followed by an aging stage and a water washing stage. The precipitate thus obtained undergoes a further transformation step of solid / solution separation. This latter step is carried out either by filtration, drying and crushing by steaming (stoving), or spray drying using a fluidized bed. Regardless of the solid / solution separation technique chosen, the powder undergoes two transformation steps specific to the application of the present invention: a step of particle size selection by dry sieving to retain only the particle size band of interest (less than 25 μm, or 25 to 45 μm), and then a final step during sintering of the powder at an appropriate temperature, which ensures grain fusion and results in a very specific powder surface finish (preferably 30 μm). 2 / g or more). These two final steps may be reversed, i.e., selection followed by sintering, or sintering followed by selection. "Sintering" is a process that involves heating a powder without melting it. In the context of the present invention, sintering is carried out at temperatures of, for example, 400°C to 600°C. The HAP thus obtained may be in powder form and may be washed one or more times.

[0033] Typically, tumor proteins / antigens are contacted with hydroxyapatite particles by passing the tumor proteins / antigens through a column of hydroxyapatite particles, such as a chromatography column. The tumor antigens may optionally be contacted 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 be, inter alia, in powder form.

[0034] If a chromatography column is used, it can, for example, be placed under pressure.

[0035] Once the hydroxyapatite particles are loaded with tumor proteins / antigens, they are suspended in an injection solution and injected into the patient from which serum / plasma samples are obtained.

[0036] Typically, injection solutions contain an organic agent that facilitates injection, such as carboxymethylcellulose.

[0037] One aspect of the present invention is an autologous vaccine comprising hydroxyapatite and / or tricalcium phosphate particles loaded with tumor antigens obtained from serum or plasma samples of cancer patients.

[0038] Typically, the autovaccine is in the form of a suspension.

[0039] Typically, tumor antigens extracted from serum or plasma samples have a mass of 60 kDa to 130 kDa, particularly 70 kDa to 110 kDa.

[0040] According to one particular embodiment, the autovaccine is obtained according to the method described above.

[0041] Thus, the autologous vaccine according to the present invention comprises particles of HAP and / or tricalcium phosphate that have been adsorbed and resuspended with patient-specific tumor antigens (obtained from a serum or plasma sample of said patient).

[0042] The vaccine is then preferably administered by injection, for example subcutaneous or intradermal injection. The vaccine can also be administered orally or by other means that allow for mucosal vaccination.

[0043] The present invention also relates to the use of an autologous vaccine obtained according to the above method for its use in therapy, in particular in the treatment of cancer in the patient from whom the tumor protein / antigen was obtained.

[0044] Another aspect of the present invention also relates to a method of treating a cancer patient, said method comprising the steps of: a) extracting proteins from a serum or plasma sample obtained from a cancer patient; b) contacting the proteins extracted in step a) with particles of hydroxyapatite and / or tricalcium phosphate; c) suspending the hydroxyapatite and / or tricalcium phosphate particles contacted with the protein in step b) in an injection solution; d) injecting the mixture obtained in step c) into the patient from whom the serum or plasma sample used in step a) was obtained. Includes:

[0045] In the context of the present invention, "cancer" may be any cancer for which immunotherapy is applicable. The cancer may be selected from a non-exhaustive list, including melanoma, carcinoma or adenocarcinoma arising from epithelial and / or glandular cells, sarcoma arising from connective or muscle tissue cells, central nervous system tumors, hematopoietic tumors such as leukemia and lymphoma. It may also include various cancers resulting from infectious diseases, the most representative of which are cervical cancer, primary cancer of the liver, and gastric cancer.

[0046] In one particular embodiment, the cancer is selected from the group consisting of osteosarcoma, B or T lymphoma, breast tumor, melanoma, angiosarcoma, mast cell tumor, fibrosarcoma, brain or central nervous system tumor, schwannoma, mesothelioma, seminoma, teratoma, and glioblastoma.

[0047] In one preferred embodiment, the cancer is selected from glioblastoma, sarcoma (such as osteosarcoma or fibrosarcoma), melanoma, carcinoma, or adenocarcinoma.

[0048] In one particular embodiment, the cancer is disseminated cancer, ie, cancer with metastasis (patients classified in the NxMx category according to the TNM classification).

[0049] Patients can receive a single or multiple injections of the vaccine. A dose generally contains 30-50 mg of hydroxyapatite and / or tricalcium phosphate and 1000-2000 μg of protein.

