Fragmented tumor antigen, method for preparing same, and use thereof
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-03
- Publication Date
- 2026-08-13
Smart Images
Figure CN2026076701_13082026_PF_FP_ABST
Abstract
Description
A fragmented tumor antigen, its preparation method and uses
[0001] This application claims priority to Chinese Patent Application No. 202510130933.0, filed on February 5, 2025, entitled "A Fragmented Tumor Antigen and Its Preparation Method and Use", 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 fragmented tumor antigen and its preparation method and uses. Background Technology
[0003] Tumor vaccines are currently a hot research topic in the field of tumor immunotherapy both domestically and internationally. As an active immunization method for tumors, they are crucial in preventing tumor development and progression. Some tumor vaccines contain only certain single tumor antigens, resulting in insufficient immunogenicity and inability to fully mobilize the body's immune system, thus limiting their anti-tumor effects. Increasing evidence suggests that cancer development is caused by multiple factors and multiple genes; therefore, the development of whole-tumor antigen vaccines is essential for tumor vaccine development. Using tumor cells or their components as the vaccine substrate has unique advantages: it retains all antigens, is less likely to be tolerated, is inexpensive, and is readily available. Tumor cells and their components are the main source of immune-activating substances for tumor vaccines.
[0004] Currently, prophylactic tumor vaccines are complex in composition, requiring at least one adjuvant and tumor antigen to be administered simultaneously. This increases the complexity of the preparation process, prolongs vaccine preparation time, and raises production costs. Furthermore, the simultaneous immunization with multiple components reduces vaccine safety, hindering the further promotion and use of tumor vaccines. Therefore, developing a prophylactic tumor vaccine with a single component, simple and time-saving preparation, low cost, significant efficacy, and high safety is crucial for effectively preventing the occurrence of tumors, including cold-cell tumors. Summary of the Invention
[0005] The purpose of this application is to provide a fragmented tumor antigen, its preparation method, and its uses, in order to improve the immunogenicity of the fragmented tumor antigen. The specific technical solution is as follows:
[0006] The first aspect of this application provides a method for preparing a fragmented tumor antigen, comprising:
[0007] (1) Obtaining a suspension of tumor cells;
[0008] (2) Irradiate the tumor cell suspension to obtain fragmented tumor antigens; wherein the irradiation dose is 5 Gy to 150 Gy.
[0009] In one embodiment of this application, the irradiation dose is 6 Gy to 110 Gy, preferably 10 Gy to 20 Gy.
[0010] In one embodiment of this application, the irradiation is performed using ultrafast laser particle FLASH radiotherapy, gamma ray irradiation, or X-ray irradiation.
[0011] In one embodiment of this application, the irradiation dose rate is 0.01 Gy / s to 9 × 10⁻⁶. 9 Gy / s, preferably 0.1 Gy / s to 1 × 10 9 Gy / s.
[0012] In one embodiment of this application, the number of cells in the tumor cell suspension is 0.01 × 10⁻⁶. 6 100 x 10 6 The number of units is preferably 0.1 × 10⁻⁶. 6 5 x 10 6 indivual.
[0013] In one embodiment of this application, the tumor cells are selected from at least one of breast cancer cells, lung cancer cells, colon cancer cells, and melanoma cells.
[0014] The second aspect of this application provides a fragmented tumor antigen prepared according to the preparation method described in the first aspect of this application.
[0015] A third aspect of this application provides a preventive tumor cell vaccine comprising the fragmented tumor antigen described in the second aspect of this application.
[0016] The fourth aspect of this application provides the use of the fragmented tumor antigen described in the second aspect of this application or the prophylactic tumor cell vaccine described in the third aspect of this application in the preparation of a medicament for the prevention of tumor diseases.
[0017] In one embodiment of this application, the tumor disease includes at least one of breast cancer, lung cancer, colon cancer, and melanoma.
[0018] The beneficial effects of this application are:
[0019] This application provides a method for preparing fragmented tumor antigens, comprising: obtaining a tumor cell suspension; irradiating the tumor cell suspension to obtain fragmented tumor antigens; wherein the irradiation dose is 5 Gy to 150 Gy. The preparation method of this application is simple, low-cost, and easy to store. The fragmented tumor antigens prepared using this method retain all tumor cell antigens. Simultaneously, the irradiated tumor cells can release damage-associated molecular patterns, enhancing tumor cell immunogenicity, promoting immune activation, further enhancing tumor-killing activity in vivo, and synergistically inhibiting tumor development and providing long-term tumor resistance.
