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 method for preparing fragmented tumor antigens and its applications

[0001] This application claims priority to two Chinese patent applications filed on March 22, 2024, with application number 2024103396080 entitled "A method for preparing a fragmented tumor antigen and its use thereof", and on October 31, 2024, with application number 2024115462825 entitled "A method for preparing a fragmented tumor antigen and its use thereof", the entire contents of which are incorporated herein by reference. Technical Field

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

[0003] Tumor vaccines are currently a research hotspot in the field of oncology both domestically and internationally. As an active immunotherapy method for cancer, they play an increasingly important role in the treatment of cancer patients. Tumor vaccines can be classified according to their antigen source into DNA vaccines, RNA vaccines, peptide vaccines, tumor cell vaccines, and dendritic cell vaccines. Among them, tumor cell vaccines involve treating autologous or allogeneic tumor cells using physical, chemical, and biological methods to render them non-tumorigenic but retain their antigenicity. These vaccines are then combined with non-specific stimulating factors for active immunotherapy in cancer patients.

[0004] Tumor metastasis is the leading cause of death in cancer patients. Currently reported tumor immunotherapy methods have limited effectiveness against metastatic tumors. For example, therapeutic vaccines (including DC cell vaccines, RNA vaccines, and autologous tumor cell vaccines) primarily inhibit the primary tumor, while their inhibitory effect on recurrent or metastatic tumors is unclear or poor. Furthermore, the preparation of existing personalized tumor vaccines is time-consuming, involves numerous steps, and is expensive, limiting their widespread clinical application. Moreover, developed tumor cell vaccines are not effective when used alone and need to be loaded onto 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 to prepare, simple to produce, low in 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 fragmented tumor antigens and their applications, so as to improve the immunogenicity of fragmented tumor antigens and enable them to have excellent anti-tumor effects in different tumor animal models, especially significantly inhibiting tumor metastasis and effectively prolonging the survival time of animals. The specific technical solution is as follows:

[0007] The first aspect of this application provides a method for preparing a fragmented tumor antigen, comprising: (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 this application, it further includes: (3) subjecting the fragmented tumor antigen to cryogenic freeze-thaw treatment.

[0009] In one embodiment of this 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 this 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 this application, the tumor cells are selected from autologous or allogeneic tumor cells.

[0012] In one embodiment of this application, the number of cells in the tumor cell suspension is 1 to 1000 × 10⁻⁶. 6 The number of units is preferably 0.5 × 10⁻⁶. 6 100 x 10 6 One, more preferably 1×10 6 20 x 10 6 indivual.

[0013] In one embodiment of this 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 this application, the incubation time is 0.01 hours to 24 hours, preferably 0.5 hours to 8 hours, and more preferably 0.5 hours to 6 hours.

[0015] In one embodiment of this 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 this application, the irradiation dose rate is 0.01 Gy / s to 1 × 10⁻⁶. 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 this 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, and more preferably X-ray.

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

[0019] A third aspect of this application provides a tumor cell vaccine comprising the fragmented tumor antigen described in the second aspect of this application.

[0020] In one embodiment of this application, the tumor cell vaccine further includes 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-based drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolites, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolites, rapamycin target molecule inhibitors, herbal medicines, nucleotide reductase inhibitors, and tyrosine kinase inhibitors.

[0022] More preferably, the polyinosinic-polycytidylic acid is selected from synthetic ribonucleic acid; the platinum-based drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cyclothioplatin, 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 metabolite is selected from at least one of cyclosporine, tacrolimus, and rapamycin; and the monoclonal antibody is selected from anti-thymocyte immune proteins, The drug comprises at least one of moromumab-CD3, dalizumab, baliximab, efazolin, and natezumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide, and mycophenolate mofetil; the antimetabolite is selected from at least one of mycophenolate mofetil, methotrexate, azathioprine, mercaptopurine, and imidazolidin; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and guanilimus; the herbal medicine is selected from at least one of triptolide and total glycosides of paeony; the nucleotide reductase inhibitor is selected from hydroxyurea; and the tyrosine kinase inhibitor is selected from leflunomide.

[0023] The fourth aspect of this application provides the use of the fragmented tumor antigen described in the second aspect of this application or the tumor cell vaccine described in the third aspect of this 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] The beneficial effects of this application are:

[0025] This application provides a method for preparing fragmented tumor antigens, comprising: obtaining a tumor cell suspension; irradiating and incubating the tumor cell suspension to prepare fragmented tumor antigens. The fragmented tumor antigens prepared using this method can maximize the preservation of tumor cell immunogenicity, overcome the tumor-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 this application is simple, low-cost, easy to store, highly effective, and safe, and has broad application prospects.

[0026] Of course, implementing any product or method of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

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

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

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

[0031] Figure 4 shows the tumorigenicity of radiographed tumor antigens without cryogenic 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 inhibition effect of FAST in the 4T-1 cold tumor distal model; Figure 5C shows the survival prolongation 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 after FAST treatment in the CT-26 thermal tumor distal model; Figure 6B shows the tumor growth inhibition effect of FAST in the CT-26 thermal tumor distal model; Figure 6C shows the survival prolongation effect of FAST in the CT-26 thermal tumor distal model.

[0034] Figure 7A shows the tumor growth inhibition effect of FAST in the B16-F10 cold tumor distal model; Figure 7B shows the survival prolongation 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 time points after FAST treatment in the 4T-1 cold tumor surgical resection model; Figure 8B shows the effect of FAST in inhibiting lung metastasis of tumors in the 4T-1 cold tumor surgical resection model; Figure 8C shows the survival prolongation effect of FAST in the 4T-1 cold tumor surgical resection model.

[0036] Figure 9A shows in vivo imaging of mice where fragmented antigens from different tumor cell sources specifically inhibited tumor growth; Figure 9B shows the specific inhibition of tumor growth by fragmented antigens from different tumor cell sources.

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

[0038] Figure 11A shows in vivo imaging of mice to demonstrate the therapeutic effect of 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 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 (Foxp3+CD4+ T) cells in tumor tissue after treatment with FAST and its corresponding components; Figure 12E shows the percentage of KI76+CD8+ T cells in tumor tissue after treatment with FAST and its corresponding components.

[0040] Figure 13A shows the tumor growth inhibition effect of different treatments such as FA combined with IR and FAST after normalization; Figure 13B shows the lung metastasis inhibition effect of different treatments such as FA combined with IR and FAST after normalization.

[0041] Figure 14A shows the tumor growth inhibition effect of cisplatin (CDDP) combined with different treatments such as IR or FA and FAST after normalization; Figure 14B shows the tumor lung metastasis inhibition effect of CDDP combined with different treatments such as IR or FA and FAST after normalization.

