Method for detecting tumor-specific t cells

Abstract

The application discloses a method for detecting tumor-specific T cells. After collecting whole cell components of tumor cells or tumor tissues, the free whole cell components or the whole cell lysate components loaded on nano / microparticles are incubated with peripheral immune cells. After the cancer-specific T cells are activated, the specific molecules of the tumor-specific T cells are detected to determine the content of the cancer-specific T cells in peripheral tissues such as peripheral blood. The whole cell lysate components are water-soluble components and non-water-soluble components, which are in a free state or loaded on nano / microparticles. The loading mode is that the water-soluble components and the non-water-soluble components of the whole cells are respectively or simultaneously loaded in the particle interior and / or respectively or simultaneously loaded on the particle surface. The detection method is flow cytometry, enzyme-linked immunospot technology, enzyme-linked immunosorbent assay technology, colloidal gold immunochromatography, gene detection technology and the like.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy and immune detection, specifically relating to a method for detecting tumor-specific T cells based on whole cells. Background Technology

[0002] The development of immunotherapy technology has been extremely rapid in recent years, especially in the field of cancer immunotherapy. With a deeper understanding of cancer, it has been discovered that the human immune system and various immune cells play a crucial role in suppressing the occurrence and development of cancer. In recent years, therapies such as PD-1 antibodies and CAR-T have been approved for clinical use, showing promising clinical results. However, cancer vaccines and PD-1 antibody immunotherapies are only effective for a subset of patients. Therefore, it is crucial to determine the effectiveness of immunotherapy drugs and the patient's prognosis before or during treatment.

[0003] Existing technology discloses a detection particle effective for detecting tumor-specific T cell content, its corresponding preparation method, a kit including the particle, and a detection method using the particle for detecting tumor-specific T cell content. This method is used to activate tumor-specific T cells in detecting the content of tumor-specific T cells based on any one or more of the following: cell secretions secreted by activated tumor-specific T cells in the test sample, the proliferation status of activated tumor-specific T cells, or cell surface markers of activated tumor-specific T cells. Immunotherapy relies on T cells activated by cancer-specific / related antigens in the immune system to kill tumor cells; therefore, the content of cancer-specific T cells in a patient is closely related to the efficacy of immunotherapy. However, currently, there is a lack of effective means to comprehensively and accurately detect the content of cancer-specific T cells in the peripheral blood of patients. Summary of the Invention

[0004] This invention provides a method for detecting tumor-specific T cells and their content in peripheral tissues, which can provide reference information for the prognosis of cancer patients. Tumor-specific T cells are activated and secrete or express certain specific molecules after co-incubation with tumor cells, whole tumor tissue cells, tumor cell lysate components, whole tumor tissue cell lysate components, or micro / nano particles loaded with lysate components. The content of tumor-specific T cells can be determined by detecting these secreted or expressed specific molecules. The key technology lies in the activation of T cells.

[0005] The present invention adopts the following technical solution:

[0006] A method for detecting tumor-specific T cells includes the following steps: incubating an activator with peripheral immune cells, and then detecting specific molecules of tumor-specific T cells to achieve the detection of tumor-specific T cells.

[0007] The method for detecting the content of tumor-specific T cells includes the following steps: incubating an activator with peripheral immune cells, then detecting specific molecules of tumor-specific T cells; and finally obtaining the content of tumor-specific T cells based on the ratio of the number of tumor-specific T cells to the number of peripheral immune cells.

[0008] In this invention, the activator includes tumor cells, whole tumor tissue cells, tumor cell lysate components, and whole tumor tissue cell lysate components, and may also include an immune adjuvant. The lysate components in the tumor cell lysate components and whole tumor tissue cell lysate components can be water-soluble or water-insoluble lysate components, preferably water-soluble and water-insoluble lysate components.

[0009] In this invention, the activator can be a free cell or a free lysate component, or a lysate component loaded on micro / nanoparticles; preferably, it is a free lysate component or a lysate component loaded on micro / nanoparticles; the lysate is loaded inside and / or on the surface of the micro / nanoparticles. The lysate component is loaded inside and / or on the surface of the micro / nanoparticles in ways including, but not limited to, non-covalent adsorption, electrostatic interaction, hydrophobic interaction, hydrogen bonding, and covalent bonding. This invention can simultaneously use micro / nanoparticles loaded with water-soluble components and micro / nanoparticles loaded with insoluble components, or it can use micro / nanoparticles simultaneously loaded with both water-soluble and insoluble components, or micro / nanoparticles loaded only with water-soluble components, or micro / nanoparticles loaded only with insoluble components.

[0010] In this invention, the incubation conditions are those under which cells can survive, such as 4℃~60℃, preferably 37℃; the incubation time is 1~100 hours, for example 5~70 hours, preferably 10~50 hours.

[0011] In this invention, the micro / nano particles can be organic, inorganic, or biological materials, such as synthetic polymers, natural polymers, or inorganic materials. The micro / nano particles are micrometer-sized or nanometer-sized particles, wherein the particle size of the nanoparticles is 1 nm to 1000 nm, preferably 30 nm to 800 nm, and more preferably 50 nm to 600 nm; the particle size of the micrometer-sized particles is 1 μm to 1000 μm, preferably 1 μm to 100 μm, more preferably 1 μm to 10 μm, and most preferably 1 μm to 5 μm. The specific preparation method of the micro / nano particles is an existing technology, such as solvent evaporation, dialysis, extrusion, or thermal melting. The shape of the micro / nano particles is not limited, and can be spherical, ellipsoidal, barrel-shaped, polygonal, linear, worm-shaped, square, triangular, butterfly-shaped, or disk-shaped.

[0012] The present invention employs a solvent evaporation method, such as a double emulsion method, to load cell lysate components onto micro / nano particles. Other methods for loading cell lysate components onto micro / nano particles can also be used. Specifically, when the activator is a cell lysate component loaded onto micro / nano particles, the preparation method involves adding an aqueous solution to an organic phase solution of the micro / nano particle material, followed by ultrasonication, stirring, or homogenization, then adding a first emulsifier solution, followed by ultrasonication, stirring, or homogenization, and finally adding a second emulsifier solution. After stirring, micro / nano particles loaded with cell lysate components are obtained, serving as the activator. Examples include the following steps:

[0013] (1) Add the aqueous solution to the organic phase solution of the polymer material, and after ultrasonic treatment, stirring or homogenization, add it to the first emulsifier solution, and then after ultrasonic treatment, stirring or homogenization, add it to the second emulsifier solution, stir, centrifuge and resuspend the precipitate to obtain the residue, or after ultrafiltration to obtain the residue.

[0014] (2) Freeze-dry the residue from step (1) and then disperse it in a dispersion; or disperse the residue from step (1) in a dispersion, add an aqueous solution, mix and let stand to obtain micro-nano particles as activators.

[0015] After adding the above-mentioned material to the second emulsifier solution, followed by stirring and centrifugation or ultrafiltration, micro / nano particles containing internally loaded lysate components or lysate components / immune adjuvants can be obtained. Further, lysate components or lysate components / immune adjuvants can be loaded onto the surface of the aforementioned micro / nano particles containing internally loaded lysate components or lysate components / immune adjuvants.

[0016] The aqueous phase solution mentioned above is a solution of lysate components, or a solution of lysate components / immunoadjuvants; the ultrasound is probe ultrasound or any other ultrasound method; the stirring is mechanical stirring, magnetic stirring, etc.; and the homogenization treatment is high-pressure homogenization treatment or high-shear homogenization treatment, etc.