[0050] Preferably, the patient will receive several injections spaced apart in time, for example, by days, weeks, or months, hi a preferred embodiment, injections are spaced one week apart for the first month and one month apart for the next four months.

[0051] Each injection can ideally be prepared from a new tumor protein sample from the patient if there are signs of vaccine escape or ineffectiveness, especially if tumor marker levels rise.

[0052] The vaccines of the present invention represent a significant evolution in protocols compared to techniques using tumor biopsies in that proteins do not need to be extracted from the intracytoplasmic compartment.

[0053] SDS-PAGE electrophoresis of proteins fixed in powders prepared for injection shows that the protein bands are different from those obtained by tumor biopsy (as in application WO2014 / 184453). Moreover, as shown below, the results obtained with the vaccine according to the invention are as good or even better.

[0054] The autologous vaccine according to the present invention can be used in combination with another anti-cancer treatment, such as radiation therapy, chemotherapy, or another immunotherapeutic agent.

[0055] The present invention is illustrated in more detail in the following examples, which are provided for illustrative purposes only and are not to be construed as limiting the scope of the invention. [Example]

[0056] The protocol of the present invention was carried out in patients with a pathology that had a very poor prognosis and was incurable with currently available therapies. Patients were explicitly asked to participate and participated in the protocol by signing an informed consent.

[0057] This protocol can be applied to disseminated cancer as a final-line treatment after failure of conventional therapy, optionally in combination with chemotherapy or other immunotherapies. It can also be envisioned to prevent recurrence of cancer for which there is no effective treatment after surgery.

[0058] Twenty-seven patients were enrolled in this protocol. Nine patients followed the immunotherapy protocol without additional chemotherapy. Ten patients had glioblastoma, two had sarcoma, and 15 had carcinoma or adenocarcinoma. Twenty-two patients had progressive disease when the immunotherapy protocol was administered; 13 of these patients were disease-free, had stable disease, or showed partial regression 5 months after initiation of the protocol. Excluding glioblastoma, 72% of patients with progressive disease at the time of vaccination achieved stable disease (SD) or partial regression (PR) 2 months after vaccination. In conclusion, although this series is heterogeneous, a significant proportion of cases demonstrated a clinical response to this final-line technique, with no adverse events. Disseminated cancer can be stabilized by this technique, demonstrating that immune responses can be achieved in humans, even in rapidly progressing, aggressive disease.

[0059] Example 1: Electrophoretic differences of proteins adsorbed to powders in contact with serum or tumor biopsies Electrophoresis of proteins contained in vaccines prepared from biopsy or serum proteins was compared.

[0060] The HAP particles are typically obtained according to the method described in Example 1 of application WO2014 / 184553.

[0061] Doses were prepared as follows:

[0062] -Start with a biopsy: Tumor tissues and all materials used to prepare the vaccine were handled aseptically in a laminar flow hood. Frozen tumor tissue (200 mg) was homogenized using a tissue bowl homogenizer. 1 ml of NaHCO3 (30 mM, pH 7) was added per ml of homogenate.

[0063] The resulting homogenate was then centrifuged at 1000 g for 15 minutes at 4°C to remove all tissue debris. The supernatant was mixed at 50% with a supersaturated solution of ammonium nitrate and allowed to stand at 4°C for 1 hour before centrifugation. The pellet was resuspended in 0.02 M phosphate buffer, pH 7. This solution was then applied to a chromatography column containing HAP powder. A column (Poly-prep Chromatography Column, Cat. 731-1550, BioRad) was loaded with 0.2 g of HAP (0-25 μm) and equilibrated with 10 volumes of phosphate buffer (20 mM, pH 7). The resuspended pellet was then added. The column was then washed with 3 ml of 100 mM NaCl solution. The powder was then suspended in 5 ml of carboxymethylcellulose (CMC) solution (2% in 20 mM NaCl). 0.5 ml of this solution was used for each vaccine injection.

[0064] -Start with serum: The procedure is much simpler: 3 cc of serum is diluted 50% in supersaturated ammonium nitrate solution, left at 4°C for 1 hour, and then centrifuged. The pellet is resuspended in 0.02 M phosphate buffer, pH 7. This solution is then passed through a chromatography column containing HAP powder. A column (Poly-prep Chromatography Column, Cat. 731-1550, BioRad) was loaded with 0.2 g of HAP (0-25 μm) and equilibrated with 10 volumes of phosphate buffer (20 mM, pH 7). The resuspended pellet was then added. The column was then washed with 3 ml of 100 mM NaCl solution. The powder was then suspended in 5 ml of carboxymethylcellulose (CMC) solution (2% in 20 mM NaCl). 0.5 ml of this solution was used for each vaccine injection.