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

[0043] Figure 16A shows the tumor growth curves after treatment with different methods, such as CDDP combined with IR and different doses of FA; Figure 16B shows the mouse survival curves after treatment with different methods, such as CDDP combined with IR and different doses of FA. Detailed Implementation

[0044] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all 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 this application provides a method for preparing a fragmented tumor antigen, comprising: (1) obtaining a tumor cell suspension; and (2) irradiating and incubating the tumor cell suspension to prepare a fragmented tumor antigen.

[0046] In this application, there is no particular limitation on the method of obtaining tumor cell suspension, as long as it can achieve the purpose of this application, such as enzymatic filtration, digestion suspension culture, etc.

[0047] The inventors discovered in their research that treating tumor cells using the irradiation method described in this application can enhance the immunogenicity of tumor cells, activate the body's anti-tumor immune response, inhibit tumor growth, and produce a good anti-tumor effect. Other treatment methods, such as ultrasound, hyperthermia, and in vitro treatment of tumor cells with chemotherapeutic drugs, ultimately produce fragmented tumor antigens that cannot control tumor growth, even when combined with Mn adjuvant.

[0048] In this application, the form of the fragmented tumor antigen is not particularly limited, as long as it achieves the purpose 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 solution. The lyophilized powder form of the fragmented tumor antigen not only fully retains the activity of the tumor antigen and facilitates storage and transportation, but also improves the safety during the preservation process and extends the shelf life of the fragmented tumor antigen.

[0049] In one embodiment of this application, it further includes: (3) subjecting the fragmented tumor antigen to cryogenic freeze-thaw treatment.

[0050] The inventors discovered in their research that fragmented tumor antigens subjected to cryogenic freeze-thaw treatment exhibit better anti-tumor effects and are safer. Furthermore, the cryogenic freezer allows for the storage of tumor antigens prepared in large quantities for subsequent use, eliminating the need for ad-hoc preparation for each use, reducing time and batch variations, and meeting the conditions for the commercialization of tumor antigens. The prepared fragmented tumor antigens, after being stored in the cryogenic freezer for 1-2 months, still retain their anti-tumor activity when used for immunization.

[0051] In one embodiment of this 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 this application, the tumor cells are derived from at least one of the following: tumor cells obtained by puncture, tumor cells obtained by surgery, circulating tumor cells, and tumor cells cultured in vitro. Selecting tumor cells from these sources can better maintain the survival of tumor antigens in the tumor vaccine, stimulate the body to produce sustained and effective immune protection, and reduce tumor heterogeneity.

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

[0054] In this application, autologous tumor cells refer to tumor cells derived from the patient themselves or from the same animal. Allogeneic tumor cells refer to tumor cells of the same genetic origin, specifically tumor cells of the same type of cancer derived from another organism.

[0055] In one embodiment of this application, the number of cells in the tumor cell suspension is 1 to 1000 × 10⁻⁶. 6 The number of units is preferably 0.5 × 10⁻⁶. 6 100 x 10 6 One, more preferably 1×10 6 20 x 10 6 For example, the number of cells in the tumor cell suspension can be 1, 10, 100, 1000, or 0.01 × 10⁻⁶. 6 0.1×10 6 0.5×10 6 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 5×10 6 8×106 10×10 6 15×10 6 1 piece, 20×10 6 30×10 6 40×10 6 50×10 6 60×10 6 70×10 6 80×10 6 1 piece, 90×10 6 100×10 6 1 piece, 200×10 6 1 piece, 300×10 6 400×10 6 500×10 6 600 x 10 6 700×10 6 800×10 6 1, 900×10 6 1000×10 6 The number of cells in a tumor cell suspension can be a range consisting of any two values ​​within this range. Controlling the number of cells in a tumor cell suspension within this range can better improve the therapeutic effect of fragmented tumor antigens in the prevention and / or treatment of tumor diseases.

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

[0057] In one embodiment of this application, the incubation time is 0.01 hours to 24 hours, preferably 0.5 hours to 8 hours, and more preferably 0.5 hours to 6 hours. For example, the incubation time can be 0.01 hours, 0.1 hours, 0.5 hours, 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 of any two values ​​within this range. Controlling the incubation time within the above range can maximize the preservation of tumor cell immunogenicity, activate the body's effective anti-tumor immune response, inhibit tumor growth, and produce a better anti-tumor effect.

[0058] In one embodiment of this 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 ​​within this range. By controlling the irradiation dose within the above range, the immunogenicity of tumor cells can be preserved to the maximum extent, 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.

[0059] In one embodiment of this application, the irradiation dose rate is 0.01 Gy / s to 1 × 10⁻⁶. 9 The irradiation dose rate is preferably 0.01 Gy / s to 100 Gy / s, more preferably 0.01 Gy / s to 40 Gy / s. For example, the irradiation dose rate 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 × 10⁻⁶ Gy / s, or 1 × 10⁻⁶ Gy / s. 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 any range of two values ​​within this range. Controlling the irradiation dose rate within this range can maximize the preservation 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 this 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, and more preferably X-ray.

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

[0062] A third aspect of this application provides a tumor cell vaccine comprising the fragmented tumor antigen described in the second aspect of this application.

[0063] In one embodiment of this application, the tumor cell vaccine further includes 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-based drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolites, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolites, rapamycin target molecule inhibitors, herbal medicines, nucleotide reductase inhibitors, and tyrosine kinase inhibitors.

[0065] More preferably, the polyinosinic-polycytidylic acid is selected from synthetic ribonucleic acid; the platinum-based drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cyclothioplatin, 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 metabolite is selected from at least one of cyclosporine, tacrolimus, and rapamycin; and the monoclonal antibody is selected from anti-thymocyte immune proteins, The drug comprises at least one of moromumab-CD3, dalizumab, baliximab, efazolin, and natezumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide, and mycophenolate mofetil; the antimetabolite is selected from at least one of mycophenolate mofetil, methotrexate, azathioprine, mercaptopurine, and imidazolidin; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and guanilimus; the herbal medicine is selected from at least one of triptolide and total glycosides of paeony; the nucleotide reductase inhibitor is selected from hydroxyurea; and the tyrosine kinase inhibitor is selected from leflunomide.

[0066] The fourth aspect of this application provides the use of the fragmented tumor antigen described in the second aspect of this application or the tumor cell vaccine described in the third aspect of this 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 this application, the tumor disease is a metastatic tumor. The tumor cell vaccine of this application can maximize the preservation of tumor cell immunogenicity, overcome the tumor-suppressive immune microenvironment, and induce a strong immune response to effectively inhibit tumor metastasis, thus producing a better anti-tumor metastasis effect.

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

[0069] In this 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 1 hour and 6 hours after irradiation can inhibit tumor growth and produce a better anti-tumor effect.

[0070] In one embodiment of this 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 acid, platinum drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolites, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolites, rapamycin target molecule inhibitors, herbal medicines, nucleotide reductase inhibitors, and tyrosine kinase inhibitors.

