Preparation method for and use of fragmented tumor antigen

Abstract

The present application provides a preparation method for a fragmented tumor antigen. The method comprises: acquiring a tumor cell suspension; and irradiating and incubating the tumor cell suspension to prepare a fragmented tumor antigen. The preparation method of the present application is simple and has a low cost and storage convenience. A fragmented tumor antigen prepared by the preparation method of the present application can significantly inhibit tumor growth in vivo after being inoculated into an individual, can also effectively inhibit metastasis and recurrence of a tumor, induces the generation of immunological memory for a continuous anti-tumor effect, and significantly improves the control of a metastatic tumor. The fragmented tumor antigen prepared by the present application, either alone or in combination, can exert an effective anti-tumor effect in vivo.

Description

A preparation method of fragmented tumor antigen and its use

[0001] This application claims priority to the Chinese patent application No. 2024103396080, filed with the Patent Office of the State Intellectual Property Office of China on March 22, 2024, with the invention title “A method for preparing a fragmented tumor antigen and its use”, and the Chinese patent application No. 2024115462825, filed with the Patent Office of the State Intellectual Property Office of China on October 31, 2024, with the invention title “A method for preparing a fragmented tumor antigen and its use”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application relates to the field of vaccine technology, and in particular to a method for preparing a fragmented tumor antigen and its use. Background Art

[0003] Tumor vaccines are currently a hot topic in the oncology field both domestically and internationally. As an active immunotherapy approach for tumors, they are playing an increasingly important role in the treatment of cancer patients. Tumor vaccines can be categorized by the source of their antigens into DNA vaccines, RNA vaccines, peptide vaccines, tumor cell vaccines, and dendritic cell vaccines. Tumor cell vaccines involve treating autologous or allogeneic tumor cells with physical, chemical, or biological methods to render them tumorigenic but retain their antigenicity. These vaccines, combined with nonspecific stimulatory factors, are used for active immunotherapy of cancer patients.

[0004] Tumor metastasis is the leading cause of death in cancer patients. Currently reported tumor immunotherapy methods have limited effects on metastatic tumors. For example, the anti-tumor effects of therapeutic vaccines (including DC cell vaccines, RNA vaccines, and autologous tumor cell vaccines, etc.) are mostly effective in inhibiting primary tumors, while the inhibitory effects on recurrent or metastatic tumors are unclear or ineffective. In addition, the preparation process of existing personalized tumor vaccines is time-consuming, involves many steps, and is expensive, which limits their widespread clinical application. Moreover, the developed tumor cell vaccines are not effective when used alone and need to be loaded on DC cells or used in combination with one or more adjuvant molecules or chemotherapy / immunotherapy drugs to effectively inhibit tumor metastasis.

[0005] Therefore, it is crucial to find a tumor treatment method that is time-saving, simple to produce, low-cost, and has a significant inhibitory effect on metastatic tumors, in order to provide reference and assistance for the clinical treatment of metastatic tumors. Summary of the Invention

[0006] The purpose of this application is to provide a method for preparing a fragmented tumor antigen and its use, so as to improve the immunogenicity of the fragmented tumor antigen and enable the fragmented tumor antigen to have excellent anti-tumor effects in different tumor animal models, especially significantly inhibiting tumor metastasis and effectively prolonging animal survival. The specific technical solution is as follows:

[0007] The first aspect of the present application provides a method for preparing a fragmented tumor antigen, which comprises: (1) obtaining a tumor cell suspension; and (2) irradiating and incubating the tumor cell suspension to prepare a fragmented tumor antigen.

[0008] In one embodiment of the present application, the method further comprises: (3) performing ultra-low temperature freeze-thaw treatment on the fragmented tumor antigen.

[0009] In one embodiment of the present application, the tumor cells are selected from at least one of breast cancer cells, colorectal cancer cells, melanoma cells, lung cancer cells, liver cancer cells, pancreatic cancer cells, kidney cancer cells, esophageal cancer cells, gastric cancer cells, prostate cancer cells, brain cancer cells, oral cancer cells, bile duct cancer cells, ovarian cancer cells, cervical cancer cells, osteosarcoma cells and testicular cancer cells.

[0010] In one embodiment of the present application, the tumor cells are derived from at least one of tumor cells obtained by puncture, tumor cells obtained by surgery, circulating tumor cells, and tumor cells cultured in vitro.

[0011] In one embodiment of the present application, the tumor cells are selected from autologous or allogeneic tumor cells.

[0012] In one embodiment of the present application, the number of cells in the tumor cell suspension is 1 to 1000×10 6 , preferably 0.5×10 6 ~100×10 6 , more preferably 1×10 6 ~20×10 6 indivual.

[0013] In one embodiment of the present application, the incubation temperature is 4°C to 50°C, preferably 30°C to 40°C, and more preferably 35°C to 40°C.

[0014] In one embodiment of the present application, the incubation time is 0.01 hour to 24 hours, preferably 0.5 hour to 8 hours, and more preferably 0.5 hour to 6 hours.

[0015] In one embodiment of the present application, the irradiation dose is 2 Gy to 200 Gy, preferably 2 Gy to 50 Gy, more preferably 6 Gy to 40 Gy, and even more preferably 8 Gy to 20 Gy.

[0016] In one embodiment of the present application, the irradiation dose rate is 0.01 Gy / s to 1×10 9 Gy / s, preferably 0.01 Gy / s to 100 Gy / s, more preferably 0.01 Gy / s to 40 Gy / s.

[0017] In one embodiment of the present application, the irradiation method is selected from at least one of electron, photon, proton, heavy ion and neutron radiation, preferably at least one of photon and proton, more preferably X-ray.

[0018] The second aspect of the present application provides a fragmented tumor antigen obtained by the preparation method described in the first aspect of the present application.

[0019] The third aspect of the present application provides a tumor cell vaccine, which includes the fragmented tumor antigen described in the second aspect of the present application.

[0020] In one embodiment of the present application, the tumor cell vaccine further comprises an immune adjuvant; the immune adjuvant is an immunomodulatory drug;

[0021] Preferably, the immunomodulatory drug is selected from at least one of polyinosinic-polycytidylic acid, platinum drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolism drugs, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolites, rapamycin target inhibitors, botanicals, ribonucleotide reductase inhibitors and tyrosine kinase inhibitors;

[0022] More preferably, the polyinosinic-acid is selected from artificially synthesized ribonucleic acid; the platinum drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cyclosulfate platinum, nedaplatin and lobaplatin; the calcineurin inhibitor is selected from at least one of cyclosporine, tacrolimus and mycophenolate mofetil; the glucocorticoid is selected from at least one of prednisone, methylprednisone and methylprednisolone; the alkylating agent is selected from at least one of cyclophosphamide and chlorambucil; the microbial metabolism drug is selected from at least one of cyclosporine, tacrolimus and rapamycin; the monoclonal antibody drug is selected from anti-thymocyte immune protein, At least one of muromonab-CD3, daclizumab, basiliximab, efalizumab and natalizumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide and mycophenolate mofetil; the antimetabolite drug is selected from at least one of mycophenolate mofetil, methotrexate, azathioprine, mercaptopurine and mizoribine; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and gustation; the botanical drug is selected from at least one of tripterygium wilfordii glycosides and total glucosides of white paeony; the ribonucleotide reductase inhibitor is selected from hydroxyurea; the tyrosine kinase inhibitor is selected from leflunomide.

[0023] The fourth aspect of the present application provides the use of the fragmented tumor antigen described in the second aspect of the present application or the tumor cell vaccine described in the third aspect of the present application in the treatment and / or prevention of tumor diseases; preferably, the tumor diseases include at least one of breast cancer, colorectal cancer, melanoma, lung cancer, liver cancer, pancreatic cancer, kidney cancer, esophageal cancer, gastric cancer, prostate cancer, brain cancer, oral cancer, bile duct cancer, ovarian cancer, cervical cancer, osteosarcoma and testicular cancer.

[0024] Beneficial effects of this application:

[0025] The present application provides a method for preparing a fragmented tumor antigen, comprising: obtaining a tumor cell suspension; irradiating and incubating the tumor cell suspension, and then preparing a fragmented tumor antigen. The fragmented tumor antigen prepared by the preparation method of the present application can maximize the retention of tumor cell immunogenicity, overcome the tumor's suppressive immune microenvironment, and induce a strong anti-tumor immune response, effectively inhibiting tumor growth and metastasis, and producing a better anti-tumor metastasis effect. Furthermore, the preparation method of the present application is simple, low-cost, easy to store, effective, and highly safe, and has broad application prospects.

[0026] Of course, it is not necessary to achieve all the advantages described above at the same time when implementing any product or method of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0028] Figure 1A shows the growth inhibitory effect of fragmented tumor antigens and their combination with manganese (Mn) adjuvant on 4T-1 distal tumors after irradiation; Figure 1B shows the growth inhibitory effect of fragmented tumor antigens and their combination with Mn adjuvant on 4T-1 distal tumors after ultrasound treatment; Figure 1C shows the growth inhibitory effect of fragmented tumor antigens and their combination with Mn adjuvant on 4T-1 distal tumors after hyperthermia treatment; Figure 1D shows the growth inhibitory effect of fragmented tumor antigens and their combination with Mn adjuvant on 4T-1 distal tumors after treatment with chemotherapy drugs (doxorubicin, DOX);

[0029] Figure 2 shows the growth inhibitory effect of fragmented tumor antigen immunotherapy (FAST) on 4T-1 distal tumors after different doses of irradiation;

[0030] Figure 3A shows the growth inhibitory effect of fragmented tumor antigens incubated at different times after irradiation on 4T-1 distal tumors; Figure 3B shows the growth inhibitory effect of mixed fragmented tumor antigens inoculated at different times after irradiation on 4T-1 distal tumors; Figure 3C shows the normalized growth inhibitory effect of fragmented tumor antigens incubated at different times after irradiation on 4T-1 tumors;

[0031] FIG4 shows the tumorigenicity of irradiated segmented tumor antigens that were not subjected to ultra-low temperature freeze-thaw treatment;

[0032] Figure 5A shows in vivo imaging of mice after FAST treatment in the 4T-1 cold tumor distal model; Figure 5B shows the tumor growth inhibitory effect of FAST in the 4T-1 cold tumor distal model; Figure 5C shows the survival extension effect of FAST in the 4T-1 cold tumor distal model; Figure 5D shows the effect of FAST in inhibiting tumor lung metastasis in the 4T-1 cold tumor distal model;