[0017] Preferably, when the aqueous phase solution is a lysate component solution, the concentration of protein and peptide is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL; when the aqueous phase solution is a lysate component / immunoadjuvant solution, the concentration of protein and peptide is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of immunoadjuvant is greater than 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. In the organic phase solution of the polymer material, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL. The first emulsifier solution is preferably an aqueous solution of polyvinyl alcohol with a concentration of 10 mg / mL to 50 mg / mL, preferably 20 mg / mL. The second emulsifier solution is preferably an aqueous solution of polyvinyl alcohol with a concentration of 1 mg / mL to 20 mg / mL, preferably 5 mg / mL. The dispersion solution is PBS buffer, physiological saline, or pure water.

[0018] Preferably, when stirring is mechanical or magnetic, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, for example, a stirring speed of 50 rpm to 1500 rpm and a stirring time of 0.5 hours to 5 hours; when ultrasonic treatment is performed, the ultrasonic power is 50W to 500W and the time is greater than 0.1 seconds, for example, 2 to 200 seconds; when homogenizing, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used, with a pressure greater than 20 psi when using a high-pressure / ultra-high-pressure homogenizer and a rotation speed greater than 1000 rpm when using a high-shear homogenizer. The nanoparticle size can be controlled by the duration of ultrasonic treatment, the stirring speed, or the pressure and time of homogenization; excessively large or small nanoparticles will lead to changes in particle size.

[0019] In this invention, the volume ratio of the aqueous phase solution to the organic phase solution of the polymer material is 1:(1.1-5000), preferably 1:(1.5-500); the volume ratio of the organic phase solution of the polymer material to the first emulsifier solution is 1:(1.1-1000), preferably 1:(1.5-500); the volume ratio of the first emulsifier solution to the second emulsifier solution is 1:(1.5-2000), preferably 1:(2-500); and the volume ratio of the dispersion to the aqueous phase solution is 1:10000-10000:1, preferably 1:100-100:1, and most preferably 1:30-30:1.

[0020] In this invention, cancer cells or tumor tissue are in a free state. The tumor includes hematologic malignancies and solid tumors, such as endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, respiratory system tumors, leukemia, skin cancer, breast cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, brain cancer, colon cancer, prostate cancer, rectal cancer, head and neck cancer, kidney cancer, bone cancer, nasal cancer, bladder cancer, thyroid cancer, esophageal cancer, cervical cancer, ovarian cancer, uterine cancer, pelvic cancer, testicular cancer, penile cancer, lymphoma, tongue cancer, gingival cancer, retinoblastoma, and sarcoma.

[0021] The enrichment solution used in this invention to dissolve insoluble pyrolysis product components includes aqueous solutions of urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerol, alkaline, acidic, protein-degrading enzymes, albumin, lecithin, inorganic salts, polyethylene glycol octylphenyl ether (Triton), dimethyl sulfoxide (DMSO), acetonitrile, ethanol, methanol, N,N-dimethylformamide (DMF), propanol, isopropanol, Tween, acetic acid, cholesterol, amino acids, glycosides, and choline. The insoluble pyrolysis product components can be dissolved in the enrichment solution or in organic solvents, such as DMSO, glycerol, acetonitrile, ethanol, methanol, DMF, isopropanol, dichloromethane, propanol, and ethyl acetate.

[0022] In this invention, after the activator activates T cells, the specific molecules secreted by the T cells are proteins, polypeptides, nucleic acids, sugars, or lipids. After expression, the specific molecules can be located in the cell membrane, cytoplasm, organelles, or cell nucleus. Tumor-specific T cells can be qualitatively or quantitatively identified by detection methods, including but not limited to flow cytometry, enzyme-linked immunospot (ELISA) technology, ELISA loading technology, colloidal gold immunochromatography, gene detection technology, and multi-cytokine detection technology.

[0023] The immune adjuvants described in this invention are immune enhancers or immune suppressors; the immune enhancers or immune suppressors are added in the following ways: loaded only within micro / nano particles, loaded only on the surface of micro / nano particles, loaded simultaneously within micro / nano particles, and loaded on the surface of micro / nano particles. Immune enhancers are used to enhance the detection of immune cells that can secrete or express pro-inflammatory cellular markers such as IFN-γ and IL-12; immune suppressors are used to enhance the detection of immune cells that can secrete or express anti-inflammatory cellular markers such as IL-10.

[0024] Functions and effects

[0025] This invention provides a method for activating cancer-specific T cells in peripheral tissues using free whole-cell components or whole-cell lysate components loaded with particles, and for detecting the content of activated cancer-specific T cells using conventional detection techniques. This can provide information support for the efficacy of cancer immunotherapy. Existing technologies using peptide antigens to stimulate and activate cancer-specific T cells result in inaccurate activation and subsequent detection of cancer-specific T cells, affecting the design of subsequent immunotherapy regimens and treatment outcomes. This invention uses free whole-cell components of cancer cells or tissues, or whole-cell components loaded into nano / micro particles, to activate cancer-specific T cells and detect the content of activated cancer-specific T cells, resulting in a broader and more accurate detection of cancer-specific T cell content. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0027] Figure 1 This is a schematic diagram of the preparation process of the activator of the present invention;

[0028] Figure 2 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0029] Figure 3 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0030] Figure 4 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0031] Figure 5 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0032] Figure 6 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0033] Figure 7 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0034] Figure 8 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0035] Figure 9 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0036] Figure 10 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0037] Figure 11 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0038] Figure 12 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0039] Figure 13 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0040] Figure 14 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0041] Figure 15 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0042] Figure 16 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0043] Figure 17 A schematic diagram of the structure of micro / nano particles carrying water-soluble and non-water-soluble cellular components;

[0044] Figure 18 The results of the experiment on melanoma in Example 1;

[0045] Figure 19 The experimental results for breast cancer in Example 2;

[0046] Figure 20 The results of the experiment for melanoma in Example 3;

[0047] Figure 21 The results are from the experiment on lung cancer in Example 4.

[0048] In the following experimental results, each data point in the tumor growth inhibition experiment graph is represented as mean ± standard error (mean ± SEM); other experimental data points are represented as mean ± standard deviation (mean ± SD). Significant differences in the tumor growth inhibition experiment were analyzed using ANOVA, while significant differences in other experiments were analyzed using... t Test analysis. * indicates that this group is compared with the control group. P <0.05 indicates a significant difference; ** indicates a significant difference between this group and the control group (P < 0.01); *** indicates a significant difference between this group and the control group. P <0.0001, indicating a significant difference. Detailed Implementation

[0049] This invention discloses a method for detecting the content of tumor-specific T cells in peripheral tissues to predict patient prognosis, which is helpful for disease diagnosis and treatment. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and products of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0050] This invention discloses a technique for detecting tumor-specific T cells and their content in peripheral tissues: activating agents are incubated with peripheral immune cells, and then the specific molecules of tumor-specific T cells are detected to achieve the detection of tumor-specific T cells; activating agents are incubated with peripheral immune cells, and then the specific molecules of tumor-specific T cells are detected, and the content of tumor-specific T cells is obtained according to the ratio of the number of tumor-specific T cells to the number of peripheral immune cells.

[0051] In this invention, the activator includes tumor cells, whole tumor tissue cells, tumor cell lysate components, and whole tumor tissue cell lysate components. The lysates in the tumor cell lysate components and whole tumor tissue cell lysate components can be water-soluble or water-insoluble lysate components, preferably water-soluble and water-insoluble lysate components.