[0020] 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
[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these accompanying drawings.
[0022] Figure 1 shows the antitumor effect of mice in each group of test example 1 inoculated with the fragmented tumor antigens prepared in Examples 1-1 to 1-3 and Example 2-2;
[0023] Figure 2A shows the antitumor effect of fragmented tumor antigens on mice in each group of test example 2 after being irradiated with different low doses (4Gy, 8Gy and 12Gy) of γ-rays.
[0024] Figure 2B shows the tumor volume growth curves of mice in each group of test example 2 after being irradiated with different low doses (4Gy, 8Gy and 12Gy) of γ-rays and treated with fragmented tumor antigens.
[0025] Figure 3A shows the antitumor effect of fragmented tumor antigens after different doses (8 Gy and 20 Gy) of γ-ray irradiation on mice in test example 3 in immunocompetent and immunodeficient mice.
[0026] Figure 3B shows the tumor volume growth curves of fragmented tumor antigens in immunocompetent and immunodeficient mice after mice in each group of test example 3 were irradiated with different doses (8 Gy and 20 Gy) of γ-rays.
[0027] Figure 4A shows the anti-tumor effect of mice bearing tumors after 26 days of small-scale, multiple-inoculation with the fragmented tumor antigen prepared in Example 3 in Test Example 4.
[0028] Figure 4B shows the tumor volume growth curves of mice in each group after tumor bearing in Test Example 4.
[0029] Figure 4C shows the body weight of mice bearing tumors 26 days after being inoculated with fragmented tumor antigen prepared in Example 3 in small, multiple small doses in Test Example 4.
[0030] Figure 5A shows the anti-tumor effect of the fragmented tumor antigen prepared in Example 4 on mice bearing tumors for 26 days after a single high-dose vaccination in Test Example 5.
[0031] Figure 5B shows the tumor volume growth curves of mice in each group after tumor bearing in Test Example 5.
[0032] Figure 5C shows the survival curve of mice bearing tumors 30 days after a single high-dose inoculation with the fragmented tumor antigen prepared in Example 4 in Test Example 5.
[0033] Figure 5D shows the body weight of mice bearing tumors 26 days after a single high-dose inoculation with the fragmented tumor antigen prepared in Example 4 in Test Example 5.
[0034] Figure 6 shows CD4 in Test Example 6. + T, CD8 + T is a tumor volume growth curve of mice bearing tumors after being inoculated with the fragmented tumor antigen prepared in Example 2-2.
[0035] Figure 7 shows the tumor volume growth curve of mice bearing tumors after being inoculated with the fragmented tumor antigen prepared in Example 2-2 following antagonism of interferon (IFN) or its receptor in Test Example 7. Detailed Implementation
[0036] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and 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.
[0037] The first aspect of this application provides a method for preparing a fragmented tumor antigen, comprising:
[0038] (1) Obtaining a suspension of tumor cells;
[0039] (2) The tumor cell suspension is irradiated to obtain fragmented tumor antigens; wherein the irradiation dose is 5 Gy to 150 Gy, preferably 6 Gy to 110 Gy, and more preferably 10 Gy to 20 Gy. For example, the irradiation dose can be 5 Gy, 6 Gy, 8 Gy, 10 Gy, 12 Gy, 16 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, 100 Gy, 120 Gy, 150 Gy, or any two values in between. Controlling the irradiation dose within the above range can maximize the preservation of tumor cell immunogenicity and achieve better anti-tumor effects.
[0040] The inventors discovered in their research that, using the preparation method described in this application, with an irradiation dose ranging from 5 Gy to 150 Gy, all tumor cell antigens can be retained. When tumor cells appear in the body, these fragmented tumor antigens can efficiently present antigens and activate antigen-presenting cells, initiating specific immune killing of tumor cells and inhibiting tumor formation. Simultaneously, irradiated tumor cells can release damage-related molecular patterns (such as calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP)) to enhance tumor cell immunogenicity, promote immune activation, further enhance tumor-killing activity in vivo, and synergistically inhibit tumor development and provide long-term resistance to tumors.
[0041] 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 or digestion suspension culture.