[0033] Figure 6A shows in vivo imaging of mice treated with FAST in the CT-26 thermal tumor distal model; Figure 6B shows the tumor growth inhibitory effect of FAST in the CT-26 thermal tumor distal model; Figure 6C shows the survival-extending effect of FAST in the CT-26 thermal tumor distal model;

[0034] FIG7A shows the tumor growth inhibitory effect of FAST in the B16-F10 cold tumor distal model; FIG7B shows the survival prolonging effect of FAST in the B16-F10 cold tumor distal model;

[0035] Figure 8A shows in vivo imaging of mice before and after surgery and at different times after FAST treatment in the 4T-1 cold tumor resection model; Figure 8B shows the effect of FAST in inhibiting lung metastasis in the 4T-1 cold tumor resection model; Figure 8C shows the survival-extending effect of FAST in the 4T-1 cold tumor resection model;

[0036] FIG9A shows in vivo imaging of mice in which fragmented antigens derived from different tumor cells specifically inhibit tumor growth; FIG9B shows that fragmented antigens derived from different tumor cells specifically inhibit tumor growth;

[0037] Figure 10A shows in vivo imaging of mice treated with FAST at different courses of treatment; Figure 10B shows the tumor growth inhibition effect of FAST treatment at different courses of treatment; Figure 10C shows the tumor lung metastasis inhibition effect of FAST treatment at different courses of treatment; Figure 10D shows that three courses of FAST treatment can significantly induce the generation of central memory cells and effector memory cells;

[0038] Figure 11A shows the in vivo imaging of mice treated with FA combined with α-PD-L1 / α-CTLA-4; Figure 11B shows the tumor growth inhibition effect of FA combined with α-PD-L1 / α-CTLA-4; Figure 11C shows the tumor lung metastasis inhibition effect of FA combined with α-PD-L1 / α-CTLA-4;

[0039] Figure 12A shows the percentage of CD3+T cells in tumor tissues after treatment with FAST and its corresponding components; Figure 12B shows the percentage of CD8+T cells in tumor tissues after treatment with FAST and its corresponding components; Figure 12C shows the percentage of CD4+T cells in tumor tissues after treatment with FAST and its corresponding components; Figure 12D shows the percentage of Treg (Foxp3+CD4+T) cells in tumor tissues after treatment with FAST and its corresponding components; Figure 12E shows the percentage of KI76+CD8+T cells in tumor tissues after treatment with FAST and its corresponding components;

[0040] FIG13A shows the normalized tumor growth inhibition effect of FA combined with different treatments such as IR and FAST; FIG13B shows the normalized lung metastasis inhibition effect of FA combined with different treatments such as IR and FAST;

[0041] FIG14A shows the normalized tumor growth inhibition effect of cisplatin (CDDP) combined with different treatments such as IR or FA and FAST; FIG14B shows the normalized tumor lung metastasis inhibition effect of CDDP combined with different treatments such as IR or FA and FAST;

[0042] Figure 15A shows the normalized tumor growth inhibitory effect of 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) + CDDP combined with different treatments such as IR or FA and FAST; Figure 15B shows the normalized tumor lung metastasis inhibitory effect of PGPC + CDDP combined with different treatments such as IR or FA and FAST;

[0043] FIG16A shows the tumor growth curves after treatment with CDDP combined with IR and different doses of FA; FIG16B shows the survival curves of mice after treatment with CDDP combined with IR and different doses of FA. DETAILED DESCRIPTION

[0044] The following will be combined with the accompanying drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0045] The first aspect of the present application provides a method for preparing a fragmented tumor antigen, which comprises: (1) obtaining a tumor cell suspension; and (2) irradiating and incubating the tumor cell suspension to prepare a fragmented tumor antigen.

[0046] In the present application, there is no particular limitation on the method of obtaining the tumor cell suspension, as long as the purpose of the present application can be achieved. For example, methods such as enzymatic filtration, digestion suspension culture, etc. can be used.

[0047] The inventors discovered that treating tumor cells with the irradiation method described in this application can enhance their immunogenicity, activate the body's anti-tumor immune response, inhibit tumor growth, and produce a significant anti-tumor effect. However, fragmented tumor antigens produced by treating tumor cells with other treatments, such as ultrasound, hyperthermia, and chemotherapy drugs in vitro, are unable to control tumor growth, even when combined with a Mn adjuvant.

[0048] In this application, the form of the fragmented tumor antigen is not particularly limited, as long as it can achieve the objectives of this application. For example, it can be a solution containing the fragmented tumor antigen, or a lyophilized powder containing the fragmented tumor antigen obtained by freeze-drying the fragmented tumor antigen solution. The fragmented tumor antigen in lyophilized powder form not only fully retains the activity of the tumor antigen, but also facilitates storage and transportation. It also improves the safety of the tumor antigen during storage and extends the shelf life of the fragmented tumor antigen.

[0049] In one embodiment of the present application, the method further comprises: (3) performing ultra-low temperature freeze-thaw treatment on the fragmented tumor antigen.

[0050] The inventors discovered in their research that fragmented tumor antigens that have undergone ultra-low temperature freeze-thaw treatment exhibit enhanced anti-tumor efficacy and are safer. Furthermore, ultra-low temperature freezers can store large quantities of prepared tumor antigens for subsequent use, eliminating the need for improvisation for each use, the time required, and batch variations. This meets the requirements for commercialization of tumor antigens. Prepared fragmented tumor antigens can maintain their anti-tumor effects even after one to two months of storage in an ultra-low temperature freezer for immunization.

[0051] In one embodiment of the present application, the tumor cells are selected from at least one of breast cancer cells, colorectal cancer cells, melanoma cells, lung cancer cells, liver cancer cells, pancreatic cancer cells, kidney cancer cells, esophageal cancer cells, gastric cancer cells, prostate cancer cells, brain cancer cells, oral cancer cells, bile duct cancer cells, ovarian cancer cells, cervical cancer cells, osteosarcoma cells and testicular cancer cells.

[0052] In one embodiment of the present application, the tumor cells are derived from at least one of biopsy-derived tumor cells, surgically-derived tumor cells, circulating tumor cells, and in vitro cultured tumor cells. Selecting tumor cells from these sources can better maintain the presence of tumor antigens in the tumor vaccine, stimulate sustained and effective immune protection, and reduce tumor heterogeneity.

[0053] In one embodiment of the present application, the tumor cells are selected from autologous or allogeneic tumor cells.

[0054] In this application, autologous tumor cells are tumor cells from the patient himself or the same animal. Allogeneic tumor cells are syngeneic tumor cells, which refer to tumor cells of the same type of cancer derived from an allogeneic source.

[0055] In one embodiment of the present application, the number of cells in the tumor cell suspension is 1 to 1000×10 6 , preferably 0.5×10 6 ~100×10 6 , more preferably 1×10 6 ~20×10 6 For example, the number of cells in the tumor cell suspension can be 1, 10, 100, 1000, 0.01×10 6 0.1×10 6 0.5×10 6 1×10 6 2×10 6 3×10 6 4×10 6 5×10 6 8×106 10×10 6 15×10 6 20×10 6 30×10 6 40×10 6 50×10 6 60×10 6 70×10 6 80×10 6 90×10 6 100×10 6 200×10 6 300×10 6 400×10 6 500×10 6 600×10 6 700×10 6 800×10 6 900×10 6 1000×10 6 Controlling the number of cells in the tumor cell suspension within the above range can better improve the therapeutic effect of the fragmented tumor antigen in preventing and / or treating tumor diseases.

[0056] In one embodiment of the present application, the incubation temperature is 4°C to 50°C, preferably 30°C to 40°C, and more preferably 35°C to 40°C. For example, the incubation temperature can be 4°C, 6°C, 8°C, 10°C, 15°C, 17°C, 20°C, 25°C, 27°C, 30°C, 35°C, 37°C, 40°C, 45°C, 47°C, 50°C, or a range consisting of any two values ​​therebetween. Controlling the incubation temperature within the above range can maximize the retention of tumor cell immunogenicity, activate the body's effective anti-tumor immune response, inhibit tumor growth, and produce better anti-tumor effects.

[0057] In one embodiment of the present application, the incubation time is 0.01 hour to 24 hours, preferably 0.5 hour to 8 hours, and more preferably 0.5 hour to 6 hours. For example, the incubation time can be 0.01 hour, 0.1 hour, 0.5 hour, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 24 hours or a range consisting of any two values ​​therebetween. By controlling the incubation time within the above range, the immunogenicity of tumor cells can be maximized, the body's effective anti-tumor immune response can be activated, tumor growth can be inhibited, and a better anti-tumor effect can be produced.

[0058] In one embodiment of the present application, the irradiation dose is 2 Gy to 200 Gy, preferably 2 Gy to 50 Gy, more preferably 6 Gy to 40 Gy, and even more preferably 8 Gy to 20 Gy. For example, the irradiation dose can be 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, 12 Gy, 14 Gy, 16 Gy, 18 Gy, 20 Gy, 22 Gy, 24 Gy, 26 Gy, 28 Gy, 30 Gy, 32 Gy, 34 Gy, 36 Gy, 38 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 70 Gy, 80 Gy, 90 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy, 200 Gy, or a range consisting of any two values ​​therebetween. Controlling the irradiation dose within the above range can maximize the retention of tumor cell immunogenicity, activate the body's effective anti-tumor immune response, inhibit tumor growth, and produce better anti-tumor effects.

[0059] In one embodiment of the present application, the irradiation dose rate is 0.01 Gy / s to 1×10 9 Gy / s, preferably 0.01 Gy / s to 100 Gy / s, more preferably 0.01 Gy / s to 40 Gy / s. For example, the dose rate of the irradiation can be 0.01 Gy / s, 0.1 Gy / s, 1 Gy / s, 10 Gy / s, 20 Gy / s, 40 Gy / s, 60 Gy / s, 80 Gy / s, 1×10 2 Gy / s, 1×10 3 Gy / s, 1×10 4 Gy / s, 1×10 5 Gy / s, 1×10 6 Gy / s, 1×10 7 Gy / s, 1×10 8 Gy / s, 1×10 9 Gy / s or a range consisting of any two values ​​therebetween. Controlling the irradiation dose rate within the above range can maximize the retention of tumor cell immunogenicity, activate the body's effective anti-tumor immune response, inhibit tumor growth, and produce better anti-tumor effects.