[0072] More preferably, the polyinosinic-polycytidylic acid is selected from synthetic ribonucleic acid; the platinum-based drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cyclothioplatin, 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 metabolite is selected from at least one of cyclosporine, tacrolimus, and rapamycin; and the monoclonal antibody is selected from anti-thymocyte immune proteins. The drug is selected from at least one of moromumab-CD3, dalizumab, baliximab, efazolin, and natezumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide, and mycophenolate mofetil; the antimetabolite is selected from at least one of mycophenolate mofetil, methotrexate, azathioprine, mercaptopurine, and imidazolidin; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and guanilimus; the herbal medicine is selected from at least one of triptolide and total glycosides of paeony; the nucleotide reductase inhibitor is selected from hydroxyurea; and the tyrosine kinase inhibitor is selected from leflunomide.

[0073] In this application, the method of combining the fragmented tumor antigen with the immunomodulatory drug is not particularly limited, as long as the purpose of this application can be achieved. For example, the immunomodulatory drug is injected on the first day of treatment, the fragmented tumor antigen is injected on the second day, and the above vaccination steps are repeated 2 to 3 times after an interval of 1 to 2 days, which constitutes one course of treatment. A total of 1 to 5 courses of treatment are administered.

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

[0075] In this application, the method of combining fragmented tumor antigen with radiotherapy is not particularly limited, as long as it achieves the purpose of this application. For example, the tumor can be treated with conventional fractionated radiotherapy first, followed by injection of fragmented tumor antigen 1-3 days later, with an interval of 1-2 days, and the above injection steps can be repeated 2-9 times, for a total of 3-10 injections of fragmented tumor antigen. This application does not particularly limit the method of radiotherapy, which is conventional clinical tumor radiotherapy, such as at least one of conventional fractionated radiotherapy, macrofractionated radiotherapy, and single-fractionated radiotherapy.

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

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

[0078] In one embodiment of this application, the fragmented tumor antigen is used in combination with a clinical oncology therapeutic drug; the clinical oncology therapeutic drug is selected from at least one of phospholipid compounds, immunomodulatory drugs, and nucleic acid analogs; 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 at least one of low-dose chemotherapy drugs, such as platinum drugs, such as cisplatin, carboplatin, oxaliplatin, cyclothioplatin, nedaplatin, and lobaplatin; the nucleic acid analog is selected from modified RNA or DNA.

[0079] In this application, there are no particular limitations on the combined use of fragmented tumor antigen and clinical tumor treatment drugs, as long as the purpose of this application can be achieved. For example, clinical tumor treatment drugs can be given on the first day of treatment, and fragmented tumor antigen can be injected on the second day. After an interval of 1 to 2 days, the above vaccination steps can be repeated 2 to 3 times, which constitutes one course of treatment. A total of 1 to 5 courses of treatment can be administered.

[0080] In this application, there is no particular limitation on the dosage of fragmented tumor antigen, as long as the purpose of this application is achieved. For example, it can be a single high-dose injection, multiple low-dose booster injections, or multiple treatment courses. A single high-dose injection can be a single injection dose of 3 × 10⁻⁶. 6 5 x 10 6 One dose is administered for each individual; multiple booster doses of low-dose vaccine can be administered in a single dose of 0.5 × 10⁻⁶. 6 2×10 6 The total number of doses is 3 to 10. Multiple courses of treatment can be 1 to 5 repeated high-dose single doses, which is equivalent to 1 to 5 courses of treatment; or 1 to 5 repeated low-dose booster doses, which is equivalent to 1 to 5 courses of treatment.

[0081] In this application, there are no specific limitations on the dosage of immunomodulatory drugs administered; the manufacturer's instructions can be consulted as long as the purpose of this application is achieved. Similarly, there are no specific limitations on the dosage of clinical oncology treatment drugs taken orally; the manufacturer's instructions can be consulted as long as the purpose of this application is achieved.

[0082] In one embodiment of this 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 this application provides the use of the tumor immunotherapy method provided in the fifth aspect of this application in the treatment and / or prevention of tumor diseases.

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

[0085] The tumor immunotherapy method described in this application eliminates the need for extraction, sequencing, and synthesis of processed fragmented tumor antigens, as well as the requirement for specialized antigen delivery technologies and equipment. The preparation process is simple and time-efficient. Furthermore, it achieves significant anti-tumor effects without the need for combined use with immune checkpoint inhibitors. The reagents used in this method are commonly used clinical drugs, offering low cost and high safety. The prepared fragmented tumor antigens can be stored at low temperatures, facilitating preservation and transportation, and exhibiting excellent efficacy with great application potential. More importantly, this tumor immunotherapy method demonstrates high immunogenicity, low off-target rate, and efficient and long-lasting activation of the immune response. It exhibits excellent anti-tumor effects in various tumor animal models, particularly significantly inhibiting tumor metastasis and effectively prolonging animal survival.

[0086] Example

[0087] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various experiments and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.

[0088] Laboratory animals and materials

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

[0090] The 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] All mouse models used in this application were constructed by our research group.

[0094] Example 1

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

[0096] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the irradiation was carried out by X-rays, the irradiation dose was 12 Gy, and the irradiation dose rate was 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 mixed thoroughly with 100 μL of PBS to prepare 1×10⁻⁶ cells. 6 A single-cell suspension;

[0099] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the irradiation was carried out by X-rays, the irradiation dose was 36 Gy, and the irradiation dose rate was 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 mixed thoroughly with 100 μL of PBS to prepare 1×10⁻⁶ cells. 6A single-cell suspension;

[0102] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the irradiation was carried out by X-rays, the irradiation dose was 12 Gy, and the irradiation dose rate was 0.1 Gy / s;

[0103] (3) The fragmented tumor antigen was subjected to cryogenic freeze-thaw treatment; the cryogenic freeze-thaw treatment temperature was -80℃ and the time was 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 mixed thoroughly with 100 μL of PBS to prepare 1×10⁻⁶ cells. 6 A single-cell suspension;

[0106] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 6 hours to obtain fragmented tumor antigens; the irradiation was carried out by X-rays, the irradiation dose was 12 Gy, and the irradiation dose rate was 0.1 Gy / s;

[0107] (3) The fragmented tumor antigen was subjected to cryogenic freeze-thaw treatment; the cryogenic freeze-thaw treatment temperature was -80℃ and the time was 12 hours.

[0108] Example 5

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

[0110] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the irradiation was carried out by X-rays, the irradiation dose was 12 Gy, and the irradiation dose rate was 0.1 Gy / s;

[0111] (3) The fragmented tumor antigen was subjected to cryogenic freeze-thaw treatment; the cryogenic freeze-thaw treatment temperature was -80℃ and the time was 12 hours.