[0042] 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 fragmented tumor antigen solution, or a mixture of the fragmented tumor antigen with a hydrogel, or a loading of the fragmented tumor antigen onto other carriers such as nanocarriers. 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 of the tumor antigen and extends the shelf life of the fragmented tumor antigen. Mixing the fragmented tumor antigen with a hydrogel, or loading the fragmented tumor antigen onto other carriers such as nanocarriers, can increase the delivery efficiency of the tumor antigen or achieve targeted delivery of the tumor antigen.
[0043] In one embodiment of this application, the tumor cell suspension, after irradiation and incubation, further includes undergoing cryogenic freeze-thaw treatment. In this application, the method of cryogenic freeze-thaw treatment is not particularly limited, as long as it achieves the purpose of this application; for example, cryogenic freeze-thaw treatment at -60℃ to -100℃ for 10 to 15 hours is performed.
[0044] 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.
[0045] In one embodiment of this application, the irradiation employs ultrafast laser particle FLASH radiotherapy, gamma ray irradiation, or X-ray irradiation. All of these irradiation methods can effectively inhibit tumor occurrence or development. For example, when ultrafast laser particle FLASH radiotherapy is used, the irradiation dose can be 15 Gy to 150 Gy; when gamma ray irradiation or X-ray irradiation is used, the irradiation dose can be 5 Gy to 20 Gy. Preferably, the irradiation uses gamma ray irradiation. The inventors have found in their research that, compared to other irradiation methods, gamma ray irradiation can maximize the preservation of tumor cell immunogenicity and has a better anti-tumor effect.
[0046] In one embodiment of this application, the irradiation dose rate is 0.01 Gy / s to 9 × 10⁻⁶. 9 Gy / s, preferably 0.1 Gy / s to 1 × 10 9 Gy / s. For example, the dose rate of the irradiation can be 0.01 Gy / s, 0.05 Gy / s, 0.1 Gy / s, 1 Gy / s, 10 Gy / s, 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, 2×10 9 Gy / s, 3×10 9 Gy / s, 4×10 9 Gy / s, 5×10 9 Gy / s, 6×10 9 Gy / s, 7×10 9 Gy / s, 8×10 9 Gy / s, 9×10 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 and result in better anti-tumor effects.
[0047] In one embodiment of this application, the number of cells in the tumor cell suspension is 0.01 × 10⁻⁶. 6 100 x 10 6 The number of units is preferably 0.1 × 10⁻⁶. 6 5 x 10 6 For example, the number of cells in the tumor cell suspension can be 0.01 × 10⁻⁶. 6 0.05×10 6 0.1×106 0.5×10 6 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 5×10 6 10×10 6 1 piece, 20×10 6 40×10 6 60×10 6 80×10 6 100×10 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 of tumor diseases.
[0048] In one embodiment of this application, the tumor cells are selected from at least one of breast cancer cells, lung cancer cells, colon cancer cells, and melanoma cells.
[0049] In this application, the source of tumor cells is not specifically limited, as long as it achieves the purpose of this application. For example, the tumor cells are selected from autologous or allogeneic tumor cells. Autologous tumor cells are tumor cells from the patient or the same animal, while allogeneic tumor cells are tumor cells with the same gene, referring to tumor cells of the same type of cancer derived from another organism. For example, the tumor cells may be 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 the above 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.
[0050] In one embodiment of this application, the irradiation of the tumor cell suspension further includes an incubation step. The incubation temperature is 25℃ to 45℃, preferably 35℃ to 40℃; the incubation time is 0.1h to 30h, preferably 0.5h to 8h. For example, the incubation temperature can be 25℃, 30℃, 35℃, 40℃, 45℃, or any two values within this range, and the incubation time can be 0.1h, 0.5h, 1h, 5h, 10h, 15h, 20h, 25h, 30h, or any two values within this range. Controlling the incubation temperature and time within the above range can maximize the preservation of tumor cell immunogenicity and produce a better anti-tumor effect.
[0051] The second aspect of this application provides a fragmented tumor antigen prepared according to the preparation method described in the first aspect of this application.
[0052] A third aspect of this application provides a preventive tumor cell vaccine comprising the fragmented tumor antigen described in the second aspect of this application.
[0053] In one embodiment of this application, the prophylactic tumor cell vaccine further includes an adjuvant. This application does not specifically limit the type of adjuvant, as long as it achieves the purpose of this application. For example, it may include a manganese adjuvant, which includes various adjuvants with manganese ions as the main active ingredient, including at least one of manganese salt adjuvants, colloidal manganese adjuvants, nano-manganese adjuvants, and hydrogel manganese adjuvants. Other adjuvants that can enhance vaccine efficacy may also be used, including at least one of aluminum adjuvants, novel oil-emulsion adjuvants, liposome adjuvants, CpG oligonucleotide adjuvants, cytokine adjuvants, nano-adjuvants, polysaccharide adjuvants, and complex system adjuvants.