[0060] In one embodiment of the present application, the irradiation method is selected from at least one of electron, photon, proton, heavy ion and neutron radiation, preferably at least one of photon and proton, more preferably X-ray.

[0061] The second aspect of the present application provides a fragmented tumor antigen obtained by the preparation method described in the first aspect of the present application.

[0062] The third aspect of the present application provides a tumor cell vaccine, which includes the fragmented tumor antigen described in the second aspect of the present application.

[0063] In one embodiment of the present application, the tumor cell vaccine further comprises an immune adjuvant; the immune adjuvant is an immunomodulatory drug;

[0064] Preferably, the immunomodulatory drug is selected from at least one of polyinosinic-polycytidylic acid, platinum drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolism drugs, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolites, rapamycin target inhibitors, botanicals, ribonucleotide reductase inhibitors and tyrosine kinase inhibitors;

[0065] More preferably, the polyinosinic-acid is selected from artificially synthesized ribonucleic acid; the platinum drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cyclosulfate platinum, nedaplatin and lobaplatin; the calcineurin inhibitor is selected from at least one of cyclosporine, tacrolimus and mycophenolate mofetil; the glucocorticoid is selected from at least one of prednisone, methylprednisone and methylprednisolone; the alkylating agent is selected from at least one of cyclophosphamide and chlorambucil; the microbial metabolism drug is selected from at least one of cyclosporine, tacrolimus and rapamycin; the monoclonal antibody drug is selected from anti-thymocyte immune protein, At least one of muromonab-CD3, daclizumab, basiliximab, efalizumab and natalizumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide and mycophenolate mofetil; the antimetabolite drug is selected from at least one of mycophenolate mofetil, methotrexate, azathioprine, mercaptopurine and mizoribine; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and gustation; the botanical drug is selected from at least one of tripterygium wilfordii glycosides and total glucosides of white paeony; the ribonucleotide reductase inhibitor is selected from hydroxyurea; the tyrosine kinase inhibitor is selected from leflunomide.

[0066] The fourth aspect of the present application provides the use of the fragmented tumor antigen according to the second aspect of the present application or the tumor cell vaccine according to the third aspect of the present application in the treatment and / or prevention of tumor diseases; preferably, the tumor diseases include at least one of breast cancer, colorectal cancer, melanoma, lung cancer, liver cancer, pancreatic cancer, kidney cancer, esophageal cancer, gastric cancer, prostate cancer, brain cancer, oral cancer, bile duct cancer, ovarian cancer, cervical cancer, osteosarcoma and testicular cancer.

[0067] In one embodiment of the present application, the tumor disease is a metastatic tumor. The tumor cell vaccine of the present application can maximize the retention of tumor cell immunogenicity, overcome the tumor suppressive immune microenvironment, and induce a strong immune response to effectively inhibit tumor metastasis, thereby producing a better anti-tumor metastasis effect.

[0068] The fifth aspect of the present application provides a method for tumor immunotherapy, which comprises: administering an effective amount of the fragmented tumor antigen described in the first aspect of the present application or the tumor cell vaccine described in the second aspect of the present application to an individual in need.

[0069] In the present application, fragmented tumor antigens obtained from different irradiation doses and / or different incubation times can be mixed and inoculated to inhibit tumor growth and produce a better anti-tumor effect. For example, mixed inoculation of fragmented tumor antigens obtained 1 hour and 6 hours after irradiation can inhibit tumor growth and produce a better anti-tumor effect.

[0070] In one embodiment of the present application, the fragmented tumor antigen is used in combination with an immunomodulatory drug as an immune adjuvant;

[0071] Preferably, the immunomodulatory drug is selected from at least one of polyinosinic-polycytidylic acid, platinum drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolism drugs, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolites, rapamycin target inhibitors, botanicals, ribonucleotide reductase inhibitors and tyrosine kinase inhibitors;

[0072] More preferably, the polyinosinic-acid is selected from artificially synthesized ribonucleic acid; the platinum drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cyclosulfate platinum, nedaplatin and lobaplatin; the calcineurin inhibitor is selected from at least one of cyclosporine, tacrolimus and mycophenolate mofetil; the glucocorticoid is selected from at least one of prednisone, methylprednisone and methylprednisolone; the alkylating agent is selected from at least one of cyclophosphamide and chlorambucil; the microbial metabolic drug is selected from at least one of cyclosporine, tacrolimus and rapamycin; the monoclonal antibody drug is selected from anti-thymocyte immune protein , muromonab-CD3, daclizumab, basiliximab, efalizumab and natalizumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide and mycophenolate mofetil; the antimetabolite drug is selected from at least one of mycophenolate mofetil, methotrexate, azathioprine, mercaptopurine and mizoribine; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and gumarilimus; the botanical drug is selected from at least one of tripterygium wilfordii glycosides and total glucosides of white paeony; the ribonucleotide reductase inhibitor is selected from hydroxyurea; the tyrosine kinase inhibitor is selected from leflunomide.

[0073] In the present application, there is no particular limitation on the manner in which the fragmented tumor antigen and the immunomodulatory drug are used in combination, as long as the purpose of the present application can be achieved. For example, the immunomodulatory drug is injected on the first day of treatment, and the fragmented tumor antigen is injected on the second day. After an interval of 1 to 2 days, the above vaccination steps are repeated 2 to 3 times, which is one course of treatment, and a total of 1 to 5 courses of vaccination are required.

[0074] In one embodiment of the present application, the fragmented tumor antigen is treated in combination with radiotherapy.

[0075] In this application, the method of combined treatment with fragmented tumor antigens and radiotherapy is not particularly limited, as long as it can achieve the purpose of this application. For example, the tumor is first subjected to conventional fractionated radiotherapy, and then the fragmented tumor antigen is injected 1-3 days later. After an interval of 1-2 days, the above vaccination steps are repeated 2-9 times, for a total of 3-10 vaccinations of the fragmented tumor antigen. This application does not specifically limit the method of radiotherapy, and the radiotherapy can be conventional clinical tumor radiotherapy, for example, at least one of conventional fractionated radiotherapy, large fractionated radiotherapy, and single radiotherapy.

[0076] In one embodiment of the present application, the fragmented tumor antigen is used in combination with a low-dose chemotherapy drug as an immune adjuvant.

[0077] In the present application, there is no particular limitation on the combined use of fragmented tumor antigens and low-dose chemotherapy drugs, as long as the purpose of the present application can be achieved. For example, a low-dose chemotherapy drug is injected on the first day of treatment, and then a fragmented tumor antigen is injected after the second day. After an interval of 1 to 2 days, the above vaccination steps are repeated 2 to 3 times, which is one course of treatment, and a total of 1 to 5 courses of vaccination are required.

[0078] In one embodiment of the present application, the fragmented tumor antigen is used in combination with a clinical tumor therapeutic drug; the clinical tumor therapeutic drug is selected from at least one of a phospholipid compound, an immunomodulatory drug and a nucleic acid analog; preferably, the phospholipid compound is selected from at least one of PGPC, 1-hexadecyl 2-(4'-carboxybutyl) glycerol 3-phosphocholine (VB201) and oxidized phosphatidylcholine (OxPC), lecithin and cephalin; the immunomodulatory drug is selected from low-dose chemotherapy drugs, such as platinum drugs, such as: cisplatin, carboplatin, oxaliplatin, cyclosulfide platinum, nedaplatin and lobaplatin; the nucleic acid analog is selected from modified RNA or DNA.

[0079] In the present application, there is no particular limitation on the combined use of fragmented tumor antigens and clinical tumor therapeutic drugs, as long as the purpose of the present application can be achieved. For example, clinical tumor therapeutic drugs are administered on the first day of treatment, and fragmented tumor antigens are injected after the second day. The above vaccination steps are repeated 2 to 3 times after an interval of 1 to 2 days, which is one course of treatment, and a total of 1 to 5 courses of vaccination are required.

[0080] In this application, there is no particular limitation on the vaccination dose of the fragmented tumor antigen, as long as the purpose of this application can be achieved. For example, a single large-dose vaccination, multiple small-dose booster vaccinations, or multiple courses of vaccination can be used. A single large-dose vaccination can be a single vaccination dose of 3×10 6 ~5×10 6 For a total of 1 vaccination, a small-dose multiple booster vaccination can be a single vaccination dose of 0.5×10 6 ~2×10 6 Multiple courses of vaccination can be repeated high-dose single vaccination 1 to 5 times, that is, 1 to 5 courses of treatment; or repeated low-dose multiple booster vaccination 1 to 5 times, that is, 1 to 5 courses of treatment.

[0081] In this application, there is no particular limitation on the dosage of immunomodulatory drugs, and the manufacturer's instructions can be referred to, as long as the purpose of this application can be achieved. There is no particular limitation on the dosage of clinical tumor treatment drugs, and the manufacturer's instructions can be referred to, as long as the purpose of this application can be achieved.

[0082] In one embodiment of the present application, the injection method of the fragmented tumor antigen or the immunomodulatory drug is selected from at least one of subcutaneous injection (sc), intraperitoneal injection (ip), intravenous injection (iv) and intratumoral injection (it).

[0083] The sixth aspect of the present application provides the use of the tumor immunotherapy method provided in the fifth aspect of the present application in the treatment and / or prevention of tumor diseases.

[0084] In one embodiment of the present application, the tumor disease includes at least one of breast cancer, colorectal cancer, melanoma, lung cancer, liver cancer, pancreatic cancer, kidney cancer, esophageal cancer, gastric cancer, prostate cancer, brain cancer, oral cancer, bile duct cancer, ovarian cancer, cervical cancer, osteosarcoma and testicular cancer.

[0085] The tumor immunotherapy method of the present application does not require the extraction, sequencing and synthesis of the processed fragmented tumor antigens, nor does it require unique antigen delivery technology and related equipment. The preparation process is simple and time-consuming. At the same time, significant anti-tumor effects can be achieved without the use of combined immune checkpoint inhibitors. The reagents selected in the present application method are commonly used clinical drugs with low cost and high safety. The prepared fragmented tumor antigens can be stored at low temperatures, which is easy to store and transport, has excellent effects, and has great application prospects. More importantly, the tumor immunotherapy method of the present application has high immunogenicity, low off-target rate, can efficiently and long-term activate immune responses, has excellent anti-tumor effects in different tumor animal models, and can especially significantly inhibit tumor metastasis and effectively prolong the survival time of animals.