[0112] Example 6

[0113] (1) B16-F10 melanoma cells were washed three times with PBS, and then 100 μL of PBS was added and mixed thoroughly to prepare 1×10⁻⁶ cells. 6 A single-cell suspension;

[0114] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the irradiation was carried out by X-rays, the irradiation dose was 12 Gy, and the irradiation dose rate was 0.1 Gy / s;

[0115] (3) The fragmented tumor antigen was subjected to cryogenic freeze-thaw treatment; the cryogenic freeze-thaw treatment temperature was -80℃ and the time was 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 mixed thoroughly with 100 μL of PBS to prepare 5×10⁻⁶ cells. 5 A single-cell suspension;

[0118] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the irradiation was carried out by X-rays, the irradiation dose was 12 Gy, and the irradiation dose rate was 0.1 Gy / s;

[0119] (3) The fragmented tumor antigen was subjected to cryogenic freeze-thaw treatment; the cryogenic freeze-thaw treatment temperature was -80℃ and the time was 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 mixed thoroughly with 100 μL of PBS to prepare 5×10⁻⁶ cells. 6 A single-cell suspension;

[0122] (2) After irradiating the single-cell suspension, it was incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the irradiation was carried out by X-rays, the irradiation dose was 12 Gy, and the irradiation dose rate was 0.1 Gy / s;

[0123] (3) The fragmented tumor antigen was subjected to cryogenic freeze-thaw treatment; the cryogenic freeze-thaw treatment temperature was -80℃ and the time was 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 mixed thoroughly with 100 μL of PBS to prepare 1×10⁻⁶ cells. 6 A single-cell suspension;

[0126] (2) The single-cell suspension was subjected to sonication and then incubated at 37°C for 1 hour to obtain fragmented tumor antigen; the sonication power was 400W and the sonication 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 mixed thoroughly with 100 μL of PBS to prepare 1×10⁻⁶ cells.6 A 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 temperature of hyperthermia treatment was 45°C and the time of hyperthermia treatment 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 mixed thoroughly with 100 μL of PBS to prepare 1×10⁻⁶ cells. 6 A 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 antigen; the final concentration of doxorubicin is 10 μM.

[0133] Test Example 1

[0134] Fifty 6-week-old female Balb / c mice were inoculated with logarithmic growth 4T-1-Luc breast cancer cells in their right hind limbs on day 0, with 1 million cells per mouse. The inoculation continued until the tumor reached 150 mm. 3 Subsequently, Balb / c mice were randomly divided into the following 10 groups and the corresponding treatments were initiated:

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

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

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

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

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

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

[0141] Group 7 (IR+Mn): Subcutaneous injection of Mn combined with the fragmented tumor antigen prepared in Example 1. On the day the treatment began, each Balb / c mouse was first subcutaneously injected with 100 μL of Mn at a concentration of 200 μg / mL in the left hind limb. The next day, 100 μL of the fragmented tumor antigen prepared in Example 1 was injected subcutaneously. After an interval of 1 day, the above injection was repeated twice.

[0142] Group 8 (Ultrasound + Mn): Subcutaneous injection of Mn combined with injection of fragmented tumor antigen prepared in Comparative Example 1. On the day the treatment began, each Balb / c mouse was first subcutaneously injected with 100 μL of Mn at a concentration of 200 μg / mL in the left hind limb. The next day, 100 μL of fragmented tumor antigen prepared in Comparative Example 1 was injected subcutaneously. After an interval of 1 day, the above injection was repeated twice.

[0143] Group 9 (thermotherapy + Mn): Subcutaneous injection of Mn combined with injection of fragmented tumor antigen prepared in Comparative Example 2. Specifically, on the day the treatment began, each Balb / c mouse was first subcutaneously injected with 100 μL of Mn at a concentration of 200 μg / mL in the left hind limb. The next day, 100 μL of fragmented tumor antigen prepared in Comparative Example 2 was injected subcutaneously. After an interval of 1 day, the above injection was repeated twice.

[0144] Group 10 (DOX+Mn) was subcutaneously inoculated with Mn in combination with fragmented tumor antigen prepared in Comparative Example 3. Specifically, on the day treatment began, each Balb / c mouse was subcutaneously injected with 100 μL of Mn at a concentration of 200 μg / mL in the left hind limb. The next day, 100 μL of fragmented tumor antigen prepared in Comparative Example 3 was injected subcutaneously. After a one-day interval, the above inoculation was repeated twice.

[0145] The growth of tumors in mice was observed periodically during the experiment.

[0146] Figure 1A shows the growth-inhibiting effect of fragmented tumor antigen and its combination with Mn adjuvant on 4T-1 distal tumors after irradiation treatment; Figure 1B shows the growth-inhibiting effect of fragmented tumor antigen and its combination with Mn adjuvant on 4T-1 distal tumors after ultrasound treatment; Figure 1C shows the growth-inhibiting effect of fragmented tumor antigen and its combination with Mn adjuvant on 4T-1 distal tumors after thermotherapy treatment; Figure 1D shows the growth-inhibiting effect of fragmented tumor antigen and its combination with 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 from ultrasound, hyperthermia, or doxorubicin treatment have no inhibitory effect on tumor growth and may even promote it. Even when combined with Mn adjuvant, tumor growth cannot be inhibited. Only irradiated fragmented tumor antigens can inhibit tumor growth and produce a good anti-tumor effect. Irradiated tumor cells, alone or in combination with Mn adjuvant, can significantly inhibit tumor growth, but Mn adjuvant failed to effectively improve the tumor control effect of irradiated fragmented tumor antigens.

[0148] Test Example 2

[0149] Fifteen 6-week-old female Balb / c mice were inoculated with logarithmic growth 4T-1-Luc breast cancer cells in their right hind limbs on day 0, with 1 million cells per mouse. The inoculation continued until the tumor reached 150 mm. 3 Subsequently, Balb / c mice were randomly divided into the following 3 groups and the corresponding treatments were initiated:

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

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

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

[0153] The growth of tumors in mice was observed periodically during the experiment.

[0154] Figure 2 shows the growth-inhibiting effect of fragmented tumor antigen immunotherapy after different doses of irradiation on distal 4T-1 tumors. As can be seen from Figure 2, both 12 Gy and 36 Gy irradiation of the fragmented tumor antigen inhibited tumor growth, producing good anti-tumor effects. Compared with the 36 Gy group, the 12 Gy irradiation group showed earlier and stronger tumor-inhibiting effects.

[0155] Test Example 3

[0156] Twenty 6-week-old female Balb / c mice were inoculated with logarithmic growth 4T-1-Luc breast cancer cells in their right hind limbs on day 0, with 1 million cells per mouse. The inoculation continued until the tumor reached 150 mm. 3Then, the Balb / c mice were randomly divided into the following 4 groups:

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

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

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

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

[0161] The growth of tumors in mice was observed periodically during the experiment.