[0054] The fourth aspect of this application provides the use of the fragmented tumor antigen described in the second aspect of this application or the prophylactic tumor cell vaccine described in the third aspect of this application in the preparation of a medicament for the prevention of tumor diseases.
[0055] The inventors discovered in their research that the fragmented tumor antigen or prophylactic tumor cell vaccine prepared using the method described in this application can retain all tumor cell antigens. When tumor cells appear in the body, this fragmented tumor antigen can efficiently present the antigen and activate antigen-presenting cells, initiating specific immune killing of tumor cells and inhibiting tumor formation. Simultaneously, irradiated tumor cells can release damage-associated molecular patterns, enhancing tumor cell immunogenicity, promoting immune activation, further enhancing tumor-killing activity in the body, and synergistically inhibiting tumor development and providing long-term resistance to tumors. Therefore, it can be used to prepare drugs for the prevention of tumor diseases.
[0056] In one embodiment of this application, the tumor disease includes at least one of breast cancer, lung cancer, colon cancer, and melanoma.
[0057] The fifth aspect of this application provides a tumor immunotherapy method comprising: administering to an individual in need an effective amount of the fragmented tumor antigen described in the second aspect of this application or the prophylactic tumor cell vaccine described in the third aspect of this application.
[0058] In this application, fragmented tumor antigens obtained from different irradiation doses can be mixed and inoculated to inhibit tumor growth and produce a good anti-tumor effect. For example, mixed inoculation of fragmented tumor antigens with irradiation doses of 20 Gy, 25 Gy, and 105 Gy can inhibit tumor growth and produce a good anti-tumor effect.
[0059] In one embodiment of this application, the injection method of the fragmented tumor antigen or the prophylactic tumor cell vaccine is selected from low-dose multiple immunizations or high-dose single immunizations. For example, it can be a high-dose single vaccination, low-dose multiple booster vaccinations, or multiple courses of vaccination. A high-dose single vaccination can be a single dose of 3 × 10⁻⁶. 6 10×10 6 One dose is administered for each individual; multiple booster doses of low-dose vaccine can be administered in a single dose of 0.2 × 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.
[0060] In one embodiment of this application, the injection method of the fragmented tumor antigen or the tumor cell vaccine is selected from at least one of subcutaneous injection (sc), intraperitoneal injection (ip), intravenous injection (iv), and intratumoral injection (it).
[0061] The tumor immunotherapy method described in this application has high immunogenicity and has the effect of inhibiting tumor development in different types of tumor animal models, including clinically refractory cold tumors.
[0062] Example
[0063] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.
[0064] Laboratory animals and materials
[0065] Female BALB / c mice were purchased from the Zhejiang Provincial Experimental Animal Center. They were 4–6 weeks old and weighed 20 ± 5 g.
[0066] The mouse 4T-1-Luc breast cancer tumor cells were donated by Wenzhou Medical University.
[0067] All mouse models used in this application were constructed by our research group.
[0068] Example 1-1
[0069] (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 1.2 × 10⁻⁶ cells. 5 A single-cell suspension;
[0070] (2) Single-cell suspensions were FLASH irradiated using a small laser plasma accelerator (CLAPA). The radiation source was 100 cm away from the single-cell suspension, the irradiation dose was 20 Gy, and the irradiation dose rate was 1 × 10⁻⁶. 9 Gy / s, to obtain fragmented tumor antigens.
[0071] Examples 1-2
[0072] (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 1.2 × 10⁻⁶ cells. 5 A single-cell suspension;
[0073] (2) Single-cell suspensions were FLASH irradiated using a small laser plasma accelerator (CLAPA). The radiation source was 100 cm away from the single-cell suspension, the irradiation dose was 25 Gy, and the irradiation dose rate was 1 × 10⁻⁶. 9 Gy / s, to obtain fragmented tumor antigens.
[0074] Examples 1-3
[0075] (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 1.2 × 10⁻⁶ cells. 5 A single-cell suspension;
[0076] (2) Single-cell suspensions were FLASH irradiated using a small laser plasma accelerator (CLAPA). The radiation source was 100 cm away from the single-cell suspension, the irradiation dose was 105 Gy, and the irradiation dose rate was 1 × 10⁻⁶. 9 Gy / s, to obtain fragmented tumor antigens.