[0052] In this invention, the activator can be free cells or free lysate components, or lysate components loaded onto micro / nanoparticles; preferably, it is a free cell lysate component or a lysate component loaded onto micro / nanoparticles; the lysate component is loaded inside and / or on the surface of the micro / nanoparticles. The lysate component is loaded inside and / or on the surface of the micro / nanoparticles in ways including, but not limited to, non-covalent adsorption, electrostatic interaction, hydrophobic interaction, hydrogen bonding, and covalent bonding. This invention can simultaneously use micro / nanoparticles loaded with water-soluble components and micro / nanoparticles loaded with insoluble components, or it can use micro / nanoparticles simultaneously loaded with water-soluble and insoluble components, micro / nanoparticles loaded only with water-soluble components, or micro / nanoparticles loaded only with insoluble components.

[0053] The present invention, when using whole cells or tumor cells for detection, can be divided into three steps: (1) collecting tumor cells or tumor tissue; (2) co-incubating the tumor cells or whole cells with a sample of peripheral tissue containing immune cells such as T cells for more than 10 minutes, for example, 16 hours; (3) detecting specific molecules that mark T cell activation using methods such as flow cytometry, ELISPOT, ELISA, and multi-cytokine detection. These specific molecules can be secreted outside the T cells, expressed on the surface of the T cells, or located inside the T cells. These specific molecules can be proteins, nucleic acids, carbohydrates, or lipids.

[0054] The present invention utilizes nano / micro particles loaded with cancer cell lysate or tumor tissue lysate components for detection, which can be divided into four steps: (1) collecting whole cells of tumor tissue or tumor cells; (2) preparing tumor cell lysate components or whole cell lysate components of tumor tissue, which can then be loaded onto micro / nano particles; (3) co-incubating the micro / nano particles or lysate components loaded with lysate components with a sample of peripheral tissue containing immune cells such as T cells for more than 10 minutes, for example, 16 hours; (4) detecting specific molecules that mark T cell activation using methods such as flow cytometry, ELISPOT, ELISA, and multi-cytokine detection. These specific molecules can be secreted outside T cells, expressed on the surface of T cells, or located inside T cells. These specific molecules can be proteins, nucleic acids, carbohydrates, or lipids.

[0055] This invention first obtains water-soluble lysates after whole-cell lysis of tumor cells or tumor tissue, and then obtains insoluble lysates and uses a specific method to solubilize the insoluble components. The free lysates can be directly used for detection or loaded onto micro / nano particles for further detection. Preferably, in practical applications, both water-soluble and insoluble components are collected simultaneously and loaded onto micro / nano particles, which improves detection accuracy.

[0056] This invention allows for the direct lysis and solubilization of whole tumor cells or tumor tissues using urea or guanidine hydrochloride solution to obtain both water-insoluble and water-soluble components that are simultaneously soluble in the solubilization solution. These components can then be used directly for detection in their free state or loaded onto micro / nano particles for further detection. Preferably, in practical applications, loading the lysate components onto micro / nano particles improves detection accuracy.

[0057] In this invention, tumor cells or whole tumor tissue cells can be used after inactivation or denaturation treatment before and after lysis, or they can be used directly without any inactivation or denaturation treatment before and after lysis. Inactivation or denaturation treatment methods can include ultraviolet irradiation, high-temperature heating, and in actual use, radiation irradiation, high pressure, freeze-drying, and formaldehyde inactivation or denaturation treatment methods can also be used. Those skilled in the art will understand that appropriate adjustments can be made according to specific circumstances during practical applications.

[0058] Furthermore, the activators include immune adjuvants, which are immunosuppressants or immune enhancers, and may be encapsulated only within microparticles, encapsulated within microparticles and simultaneously loaded on the surface of microparticles, or loaded only on the surface of microparticles.

[0059] A schematic diagram of the preparation process of the activator of this invention is shown below. Figure 1 The schematic diagram of the structure of the micro / nano particles loaded with whole cells is shown below. Figures 1 to 17 As shown; in actual use, it can be either using only one specific structure of micro-nano particles, or using two or more different structures of micro-nano particles at the same time. Figures 2-5 Both the surface and interior of the micro- and nano-particles contain immune adjuvants; Figures 6-9 In this context, the immune adjuvant is only distributed within the micro- and nano-particles; Figures 10-13 Micro- and nano-sized particles contain immune adjuvants only on their outer surface; Figures 14-17 Neither the interior nor the outer surface of the micro / nano particles contain immune adjuvants; Figure 2 , Figure 6 , Figure 10 and Figure 14 When the water-soluble or non-water-soluble components of the cells or tissues loaded by the micro- or nano-particles are distributed inside the micro- or nano-particles, they do not form a distinct core. Figure 3 , Figure 7 , Figure 11 and Figure 15 When the water-soluble or non-water-soluble components of the cells or tissues loaded by the micro- or nano-particles are distributed inside the micro- or nano-particles, they form a core. The core can be generated during the preparation process or formed by using polymers or inorganic salts. Figure 4 , Figure 8 , Figure 12 and Figure 16 When the water-soluble or non-water-soluble components of the cells or tissues loaded by the micro- or nano-particles are distributed inside the micro- or nano-particles, multiple core parts are formed. The core can be generated during the preparation process or formed by using polymers or inorganic salts. Figure 5 , Figure 9 , Figure 13 and Figure 17When water-soluble or water-insoluble components of cells or tissues encapsulated in micro / nano particles are distributed within the micro / nano particles, they are located on the outer layer of the formed core. In each figure, 1 represents water-soluble components of cells or tissues, 2 represents water-insoluble components of cells or tissues, 3 represents immune adjuvants, 4 represents micro / nano particles, and 5 represents the core portion of micro / nano particles; a indicates that the micro / nano particles are loaded with both water-soluble components of cells or tissues both internally and on their surface; b indicates that the micro / nano particles are loaded with both water-insoluble components of cells or tissues both internally and on their surface; c indicates that the micro / nano particles are loaded with water-insoluble components of cells or tissues internally and on their surface with water-soluble components; d indicates that the micro / nano particles are loaded with both water-soluble components of cells or tissues internally and on their surface with water-insoluble components; e indicates that the micro / nano particles are loaded with both water-soluble and water-insoluble components of cells or tissues internally. The surface of micro / nano particles can simultaneously carry water-soluble and insoluble components from cells or tissues. f represents the water-soluble and insoluble components from cells or tissues simultaneously carried inside the micro / nano particles, while the surface of the micro / nano particles only carries water-soluble components from cells or tissues. g represents the water-soluble and insoluble components from cells or tissues simultaneously carried inside the micro / nano particles, while the surface of the micro / nano particles only carries insoluble components from cells or tissues. h represents the insoluble components from cells or tissues simultaneously carried inside the micro / nano particles, while the surface of the micro / nano particles simultaneously carries water-soluble and insoluble components from cells or tissues. i represents the water-soluble components from cells or tissues simultaneously carried inside the micro / nano particles, while the surface of the micro / nano particles simultaneously carries water-soluble and insoluble components from cells or tissues.

[0060] In some embodiments, cell lysate components can be first encapsulated within micro / nanoparticles, along with an immune adjuvant; then, the cell lysate components can be loaded onto the surface of the micro / nanoparticles, with the immune adjuvant also loaded onto the micro / nanoparticle surface. In practical applications, tumor cells or whole tumor tissues can be directly lysed using a solubilizing solution (such as an 8M urea aqueous solution or a 6M guanidine hydrochloride aqueous solution) to directly dissolve the cell lysate components, which are then loaded onto micro / nanoparticles.