[0086] Example

[0087] The following examples and comparative examples are provided to more specifically illustrate the embodiments of the present application. Various experiments and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.

[0088] Experimental animals and materials

[0089] Female Balb / c mice were purchased from Zhejiang Provincial Experimental Animal Center, aged 4 to 6 weeks and weighing 20 ± 5 g.

[0090] Mouse 4T-1-Luc breast cancer tumor cells were donated by Wenzhou Medical University.

[0091] CT-26 colon cancer cells were purchased from ATCC.

[0092] B16-F10 melanoma cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0093] The mouse models used in this application were all constructed by our research group.

[0094] Example 1

[0095] (1) 4T-1-Luc breast cancer cells were collected by trypsin digestion, washed three times with phosphate buffered saline (PBS), and then added with 100 μL PBS and mixed evenly to prepare 1×10 6 Single cell suspension;

[0096] (2) The single cell suspension was irradiated and incubated at 37°C for 1 hour to obtain fragmented tumor antigens; X-ray irradiation was performed with an irradiation dose of 12 Gy and an irradiation dose rate of 0.1 Gy / s.

[0097] Example 2

[0098] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 1×10 6 Single cell suspension;

[0099] (2) The single cell suspension was irradiated and incubated at 37°C for 1 hour to obtain fragmented tumor antigens; X-ray irradiation was performed with an irradiation dose of 36 Gy and an irradiation dose rate of 0.1 Gy / s.

[0100] Example 3

[0101] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 1×10 6Single cell suspension;

[0102] (2) irradiating the single cell suspension and incubating at 37°C for 1 hour to obtain fragmented tumor antigens; irradiation was performed using X-rays with a dose of 12 Gy and a dose rate of 0.1 Gy / s;

[0103] (3) The fragmented tumor antigen is subjected to ultra-low temperature freeze-thaw treatment; the temperature of the ultra-low temperature freeze-thaw treatment is -80°C and the time is 12 hours.

[0104] Example 4

[0105] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 1×10 6 Single cell suspension;

[0106] (2) The single cell suspension was irradiated and incubated at 37°C for 6 hours to obtain fragmented tumor antigens; the irradiation was performed using X-rays with a dose of 12 Gy and a dose rate of 0.1 Gy / s;

[0107] (3) The fragmented tumor antigen is subjected to ultra-low temperature freeze-thaw treatment; the temperature of the ultra-low temperature freeze-thaw treatment is -80°C and the time is 12 hours.

[0108] Example 5

[0109] (1) CT-26 colon cancer cells were washed three times with PBS, and then 100 μL PBS was added and mixed evenly to prepare 1×10 6 Single cell suspension;

[0110] (2) irradiating the single cell suspension and incubating at 37°C for 1 hour to obtain fragmented tumor antigens; irradiation was performed using X-rays with a dose of 12 Gy and a dose rate of 0.1 Gy / s;

[0111] (3) The fragmented tumor antigen is subjected to ultra-low temperature freeze-thaw treatment; the temperature of the ultra-low temperature freeze-thaw treatment is -80°C and the time is 12 hours.

[0112] Example 6

[0113] (1) Wash B16-F10 melanoma cells three times with PBS, add 100 μL PBS and mix well to prepare 1×10 6 Single cell suspension;

[0114] (2) irradiating the single cell suspension and incubating at 37°C for 1 hour to obtain fragmented tumor antigens; irradiation was performed using X-rays with a dose of 12 Gy and a dose rate of 0.1 Gy / s;

[0115] (3) The fragmented tumor antigen is subjected to ultra-low temperature freeze-thaw treatment; the temperature of the ultra-low temperature freeze-thaw treatment is -80°C and the time is 12 hours.

[0116] Example 7

[0117] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 5×10 5 Single cell suspension;

[0118] (2) irradiating the single cell suspension and incubating at 37°C for 1 hour to obtain fragmented tumor antigens; irradiation was performed using X-rays with a dose of 12 Gy and a dose rate of 0.1 Gy / s;

[0119] (3) The fragmented tumor antigen is subjected to ultra-low temperature freeze-thaw treatment; the temperature of the ultra-low temperature freeze-thaw treatment is -80°C and the time is 12 hours.

[0120] Example 8

[0121] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 5×10 6 Single cell suspension;

[0122] (2) irradiating the single cell suspension and incubating at 37°C for 1 hour to obtain fragmented tumor antigens; irradiation was performed using X-rays with a dose of 12 Gy and a dose rate of 0.1 Gy / s;

[0123] (3) The fragmented tumor antigen is subjected to ultra-low temperature freeze-thaw treatment; the temperature of the ultra-low temperature freeze-thaw treatment is -80°C and the time is 12 hours.

[0124] Comparative Example 1

[0125] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 1×10 6 Single cell suspension;

[0126] (2) The single cell suspension was ultrasonically treated and then incubated at 37°C for 1 hour to obtain fragmented tumor antigens; the ultrasonic power was 400 W and the ultrasonic treatment time was 30 minutes.

[0127] Comparative Example 2

[0128] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 1×106 Single cell suspension;

[0129] (2) The single cell suspension was subjected to hyperthermia treatment and then incubated at 37°C for 1 hour to obtain fragmented tumor antigens; the hyperthermia temperature was 45°C and the hyperthermia time was 1 hour.

[0130] Comparative Example 3

[0131] (1) 4T-1-Luc breast cancer tumor cells were collected by trypsin digestion, washed three times with PBS, and then added with 100 μL PBS and mixed evenly to prepare 1×10 6 Single cell suspension;

[0132] (2) Add doxorubicin to the single cell suspension for 6 hours and then incubate at 37°C for 1 hour to obtain fragmented tumor antigens; the final concentration of doxorubicin is 10 μM.

[0133] Test Example 1

[0134] Fifty six-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells per mouse on day 0. The cells were then inoculated until the tumor grew to 150 mm. 3 Afterwards, Balb / c mice were randomly divided into the following 10 groups and started the corresponding treatment:

[0135] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day of tumor bearing;

[0136] Group 2 (IR): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 1 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0137] Group 3 (ultrasound): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Comparative Example 1 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0138] Group 4 (hyperthermia): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Comparative Example 2 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0139] Group 5 (DOX): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Comparative Example 3 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0140] Group 6 (Mn): Each Balb / c mouse was subcutaneously injected with 100 μL of 200 μg / mL Mn into the left hind limb on the 5th, 7th, and 9th day of tumor bearing.

[0141] Group 7 (IR+Mn): Subcutaneous inoculation of Mn combined with the fragmented tumor antigen prepared in Example 1. That is, on the first day of treatment, each Balb / c mouse was subcutaneously injected with 100 μL of 200 μg / mL Mn into the left hind limb. The next day, 100 μL of the fragmented tumor antigen prepared in Example 1 was subcutaneously injected. The above inoculations were repeated two more times after an interval of one day.

[0142] Group 8 (ultrasound + Mn): Subcutaneous inoculation of Mn combined with the fragmented tumor antigen prepared in Comparative Example 1. That is, on the first day of treatment, each Balb / c mouse was subcutaneously injected with 100 μL of 200 μg / mL Mn into the left hind limb. The next day, 100 μL of the fragmented tumor antigen prepared in Comparative Example 1 was subcutaneously injected. The above inoculations were repeated two more times after an interval of one day.

[0143] Group 9 (hyperthermia + Mn): Subcutaneous inoculation of Mn combined with the fragmented tumor antigen prepared in Comparative Example 2. That is, on the first day of treatment, each Balb / c mouse was subcutaneously injected with 100 μL of 200 μg / mL Mn into the left hind limb. The next day, 100 μL of the fragmented tumor antigen prepared in Comparative Example 2 was subcutaneously injected. The above inoculations were repeated two more times after an interval of one day.

[0144] Group 10 (DOX + Mn) was subcutaneously inoculated with Mn combined with the fragmented tumor antigen prepared in Comparative Example 3. That is, on the first day of treatment, each Balb / c mouse was subcutaneously injected with 100 μL of 200 μg / mL Mn into the left hind limb. On the second day, 100 μL of the fragmented tumor antigen prepared in Comparative Example 3 was subcutaneously injected. The above inoculation was repeated 2 times after an interval of 1 day.

[0145] The growth of mouse tumors was observed regularly during the experiment.

[0146] Figure 1A shows the growth inhibitory effect of fragmented tumor antigens and their combined Mn adjuvant on 4T-1 distal tumors after irradiation treatment; Figure 1B shows the growth inhibitory effect of fragmented tumor antigens and their combined Mn adjuvant on 4T-1 distal tumors after ultrasound treatment; Figure 1C shows the growth inhibitory effect of fragmented tumor antigens and their combined Mn adjuvant on 4T-1 distal tumors after hyperthermia treatment; Figure 1D shows the growth inhibitory effect of fragmented tumor antigens and their combined Mn adjuvant on 4T-1 distal tumors after treatment with chemotherapy drugs (doxorubicin, DOX).

[0147] As shown in Figures 1A to 1D, fragmented tumor antigens obtained by ultrasound, thermotherapy, or doxorubicin treatment had no inhibitory effect on tumor growth and even promoted it. Even in combination with a Mn adjuvant, this did not inhibit tumor growth. Only irradiated fragmented tumor antigens inhibited tumor growth, producing a strong anti-tumor effect. Irradiated tumor cells, either alone or in combination with a Mn adjuvant, significantly inhibited tumor growth, but the Mn adjuvant did not effectively enhance the tumor-control efficacy of irradiated fragmented tumor antigens.

[0148] Test Example 2

[0149] Fifteen 6-week-old female Balb / c mice were inoculated with logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The number of cells inoculated was 1 million per mouse. The cells were kept in the hind limb until the tumor grew to 150 mm. 3 Afterwards, Balb / c mice were randomly divided into the following three groups and started corresponding treatment:

[0150] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day of tumor bearing;

[0151] Group 2 (FAST-12 Gy): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 1 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0152] Group 3 (FAST-36Gy): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 2 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0153] The growth of mouse tumors was observed regularly during the experiment.