[0077] Example 2-1
[0078] (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 1.2 × 10⁻⁶ cells. 5 A single-cell suspension;
[0079] (2) The single-cell suspension was irradiated with Co-60 γ rays. The radiation source was 200 cm away from the single-cell suspension. The irradiation dose was 8 Gy and the irradiation dose rate was 3.3 Gy / s, resulting in fragmented tumor antigens.
[0080] Example 2-2
[0081] (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 1.2 × 10⁻⁶ cells.5 A single-cell suspension;
[0082] (2) The single-cell suspension was irradiated with Co-60 γ rays. The radiation source was 200 cm away from the single-cell suspension, the irradiation dose was 20 Gy, and the irradiation dose rate was 3.3 Gy / s. Fragmented tumor antigens were obtained by irradiation.
[0083] Example 2-3
[0084] (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 1.2 × 10⁻⁶ cells. 5 A single-cell suspension;
[0085] (2) The single-cell suspension was irradiated with Co-60 γ rays. The radiation source was 200 cm away from the single-cell suspension. The irradiation dose was 12 Gy and the irradiation dose rate was 3.3 Gy / s. Fragmented tumor antigens were obtained by irradiation.
[0086] Example 3
[0087] (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;
[0088] (2) The single-cell suspension was irradiated with X-rays at a dose of 12 Gy and a dose rate of 0.1 Gy / s to obtain fragmented tumor antigens.
[0089] Example 4
[0090] (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 3×10⁻⁶ cells. 6 A single-cell suspension;
[0091] (2) The single-cell suspension was irradiated with X-rays at a dose of 12 Gy and a dose rate of 0.1 Gy / s to obtain fragmented tumor antigens.
[0092] Comparative Example 1
[0093] (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 1.2 × 10⁻⁶ cells. 5 A single-cell suspension;
[0094] (2) The single-cell suspension was irradiated with Co-60 γ rays. The radiation source was 200 cm away from the single-cell suspension. The irradiation dose was 4 Gy and the irradiation dose rate was 3.3 Gy / s, resulting in fragmented tumor antigen.
[0095] The preparation parameters for the examples and comparative examples are shown in Table 1.
[0096] Table 1
[0097] Test Example 1
[0098] Ten 6-week-old female BALB / c mice were randomly divided into two groups:
[0099] Group 1 (FLASH high-dose radiation): Each BALB / c mouse was subcutaneously injected with fragmented tumor antigens prepared in Examples 1-1 to 1-3 in the right upper limb. Specifically: on day 0, 100 μL of fragmented tumor antigen obtained from 20 Gy irradiation in Example 1-1 was injected; on day 1, 100 μL of fragmented tumor antigen obtained from 25 Gy irradiation in Example 1-2 was injected; on day 8, 100 μL of fragmented tumor antigen obtained from 105 Gy irradiation in Example 1-3 was injected; and on day 15, 1×10⁻⁶ fragmented tumor antigen was injected subcutaneously in the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0100] Group 2 (Conventional Dose Rate High-Dose Radiation): Each BALB / c mouse was subcutaneously injected with the fragmented tumor antigen prepared in Example 2-2 into its right upper limb. Specifically, on day 0, 100 μL of the fragmented tumor antigen obtained from 20 Gy irradiation in Example 2-2 was injected; on day 1, 100 μL of the fragmented tumor antigen obtained from 20 Gy irradiation in Example 2-2 was injected; on day 8, 100 μL of the fragmented tumor antigen obtained from 20 Gy irradiation in Example 2-2 was injected; and on day 15, 1×10⁻⁶ mc² was subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors.
[0101] The growth of tumors in mice was observed regularly during the experiment. Figure 1 shows the growth of tumors in mice on day 29 after tumor implantation in the left upper limb. As can be seen from Figure 1, the fragmented tumor antigens prepared in Examples 1-1 to 1-3 and Example 2-2 can effectively immunize the subsequently injected tumor cells to induce tumor formation. Among them, the fragmented tumor antigen prepared in Example 2-2 has a better anti-tumor effect.