[0061] The method for loading cell lysate components onto micro / nanoparticles is solvent evaporation, but any other method that can load cell lysate components onto micro / nanoparticles can also be used. In some embodiments, the nanoparticles are prepared using the double emulsion method within the solvent evaporation process. The micro / nanoparticle preparation material used is a polymer, such as the organic polymer polylactic-co-glycolic acid copolymer (PLGA) with a molecular weight of 24 kDa-38 kDa. PLGA is a biodegradable material and has been approved by the FDA for use as a pharmaceutical dressing, making it suitable as a micro / nanoparticle preparation material. The immunoadjuvant used is poly(I:C) or CpG.

[0062] An aqueous solution is added to an organic phase solution of a polymer material. After ultrasonication, stirring, or homogenization, the mixture is added to a first emulsifier solution, followed by ultrasonication, stirring, or homogenization. Then, a second emulsifier solution is added, and after stirring, centrifugation, resuspending the precipitate, or ultrafiltration, micro / nano particles are obtained as activators. The resulting micro / nano particles are internally loaded with cell lysates, or cell lysates / immunoadjuvants.

[0063] Furthermore, cell lysates, or cell lysates / immune adjuvants, are loaded onto the surface of the aforementioned micro / nano particles internally. This specifically includes the following steps:

[0064] (1) Add the aqueous solution to the organic phase solution of the polymer material, and then add it to the first emulsifier solution after ultrasonication, stirring or homogenization. After ultrasonication, stirring or homogenization, add it to the second emulsifier solution, stir, and then perform ultrafiltration purification or centrifugation to resuspend the precipitate to obtain the residue.

[0065] (2) Freeze-dry the residue of step (1) and redisperse it in a dispersion solution or disperse the residue of step (1) in a dispersion solution; then add an aqueous solution, mix and let stand to obtain micro-nano particles as activators, which are loaded with cell lysate components.

[0066] The aqueous phase solution mentioned above is a cell lysate solution, or a cell lysate / immune adjuvant solution; the stirring is mechanical stirring, magnetic stirring, etc.; the homogenization treatment is high-pressure homogenization treatment, high-shear homogenization treatment.

[0067] Preferably, when the aqueous phase solution is a cell lysate solution, the protein / peptide concentration is greater than 1 ng / mL, preferably greater than 1 mg / mL; when the aqueous phase solution is a cell lysate / immunoadjuvant solution, the protein / peptide concentration is greater than 1 ng / mL, preferably greater than 1 mg / mL, and the immunoadjuvant concentration is greater than 0.01 ng / mL, preferably greater than 0.01 mg / mL. In the organic phase solution of the polymer material, the solvent is DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, chloroform, dichloromethane, propanol, ethyl acetate, preferably dichloromethane, and the concentration of the polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL. The first emulsifier solution is preferably an aqueous solution of polyvinyl alcohol with a concentration of 10 mg / mL to 50 mg / mL, preferably 20 mg / mL. The second emulsifier solution is preferably an aqueous solution of polyvinyl alcohol with a concentration of 3 mg / mL to 9 mg / mL, preferably 5 mg / mL. The dispersion is PBS buffer, physiological saline, or pure water.

[0068] Preferably, when stirring is performed mechanically or magnetically, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, for example, the stirring speed is 1000 rpm to 12000 rpm and the stirring time is 0.5 h to 5 h; when ultrasonic treatment is performed, the time is greater than 0.1 seconds, for example, 2 seconds to 60 minutes; when homogenizing, the pressure of the high-pressure homogenizer is greater than 100 psi and the speed of the high-shear homogenizer is greater than 1000 rpm. During nano-sizing, the duration of ultrasonic treatment, stirring speed, homogenization pressure, shear rate, and time can control the size of the prepared nanoparticles; excessively large or small particles will lead to changes in particle size.

[0069] In this invention, the volume ratio of the aqueous phase solution to the organic phase solution of the polymer material is 1:(1.1-5000), preferably 1:10; the volume ratio of the organic phase solution of the polymer material to the first emulsifier solution is 1:(1.1-1000), preferably 1:2.5; the volume ratio of the first emulsifier solution to the second emulsifier solution is 1:(1.5-2000), preferably 1:10; the volume ratio of the dispersion to the aqueous phase solution is 1:10000-10000:1, preferably 1:100-100:1, and most preferably 1:30-30:1; for example, 10:1.

[0070] Preferably, the residue of step (1) is freeze-dried by resuspending the residue of step (1) in an aqueous solution of a freeze-drying protectant and then freeze-drying it; the freeze-drying protectant is preferably trehalose or sucrose, with a concentration of 2-8 wt%, preferably 3-6 wt%.

[0071] The particle size of the micro-nano particles is in the nanometer or micrometer range, which can ensure that the particles are phagocytosed by antigen-presenting cells and improve phagocytosis efficiency; the particle size of the nanoparticles is 1nm-1000nm, more preferably 30nm-1000nm, and most preferably 50nm-600nm; the particle size of the microparticles is 1μm-1000μm, more preferably 1μm-100μm, more preferably 1μm-10μm, and most preferably 1μm-5μm.

[0072] In this invention, urea or guanidine hydrochloride aqueous solution is used as the lysis solution. In actual use, any other substance aqueous solution can also be used as the lysis solution, such as sodium deoxycholate, SDS, alkaline solution with pH greater than 7, acidic solution with pH less than 7, albumin, lecithin, high concentration of inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, acetic acid, cholesterol, amino acids, glycosides, and choline.

[0073] In this invention, nanoparticles are prepared using a double emulsion method, but any other commonly used micro / nanoparticle preparation method can also be used in practice. The material used for preparing micro / nanoparticles is PLGA, but any other material capable of preparing micro / nanoparticles can also be used. In some embodiments of this invention, nanoparticles are used, while in others, micron-sized particles are used. Those skilled in the art can choose to use micro / nanoparticles based on the actual situation. In some embodiments of this invention, flow cytometry is used for detection, while in others, enzyme-linked immunospot assay (ELISPOT) or enzyme-linked immunosorbent assay (ELISA) is used. In practice, other methods such as multi-cytokine detection can also be used depending on the actual situation. In some embodiments of this invention, the specific molecule for tumor-specific T cells is interferon-γ (IFN-γ). In practical applications, any other specific molecule can be used, including secretory and membrane-bound molecules such as proteins, nucleic acids, carbohydrates, and lipids. The specific cytokines detected in this embodiment are pro-inflammatory, but in practical applications, anti-inflammatory cytokines such as IL-10 and TGF-β can also be used.

[0074] In this invention, poly(I:C) and CpG are used as immune adjuvants. In practice, immune adjuvants may not be added, or any other immune adjuvants with immune-enhancing / suppressing functions may be added, such as pattern recognition receptor agonists, BCG cell wall skeletons, BCG methanol extract residues, BCG muramyl dipeptide, Mycobacterium tumefaciens, polyantigen A, mineral oil, virus-like particles, immune-enhancing regenerated influenza virus bodies, cholera enterotoxin, saponins and their derivatives, BCG, Resiquimod, thymosin, newborn bovine liver bioactive peptides, miquimod, polysaccharides, curcumin, and immune adjuvant poly(I:C). ICLC, Corynebacterium brevicornuate vaccine, hemolytic streptococcal preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylated acid, alum, aluminum phosphate, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM-CSF, MF59, double-stranded RNA, double-stranded DNA, aluminum hydroxide, CAF01, ginseng, astragalus, and other effective components of traditional Chinese medicine. Regarding immune adjuvants, they may or may not be added in this invention. When added, the immune adjuvant is at least one of the following: microbial-derived immune adjuvants, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, and traditional Chinese medicine.