[0154] Figure 2 shows the growth inhibition effect of fragmented tumor antigen immunotherapy on distal 4T-1 tumors after different irradiation doses. As shown in Figure 2, fragmented tumor antigens irradiated with either 12Gy or 36Gy can inhibit tumor growth, producing a strong anti-tumor effect. Compared with the 36Gy group, the 12Gy group showed an earlier onset of tumor growth inhibition and a stronger ability to suppress tumor growth.

[0155] Test Example 3

[0156] Twenty 6-week-old female Balb / c mice were inoculated with logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The number of cells inoculated was 1 million per mouse. After the tumor grew to 150 mm 3Then, Balb / c mice were randomly divided into the following 4 groups:

[0157] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day of tumor bearing;

[0158] Group 2 (FA-1h): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 3 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0159] Group 3 (FA-6h): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 4 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0160] Group 4 (FA(1+6)h): Each Balb / c mouse was subcutaneously inoculated with 50 μL of the fragmented tumor antigen prepared in Example 3 and 50 μL of the fragmented tumor antigen prepared in Example 4 on the 6th, 8th, and 10th day of tumor bearing, respectively, on the left hind limb.

[0161] The growth of mouse tumors was observed regularly during the experiment.

[0162] Figure 3A shows the inhibitory effect of fragmented tumor antigens incubated at different times after irradiation on the growth of distal 4T-1 tumors. As shown in Figure 3A, fragmented tumor antigens incubated for 1 hour and 6 hours after 12 Gy irradiation both inhibited tumor growth and produced a strong anti-tumor effect, with the fragmented tumor antigens incubated for 1 hour achieving a stronger inhibitory effect. Figure 3B shows the inhibitory effect of mixed fragmented tumor antigens inoculated at different times after irradiation on the growth of distal 4T-1 tumors. The CON group in Figures 3A and 3B represents two mouse experiments conducted at different times. To compare the tumor inhibitory effects between groups, the data from the two experiments were normalized to the CON group. Figure 3C shows the normalized inhibitory effect of fragmented tumor antigens inoculated at different times after irradiation on the growth of 4T-1 tumors. As shown in Figure 3C, mixed fragmented tumor antigens inoculated 1 hour and 6 hours after irradiation achieved a stronger inhibitory effect on tumor growth and produced a stronger anti-tumor effect.

[0163] Test Example 4

[0164] Four 6-week-old female Balb / c mice were subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 1 into the left hind limb of each Balb / c mouse on day 0. Subcutaneous inoculations were repeated on days 6, 8, and 10 of tumor development. The growth of the orthotopic tumors in the mice was regularly observed during the experiment.

[0165] Figure 4 shows that the fragmented tumor antigens that were not subjected to ultra-low temperature freeze-thaw treatment formed a tumor at the inoculation site 26 days after inoculation, indicating that the fragmented tumor antigens that were only irradiated have a certain tumorigenic risk and are relatively low in safety.

[0166] Test Example 5

[0167] Thirty-two 6-week-old female Balb / c mice were inoculated with logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The number of cells inoculated was 1 million per mouse. The cells were inoculated after the tumors grew to 150 mm. 3 Afterwards, Balb / c mice were randomly divided into the following 4 groups and started corresponding treatment:

[0168] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day of tumor bearing;

[0169] Group 2 (FA): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 3 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0170] Group 3 (CTX): Each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL cyclophosphamide (CTX) on the 5th, 7th, and 9th day of tumor bearing.

[0171] Group 4 (FAST): Intraperitoneal injection of CTX combined with subcutaneous inoculation of fragmented tumor antigen in the left hind limb, that is, on the first day of treatment, each Balb / c mouse was first intraperitoneally injected with 100 μL of 10 mg / mL CTX, and on the next day, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left hind limb, and then the above inoculation was repeated twice.

[0172] During the experiment, the tumor growth of mice was regularly monitored, the number of days of survival of mice was recorded for survival analysis, and the lung metastasis of mice was counted.

[0173] Figure 5A shows the in vivo imaging of mice after FAST treatment in the 4T-1 cold tumor distal model, Figure 5B shows the tumor growth inhibitory effect of FAST in the 4T-1 cold tumor distal model, Figure 5C shows the survival extension effect of FAST in the 4T-1 cold tumor distal model, and Figure 5D shows the effect of FAST in inhibiting tumor lung metastasis in the 4T-1 cold tumor distal model.

[0174] As can be seen from Figures 5A to 5C, the fragmented tumor antigen obtained in Example 3 can effectively inhibit the growth of 4T-1 distal tumors and prolong the survival of mice. Compared with the CON group, the CTX group, FA group, and FAST group can all effectively inhibit the growth of distal tumors in mice and increase the survival of mice, among which the FAST group has the best effect (n≥5, ***P<0.001). As can be seen from Figure 5D, compared with CON, FA and FAST can significantly inhibit tumor lung metastasis, and the FAST group has a better effect in inhibiting tumor lung metastasis.

[0175] Test Example 6

[0176] Twenty-four 6-week-old female Balb / c mice were inoculated with 1 million CT-26 colon cancer cells in the right hind limb. 3 Afterwards, Balb / c mice were randomly divided into the following 4 groups and started corresponding treatment:

[0177] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day of tumor bearing;

[0178] Group 2 (FA): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 5 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0179] Group 3 (CTX): Each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on the 5th, 7th, and 9th day of tumor bearing;

[0180] Group 4 (FAST): Intraperitoneal injection of CTX combined with subcutaneous inoculation of fragmented tumor antigen on the left side, that is, on the first day of treatment, each Balb / c mouse was first intraperitoneally injected with 100 μL of 10 mg / mL CTX, and on the next day, 100 μL of the fragmented tumor antigen prepared in Example 5 was subcutaneously injected into the left hind limb. The above inoculation was repeated 2 times after an interval of 1 day.

[0181] During the experiment, the tumor growth of mice was regularly monitored, in vivo imaging was performed to show the tumor size and lung metastasis level of mice, and the number of days the mice survived was recorded for survival analysis.

[0182] Figure 6A shows the in vivo imaging of mice after FAST treatment in the CT-26 thermal tumor distal model, Figure 6B shows the tumor inhibitory effect of FAST in the CT-26 thermal tumor distal model, and Figure 6C shows the survival extension effect of FAST in the CT-26 thermal tumor distal model.

[0183] As can be seen from Figure 6A, the fragmented tumor antigen obtained in Example 5 can effectively inhibit the growth of distal tumors of CT-26 hot tumors and prolong the survival time of mice. As can be seen from Figures 6B and 6C, 20 days after inoculation, compared with the CON group, the CTX group, FA group and FAST group can all effectively inhibit the growth of distal tumors in mice (n≥5, ***P<0.001) and increase the survival time of mice. Among them, the FAST group had the best effect, which could completely regress the tumor. Even on the 65th day, the survival rate of mice in the FAST group was still 100%.

[0184] Test Example 7

[0185] Forty six-week-old female C57BL / 6 mice were inoculated with logarithmically growing B16-F10 melanoma cells in the right hind limbs at a rate of 1 million cells per mouse. The mice were inoculated with B16-F10 melanoma cells ... 3 Afterwards, C57BL / 6 mice were randomly divided into the following 4 groups and started corresponding treatment:

[0186] Group 1 (CON): Each C57BL / 6 mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day of tumor bearing;

[0187] Group 2 (FA): Each C57BL / 6 mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 6 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0188] Group 3 (CTX): Each C57BL / 6 mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on the 5th, 7th, and 9th day of tumor bearing;

[0189] Group 4 (FAST): Intraperitoneal injection of CTX combined with subcutaneous inoculation of fragmented tumor antigen on the left side, that is, on the first day of treatment, each C57BL / 6 mouse was first intraperitoneally injected with 100 μL of 10 mg / mL CTX, and on the next day, 100 μL of the fragmented tumor antigen prepared in Example 6 was subcutaneously injected into the left hind limb. The above inoculation was repeated 2 times after an interval of 1 day.

[0190] During the experiment, the tumor growth of mice was detected regularly, and the number of days the mice survived was recorded for survival analysis.

[0191] Figure 7A shows the tumor growth inhibitory effect of FAST in the B16-F10 cold tumor distal model, and Figure 7B shows the survival prolonging effect of FAST in the B16-F10 cold tumor distal model.

[0192] Cold tumors typically express low levels of immunostimulatory molecules and accumulate a large number of immunosuppressive cells in their microenvironment, which can effectively evade the surveillance and attack of the immune system, thereby reducing the therapeutic effect and increasing the difficulty of tumor treatment. As can be seen in Figure 7A, compared with the CON group, the FAST group can effectively inhibit the growth of the distal cold tumor B16-F10 in mice (n ≥ 5, ***P < 0.001), while the tumor inhibition effect of the CTX group and the FA group was not significant. Figure 7B shows that compared with the CON group, FAST and CTX can increase the survival time of mice, among which the survival extension effect of the FAST group is better.

[0193] Test Example 8

[0194] Forty six-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells per mouse on day 0. The cells were then inoculated until the tumor grew to 150 mm. 3 Afterwards, Balb / c mice were anesthetized with a gas anesthesia machine, and most of the tumor tissue was surgically removed. The Balb / c mice after tumor removal were randomly divided into the following four groups and subjected to in vivo imaging. The corresponding treatment was started on the second day after surgery:

[0195] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from day 8 to day 13 after tumor loading;

[0196] Group 2 (FA): Each Balb / c mouse was subcutaneously inoculated with the fragmented tumor antigen prepared in Example 3 on the left hind limb on the 9th, 12th, and 14th day after tumor bearing.

[0197] Group 3 (CTX): Each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on the 8th, 10th, and 13th day of tumor bearing;

[0198] Group 4 (FAST): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously inoculated with fragmented tumor antigen in the left hind limb. That is, on the first day of treatment, each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX, and on the second day, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left hind limb. The above inoculation was repeated twice.

[0199] During the experiment, the tumor growth of mice was regularly detected, lung metastasis of mice was observed, and the number of days of survival of mice was recorded for survival analysis.

[0200] Figure 8A shows the in vivo imaging of mice before and after surgery and at different times after FAST treatment in the 4T-1 cold tumor surgical resection model. Figure 8B shows the effect of FAST in inhibiting tumor lung metastasis in the 4T-1 cold tumor surgical resection model. Figure 8C shows the survival-extending effect of FAST in the 4T-1 cold tumor surgical resection model.