[0065] For both electrophoresis runs, proteins adsorbed onto the powder are desorbed prior to electrophoresis. 10 mg of vaccine dose powder is centrifuged at 500 g for 2 minutes. The supernatant is removed and the pellet is resuspended in 0.5 ml of 0.5 M NaCl. The powder is vortexed to resuspend and centrifuged at 500 g. 10 μl of the supernatant is then diluted 50% in detergent and heated to 70°C for 5 minutes before being loaded into the electrophoresis well.

[0066] result: There are notable differences in the electrophoretic profiles obtained with the two methods. Most notably, there is a reduction in the number of bands in the blood-derived electrophoresis compared to the biopsy-derived one (Figures 1 and 2). Furthermore, the blood-derived electrophoresis is much more homogeneous, indicating better reproducibility of the purification.

[0067] Example 2: Results of administering the vaccine according to the present invention to patients with adenocarcinoma This patient presented with endometrial adenocarcinoma (TNM 7a) treated preoperatively and postoperatively with two cycles of paclitaxel and carboplatin. Eighteen months after the patient presented with pulmonary miliary opacities and secondary metastases to the retroperitoneal site, she underwent hormonal therapy (megestrol acetate) and began receiving vaccinations. Three months after vaccination (MRI, PET), she demonstrated stabilization of the pulmonary lesions and a slight regression in the volume of the retroperitoneal mass. This continued until 18 months later, when the retroperitoneal mass had virtually disappeared and the pulmonary miliary opacities, although still present, had regressed. This partial regression remains stable after two and a half years of observation.

[0068] This example demonstrates that the clinical efficacy of vaccination with serum proteins can be maintained for a significant period of time, exceeding 30 months.

[0069] Example 3: Results of administration of the vaccine according to the invention to patients with colon adenocarcinoma This patient presented with colon adenocarcinoma that was treated surgically and with adriamycin. Three years later, lung and liver metastases were noted, but they were limited to the lung and liver lobes, allowing for lung and liver surgery followed by chemotherapy. Five years later, chemotherapy with gemcitabine, capecitabine, and bevacizumab was initiated after multiple liver and retroperitoneal lymph node metastases recurred. Despite chemotherapy, the disease continued to progress. After cessation of chemotherapy following a pulmonary embolism, the tumor marker (ACE) rose sharply. Treating the patient with vaccination with serum proteins (four injections at weekly intervals during the first month, followed by one injection per month) resulted in a significant decrease in the marker level. The marker then rose again three months later, and the same positron emission tomography (PET) scan performed before vaccination suggested tumor escape. Therefore, a separate preparation was produced using a different serum sample and injected every three weeks, resulting in the marker falling below normal within three months. PET revealed necrosis of liver metastases 1 year after initiation of immunotherapy.

[0070] Conclusions: Blood-derived vaccines inhibit cell proliferation detectable by cell proliferation markers as a first step, followed by a delayed effect detectable by PET over a much longer period of several months to a year.

[0071] Example 4: Anti-glioblastoma serum antibodies after vaccination with blood-prepared vaccines. This patient received the vaccination 6 months after the discovery and surgical biopsy of a stage IV glioblastoma in the right temporal region. First-line therapy was the Stupp protocol based on Temodar alternating with radiation therapy (Stupp, R. et al., Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma). ,N Engl J Med 2005;352:987-996). Vaccination with serum proteins was administered weekly for four injections per month, followed by monthly injections for the patient's lifetime. The patient maintained complete remission for 10 months and progressed for two months before death (RECIST criteria). Sections of the patient's tumor biopsy were tested for the presence of serum antibodies (IgG) to determine whether antibodies against tumor cells were present.

[0072] Paraffin-embedded tissue sections, 5 μm thick, were used. They were deparaffinized with xylol and rehydrated with an ethanol-reducing solution. Endogenous peroxidase was inhibited with H2O2. Patient serum, diluted 1 / 100 in Hepes buffer, served as the primary antibody in a marker using peroxidase-labeled anti-human IgG as the secondary antibody. Peroxidase was then developed using DAB (3,3'-diaminobenzidine), which is oxidized in the presence of hydrogen peroxide, producing an alcohol-insoluble brown deposit (Figure 3). Negative controls consisted of sections treated under the same conditions without the primary antibody (diluted serum).