[0075] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0076] Unless otherwise specified, the specific methods used in the embodiments of this invention are all conventional methods; the materials and reagents used are all commercially available. The micro / nano particle structures, preparation methods, co-incubation methods with T cells in peripheral tissues, and strategies for detecting activated T cells involved in the embodiments of this invention are only representative methods. Other micro / nano particle structures, preparation methods, co-incubation methods with T cells in peripheral tissues, and strategies for detecting activated T cells can also be described using the methods described in this invention. The embodiments only list the application of this invention in some cancers, but this invention can also be used in other cancers. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on the technical concept of this invention and existing technologies, and are not limited to the specific descriptions in the embodiments of this invention.

[0077] Example 1: Detection of cancer-specific T cells in peripheral tissues of melanoma-bearing mice

[0078] This embodiment uses mouse melanoma as a cancer model to illustrate the detection of tumor-specific T cells in peripheral tissues and the determination of their content using free whole-cell lysates of cancer cells. Because mice have limited peripheral blood and a limited number of peripheral immune cells, while the spleen has abundant blood flow and contains sufficient peripheral immune cells, this embodiment uses peripheral immune cells from the mouse spleen for relevant detection. Immune cells in the spleen are peripheral immune cells, as are immune cells in peripheral blood. In clinical applications, peripheral immune cells from human peripheral blood can be used for detection.

[0079] In this embodiment, B16-F10 mouse melanoma cells were used as a tumor cell model. First, B16-F10 cells were lysed to prepare B16-F10 cell lysate components; then, the free tumor cell lysate components were co-incubated overnight with peripheral immune cells; finally, flow cytometry was used to analyze the specific molecule gamma interferon of tumor-specific T cells. Details are as follows:

[0080] (1) Lysis of tumor cells and collection of their components

[0081] B16-F10 cells were collected, the culture medium was removed, and the cells were frozen at -80°C. After adding ultrapure water, the cells were repeatedly frozen and thawed three times, and simultaneously subjected to sonication at 150W to destroy and lyse the cells. After cell lysis, the lysate was centrifuged at 12000 RPM for 5 minutes, and the supernatant was collected as the water-soluble lysate component of B16-F10. The precipitate was dissolved in 8M urea aqueous solution to obtain the water-insoluble lysate component of B16-F10.

[0082] (2) Free tumor cell lysate components co-incubated with peripheral immune cells

[0083] Female C57BL / 6 mice aged 6-8 weeks were selected to prepare melanoma-bearing mice. On day 0, each mouse was subcutaneously inoculated with 150,000 B16-F10 cells on the lower right back. On days 4, 7, 10, 15, and 20, mice were subcutaneously injected with either a nano-cancer vaccine loaded with whole-cell components of existing cancer cells or PBS. Tumor volume was recorded every three days starting from day 6. Tumor volume was calculated using the formula v = 0.52 * a * b. 2 The tumor volume was calculated, where v is the tumor length and b is the tumor width. C57BL / 6 mice in the PBS group and the vaccine-treated group were sacrificed on days 18 and 24, respectively, and peripheral immune cells from their spleens were collected for parallel experiments.

[0084] Cells were resuspended in DMEM medium containing 10% FBS at a concentration of 4 × 10⁻⁶. 610 cells / mL; then add 10% volume (based on culture medium) of water-soluble lysate (40 mg / mL) and 1% volume (based on culture medium) of non-water-soluble lysate (30 mg / mL); incubate at 37ºC with 5% CO2 for 20 hours; then collect the incubated mouse spleen cells after centrifugation at 400g.

[0085] (3) Flow cytometry was used to detect activated cancer-specific T cells.

[0086] First, the collected mouse spleen cells were treated with Fc blocks to avoid nonspecific cell loading. Then, extracellular staining of mouse spleen cells was performed using CD3, CD4, and CD8 antibodies. After cell fixation and permeabilization, intracellular staining of mouse spleen cells was performed using FN-γ antibody. Subsequently, mouse spleen cells were analyzed using the FACS AriaTMIII system, and the results were analyzed using FlowJo 10 software. CD4... + T cells that are activated and can secrete IFN-γ are present in all CD4 cells. + The proportion of T cells and CD8 + T cells that are activated and can secrete IFN-γ are present in all CD8 cells. + The proportion of T cells.

[0087] (4) Experimental results

[0088] Figure 18 The above experimental results are as follows: a) represents the inhibitory effect of cancer vaccine treatment on tumor growth rate (n≥8); b) represents the flow cytometry analysis of cancer-specific CD8+ activated in peripheral spleen immune cells after incubation with tumor tissue lysates. + T cells account for a significant portion of CD8+ cells in the spleen. + The cell ratio, c represents the cancer-specific CD4+ activated after incubation of peripheral spleen immune cells with tumor tissue lysates by flow cytometry. + T cells account for a significant portion of CD4+ immune cells in the spleen. + Cell ratio. For example... Figure 18 As shown, compared with the PBS blank control group, the vaccine-treated mice showed significantly more activated T cells after co-incubation with tumor cell lysates, indicating that the peripheral tissues of mice treated with cancer vaccines had a significantly increased content of cancer-specific T cells. Therefore, the free whole cells described in this invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients.

[0089] Example 2: Detection of cancer-specific T cells in peripheral tissues of breast cancer-bearing mice

[0090] This embodiment uses mouse breast cancer as a cancer model to illustrate the detection of tumor-specific T cells in peripheral tissue and the determination of their content using free tumor tissue whole-cell lysates. Because mice have limited peripheral blood and a limited number of peripheral immune cells, while the spleen has abundant blood flow and contains sufficient peripheral immune cells, this embodiment uses mouse spleen peripheral immune cells for relevant detection. In clinical applications, peripheral immune cells from human peripheral blood can be used for detection.

[0091] In this embodiment, 4T1 mouse mammary tumor cells were used as the tumor cell model. First, whole-cell tumor tissue was lysed to prepare water-soluble and insoluble components. Then, the free whole-cell tumor lysate components were co-incubated overnight with peripheral immune cells. Finally, flow cytometry was used to analyze the specific molecule gamma interferon of tumor-specific T cells.

[0092] (1) Lysis of tumor tissue and collection of its components

[0093] 400,000 4T1 mammary tumor cells were subcutaneously injected into the back of each BALB / c mouse. The tumors in each mouse grew to a volume of 200 mm². 3 -1500 mm 3 Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and filtered through a cell filter with pure water, followed by five freeze-thaw cycles. After cell lysis in the tumor tissue, the cell lysate was centrifuged at 3000g for 20 minutes, and the supernatant was collected as the water-soluble component of the whole tumor cells. The insoluble component was obtained by dissolving the precipitate in an 8M urea solution.

[0094] (2) Co-incubation of free whole-cell components with peripheral immune cells

[0095] Female BALB / c mice aged 6-8 weeks were selected to prepare breast tumor-bearing mice. On day 0, each mouse was subcutaneously inoculated with 400,000 B16-F10 cells on the lower right back. On days 4, 7, 10, 15, and 20, mice were subcutaneously injected with either a nano-cancer vaccine loaded with whole-cell components of cancer cells or PBS. Tumor volume was recorded every three days starting from day 6. Tumor volume was calculated using the formula v = 0.52 * a * b. 2 The tumor volume was calculated, where v is the tumor volume, a is the tumor length, and b is the tumor width. On day 24, mice in the vaccine-treated group and the PBS-treated group were sacrificed, and immune cells from their spleens were collected.