[0162] Figure 3A shows the growth-inhibiting effect of fragmented tumor antigens incubated for different times after irradiation on distal 4T-1 tumors. As can be seen from Figure 3A, fragmented tumor antigens incubated for 1 h and 6 h after 12 Gy irradiation can inhibit tumor growth and produce good anti-tumor effects, with the fragmented tumor antigens incubated for 1 h showing a better inhibitory effect. Figure 3B shows the growth-inhibiting effect of mixed fragmented tumor antigens incubated for different times after irradiation on distal 4T-1 tumors. The CON group in Figures 3A and 3B represents two mouse experiments at different time points. To compare the tumor inhibition effects between groups, the data from the two experiments were normalized to the CON group. Figure 3C shows the normalized growth-inhibiting effect of fragmented tumor antigens incubated at different time points after irradiation on 4T-1 tumors. As can be seen from Figure 3C, mixed inoculation of fragmented tumor antigens after 1 h and 6 h after irradiation can inhibit tumor growth and produce better anti-tumor effects.

[0163] Test Example 4

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

[0165] Figure 4 shows that tumors formed at the inoculation site 26 days after inoculation with fragmented tumor antigens that had not undergone cryogenic freeze-thaw treatment, indicating that fragmented tumor antigens that were only irradiated have a certain tumorigenic risk and relatively low safety.

[0166] Test Example 5

[0167] Thirty-two 6-week-old female Balb / c mice were inoculated with logarithmic growth 4T-1-Luc breast cancer cells in their right hind limbs on day 0, with 1 million cells per mouse. The inoculation continued until the tumor reached 150 mm. 3 Subsequently, Balb / c mice were randomly divided into the following 4 groups and the corresponding treatments were initiated:

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

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

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

[0171] Group 4 (FAST): Intraperitoneal injection of CTX combined with subcutaneous inoculation of fragmented tumor antigen in the left hind limb. On the day treatment began, each Balb / c mouse was first injected intraperitoneally 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 injected subcutaneously in the left hind limb. The above inoculation was then repeated twice.

[0172] In the experiment, the growth of tumors in mice was monitored regularly, the number of days the mice survived was recorded for survival analysis, and the lung metastasis in mice was statistically analyzed.

[0173] 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 inhibition effect of FAST in the 4T-1 cold tumor distal model. Figure 5C shows the survival prolongation 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.

[0174] As shown in Figures 5A to 5C, the fragmented tumor antigen obtained in Example 3 effectively inhibited the growth of 4T-1 distal tumors and prolonged the survival time of mice. Compared with the CON group, the CTX group, FA group, and FAST group all effectively inhibited the growth of distal tumors in mice and increased the survival time of mice, with the FAST group showing the best effect (n≥5, ***P<0.001). As shown in Figure 5D, compared with CON, FA and FAST significantly inhibited tumor lung metastasis, with the FAST group showing better inhibition of tumor lung metastasis.

[0175] Test Example 6

[0176] Twenty-four 6-week-old female Balb / c mice were used, and logarithmic growth CT-26 colon cancer cells were inoculated into the right hind limb of each mouse, with 1 million cells per mouse. Tumors were allowed to grow to 150 mm in diameter. 3 Subsequently, Balb / c mice were randomly divided into the following 4 groups and the corresponding treatments were initiated:

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

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

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

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

[0181] In the experiment, the growth of mouse tumors was monitored regularly, in vivo imaging was performed to show the size of mouse tumors and the level of lung metastasis, and the survival days of mice were recorded for survival analysis.

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

[0183] As shown in Figure 6A, the fragmented tumor antigen obtained in Example 5 can effectively inhibit the growth of distal tumors in CT-26 thermal tumors and prolong the survival time of mice. As shown in Figures 6B and 6C, 20 days after inoculation, compared with the CON group, the CTX group, FA group and FAST group can 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 has the best effect, which can completely regress the tumor. Even on day 65, the survival rate of mice in the FAST group is still 100%.

[0184] Test Example 7

[0185] Forty 6-week-old female C57BL / 6 mice were used, and logarithmic growth B16-F10 melanoma cells were inoculated into the right hind limb of each mouse, with 1 million cells per mouse. The inoculation continued until the tumor reached 150 mm. 3 Subsequently, the C57BL / 6 mice were randomly divided into the following 4 groups, and the corresponding treatments were initiated:

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

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

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

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

[0190] During the experiment, the growth of tumors in mice was monitored periodically, and the number of days the mice survived was recorded for survival analysis.

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

[0192] Cold tumors typically express low levels of immunostimulatory molecules, while their microenvironment accumulates a large number of immunosuppressive cells, effectively evading the surveillance and attack of the immune system, thus reducing treatment efficacy and increasing the difficulty of tumor treatment. As shown in Figure 7A, compared with the CON group, the FAST group effectively inhibited the growth of distal cold tumors B16-F10 in mice (n≥5, ***P<0.001), while the tumor inhibition effects of the CTX and FA groups were not significant. Figure 7B shows that compared with the CON group, FAST and CTX increased the survival time of mice, with the FAST group showing a better survival extension effect.

[0193] Test Example 8

[0194] Forty 6-week-old female Balb / c mice were inoculated with logarithmic growth 4T-1-Luc breast cancer cells in their left hind limbs on day 0, with 1 million cells per mouse. The inoculation continued until the tumor reached 150 mm. 3 After anesthetizing Balb / c mice with an air anesthesia machine, most of the tumor tissue was surgically removed. The Balb / c mice after tumor removal were randomly divided into the following 4 groups, and in vivo imaging was performed. The corresponding treatment began on the second day after surgery:

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

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

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

[0198] Group 4 (FAST): Each Balb / c mouse was intraperitoneally injected with CTX in combination with subcutaneous inoculation of fragmented tumor antigen in the left hind limb. Specifically, on the day treatment began, 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 then repeated twice.

[0199] During the experiment, the growth of tumors in mice was monitored regularly, lung metastasis was observed, and the survival days of mice were recorded for survival analysis.

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

[0201] As shown in Figure 8A, compared with the CON group, the fragmented tumor antigen, CTX, and FAST obtained in Example 3 all effectively inhibited the recurrence of distal tumors in 4T-1 mice after surgery. At 21 and 28 days post-surgery, all distal tumors in the saline group mice recurred and grew, while very small tumors appeared in the distal tumors of the fragmented tumor antigen group mice. The distal tumors in the CTX and FAST immunization groups mice showed virtually no recurrence. At 40 days post-surgery, no tumors were observed in the distal tumors of the FAST immunization group mice. As shown in Figures 8B and 8C, compared with saline, the fragmented tumor antigen significantly reduced lung metastasis and prolonged the survival time of some mice; while CTX alone or FAST immunization effectively inhibited the generation of distal lung tumor metastasis and significantly prolonged the survival time of mice, with FAST showing better therapeutic effects.

[0202] Test Example 9

[0203] Eighteen 6-week-old female Balb / c mice were subcutaneously inoculated with logarithmic growth CT-26 colon cancer cells in their left hind limbs on day 0, with 1 million cells per mouse. Inoculation continued until the tumor reached 100 mm. 3 Subsequently, Balb / c mice were randomly divided into the following 3 groups and the corresponding treatments were initiated:

[0204] Group 1 (CON): Each Balb / c mouse was subcutaneously injected with 100 μL of physiological saline in the left hind limb. The injection was performed 3 times, with one day between the 6th and 10th day of tumor bearing.