[0102] Test Example 2
[0103] Fifteen 6-week-old female BALB / c mice were randomly divided into 3 groups:
[0104] Group 1 (4 Gy): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Comparative Example 1 into the right hind limb. After a 7-day interval, the above injection was repeated twice. On the second day after the last immunization (i.e., the last injection), 1 × 10⁻⁶ g of the fragmented tumor antigen was subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0105] Group 2 (8 Gy): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 2-1 into the right hind limb. After a 7-day interval, the above injection was repeated twice. On the second day after the last immunization, 1 × 10⁻⁶ g of the fragmented tumor antigen was subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0106] Group 3 (12 Gy): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Examples 2-3 into the right hind limb. After a 7-day interval, the above injection was repeated twice. On the second day after the last immunization, 1 × 10⁻⁶ g of the fragmented tumor antigen was subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors.
[0107] The growth of tumors in mice was observed periodically during the experiment. Figure 2A shows the tumor growth of mice one week after tumor implantation, and Figure 2B shows the tumor volume growth curves of mice in each group from 12 to 26 days after tumor implantation. The curves for 8 Gy and 12 Gy overlap in Figure 2B. Figures 2A and 2B show that the fragmented tumor antigen prepared in Example 2-1 completely inhibited tumor development, and the fragmented tumor antigen prepared in Example 2-3 completely inhibited tumor development and progression. The fragmented tumor antigen prepared in Comparative Example 1 slowed the tumor growth rate but did not significantly inhibit tumor growth, especially in the later stages. The results indicate that fragmented tumor antigens with radiation doses within the scope of this application have a better effect on inhibiting tumor growth.
[0108] Test Example 3
[0109] Eighteen 6-week-old female BALB / c mice were randomly divided into 3 groups:
[0110] Group 1 (CON): Each BALB / c mouse was subcutaneously injected with 100 μL of physiological saline in its right hind limb. This injection was repeated twice after a 7-day interval. On the second day after the last immunization, 1 × 10⁻⁶ μL of saline was subcutaneously injected into the left upper limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0111] Group 2 (low-dose radiation): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 2-1 into the right hind limb. This injection was repeated twice after a 7-day interval. On the second day after the last immunization, 1 × 10⁻⁶ μL of the fragmented tumor antigen was subcutaneously injected into the left upper limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0112] Group 3 (High-dose radiation): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 2-2 into the right hind limb. This injection was repeated twice after a 7-day interval. On the second day after the last immunization, 1 × 10⁻⁶ μL of the fragmented tumor antigen was subcutaneously injected into the left upper limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors.
[0113] Six nude mice were used as a separate group, designated as Group 4 (high-dose radiation nude mice). Each mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 2-2 into its right hind limb. This injection was repeated twice after a 7-day interval. On the second day after the last immunization, 1 × 10⁻⁶ μL of the fragmented tumor antigen was subcutaneously injected into the left upper limb of each mouse. 6 4T-1 tumor cells were used to bear tumors.
[0114] The growth of tumors in mice was observed regularly during the experiment. Figure 3A shows the growth of tumors in mice 25 days after tumor implantation, and Figure 3B shows the tumor volume growth curves of mice in each group from 13 to 25 days after tumor implantation. As can be seen from Figures 3A and 3B, compared with Group 1, the fragmented tumor antigen prepared in Example 2-2 completely inhibited tumor development. Although the fragmented tumor antigen prepared in Example 2-1 could inhibit tumor development in mice, the dosage was too small to completely kill all tumor cells, resulting in tumor formation at the inoculation site. However, the fragmented tumor antigen prepared in Example 2-2 did not show tumor-suppressive activity in immunodeficient nude mice, suggesting that T cells are crucial in the anti-tumor effect of this prophylactic tumor cell vaccine.
[0115] Test Example 4
[0116] Ten 6-week-old female BALB / c mice were randomly divided into two groups:
[0117] Group 1 (CON): Each BALB / c mouse was subcutaneously injected with 100 μL of physiological saline in its left hind limb. This injection was repeated twice after a 5-day interval. On the second day after the last immunization, 1 × 10⁻⁶ μL of saline was subcutaneously injected in the right hind limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0118] Group 2 (Radiation): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 3 into the left hind limb. This injection was repeated twice after a 5-day interval. On the second day after the last immunization, 1 × 10⁻⁶ μL of the fragmented tumor antigen was subcutaneously injected into the right hind limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0119] The growth of mouse tumors was observed periodically during the experiment. Figure 4A shows the growth of mouse tumors 26 days after tumor implantation, Figure 4B shows the tumor volume growth curves of each group of mice after tumor implantation, and Figure 4C shows the changes in mouse body weight 26 days after tumor implantation. As can be seen from Figures 4A and 4B, the fragmented tumor antigen prepared in Example 3 can completely and effectively inhibit the occurrence and development of 4T-1 tumors. No visible tumors appeared in any of the mice, and a significant tumor inhibition effect was maintained for 26 days after tumor implantation. Until the end of the observation period, no visible tumors appeared in any of the mice, confirming that the fragmented tumor antigen prepared in Example 3 of this application can significantly prevent the occurrence and development of cold tumor 4T-1 in the long term. Furthermore, Figure 4C shows that there was no difference in body weight among all groups of mice during the observation period (NS indicates no significant difference), meaning that the fragmented tumor antigen prepared in Example 3 of this application does not affect mouse growth, indicating that the fragmented tumor antigen prepared in this application is safe and reliable.