[0096] Cells were resuspended in RPMI 1640 medium containing 10% FBS at a concentration of 4 × 10⁻⁶. 6Mouse spleen cells were collected at 1000 g / mL. 5% (by volume) of the water-soluble component (35 mg / mL) and 1% (by volume) of the non-water-soluble component dissolved in 8 M urea (35 mg / mL) were added. The mixture was incubated at 37ºC with 5% CO2 for 18 hours. The incubated mouse spleen cells were then collected after centrifugation at 400 g.

[0097] (3) Flow cytometry was used to detect activated cancer-specific T cells.

[0098] Mouse spleen cells were first treated with Fc blocks to avoid nonspecific cell loading. Then, extracellular staining of mouse spleen cells was performed using CD3, CD4, and CD8 antibodies. After cell fixation and permeabilization, intracellular staining of mouse spleen cells was performed using FN-γ antibody. Subsequently, mouse spleen cells were analyzed using the FACS AriaTMIII system, and the results were analyzed using FlowJo 10 software. CD4... + T cells that are activated and can secrete IFN-γ are present in all CD4 cells. + The proportion of T cells and CD8 + T cells that are activated and can secrete IFN-γ are present in all CD8 cells. + The proportion of T cells.

[0099] (4) Experimental results

[0100] Figure 19 The above are experimental results for breast cancer. a) shows the inhibitory effect of cancer vaccine treatment on tumor growth rate (n≥9); b) shows the activation of cancer-specific CD8+ in peripheral immune cells of the spleen after incubation with tumor tissue lysates. + T cells account for a significant portion of CD8+ immune cells in the spleen. + The ratio of cells; c, cancer-specific CD4 activated in peripheral spleen immune cells after incubation with tumor tissue lysates. + T cells account for a significant portion of CD4+ immune cells in the spleen. + Cell ratio.

[0101] like Figure 19 As shown, compared with the PBS blank control group, the number of activated T cells in the peripheral immune cells of the vaccine-treated mice was significantly higher after co-incubation with whole-cell lysates of tumor tissue. This indicates that the content of cancer-specific T cells in the peripheral tissues of mice treated with cancer vaccines is significantly increased. Therefore, the free whole cells described in this invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients.

[0102] Example 3: Melanoma tumor tissue lysate loaded onto nanoparticles to activate cancer-specific T cells in peripheral tissues.

[0103] This embodiment uses a mouse melanoma model to illustrate how to prepare nanoparticles loaded with whole-cell components of melanoma tumor tissue and how to use these nanoparticles to activate cancer-specific T cells in peripheral tissues. After activation, the content of activated cancer-specific T cells was detected using enzyme-linked immunosorbent assay (ELISA).

[0104] In this embodiment, enzyme-linked immunosorbent assay (ELISA) was used to detect IFN-γ secreted by activated T cells. In practical applications, other methods such as enzyme-linked immunospot assay and flow cytometry can also be used to detect other substances secreted by activated T cells or other substances expressed on the cell membrane surface.

[0105] In this embodiment, mouse B16-F10 melanoma cells were seeded into C57BL / 6 mice. Tumor tissue was then harvested, and the water-soluble components from the tumor tissue lysate and the previously insoluble components dissolved in 8M urea were simultaneously loaded onto the interior and surface of nanoparticles. Then, using PLGA as the nanoparticle framework material, nanoparticles loaded with both the water-soluble and insoluble components of the tumor tissue lysate were prepared using a solvent evaporation method. These nanoparticles were then used to activate cancer-specific T cells in mouse peripheral tissues.

[0106] (1) Lysis of tumor tissue and collection of its components

[0107] 150,000 B16-F10 melanoma cells were subcutaneously injected into the back of each C57BL / 6 mouse. Tumors in each mouse grew to a volume of 200 mm². 3 -1500 mm 3 Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and filtered through a cell filter. Pure water was added, and the mixture was subjected to a freeze-thaw cycle five times, with each thaw cycle involving sonication at 150W for two minutes. After cell lysis in the tumor tissue, the cell lysate was centrifuged at a speed greater than 100 g for 5 minutes. The supernatant was collected as the water-soluble component of the tumor tissue. An 8M urea aqueous solution was added to the resulting precipitate to dissolve the precipitate, thus converting the previously insoluble components into those soluble in the 8M urea aqueous solution. The water-soluble components of the tumor tissue lysate and the previously insoluble components dissolved in 8M urea serve as the raw material for preparing the nanoparticles.

[0108] (2) Preparation of nanoparticles loaded with whole-cell components

[0109] In this embodiment, the nanoparticles loaded with cell components and the blank control nanoparticles were prepared using the double emulsion method in the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 24 kDa-38 kDa, and the preparation method was as described above. In addition, nanoparticles simultaneously loaded with four melanoma polypeptide antigens were also prepared in this embodiment. The four polypeptide antigens used were Melan-A: 26-35 (L27: GILTV), Melan-A: 51-73 (RR23: RNGYRALMDKSLHVGTQCALTRR); gp100: 25-33 (EGSRNQDWL) and gp100: 44-59 (WNRQLYPEWTEAQRLD). The concentration of each polypeptide was 5 mg / mL during nanoparticle preparation. Details are as follows:

[0110] 200 μL of the above-mentioned water-soluble component solution (30 mg / mL) and 200 μL of the non-water-soluble component solution (30 mg / mL) were added to 1 mL of PLGA (100 mg) dichloromethane solution and sonicated for 30 s. Then, the mixture was added to 2.5 mL of polyvinyl alcohol aqueous solution (20 mg / mL) and sonicated for 30 s. Then, the mixture was added to 50 mL of polyvinyl alcohol aqueous solution (5 mg / mL) and stirred until the organic solvent (dichloromethane) evaporated completely. The mixture was then centrifuged at 12000 rpm for 10 min. The supernatant was collected and the precipitate was resuspended in 20 mL of trehalose aqueous solution (4 wt%). After being freeze-dried at -80 °C, the precipitate was resuspended in 10 mL of physiological saline and then mixed with 0.5 mL of the above-mentioned water-soluble component solution (30 mg / mL) and 0.5 mL of the non-water-soluble component solution (30 mg / mL). The mixture was allowed to stand for 60 s to obtain the activator of the cleavage components loaded inside and on the surface of the nanoparticles.

[0111] The preparation of empty nanoparticles and nanoparticles loaded with four polypeptide antigens is the same as above, except that the lysis components are not added or are replaced.

[0112] Before loading the lysate component onto the nanoparticle surface, the average nanoparticle size was approximately 280 nm. After loading the lysate component, the nanoparticle size was approximately 300 nm. Each 1 mg of PLGA nanoparticles was loaded with 150 μg of lysate component. The blank nanoparticle size was approximately 250 nm. The nanoparticles loaded with peptide antigen had a size of approximately 290 nm, and the total peptide loading per 1 mg of PLGA nanoparticles was approximately 50 μg of peptide antigen.

[0113] (3) Nanoparticles activate cancer-specific T cells

[0114] Female C57BL / 6 mice aged 6-8 weeks were selected to prepare melanoma-bearing mice. On day 0, each mouse was subcutaneously inoculated with 150,000 B16-F10 melanoma cells on the lower right back. On days 4, 7, 10, 15, and 20, mice were subcutaneously injected with either a nano-cancer vaccine loaded with whole-cell components of cancer cells or PBS. Tumor volume was recorded every three days starting from day 6. Tumor volume was calculated using the formula v = 0.52 * a * b. 2 The tumor volume was calculated, where v is the tumor length and b is the tumor width. Mice in the PBS group and the vaccine-treated group were sacrificed on days 18 and 24, respectively, and peripheral immune cells were collected from their spleens.