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

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

[0207] The growth of tumors in mice was monitored periodically during the experiment.

[0208] Figure 9A shows in vivo imaging of mice where fragmented antigens from different tumor cell sources specifically inhibited tumor growth, and Figure 9B shows the specific inhibition of tumor growth by fragmented antigens from different tumor cell sources.

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

[0210] Test Case 10

[0211] Twenty 6-week-old female Balb / c mice were used. On day 0, logarithmic growth 4T-1-Luc breast cancer cells were inoculated into the right hind limb of each mouse, with 1 million cells per mouse. The inoculation was carried out when the tumor grew to 100 mm. 3 Afterwards, they were randomly divided into the following 4 groups and began corresponding treatments:

[0212] Group 1 (CON): Each Balb / c mouse was subcutaneously injected with 100 μL of physiological saline in the left hind limb for 6 consecutive days from day 5 to day 10 after tumor bearing; then the treatment was repeated for 2 more cycles.

[0213] Group 2 (FAST(One)): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously injected with the fragmented tumor antigen prepared in Example 3 in the left hind limb for FAST immunization. Specifically, on the day the treatment began, 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 in the left hind limb. The above injection was repeated twice after an interval of 1 day.

[0214] Group 3 (FAST(Two)): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously injected with the fragmented tumor antigen prepared in Example 3 in the left hind limb for FAST immunization. Specifically, on the day the treatment began, each Balb / c mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX, and the next day, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected in the left hind limb. After an interval of 1 day, the above injection was repeated twice. Three immunizations constituted one course of treatment. After one course of treatment was completed, another course of treatment was repeated.

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

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

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

[0218] As shown in Figures 10A and 10B, compared with the CON group, 1, 2, and 3 courses of FAST treatment significantly inhibited contralateral tumor growth, with the 3-course FAST group showing the best contralateral tumor inhibition. Figure 10C shows that 1, 2, and 3 courses of FAST treatment significantly inhibited the development of distant metastases, with the 3-course FAST group showing the best effect. Figure 10D shows that, compared with the CON group, the FAST(Three) group showed a significant increase in central memory cells and effector memory cells, indicating that 3 courses of FAST immunization can produce a long-lasting anti-tumor effect (n≥4, **P<0.01). These results demonstrate that the tumor immunotherapy method developed in this application can not only effectively inhibit distant tumors and metastases, but also generate immune memory through repeated immunization, resulting in a long-lasting anti-tumor effect.

[0219] Test Example 11

[0220] Sixty 6-week-old female Balb / c mice were used. On day 0, logarithmic growth 4T-1-Luc breast cancer cells were inoculated into the right hind limb of each mouse, with 1 million cells per mouse. The inoculation was carried out when the tumor grew to 100 mm. 3 Subsequently, Balb / c mice were randomly divided into the following 10 groups and the corresponding treatments were initiated:

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

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

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

[0224] Group 4 (FAST): Each Balb / c mouse was intraperitoneally injected with CTX and subcutaneously injected with fragmented tumor antigen in the left hind limb. Specifically, on the day the treatment began, 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 in the left hind limb. The above injection was repeated twice after an interval of 1 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 days 5, 7, 9 and 11 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 days 5, 7, 9 and 11 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 days 5, 7, 9, and 11 of tumor bearing; at the same time, each mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on days 5, 7, and 9 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 days 5, 7, 9, and 11 of tumor bearing; at the same time, each mouse was intraperitoneally injected with 100 μL of 10 mg / mL CTX on days 5, 7, and 9 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 days 5, 7, 9, and 11 of tumor bearing; at the same time, each Balb / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 3 on the left hind limb on days 6, 8, and 10 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 days 5, 7, 9, and 11 of tumor bearing; simultaneously, each Balb / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 3 on the left hind limb on days 6, 8, and 10 of tumor bearing.

[0231] The growth of tumors in mice was monitored regularly during the experiment, and the lung metastasis in mice was observed.

[0232] Figure 11A shows in vivo imaging of mice to demonstrate the therapeutic effect of FA combined with α-PD-L1 / α-CTLA-4; 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 shown in Figures 11A and 11B, injection of CTX or fragmented tumor antigen alone can inhibit tumor growth, while FAST immunization can significantly inhibit tumor growth. In tumor-bearing mice, we treated them with FA in combination with PD-L1 or CTLA-4 inhibitors and found that the combination with 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). Simultaneously, Figure 11C, the results of detecting distant lung metastases, also showed that CTX and α-PD-L1 immunization alone could not inhibit the occurrence and development of distant metastases, while fragmented tumor antigen alone or FAST immunization could effectively inhibit the production of distant metastases. The combination of FA and α-PD-L1 did not further reduce the risk of lung metastases, while the combination with α-CTLA-4 reduced lung metastases to some extent (Figure 11C). This indicates that FA alone with FAST has shown a significant anti-tumor effect, 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 6-week-old female Balb / c mice were used, and logarithmic growth 4T-1-Luc breast cancer cells were inoculated into the right hind limb of each mouse, with 1 million cells per mouse. The inoculation continued until the tumor reached 200 mm. 3 Afterwards, they were randomly divided into the following 4 groups and began corresponding treatments:

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

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

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

[0239] Group 4 (FAST): Each Balb / c mouse was intraperitoneally injected with CTX combined with subcutaneous inoculation of fragmented tumor antigen in the left hind limb. Specifically, on the day treatment began, 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 after an interval of 1 day.

[0240] Distal tumor tissues from mice in each group were collected and stored in physiological saline at 4°C. The tissues were then cut into small pieces, and 0.25g of each piece was placed in complete culture medium containing collagenase (1μg / ml) and hyaluronidase (0.1μg / ml). The tissues were homogenized twice using a tissue homogenizer and then digested overnight in a shaker at 37°C. The resulting single-cell suspension was prepared by filtering through a 100μm cell sieve. The cells were fixed, and a flow cytometry-labeled surface marker was 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 cells) 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 other groups, the FAST group significantly increased the percentage of immune cells within the tumor (Figure 12A). Further analysis revealed increased levels of CD4+ T cells and CD8+ T cells (Figures 12B and 12C), and increased levels of Treg cells (FOX3). + CD4 +The decreased proportion of T cells (Figure 12D) indicates that FAST can promote the infiltration of immune cells within the tumor, increase T cell content, inhibit Treg cells, and promote immune activation. Simultaneously, KI67 in tumor tissue... + CD8 + The proportion of T cells increased significantly (Figure 12E), indicating that FAST can exert its anti-tumor effect by activating the body's adaptive immune response.