[0201] As can be seen from Figure 8A, compared with the CON group, the fragmented tumor antigens, CTX and FAST obtained in Example 3 can effectively inhibit the recurrence of 4T-1 distal tumors after surgery. 21 days and 28 days after surgery, all distal tumors of mice in the normal saline group relapsed and grew, very small tumors appeared in the distal part of the mice in the fragmented tumor antigen group, and there was basically no recurrence of distal tumors in the mice in the CTX and FAST immunization groups. No tumor was observed in the distal part of the mice in the FAST immunization group 40 days after surgery. As can be seen from Figures 8B and 8C, compared with normal saline, fragmented tumor antigens can significantly reduce tumor lung metastasis and prolong the survival time of some mice; CTX alone or FAST immunization can effectively inhibit the occurrence of distal lung tumor metastasis and significantly prolong the survival time of mice, and FAST treatment effect is better.

[0202] Test Example 9

[0203] Eighteen 6-week-old female Balb / c mice were subcutaneously inoculated with logarithmically growing CT-26 colon cancer cells in the left hind limb on day 0. The number of cells inoculated was 1 million per mouse. After the tumor grew to 100 mm 3 Afterwards, Balb / c mice were randomly divided into the following three groups and started corresponding treatment:

[0204] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline into the left hind limb three times with an interval of 1 day from the 6th to the 10th day of tumor bearing;

[0205] Group 2 (FA(4T1)): Each Balb / c mouse was subcutaneously inoculated with the fragmented tumor antigen prepared in Example 3 on the left hind limb on the 6th, 8th, and 10th day after tumor bearing.

[0206] Group 3 (FA(CT26)): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 5 on the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0207] The tumor growth of mice was monitored regularly during the experiment.

[0208] FIG9A shows the in vivo imaging of mice in which fragmented antigens derived from different tumor cells specifically inhibit tumor growth, and FIG9B shows the in vivo imaging of mice in which fragmented antigens derived from different tumor cells specifically inhibit tumor growth.

[0209] As shown in Figure 9A, compared with the CON group, FA(CT26) inoculation significantly inhibited the growth of the contralateral CT-26 tumor, while FA(4T1) inoculation did not. The tumor growth curve in Figure 9B also shows that FA(CT26) significantly inhibited the growth of the contralateral CT-26 tumor relative to the CON group, while FA(4T1) inoculation did not. Furthermore, FA(CT26) also effectively inhibited the growth of the contralateral CT-26 tumor relative to the FA(4T1) group (n ≥ 4, *P < 0.05, **P < 0.01, ns indicates no difference).

[0210] Test Example 10

[0211] Twenty 6-week-old female Balb / c mice were inoculated with logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The number of cells inoculated was 1 million per mouse. After the tumor grew to 100 mm 3 Afterwards, they were randomly divided into the following 4 groups and started corresponding treatment:

[0212] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline into the left hind limb for 6 consecutive days from the 5th to the 10th day after tumor bearing; and then repeated for 2 more courses.

[0213] Group 2 (FAST (One)): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously inoculated with the fragmented tumor antigen prepared in Example 3 in the left hind limb for FAST immunization. That is, on the first day of treatment, each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX. On the second day, each Balb / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 3 in the left hind limb. This inoculation was repeated two more times after an interval of one day.

[0214] Group 3 (FAST (Two)): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously inoculated with the fragmented tumor antigen prepared in Example 3 in the left hind limb for FAST immunization. That is, on the first day of treatment, each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX. On the second day, each Balb / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 3 in the left hind limb. This inoculation was repeated two more times after an interval of one day. Three immunizations constituted one course of treatment. After one course of treatment, another course of treatment was repeated.

[0215] Group 4 (FAST (Three)): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously inoculated with the fragmented tumor antigen prepared in Example 3 in the left hind limb for FAST immunization. That is, on the first day of treatment, each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX, and on the next day, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left hind limb. The above vaccination was repeated two times with an interval of 1 day. Three immunizations constituted one course of treatment. After one course of vaccination was completed, two more courses of treatment were repeated.

[0216] During the experiment, the growth of mouse tumors was observed regularly, and the lung metastasis of the mouse tumors and the immune memory level of FAST therapy were detected.

[0217] Figure 10A shows the in vivo imaging of mice treated with FAST at different courses of treatment, Figure 10B shows the tumor inhibitory effect of FAST treatment at different courses of treatment, Figure 10C shows the tumor lung metastasis inhibitory effect of FAST treatment at different courses of treatment, and Figure 10D shows that three courses of FAST treatment can significantly induce the production of central memory cells and effector memory cells.

[0218] As can be seen from Figures 10A and 10B, compared with the CON group, vaccination with 1, 2, and 3 courses of FAST can significantly inhibit the growth of the contralateral tumor, and the contralateral tumor inhibition effect of the FAST group with 3 courses of vaccination is the best. As can be seen from Figure 10C, vaccination with 1, 2, and 3 courses of FAST can significantly inhibit the production of distal metastases, and the effect of the FAST group with 3 courses of vaccination is the best. As can be seen from Figure 10D, compared with the CON group, a significant increase in central memory cells and effector memory cells can be detected in the FAST (Three) group, indicating that 3 courses of FAST immunization can produce long-term anti-tumor effects (n≥4, **P<0.01). The above results show that the tumor immunotherapy method developed in this application can not only effectively inhibit distal tumors and metastases, but also multiple immunizations can produce immune memory and exert long-term anti-tumor effects.

[0219] Test Example 11

[0220] Sixty six-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The cells were then inoculated and the mice were inoculated until the tumors grew to 100 mm. 3 Afterwards, Balb / c mice were randomly divided into the following 10 groups and started the corresponding treatment:

[0221] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day after tumor loading;

[0222] Group 2 (FA): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 3 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0223] Group 3 (CTX): Each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on the 5th, 7th, and 9th day of tumor bearing;

[0224] Group 4 (FAST): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously inoculated with fragmented tumor antigen into the left hind limb. That is, on the first day of treatment, each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX. On the second day, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left hind limb. This inoculation was repeated two more times after an interval of one day.

[0225] Group 5 (α-PD-L1): Each Balb / c mouse was intraperitoneally injected with 100 μL of programmed cell death-ligand 1 (PD-L1) (B7-H1; BIOCELL, Catalog #BE0101) on the 5th, 7th, 9th, and 11th day of tumor bearing.

[0226] Group 6 (α-CTLA-4): Each Balb / c mouse was intraperitoneally injected with 100 μL of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (CD152; BIOCELL, Catalog#: BP0164) on the 5th, 7th, 9th, and 11th day of tumor bearing.

[0227] Group 7 (α-PD-L1 + CTX): Each Balb / c mouse was intraperitoneally injected with 100 μL of anti-PD-L1 (B7-H1; BIOCELL, Catalog #BE0101) on the 5th, 7th, 9th, and 11th day of tumor bearing. Simultaneously, each mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on the 5th, 7th, and 9th day of tumor bearing.

[0228] Group 8 (α-CTLA-4 + CTX): Each Balb / c mouse was intraperitoneally injected with 100 μL of anti-CTLA-4 (CD152; BIOCELL, Catalog #: BP0164) on the 5th, 7th, 9th, and 11th day of tumor bearing. Simultaneously, each mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on the 5th, 7th, and 9th day of tumor bearing.

[0229] Group 9 (α-PD-L1+FA): Each Balb / c mouse was intraperitoneally injected with 100 μL of anti-PD-L1 on the 5th, 7th, 9th, and 11th day of tumor bearing. Simultaneously, each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 3 on the 6th, 8th, and 10th day of tumor bearing.

[0230] Group 10 (α-CTLA-4 + FA): Each Balb / c mouse was intraperitoneally injected with 100 μL of anti-CTLA-4 on the 5th, 7th, 9th, and 11th day of tumor bearing. Simultaneously, each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 3 on the 6th, 8th, and 10th day of tumor bearing.

[0231] During the experiment, the tumor growth of mice was regularly monitored and the lung metastasis of mice was observed.

[0232] Figure 11A shows the in vivo imaging of mice treating with FA combined with α-PD-L1 / α-CTLA-4, and Figure 11B shows the tumor growth inhibition effect of FA combined with PD-L1 / CTLA-4. Figure 11C shows the tumor lung metastasis inhibition effect of FA and FA combined with α-PD-L1 / α-CTLA-4.

[0233] As can be seen from Figures 11A and 11B, injection of CTX or fragmented tumor antigens alone can inhibit tumor growth, and FAST immunization can significantly inhibit tumor growth. We combined FA with PD-L1 or CTLA-4 inhibitors to treat tumor-bearing mice and found that combined ICI (α-PD-L1 or α-CTLA-4) did not significantly enhance the inhibitory effect of FA on distant tumor growth (Figures 11A and 11B). At the same time, Figure 11C shows the results of the detection of distal lung tissue metastases. CTX and α-PD-L immunization alone cannot inhibit the occurrence and development of distal metastases, while fragmented tumor antigen alone or FAST immunization can effectively inhibit the generation of distal metastases. The combination of FA and α-PD-L1 did not further reduce the risk of lung metastasis, while the combination of α-CTLA-4 reduced lung metastasis to a certain extent (Figure 11C). This shows that FA alone has shown a significant anti-tumor effect when administered with FAST, and FA combined with α-PD-L1 did not produce a synergistic anti-tumor effect, that is, FAST alone can achieve the therapeutic effect of FA combined with immunosuppressants.

[0234] Test Example 12

[0235] Twenty six-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb. 3 Afterwards, they were randomly divided into the following 4 groups and started corresponding treatment:

[0236] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline on the left hind limb for 6 consecutive days from the 5th to the 10th day after tumor loading;

[0237] Group 2 (FA): Each Balb / c mouse was subcutaneously inoculated with 100 μL of the fragmented tumor antigen prepared in Example 3 into the left hind limb on the 6th, 8th, and 10th day of tumor bearing.

[0238] Group 3 (CTX): Each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on the 5th, 7th, and 9th day of tumor bearing;

[0239] Group 4 (FAST): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously inoculated with fragmented tumor antigen into the left hind limb. That is, on the first day of treatment, each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX. On the second day, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left hind limb. The above inoculations were repeated 2 more times after 1 day.