[0115] Cells were resuspended in RPMI 1640 medium containing 10% FBS at a concentration of 5 × 10⁻⁶. 6 Cells / mL. Nanoparticles loaded with water-soluble and insoluble components were added to the sample to a final concentration of 300 μg / mL; or nanoparticles loaded with peptide antigens were added to a final concentration of 300 μg / mL; or an equal volume of blank nanoparticles were added; or an equal volume of free whole-cell lysate was added; or an equal volume of free peptide antigen was added. The samples were then incubated at 37ºC (5% CO2) for 72 hours. Afterwards, the samples were centrifuged at 400g for 5 minutes, and the supernatant was collected. The concentration of IFN-γ in the supernatant was analyzed using ELISA.

[0116] In ELISA assays, activated tumor-specific T cells secrete specific cellular secretions such as IFN-γ. The concentration of these secretions indicates the number of activated cancer-specific T cells.

[0117] (4) Experimental results

[0118] Figure 20 The results of the above-mentioned melanoma experiments are as follows: a) Experiment on the inhibitory effect of cancer vaccine treatment on tumor growth rate (n≥8); b) Analysis of the content of activated cancer T cells in peripheral immune cells of the spleen after incubation with free tumor tissue lysates or nanoparticles loaded with tumor tissue lysates using ELISA.

[0119] like Figure 20As shown, compared with the PBS blank control group and the blank nanoparticle treatment group in the vaccine treatment group, the peripheral immune cells of mice in the vaccine treatment group showed significantly more activated T cells after co-incubation with free tumor tissue whole cell components, or nanoparticles loaded with tumor tissue whole cell components, or free polypeptide antigens, or nanoparticles loaded with polypeptide antigens. This indicates that the content of cancer-specific T cells in the peripheral tissues of mice treated with cancer vaccines is significantly increased. Therefore, the free tumor tissue whole cell components described in this invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients. When using free tumor tissue whole cell components and free polypeptide antigens to stimulate and activate T cells, the free whole cell components can stimulate and activate more T cells; when using nanoparticles loaded with whole cell lysate components and nanoparticles loaded with polypeptide antigens to stimulate and activate T cells, the nanoparticles loaded with whole cell lysate components can stimulate and activate more T cells; moreover, the nanoparticles loaded with whole cell lysate components stimulate and activate more T cells than the nanoparticles loaded with free whole cell lysate components.

[0120] Example 4: Loading lung cancer tumor tissue lysate and immune adjuvants onto microparticles to activate cancer-specific T cells in peripheral tissues.

[0121] This embodiment uses mouse lung cancer to illustrate the preparation of microparticles loaded with lung cancer tumor tissue lysate components and an immune adjuvant, as well as microparticles loaded only with lung cancer tumor tissue lysate components, to activate tumor-specific T cells in peripheral tissues. The content of tumor-specific T cells was detected using enzyme-linked immunospot assay (ELISPOT). This embodiment also tested the effects of CpG or poly I:C as immune adjuvants and without the addition of immune adjuvants.

[0122] This embodiment uses enzyme-linked immunospot assay (ELISPOT) to detect the specific molecule IFN-γ of activated tumor-specific T cells. In practical applications, other methods such as flow cytometry and ELISA can also be used to detect the specific molecules of other tumor-specific T cells.

[0123] In this embodiment, mouse LLC lung tumor cells were seeded into C57BL / 6 mice. Tumor tissue was then harvested, and the water-soluble components of the tumor tissue lysate and the insoluble components dissolved in 6M guanidine hydrochloride were obtained. Microparticles loaded with either the water-soluble or insoluble components of the tumor tissue lysate were prepared using PLGA as the microparticle framework material via a solvent evaporation method. The water-soluble and insoluble components of the tumor tissue lysate were simultaneously loaded inside and on the surface of the microparticles, respectively. In microparticles containing an immunoadjuvant, the immunoadjuvant was only encapsulated inside the microparticles. These microparticles were then used to detect tumor-specific T cells in mouse peripheral tissues.

[0124] (1) Lysis of tumor tissue and collection of its components

[0125] 2,000,000 LLC lung tumor cells were subcutaneously injected into the lateral dorsal region of each C57BL / 6 mouse. The injected tumors in each mouse grew to a volume of 200 mm². 3 -1500 mm 3 Mice were euthanized and tumor tissue was harvested. Tumor tissue pieces of the same size were cut into small, ground, and filtered through a cell filter. Pure water was added, and the whole tumor cells were subjected to routine UV irradiation and heating for inactivation and denaturation. The mixture was then subjected to five freeze-thaw cycles, with sonication at 250W for one minute each time during thawing. After the whole tumor cells were lysed, the cell lysate was centrifuged at 5000 rpm for 15 minutes. The supernatant was collected as the water-soluble component of the whole tumor cells. The insoluble component was obtained by dissolving the precipitate in 6M guanidine hydrochloride aqueous solution.

[0126] (2) Preparation of micron-sized particles loaded with whole-cell components

[0127] In this embodiment, the micron-sized particles loaded with cell lysates were prepared using the double emulsion method in the solvent evaporation process. The PLGA material used for preparing the micron-sized particles had a molecular weight of 24 kDa-38 kDa, and the preparation method was as described above, specifically as follows:

[0128] Add 150 μL of the above water-soluble component solution (60 mg / mL) or 200 μL of the non-water-soluble component solution (10 mg / mL) to 2 mL of PLGA (50 mg) dichloromethane solution, stir for 150 s, then mix with 10 mL of polyvinyl alcohol aqueous solution (15 mg / mL), sonicate for 50 s, then mix with 300 mL of polyvinyl alcohol aqueous solution (8 mg / mL), stir until the organic solvent (dichloromethane) has evaporated completely; then centrifuge at 10000 rpm for 30 min, take out the supernatant, resuspend the precipitate in 20 mL of sucrose aqueous solution (5 wt%), freeze dry at -80℃, resuspend in 5 mL of physiological saline, then mix with 3 mL of the above water-soluble component solution (10 mg / mL) and 0.5 mL of the non-water-soluble component solution (40 mg / mL), let stand for 20 min to obtain the activator of the cleavage component loaded inside and on the surface of the nanoparticles.

[0129] Mix 150 μL of the above-mentioned water-soluble component solution (60 mg / mL) or 200 μL of the non-water-soluble component solution (10 mg / mL) with 100 μL of immunoadjuvant (CpG or poly I:C) solution (0.25 mg / mL), then add 2 mL of PLGA (50 mg) dichloromethane solution and stir for 150 s. Next, mix with 10 mL of polyvinyl alcohol aqueous solution (15 mg / mL) and sonicate for 50 s. Then, mix with 300 mL of polyvinyl alcohol aqueous solution (8 mg / mL) and stir until the organic solvent (dichloromethane) has completely evaporated. Centrifuge at 10,000 rpm for 30 min, collect the supernatant, resuspend the precipitate in 20 mL of sucrose aqueous solution (5 wt%), freeze-dry at -80℃, and resuspend in 5 mL of physiological saline. Then, mix with 2 mL of the above-mentioned water-soluble component solution (10 mg / mL) and 0.5 mL of the non-water-soluble component solution (40 mg / mL), and let stand for 20 minutes. min, activators of cleavage components loaded inside and on the surface of nanoparticles were obtained.

[0130] Before loading cell lysate components onto the surface of micron particles, the average particle size of the micron particles was about 2.0 μm. After loading cell lysate components onto the surface of the micron particles, the particle size of the micron particles was about 2.1 μm. Each 1 mg PLGA micron particles was loaded with 160 μg of cell lysate components.