[0243] Meanwhile, we combined the fragmented tumor antigen (FA) prepared in this application with other treatment methods or drugs and evaluated the anti-tumor effect of the combined application with FAST, as detailed in Test Examples 13 to 16.

[0244] Test Example 13

[0245] Twenty 6-week-old female Balb / c mice were used. On day 0, each mouse was inoculated with logarithmic growth 4T-1-Luc breast cancer cells in its right hind limb, with 1 million cells per mouse. The inoculation was continued until the tumor grew to 200 mm. 3 The subjects were randomly divided into the following 4 groups and began corresponding treatments:

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

[0247] Group 2 (IR): On days 8, 10 and 12 after tumor bearing, the tumors of each Balb / c mouse were irradiated with 8 Gy of X-rays for a total of 3 times;

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

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

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

[0251] Figure 13A shows the tumor growth inhibition effect of different treatments such as FA combined with IR and FAST after normalization treatment. Figure 13B shows the tumor lung metastasis inhibition effect of different treatments such as FA combined with IR and FAST after normalization treatment.

[0252] As shown in Figure 13A, compared with the CON group, IR alone and IR+FA in the mid-to-late stages of treatment achieved tumor growth inhibition effects comparable to FAST. As shown in Figure 13B, compared with the CON group, IR had no effect on lung metastasis, i.e., it could not effectively inhibit lung metastasis; compared with the CON group, FA combined with IR significantly reduced lung metastasis, and the inhibitory effect was comparable to 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 logarithmic growth 4T-1-Luc breast cancer cells in their right hind limbs on day 0, with 1 million cells per mouse. The inoculation continued until the tumor reached 200 mm. 3 The subjects were randomly divided into the following 5 groups and began corresponding treatments:

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

[0256] Group 2 (CDDP): Balb / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17 and 19 after tumor bearing, for a total of 3 times;

[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 bearing; at the same time, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left side of the mice on days 20, 22 and 24 after tumor bearing.

[0258] Group 4 (CDDP+IR+FA): On days 8, 10, and 12 post-tumor implantation, the tumors of each Balb / c mouse were irradiated with 8 Gy of X-rays, for a total of 3 times. After a 2-day rest period, BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 post-tumor implantation. Simultaneously, on days 16, 18, and 20 post-tumor implantation, 100 μL of the fragmented tumor antigen prepared in Example 3 was subcutaneously injected into the left side of the mice.

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

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

[0261] Figure 14A shows the tumor growth inhibition effect of CDDP combined with different treatments such as IR or FA and FAST after normalization. Figure 14B shows the tumor lung metastasis inhibition effect of CDDP combined with different treatments such as IR or FA and FAST after normalization.

[0262] As shown in Figure 14A, compared with the CON group, the CDDP group and the CDDP+FA group could partially inhibit tumor growth; the CDDP+IR+FA group could significantly inhibit tumor growth, achieving a tumor growth inhibition effect comparable to 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 group and the CDDP+IR+FA group could effectively reduce lung metastasis, with inhibition effects similar to FAST (n≥5, *P<0.05, ***P<0.001, ****P<0.0001). Furthermore, the lung metastasis inhibition effect of the CDDP+IR+FA group showed less fluctuation and higher stability.

[0263] Test Example 15

[0264] Twenty-five 6-week-old female Balb / c mice were inoculated with logarithmic growth 4T-1-Luc breast cancer cells in their right hind limbs on day 0, with 1 million cells per mouse. The inoculation continued until the tumor reached 200 mm. 3 The subjects were randomly divided into the following 5 groups and began corresponding treatments:

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

[0266] Group 2 (PGPC+CDDP): CDDP was administered intraperitoneally to Balb / c mice on days 15, 17 and 19 after tumor bearing (5 mg / kg), for a total of 3 times; PGPC was subcutaneously injected into the left hind limb of Balb / c mice on days 16, 18 and 20 after tumor bearing, for a total of 3 times.

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

[0268] Group 4 (PGPC+CDDP+IR+FA): On days 8, 10, and 12 post-tumor implantation, the tumors of each Balb / c mouse were irradiated with 8 Gy of X-rays, for a total of 3 times. After a 2-day rest period, on days 15, 17, and 19 post-tumor implantation, BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg). Simultaneously, on days 16, 18, and 20 post-tumor implantation, 100 μL of the fragmented tumor antigens FA and PGPC prepared in Example 3 were subcutaneously injected into the left side of the mice.

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

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

[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. 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 group, PGPC+CDDP+FA group, and PGPC+CDDP+IR+FA group all inhibited tumor growth, with the PGPC+CDDP+FA group showing a better tumor growth inhibition effect than the PGPC+CDDP group. The PGPC+CDDP+IR+FA group exhibited the strongest tumor inhibition effect among the three treatments, achieving a tumor growth inhibition effect comparable to the FAST group in the later stages of treatment. As shown in Figure 15B, compared with the CON group, the PGPC+CDDP group, PGPC+CDDP+FA group, and PGPC+CDDP+IR+FA group all significantly reduced lung metastasis, with inhibition effects comparable to the FAST group (n≥5, *P<0.05, **P<0.01, ***P<0.001). Furthermore, the PGPC+CDDP group showed better lung metastasis inhibition and higher stability.

[0273] Test Example 16

[0274] Based on test example 14, we selected the CDDP+IR+FA treatment regimen, which has good anti-tumor effects, and evaluated the FA inoculation dosage. The specific procedures are as follows:

[0275] Twenty 6-week-old female Balb / c mice were used. On day 0, each mouse was inoculated with logarithmic growth 4T-1-Luc breast cancer cells in its right hind limb, with 1 million cells per mouse. The inoculation was continued until the tumor grew to 200 mm. 3 The subjects were randomly divided into the following 4 groups and began corresponding treatments:

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

[0277] Group 2 (CDDP+IR+FA-50w): On days 8, 10, and 12 post-tumor implantation, the tumors of each Balb / c mouse were irradiated with 8 Gy of X-rays, for a total of 3 times. After a 2-day rest period, BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 post-tumor implantation. Simultaneously, on days 16, 18, and 20 post-tumor implantation, 100 μL of fragmented tumor antigen prepared from 50w tumor cells according to the method described in Example 7 was subcutaneously injected into the left side of the mice, for a total of 3 times.

[0278] Group 3 (CDDP+IR+FA-100w): On days 8, 10, and 12 post-tumor implantation, the tumors of each Balb / c mouse were irradiated with 8 Gy of X-rays, for a total of 3 times. After a 2-day rest period, BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 post-tumor implantation. Simultaneously, on days 16, 18, and 20, 100 μL of fragmented tumor antigen prepared from 100w tumor cells according to the method described in Example 3 was subcutaneously injected into the left side of the mice, for a total of 3 times.