[0073] It should be noted that although the labeling shows that the majority of tumor cells are positive, some are not, suggesting that the vaccine elicited a response against tumor cells and that the time between biopsy and vaccine production meant that not all clones were represented in the vaccine.

[0074] The histological results indicate that vaccination with serum proteins induces a humoral anti-tumor immune response.

[0075] Example 5: Difference between the rate of positive ELISPOTs induced by vaccines prepared from tumor biopsies and those prepared from serum We compared CD8 stimulation induced by vaccines prepared from blood or biopsies. This was performed in a series of patients with various cancerous lesions, from whom biopsy or serum samples were taken, depending on the availability of biopsies. Note that the stimulation of cellular immunity detected by Elispot does not necessarily correlate with clinical outcome. Elispot does not detect all CD8 stimulation states. ELISPOT tests were performed in each patient at the fifth injection, i.e., 3 months after the first vaccination.

[0076] Ten ml of blood is collected in a citrate tube and centrifuged in Ficoll 400 within 4 hours of sampling. Nucleated cells are then washed with RPMI medium and collected at 2 × 10 5 The cells are then cultured at a concentration of 1000 cells / well in wells coated with vaccine protein. The vaccine protein is obtained as described above for performing vaccine electrophoresis. 10 mg of protein-loaded powder is washed with 0.5 ml of 0.2 M NaCl. For plate coating, the solution is returned to 0.02 M. The cells are then cultured overnight in RPMI supplemented with 5% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells are then washed for 10 minutes with PBS containing 0.1% Tween 20 surfactant. The wells are then incubated for 1 hour in 1 ml of PBS containing 0.1% BSA and a 1 / 1000 dilution of peroxidase-labeled anti-interferon gamma antibody. The peroxidase is then developed as described in Example 4.

[0077] Fifteen patients were vaccinated with vaccines prepared from serum, and 13 were vaccinated with vaccines prepared from tumor biopsies. ELISPOT was performed after the fifth injection, i.e., three months after vaccination. The negative control was blood from an unvaccinated patient with the same type of tumor. 14 of the 15 patients vaccinated with serum had a positive ELISPOT, while only 6 of the 13 patients vaccinated with biopsies had a positive ELISPOT. This is a statistically significant difference.

[0078] In light of these varied results, the compositional differences visible by electrophoresis, and the clinical and biological results observed, the vaccine obtained using this method is surprisingly effective.

Claims

1. A method for producing an autologous cancer vaccine comprising hydroxyapatite and / or tricalcium phosphate particles loaded with patient-specific tumor proteins / antigens, said method comprising the following steps: a) extracting tumor proteins / antigens present in serum or plasma obtained from a cancer patient; and b) contacting the tumor proteins / antigens extracted in step a) with particles of hydroxyapatite and / or tricalcium phosphate to obtain an autologous vaccine A method comprising:

2. 10. The method of claim 1, wherein the patient is a human.

3. 10. The method of claim 1, wherein the patient is a dog, horse, or cat.

4. 4. The method of any one of claims 1 to 3, wherein the cancer is selected from melanoma, carcinoma, adenocarcinoma, sarcoma, central nervous system tumors, leukemia, lymphoma, and cancers of infectious origin.

5. 4. The method of any one of claims 1 to 3, wherein the cancer is selected from osteosarcoma, B or T lymphoma, breast tumor, melanoma, angiosarcoma, mast cell tumor, fibrosarcoma, brain or central nervous system tumor, schwannoma, mesothelioma, seminoma, teratoma, and blastoma.

6. 4. The method of any one of claims 1 to 3, wherein the cancer is glioblastoma, sarcoma, melanoma, carcinoma, or adenocarcinoma.

7. The method according to any one of claims 1 to 6, wherein the cancer is a cancer that exhibits metastasis.

8. An autologous vaccine comprising particles of hydroxyapatite and / or tricalcium phosphate carrying patient-specific tumor proteins / antigens, the autologous vaccine being obtained by a manufacturing method described in any one of claims 1 to 7.

9. 9. The autologous vaccine of claim 8 for use in the treatment of cancer in a patient from whom a serum and / or plasma sample has been obtained.

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