[0131] (3) Microparticles activate cancer-specific T cells

[0132] Female C57BL / 6 mice aged 6-8 weeks were selected to prepare melanoma-bearing mice. On day 0, each mouse was subcutaneously inoculated with 2,000,000 LLC lung tumor cells on the lower right back. On days 4, 7, 10, 15, and 20, mice were subcutaneously injected with either a nano-cancer vaccine loaded with whole-cell components of existing cancer cells or PBS. Tumor volume was recorded every three days starting from day 6. Tumor volume was calculated using the formula v = 0.52 * a * b. 2 The tumor volume was calculated, where v is the tumor volume, a is the tumor length, and b is the tumor width. On day 24, mice in the vaccine-treated group and the PBS-treated group were sacrificed, and immune cells from their spleens were collected.

[0133] Cells were resuspended in RPMI 1640 medium containing 10% FBS at a concentration of 5 × 10⁻⁶. 6Cells / mL. 100 μL of the above-mentioned spleen cells were added to a 96-well plate pre-coated with IFN-γ antibody a (capture antibody) and blocked with culture medium for at least 1 hour. 25 μg of microparticles loaded with water-soluble components and 25 μg of microparticles loaded with insoluble components were added, and the plate was incubated at 37ºC with 5% CO2 for 72 hours. Afterward, the mixture of cells and microparticles was discarded, the 96-well plate was washed, and IFN-γ antibody b (detection antibody) was added and incubated at 37ºC (5% CO2) for at least 2 hours. The solution containing IFN-γ antibody b was discarded, the 96-well plate was washed, and colorimetric methods were used to form spots on the surface of the 96-well plate. Data were read and experimental results were analyzed using an ELISPOT analyzer.

[0134] In the ELISPOT assay, activated tumor-specific T cells secrete cytokines such as IFN-γ, which bind to antibody a loaded onto a 96-well plate. The addition of antibody b creates a double-antibody sandwich structure, and the detection antibody is attached to an enzyme that aids in color development. After substrate addition and color development, a spot forms at the location of each activated cell. Each spot represents an activated tumor-specific T cell; therefore, by measuring the number of spots formed in each well of the 96-well plate, the number of tumor-specific T cells in the sample can be determined.

[0135] (4) Experimental results

[0136] Figure 21 The results of the above-mentioned lung cancer experiments are as follows: a) Experiment on the inhibitory effect of cancer vaccine treatment on tumor growth rate (n≥9); b) Analysis of the content of activated cancer T cells in peripheral immune cells of the spleen after incubation with micron-sized particles loaded with tumor tissue lysates using the ELISPOT method.

[0137] like Figure 21As shown, compared with the PBS blank control group, the number of activated T cells in the peripheral immune cells of the vaccine-treated mice was significantly higher after co-incubation with microparticles loaded with whole tumor cell components or microparticles loaded with whole tumor cell components and an immune adjuvant. This indicates that the content of cancer-specific T cells in the peripheral tissues of mice treated with cancer vaccines is significantly increased. Moreover, regardless of whether CpG or Poly(I:C) is used as an immune adjuvant, microparticles loaded with whole tumor cell components and an immune adjuvant can activate more T cells after co-incubation with peripheral immune cells than microparticles loaded with whole tumor cell components. These results indicate that the addition of immune adjuvants can activate more cancer antigen-specific T cells. Therefore, the free whole cells described in this invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients.

[0138] The content of cancer-specific T cells in peripheral tissues such as peripheral blood of cancer patients is positively correlated with patient prognosis. This invention collects whole cells from tumor cells or tumor tissue, then co-incubates these cells with peripheral immune cells using either free whole cells or cell lysates loaded onto nano / micro particles. Once the cancer-specific T cells are activated, the content of cancer-specific T cells in peripheral tissues such as peripheral blood can be determined by detecting the specific molecules associated with these cells. The inventiveness of this invention lies in using cancer cells, whole cells of tumor tissue, cancer cell lysate components, or whole-cell lysate components of tumor tissue as activators to detect tumor-specific T cells in peripheral immune cells. The particle loading, cell incubation, and specific secretion detection methods involved are all existing technologies in this field.

Claims

1. The use of an activator in the preparation of a reagent for detecting tumor-specific T cells or their content in peripheral immune cells, said activator comprising tumor cell lysate components and / or tumor tissue whole-cell lysate components, characterized in that, The pyrolysis component is a pyrolysis component loaded on micro / nano particles; the pyrolysis component is a water-soluble pyrolysis component and an insoluble pyrolysis component, and the insoluble pyrolysis component is dissolved by a solubilizing solution, wherein the solubilizing solution is an aqueous solution of urea or an aqueous solution of guanidine hydrochloride; the micro / nano particles are micron particles or nanoparticles.

2. The application according to claim 1, characterized in that, The pyrolysis components are loaded inside and / or on the surface of micro / nano particles; the micro / nano particle preparation material is an organic material, an inorganic material, or a biological material.

3. The application according to claim 1, characterized in that, The activators also include immune adjuvants.

4. The application according to claim 3, characterized in that, The immune adjuvant is poly(I:C) or CpG.

5. The application according to claim 1, characterized in that, The reagent for detecting tumor-specific T cells or their content in peripheral immune cells includes the following steps: incubating an activator with peripheral immune cells, then detecting specific molecules of tumor-specific T cells; and finally obtaining the tumor-specific T cell content based on the ratio of the number of tumor-specific T cells to the number of peripheral immune cells.

6. The application according to claim 5, characterized in that, The incubation conditions are those under which the cells can survive; the tumors include hematologic malignancies and solid tumors; the specific molecules are proteins, polypeptides, nucleic acids, sugars, or lipids.

7. A method for detecting the content of tumor-specific T cells for non-disease diagnostic purposes, comprising the following steps: incubating an activator with peripheral immune cells, then detecting specific molecules of tumor-specific T cells; and then obtaining the content of tumor-specific T cells based on the ratio of the number of tumor-specific T cells to the number of peripheral immune cells; wherein the activator comprises tumor cell lysate components and / or tumor tissue whole-cell lysate components, characterized in that... The pyrolysis component is a pyrolysis component loaded on micro / nano particles; the pyrolysis component is a water-soluble pyrolysis component and an insoluble pyrolysis component, and the insoluble pyrolysis component is dissolved by a solubilizing solution, wherein the solubilizing solution is an aqueous solution of urea or an aqueous solution of guanidine hydrochloride; the micro / nano particles are micron particles or nanoparticles.

8. The detection method according to claim 7, characterized in that, The pyrolysis components are loaded inside and / or on the surface of micro / nanoparticles; the micro / nanoparticles are prepared from organic, inorganic, or biological materials; and the micro / nanoparticles are micron-sized or nano-sized particles.

9. The detection method according to claim 7, characterized in that, The incubation conditions are those under which the cells can survive; the tumors include hematologic malignancies and solid tumors; the specific molecules are proteins, polypeptides, nucleic acids, sugars, or lipids.

10. The use of an activator in detecting tumor-specific T cells or their content in peripheral immune cells for non-disease diagnostic purposes; said activator comprising tumor cell lysate components and / or tumor tissue whole-cell lysate components, characterized in that, The pyrolysis component is a pyrolysis component loaded on micro / nano particles; the pyrolysis component is a water-soluble pyrolysis component and an insoluble pyrolysis component, and the insoluble pyrolysis component is dissolved by a solubilizing solution, wherein the solubilizing solution is an aqueous solution of urea or an aqueous solution of guanidine hydrochloride; the micro / nano particles are micron particles or nanoparticles.

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