[0120] Test Example 5
[0121] Ten 6-week-old female BALB / c mice were randomly divided into two groups:
[0122] Group 1 (CON): Each BALB / c mouse was subcutaneously injected with 100 μL of physiological saline on the left hind limb; on the second day after immunization, BALB / c mice were subcutaneously injected with 1 × 10⁻⁶ oz. of saline on the right side. 6 4T-1 tumor cells were used to bear tumors;
[0123] Group 2 (Radiation): 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 day 2 post-immunization, 1 × 10⁻⁶ μL of the fragmented tumor antigen was subcutaneously injected into the right hind limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors.
[0124] The growth of tumors in mice was observed regularly during the experiment. Figure 5A shows the growth of tumors in mice 26 days after tumor implantation, Figure 5B shows the tumor volume growth curves of each group of mice after tumor implantation, Figure 5C shows the survival curve of tumor-bearing mice after 30 days, and Figure 5D shows the changes in body weight of mice 26 days after tumor implantation. As can be seen from Figures 5A and 5B, the fragmented tumor antigen prepared in Example 4 can completely and effectively inhibit the occurrence of 4T-1 tumors with a single immunization. No tumors appeared in any of the mice, and the significant tumor inhibition effect was maintained for 26 days after tumor implantation. No observable tumors were observed in any of the mice until the end of the observation period, confirming that the fragmented tumor antigen prepared in Example 4 of this application can significantly inhibit the occurrence of cold tumor 4T-1 with a single immunization.
[0125] Test Example 6
[0126] Twenty 6-week-old female BALB / c mice were randomly divided into 4 groups:
[0127] Group 1 (CON): Each BALB / c mouse was subcutaneously injected with 100 μL of physiological saline in its right hind limb; on day 2 post-immunization, BALB / c mice were subcutaneously injected with 1 × 10⁻⁶ oz. of saline in its left upper limb. 6 4T-1 tumor cells were used to bear tumors;
[0128] Group 2 (High Dose): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 2-2 into the right hind limb; on day 2 post-immunization, 1 × 10⁻⁶ μL of the fragmented tumor antigen was subcutaneously injected into the left upper limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0129] Group 3 (Anti-CD4): Each BALB / c mouse was injected intraperitoneally with 100 μL of CD4 antibody (purchased from Lelnco Technologies). On the second day, 100 μL of the fragmented tumor antigen prepared in Example 2-2 was subcutaneously injected into the right hind limb of the BALB / c mouse. On the second day after immunization, 1 × 10⁻⁶ cells were subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0130] Group 4 (Anti-CD8): Each BALB / c mouse was injected intraperitoneally with 100 μL of CD8 antibody (purchased from Lelnco Technologies). On the second day, 100 μL of the fragmented tumor antigen prepared in Example 2-2 was subcutaneously injected into the right hind limb of the BALB / c mouse. On the second day after immunization, 1 × 10⁻⁶ cells were subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors.
[0131] The growth of tumors in mice was observed regularly during the experiment. Figure 6 shows the tumor volume growth curves of each group of mice after tumor bearing. As can be seen from Figure 6, after injecting CD4 and CD8 antibodies to deplete the corresponding T cells, the effect of the fragmented tumor antigen prepared in Example 2-2 in preventing tumor development was completely neutralized. The tumor growth was no different from that of the first group of mice. This result, together with the result in Figure 3A, confirms that T cells (CD4... + T and CD8 + The decisive role of T cells in the anti-tumor effect of this tumor cell vaccine.