[0240] Distal tumor tissue was collected from each group of mice and stored in normal saline at 4°C. The tissue was cut into small pieces and 0.25 g of tumor tissue was placed in complete culture medium containing collagenase (1 μg / ml) and hyaluronidase (0.1 μg / ml). The tissue was ground twice using a tissue grinder and then digested overnight in a 37°C shaker. After filtering through a 100 μm cell sieve, a single-cell suspension of tumor tissue was prepared. Fixed cells were labeled with flow cytometry-related antibodies and surface markers were added to analyze the percentage of immune cells in the tumor tissue. The flow cytometry antibodies are as follows: anti-CD45-APC-CY7 (BD, NO:557659); anti-CD45-AF700 (BD, NO:560510); anti-CD3-FITC (BD, NO:553061); anti-FOXP3-PE (BD, NO:560408); anti-CD62L-PE (BD, NO:553151); anti-CD4-Percp-cy5.5 (BD, NO:550954); anti-Ki67-APC (Thermo, NO:17-5698-82); anti-CD44-APC (BD, NO:559250); anti-CD8e-APC-CY7 (BD, NO:557654).

[0241] Figure 12A shows the percentage of CD3+T cells in tumor tissue after treatment with FAST and its corresponding components, Figure 12B shows the percentage of CD8+T cells in tumor tissue after treatment with FAST and its corresponding components, Figure 12C shows the percentage of CD4+T cells in tumor tissue after treatment with FAST and its corresponding components, Figure 12D shows the percentage of Treg cells (Foxp3+CD4+T) in tumor tissue after treatment with FAST and its corresponding components, and Figure 12E shows the percentage of KI76+CD8+T cells in tumor tissue after treatment with FAST and its corresponding components.

[0242] Compared with the other groups, the FAST group significantly increased the percentage of immune cells in the tumor (Figure 12A). Further analysis revealed that the levels of CD4+ T cells and CD8+ T cells increased (Figures 12B and 12C), and Treg cells (FOX3 + CD4 +The ratio of KI67 in tumor tissue decreased (Figure 12D), indicating that FAST can promote the infiltration of immune cells in tumors, enhance the content of T cells and inhibit Treg cells, thereby promoting immune activation. + CD8 + The proportion of T cells increased significantly (Figure 12E), indicating that FAST can exert anti-tumor effects by activating the body's adaptive immune response.

[0243] At the same time, we used the fragmented tumor antigen (FA) prepared in this application in combination with other treatment methods or drugs, and evaluated the anti-tumor effect of the combined application with FAST. For details, see Test Examples 13 to 16.

[0244] Test Example 13

[0245] Twenty six-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The cells were then inoculated until the tumor grew to 200 mm. 3 They were randomly divided into the following 4 groups and started corresponding treatment:

[0246] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline into the left hind limb on days 16, 18, and 20 after tumor loading;

[0247] Group 2 (IR): On days 8, 10, and 12 after tumor loading, the tumors of each Balb / c mouse received 8 Gy of X-ray irradiation for a total of three times;

[0248] Group 3 (IR+FA): On days 8, 10, and 12 after tumor implantation, the tumors of each Balb / c mouse received 8 Gy of X-ray irradiation for a total of three times. After a three-day rest, the mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 3 on the left side on days 16, 18, and 20 after tumor implantation.

[0249] Group 4 (FAST): Each Balb / c mouse received 100 μL of the fragmented tumor antigen prepared in Example 3 subcutaneously injected into the left hind limb. This was performed three times on days 16, 18, and 20 after tumor implantation. Mice were intraperitoneally injected with 100 μL of 10 mg / mL CTX as an adjuvant three times, one day prior to administration of the fragmented tumor antigen.

[0250] During the experiment, the tumor growth of each group of mice was regularly monitored, and the anti-tumor effects of different treatments were compared with the effects of FAST treatment.

[0251] FIG13A shows the normalized tumor growth inhibitory effect of FA combined with different treatments such as IR and FAST, and FIG13B shows the normalized tumor lung metastasis inhibitory effect of FA combined with different treatments such as IR and FAST.

[0252] As shown in Figure 13A, compared with the CON group, IR alone and IR + FA achieved comparable tumor growth inhibition as FAST in the middle and late stages of treatment. As shown in Figure 13B, compared with the CON group, IR had no effect on lung metastasis, meaning it could not effectively inhibit lung metastasis. Compared with the CON group, FA combined with IR significantly reduced lung metastasis, with an inhibitory effect comparable to that of FAST (n ≥ 5, ***P < 0.001, ****P < 0.0001, ns indicates no difference).

[0253] Test Example 14

[0254] Twenty-five 6-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The cells were then inoculated and the mice were inoculated until the tumors grew to 200 mm. 3 They were randomly divided into the following 5 groups and started corresponding treatment:

[0255] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline into the left hind limb on days 16, 18, and 20 after tumor loading;

[0256] Group 2 (CDDP): Balb / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) three times on days 15, 17, and 19 after tumor loading;

[0257] Group 3 (CDDP+FA): Balb / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 after tumor implantation. Simultaneously, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left flank of the mice on days 20, 22, and 24 after tumor implantation.

[0258] Group 4 (CDDP+IR+FA): On days 8, 10, and 12 after tumor implantation, the tumors of each Balb / c mouse received 8 Gy of X-ray irradiation for a total of three times. After a two-day rest, the BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 after tumor implantation. Concurrently, on days 16, 18, and 20 after tumor implantation, the mice were subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 3 on the left flank.

[0259] Group 5 (FAST): Each Balb / c mouse received 100 μL of the fragmented tumor antigen prepared in Example 3 subcutaneously injected into the left hind limb. This was performed three times on days 16, 18, and 20 after tumor implantation. A total of three injections were also performed. One day prior to the administration of the fragmented tumor antigen, 100 μL of 10 mg / mL CTX was injected intraperitoneally as an adjuvant.

[0260] During the experiment, the growth of in situ tumors in each group of mice was regularly monitored, and the anti-tumor effects of different treatments were compared with the effects of FAST treatment.

[0261] FIG14A shows the normalized tumor growth inhibitory effect of CDDP combined with different treatments such as IR or FA and FAST, and FIG14B shows the normalized tumor lung metastasis inhibitory effect of CDDP combined with different treatments such as IR or FA and FAST.

[0262] As shown in Figure 14A, compared with the CON group, the CDDP and CDDP+FA groups partially inhibited tumor growth. The CDDP+IR+FA group significantly inhibited tumor growth, achieving an inhibitory effect comparable to that of the FAST group. As shown in Figure 14B, compared with the CON group, the CDDP group had no effect on lung metastasis. Both the CDDP+FA and CDDP+IR+FA groups effectively reduced lung metastasis, with inhibitory effects similar to those of FAST (n ≥ 5, *P < 0.05, ***P < 0.001, ****P < 0.0001). Furthermore, the CDDP+IR+FA group exhibited less volatile and more stable inhibitory effects on lung metastasis.

[0263] Test Example 15

[0264] Twenty-five 6-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The cells were then inoculated and the mice were inoculated until the tumors grew to 200 mm. 3 They were randomly divided into the following 5 groups and started corresponding treatment:

[0265] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline into the left hind limb on days 16, 18, and 20 after tumor loading;

[0266] Group 2 (PGPC+CDDP): CDDP (5 mg / kg) was intraperitoneally injected into Balb / c mice on days 15, 17, and 19 after tumor implantation, for a total of three times; PGPC was subcutaneously inoculated into the left hind limb of Balb / c mice on days 16, 18, and 20 after tumor implantation, for a total of three times;

[0267] Group 3 (PGPC+CDDP+FA): CDDP (5 mg / kg) was intraperitoneally injected into Balb / c mice on days 15, 17, and 19 after tumor implantation, for a total of three times. Simultaneously, 100 μL of the fragmented tumor antigen FA and PGPC prepared in Example 3 were subcutaneously inoculated into the left hind limb of Balb / c mice on days 16, 18, and 20 after tumor implantation, for a total of three inoculations.

[0268] Group 4 (PGPC+CDDP+IR+FA): On days 8, 10, and 12 after tumor implantation, the tumors of each Balb / c mouse received 8 Gy of X-ray irradiation for a total of three times. After a two-day rest, the BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 after tumor implantation. Simultaneously, on days 16, 18, and 20 after tumor implantation, the mice were subcutaneously injected with 100 μL of the fragmented tumor antigen FA and PGPC prepared in Example 3 on the left flank.

[0269] Group 5 (FAST): Each Balb / c mouse received 100 μL of the fragmented tumor antigen prepared in Example 3 subcutaneously injected into the left hind limb. This was performed three times on days 16, 18, and 20 after tumor implantation. A total of three injections were also performed. One day prior to the administration of the fragmented tumor antigen, 100 μL of 10 mg / mL CTX was injected intraperitoneally as an adjuvant.

[0270] During the experiment, the growth of in situ tumors in each group of mice was regularly monitored, and the anti-tumor effects of different treatments were compared with the effects of FAST treatment.

[0271] Figure 15A shows the tumor growth inhibition effect of PGPC+CDDP combined with different treatments such as IR or FA and FAST after normalization, and Figure 15B shows the tumor lung metastasis inhibition effect of PGPC+CDDP combined with different treatments such as IR or FA and FAST after normalization.

[0272] As shown in Figure 15A, compared with the CON group, the PGPC+CDDP, PGPC+CDDP+FA, and PGPC+CDDP+IR+FA groups all inhibited tumor growth, with the PGPC+CDDP+FA group exhibiting a greater inhibitory effect than the PGPC+CDDP group. The PGPC+CDDP+IR+FA group exhibited the strongest tumor inhibition among the three treatments, achieving a tumor growth inhibition effect comparable to that of the FAST group in the later stages of treatment. As shown in Figure 15B, compared with the CON group, the PGPC+CDDP, PGPC+CDDP+FA, and PGPC+CDDP+IR+FA groups all significantly reduced lung metastasis, with inhibitory effects comparable to those of the FAST group (n ≥ 5, *P < 0.05, **P < 0.01, ***P < 0.001). Furthermore, the PGPC+CDDP group exhibited a more robust and stable inhibitory effect on lung metastasis.