[0279] Group 4 (CDDP+IR+FA-500w): On days 8, 10, and 12 post-tumor implantation, the tumors of each Balb / c mouse were irradiated with 8 Gy of X-rays, for a total of 3 times. After a 2-day rest period, BALB / c mice were intraperitoneally injected with cisplatin (CDDP, 5 mg / kg) on ​​days 15, 17, and 19 post-tumor implantation. Simultaneously, on days 16, 18, and 20, 100 μL of fragmented tumor antigen prepared from 500w tumor cells according to the method described in Example 8 was subcutaneously injected into the left side of the mice, for a total of 3 times.

[0280] During the experiment, the growth of in situ tumors in each group of mice was monitored periodically, and the survival days of the mice were recorded for survival analysis.

[0281] Figure 16A shows the tumor growth curves after treatment with CDDP combined with IR and different doses of FA, and Figure 16B shows the mouse survival curves 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, and there was no difference in tumor growth inhibition among the 50W, 100W, and 500W groups treated with FA three times combined with CDDP+IR. As shown in Figure 16B, compared with the CON group, CDDP+IR combined with different doses of FA significantly prolonged mouse survival time; there was no difference in mouse survival among the 100W and 500W groups treated with FA three times combined with CDDP+IR, but the survival time of mice treated with 100W and 500W combined with CDDP+IR was longer than that of mice treated with 50W combined with CDDP+IR. This indicates that CDDP+IR combined with different doses of FA can produce similar tumor growth inhibition effects, but combining it with a larger dose of FA results in better survival benefits.

[0283] The fragmented tumor antigen prepared using the method described in this application can maximize the preservation of tumor cell immunogenicity, overcome the tumor-suppressive immune microenvironment, and induce a strong immune response, effectively inhibiting tumor growth and metastasis, and producing a better anti-tumor metastasis effect. Furthermore, the preparation method described in this application is simple, low-cost, easy to store, highly effective, and safe, and has broad application prospects.

[0284] Treating tumor-bearing mice with the tumor immunotherapy method described in this application preserves all the antigens and immunologically active substances of tumor cells, enhancing the immunogenicity of tumor antigens, efficiently presenting tumor antigens to antigen-presenting cells (such as APCs) and activating them, initiating the specific killing function of cytotoxic T lymphocytes, and exerting an anti-tumor effect. Simultaneously, radiation can induce immunogenic cell death such as pyroptosis / ferroptosis in tumor cells, releasing damage-related molecular patterns (such as CRT, HMGB1, ATP, etc.), further enhancing the in vivo adaptive immune response and improving the inhibitory effect on distant tumors. Furthermore, the immunomodulatory drug CTX can inhibit the proliferation and function of Treg cells in the tumor microenvironment, weaken the suppressive tumor immune microenvironment, and increase the tumor infiltration of cytotoxic T lymphocytes (CTLs). The different components in the tumor immunotherapy method of this application can synergistically inhibit tumor growth, metastasis, and recurrence, especially significantly inhibiting tumor metastasis; at the same time, the tumor immunotherapy method can prolong the survival time and improve the survival rate of mice, exhibiting a sustained anti-tumor effect. The tumor immunotherapy method of this application, used alone, has an anti-tumor effect comparable to that of immune checkpoint inhibitors in combination. In summary, the tumor immunotherapy method described in this application exhibits high immunogenicity and low off-target rate, effectively and sustainably activating immune responses, significantly inhibiting tumor growth and metastasis, and prolonging survival time. Furthermore, the fragmented tumor antigens prepared using the method described in this application can also be used in combination with conventional tumor treatments (such as radiotherapy, chemotherapy, and other clinical drugs) to jointly inhibit tumor growth, reduce tumor metastasis, or prolong survival time from different perspectives, providing diversified methods for tumor treatment and aiding in strategy optimization.

[0285] Furthermore, the tumor immunotherapy method described in this application does not require extraction, sequencing, or specific processing of the treated tumor antigens, nor does it require special antigen delivery technologies and related equipment. The preparation process is simple and time-saving. At the same time, it can achieve significant anti-tumor effects without the need for combined use with immune checkpoint inhibitors. The reagents used in this method are commonly used clinical drugs, which are inexpensive and safe. The prepared fragmented tumor antigens can be stored at low temperatures, making them easy to preserve and transport. They have excellent effects and great application prospects.

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

Claims

1. A method of making a fragmented tumor antigen, comprising: (1) obtaining a tumor cell suspension; (2) after irradiation and incubation of the tumor cell suspension, preparing fragmented tumor antigens.

2. The method of claim 1, further comprising: (3) subjecting the fragmented tumor antigens to ultralow-temperature freeze-thaw treatment.

3. The production 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, cholangiocarcinoma cells, ovarian cancer cells, cervical cancer cells, osteosarcoma cells, and testicular cancer cells.

4. The production 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 in vitro cultured tumor cells.

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

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

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

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

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

10. The production method according to any one of claims 1 to 2, wherein The dose rate of the irradiation is 0.01 Gy / s to 1 x 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 production method according to any one of claims 1 to 2, wherein The mode of the irradiation is selected from at least one of electron, photon, proton, heavy ion, and neutron radiation, preferably at least one of photon and proton, and more preferably X-ray.

12. A fragmented tumor antigen prepared 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 of claim 13, wherein, The tumor cell vaccine further comprises an immunoadjuvant; the immunoadjuvant is an immunomodulatory drug; Preferably, the immunomodulatory drug is selected from at least one of polyinosinic acid-polycytidylic acid, platinum drugs, calcineurin inhibitors, glucocorticoids, alkylating agents, microbial metabolite drugs, polyclonal antibody drugs, monoclonal antibody drugs, antiproliferative drugs, antimetabolite drugs, rapamycin target molecule inhibitors, plant drugs, nucleotide reductase inhibitors, and tyrosine kinase inhibitors. More preferably, the polyribonucleotide is selected from an artificially synthesized ribonucleic acid; the platinum-based drug is selected from at least one of cisplatin, carboplatin, oxaliplatin, cycloplatin, nedaplatin, and lobaplatin; the calcineurin inhibitor is selected from at least one of cyclosporine, tacrolimus, and mycophenolate; 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 metabolite is selected from at least one of cyclosporine, tacrolimus, and rapamycin; the monoclonal antibody drug is selected from at least one of anti-thymocyte immunoglobulin, muromonab-CD3, daclizumab, basiliximab, efalizumab, and natalizumab; the antiproliferative drug is selected from at least one of azathioprine, leflunomide, and mycophenolate; the antimetabolite drug is selected from at least one of mycophenolate, methotrexate, azathioprine, mercaptopurine, and mizoribine; the rapamycin target molecule inhibitor is selected from at least one of sirolimus and everolimus; the plant drug is selected from at least one of tripterygium glycosides and total paeonol glycosides; the nucleotide reductase inhibitor is selected from hydroxyurea; and 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 13-14 in the treatment and / or prevention of a tumor disease; preferably, the tumor disease comprises 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, cholangiocarcinoma, ovarian cancer, cervical cancer, osteosarcoma, and testicular cancer.

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