[0132] Test Example 7
[0133] Twenty 6-week-old female BALB / c mice were randomly divided into 4 groups:
[0134] Group 1 (CON): Each BALB / c mouse was subcutaneously injected with 100 μL of physiological saline in its right hind limb; on day 2 post-immunization, BALB / c mice were subcutaneously injected with 1 × 10⁻⁶ oz. of saline in its left upper limb. 6 4T-1 tumor cells were used to bear tumors;
[0135] Group 2 (High Dose): Each BALB / c mouse was subcutaneously injected with 100 μL of the fragmented tumor antigen prepared in Example 2-2 into the right hind limb; on day 2 post-immunization, 1 × 10⁻⁶ μL of the fragmented tumor antigen was subcutaneously injected into the left upper limb of each BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0136] Group 3 (Anti-INFγ): Each BALB / c mouse was injected intraperitoneally with 100 μL of INF-γ antibody (purchased from Lelnco Technologies). On the second day, 100 μL of the fragmented tumor antigen prepared in Example 2-2 was subcutaneously injected into the right hind limb of the BALB / c mouse. On the second day after immunization, 1 × 10⁻⁶ INF-γ was subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0137] Group 4 (Anti-IFNAR1): Each BALB / c mouse was injected intraperitoneally with 100 μL of IFNAR1 antibody (purchased from Lelnco Technologies). On the second day, 100 μL of the fragmented tumor antigen prepared in Example 2-2 was subcutaneously injected into the right hind limb of the BALB / c mouse. On the second day after immunization, 1 × 10⁻⁶ IFNAR1 antibody was subcutaneously injected into the left upper limb of the BALB / c mouse. 6 4T-1 tumor cells were used to bear tumors;
[0138] The growth of tumors in mice was observed regularly during the experiment. Figure 7 shows the tumor volume growth curves of each group of mice after tumor bearing. As can be seen from Figure 7, when the INF-γ antibody was injected to deplete the corresponding receptor, it completely neutralized the effect of the fragmented tumor antigen prepared in Example 2-2 in preventing tumor occurrence, and the tumor growth was no different from that of the mice in Group 1. When the IFNAR1 antibody was injected to deplete the corresponding receptor, it partially neutralized the effect of the fragmented tumor antigen prepared in Example 2-2 in preventing tumor occurrence. This result indicates the important role of IFN in the anti-tumor activity of this tumor cell vaccine.
[0139] In summary, the preparation method of this application is simple, low-cost, and easy to store. The fragmented tumor antigen prepared by the method of this application retains all the antigens of tumor cells. At the same time, the irradiated tumor cells can release damage-related molecular patterns to enhance the immunogenicity of tumor cells, promote immune activation, further enhance the tumor killing effect in vivo, and inhibit tumor occurrence together with the tumor antigen and resist tumors for a long time.
[0140] 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 for preparing a fragmented tumor antigen, comprising: (1) Obtaining a suspension of tumor cells; (2) Irradiate the tumor cell suspension to obtain fragmented tumor antigens; The irradiation dose is 5 Gy to 150 Gy.
2. The preparation method according to claim 1, wherein, The irradiation dose is 6 Gy to 110 Gy, preferably 10 Gy to 20 Gy.
3. The preparation method according to claim 1, wherein, The irradiation is performed using ultrafast laser particle FLASH radiotherapy, gamma ray irradiation, or X-ray irradiation.
4. The preparation method according to claim 1, wherein, The irradiation dose rate is 0.01 Gy / s to 9 × 10⁻⁶. 9 Gy / s, preferably 0.1 Gy / s to 1 × 10 9 Gy / s.
5. The preparation method according to claim 1, wherein, The number of cells in the tumor cell suspension was 0.01 × 10⁻⁶. 6 100 x 10 6 The number of units is preferably 0.1 × 10⁻⁶. 6 5 x 10 6 indivual.
6. The preparation method according to claim 1, wherein, The tumor cells are selected from at least one of breast cancer cells, lung cancer cells, colon cancer cells, and melanoma cells.
7. The fragmented tumor antigen prepared by any one of claims 1 to 6.
8. A prophylactic tumor cell vaccine comprising the fragmented tumor antigen of claim 7.
9. Use of the fragmented tumor antigen according to claim 7 or the prophylactic tumor cell vaccine according to claim 8 in the preparation of a medicament for the prevention of tumor diseases.
10. The use according to claim 9, wherein, The tumor diseases include at least one of breast cancer, lung cancer, colon cancer, and melanoma.