[0273] Test Case 16

[0274] Based on Test Example 14, we selected the CDDP+IR+FA treatment method with good anti-tumor effect and evaluated the FA inoculation dose. The specific operation is as follows:

[0275] Twenty six-week-old female Balb / c mice were inoculated with 1 million logarithmically growing 4T-1-Luc breast cancer cells in the right hind limb on day 0. The cells were then inoculated until the tumor grew to 200 mm. 3 They were randomly divided into the following 4 groups and started corresponding treatment:

[0276] Group 1 (CON): Each Balb / c mouse was subcutaneously inoculated with 100 μL of normal saline into the left hind limb on days 16, 18, and 20 after tumor loading;

[0277] Group 2 (CDDP+IR+FA-50w): On days 8, 10, and 12 after tumor implantation, the tumors of each Balb / c mouse received 8 Gy of X-ray irradiation for a total of three times. After a two-day rest, the BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 after tumor implantation. Simultaneously, on days 16, 18, and 20 after tumor implantation, the mice were subcutaneously injected with 100 μL of fragmented tumor antigen prepared from 50w tumor cells according to the method of Example 7 for a total of three times.

[0278] Group 3 (CDDP+IR+FA-100w): On days 8, 10, and 12 after tumor implantation, the tumors of each Balb / c mouse received 8 Gy of X-ray irradiation for a total of three times. After a two-day rest, the BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 after tumor implantation. Simultaneously, on days 16, 18, and 20, the mice were subcutaneously injected with 100 μL of fragmented tumor antigen prepared from 100w tumor cells according to the method of Example 3 for a total of three times.

[0279] Group 4 (CDDP+IR+FA-500w): On days 8, 10, and 12 after tumor implantation, the tumors of each Balb / c mouse received 8 Gy of X-ray irradiation for a total of three times. After a two-day rest, the BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 after tumor implantation. Simultaneously, on days 16, 18, and 20, the mice were subcutaneously injected with 100 μL of fragmented tumor antigen prepared from 500w tumor cells according to the method of Example 8 on the left flank for a total of three times.

[0280] During the experiment, the growth of in situ tumors in each group of mice was regularly detected, and the number of days the mice survived was recorded for survival analysis.

[0281] FIG16A shows the tumor growth curve after treatment with CDDP combined with IR and different doses of FA, and FIG16B shows the survival curve of mice after treatment with CDDP combined with IR and different doses of FA.

[0282] As shown in Figure 16A, compared with the CON group, CDDP+IR combined with different doses of FA significantly inhibited tumor growth. There was no difference in tumor growth inhibition between the 50W, 100W, and 500W groups treated with three doses of FA combined with CDDP+IR. Figure 16B shows that compared with the CON group, CDDP+IR combined with different doses of FA significantly prolonged mouse survival. There was no difference in mouse survival between the 100W and 500W groups treated with three doses of FA combined with CDDP+IR. However, mice treated with 100W and 500W combined with CDDP+IR had longer survival than mice treated with 50W combined with CDDP+IR. This suggests that CDDP+IR combined with different doses of FA can produce similar tumor growth inhibitory effects, but combining with higher doses of FA can provide a greater survival benefit.

[0283] The fragmented tumor antigens produced using the preparation method of this application can maximize the retention of tumor cell immunogenicity, overcome the tumor's suppressive immune microenvironment, induce a strong immune response, effectively inhibit tumor growth and metastasis, and produce a better anti-tumor metastasis effect. Furthermore, the preparation method of this application is simple, low-cost, easy to store, effective, and highly safe, with broad application prospects.

[0284] The tumor immunotherapy method of the present application is used to treat tumor-bearing mice, which retains all the antigens and immunoreactive substances of the tumor cells, can improve the immunogenicity of tumor antigens, efficiently present tumor antigens to antigen-presenting cells (such as APCs) and activate them, start the specific killing function of cytotoxic T lymphocytes, and exert an anti-tumor effect. At the same time, radiation can induce immunogenic cell death such as pyroptosis / ferroptosis in tumor cells, release damage-related molecular patterns (such as CRT, HMGB1, ATP, etc.), further enhance the adaptive immune response in the body, and improve the inhibitory effect of distal tumors. In addition, the immunomodulatory drug CTX can inhibit the proliferation and function of Treg cells in the tumor microenvironment, weaken the inhibitory tumor immune microenvironment, and increase the tumor infiltration of cytotoxic T lymphocytes (CTLs). The different components in the tumor immunotherapy method of the present application can synergistically inhibit tumor growth, metastasis and recurrence, especially significantly inhibit tumor metastasis; at the same time, the tumor immunotherapy method can prolong the survival time of mice, improve the survival rate of mice, and has a lasting anti-tumor effect. The tumor immunotherapy method of the present application has an anti-tumor effect comparable to that of immune checkpoint inhibitors when used alone. In summary, the tumor immunotherapy method of the present application has high immunogenicity and low off-target rate, can effectively and long-term activate the immune response, significantly inhibit tumor growth and metastasis, and prolong survival time. Furthermore, the fragmented tumor antigens prepared by the preparation method of the present application can also be used in combination with conventional tumor treatment methods (such as radiotherapy, chemotherapy and other clinical drugs) to jointly inhibit tumor growth, reduce tumor metastasis or prolong survival time from different angles, providing a diversified method for tumor treatment and helping to optimize the strategy.

[0285] In addition, the tumor immunotherapy method of the present application does not require the extraction, sequencing, and specific processing and production of the processed tumor antigens, nor does it require special antigen delivery technology and related equipment. The preparation process is simple and time-saving. At the same time, significant anti-tumor effects can be achieved without the need for combined use with immune checkpoint inhibitors. The reagents selected in the method of the present application are commonly used clinical drugs with low cost and high safety. The prepared fragmented tumor antigens can be stored at low temperatures, are easy to preserve and transport, have excellent effects, and have great application prospects.

[0286] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A method for preparing a fragmented tumor antigen, comprising: (1) Obtain tumor cell suspension; (2) After irradiating and incubating the tumor cell suspension, fragmented tumor antigens are prepared.

2. The preparation method according to claim 1, further comprising: (3) The fragmented tumor antigen is subjected to ultra-low temperature freeze-thaw treatment.

3. The preparation method according to any one of claims 1 to 2, wherein The tumor cells are selected from at least one of breast cancer cells, colorectal cancer cells, melanoma cells, lung cancer cells, liver cancer cells, pancreatic cancer cells, kidney cancer cells, esophageal cancer cells, gastric cancer cells, prostate cancer cells, brain cancer cells, oral cancer cells, bile duct cancer cells, ovarian cancer cells, cervical cancer cells, osteosarcoma cells and testicular cancer cells.

4. The preparation method according to any one of claims 1 to 2, wherein The tumor cells are derived from at least one of tumor cells obtained by puncture, tumor cells obtained by surgery, circulating tumor cells and tumor cells cultured in vitro.

5. The preparation method according to any one of claims 1 to 2, wherein The tumor cells are selected from autologous or allogeneic tumor cells.

6. The preparation method according to any one of claims 1 to 2, wherein The number of cells in the tumor cell suspension is 1 to 1000×10 6 , preferably 0.5×10 6 ~100×10 6 , more preferably 1×10 6 ~20×10 6 indivual.

7. The preparation method according to any one of claims 1 to 2, wherein The incubation temperature is 4°C to 50°C, preferably 30°C to 40°C, and more preferably 35°C to 40°C.

8. The preparation method according to any one of claims 1 to 2, wherein The incubation time is 0.01 hour to 24 hours, preferably 0.5 hour to 8 hours, more preferably 0.5 hour to 6 hours.

9. The preparation method according to any one of claims 1 to 2, wherein The irradiation dose is 2 Gy to 200 Gy, preferably 2 Gy to 50 Gy, more preferably 6 Gy to 40 Gy, and even more preferably 8 Gy to 20 Gy.

10. The preparation method according to any one of claims 1 to 2, wherein The irradiation dose rate is 0.01Gy / s~1×10 9 Gy / s, preferably 0.01 Gy / s to 100 Gy / s, more preferably 0.01 Gy / s to 40 Gy / s.

11. The preparation method according to any one of claims 1 to 2, wherein The irradiation method is selected from at least one of electron, photon, proton, heavy ion and neutron radiation, preferably at least one of photon and proton, more preferably X-ray.

12. A fragmented tumor antigen obtained by the preparation method according to any one of claims 1 to 11.

13. A tumor cell vaccine comprising the fragmented tumor antigen according to claim 12.

14. The tumor cell vaccine according to claim 13, wherein The tumor cell vaccine also includes an immune adjuvant; the immune adjuvant is an immunomodulatory drug; Preferably, the immunomodulatory drug is selected from at least one of polyinosinic-polycytidylic acid, platinum drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolism drugs, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolites, rapamycin target inhibitors, botanicals, ribonucleotide reductase inhibitors and tyrosine kinase inhibitors; More preferably, the polyinosinic-acid is selected from artificially synthesized ribonucleic acid; the platinum drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cyclosulfate platinum, nedaplatin and lobaplatin; the calcineurin inhibitor is selected from at least one of cyclosporine, tacrolimus and mycophenolate mofetil; the glucocorticoid is selected from at least one of prednisone, methylprednisone and methylprednisolone; the alkylating agent is selected from at least one of cyclophosphamide and chlorambucil; the microbial metabolism drug is selected from at least one of cyclosporine, tacrolimus and rapamycin; the monoclonal antibody drug is selected from anti-thymocyte immune protein, At least one of muromonab-CD3, daclizumab, basiliximab, efalizumab and natalizumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide and mycophenolate mofetil; the antimetabolite drug is selected from at least one of mycophenolate mofetil, methotrexate, azathioprine, mercaptopurine and mizoribine; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and gustation; the botanical drug is selected from at least one of tripterygium wilfordii glycosides and total glucosides of white paeony; the ribonucleotide reductase inhibitor is selected from hydroxyurea; the tyrosine kinase inhibitor is selected from leflunomide.

15. Use of the fragmented tumor antigen according to claim 12 or the tumor cell vaccine according to any one of claims 13 to 14 in the treatment and / or prevention of tumor diseases; preferably, the tumor diseases include at least one of breast cancer, colorectal cancer, melanoma, lung cancer, liver cancer, pancreatic cancer, kidney cancer, esophageal cancer, gastric cancer, prostate cancer, brain cancer, oral cancer, bile duct cancer, ovarian cancer, cervical cancer, osteosarcoma and testicular cancer.

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