Method for detecting tumor-specific T cells

Abstract

The objective of the present invention is to provide a method for detecting tumor-specific T cells. [Solution] The present invention discloses a method for detecting tumor-specific T cells. After collecting tumor cells or whole cells from tumor tissue, free whole cells or cell lysates are loaded onto nanoparticles / microparticles and co-cultured with peripheral immune cells. After activation of the cancer-specific T cells, the specific molecules of the tumor-specific T cells are detected, thereby confirming the presence of cancer-specific T cells in peripheral tissues such as peripheral blood. The components of the whole cell lysate of the present invention are water-soluble and water-insoluble components, which are either free or loaded onto nanoparticles or microparticles. The loading method involves the water-soluble and water-insoluble components of the whole cells being encapsulated separately or simultaneously within the particles and / or loaded separately or simultaneously on the surface of the particles. Detection methods include flow cytometry, enzyme-linked immunospot assay, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, and gene detection techniques.

Description

[Technical field]

[0001] The present invention is in the field of immunotherapy and immunodetection, and in particular relates to a whole cell-based method for detecting tumor-specific T cells. [Background technology]

[0002] In recent years, immune technology has developed rapidly, especially in the field of cancer immunotherapy. As our understanding of cancer deepens, it has become clear that the human immune system and various immune cells play an important role in the process of suppressing the occurrence and progression of cancer. In recent years, PD-1 antibodies and CAR-T cell therapies have been approved one after another and have been applied clinically, with good clinical effects, but cancer immunotherapies such as cancer vaccines and PD-1 antibodies are only effective in a small proportion of patients. It is very important to know how to judge the efficacy of immunotherapy drugs and the prognosis of patients before or during administration.

[0003] The prior art discloses detection particles that can be effectively used for detecting tumor-specific T cell content, a preparation method thereof, a kit containing the same, and a detection method using the same to detect tumor-specific T cell content, and the detection is performed based on one or more of the following: cell secretions secreted from activated tumor-specific T cells in a test sample, the proliferation state of activated tumor-specific T cells, or cell surface markers of activated tumor-specific T cells, and is used to activate tumor-specific T cells when detecting the tumor-specific T cell content. Since immunotherapy relies on the immune system to kill tumor cells by T cells activated by cancer-specific / associated antigens, the content of cancer-specific T cells in a patient's body is closely related to the effectiveness of immunotherapy. However, currently, there is no effective means for comprehensively and accurately detecting the content of cancer-specific T cells in a patient's peripheral blood. Summary of the Invention [Problem to be solved by the invention]

[0004] The present 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 and tumor cells, whole cells of tumor tissue, tumor cell lysate components, whole cell lysate components of tumor tissue or lysate components of tumor tissue are activated after co-culture and secrete or express some specific molecules, and the content of tumor cell-specific T cells can be measured by detecting these secreted or expressed specific molecules, and the key technology is the activation of T cells. [Means for solving the problem]

[0005] The present invention provides the following technical solution: A method for detecting tumor-specific T cells, comprising the steps of incubating an activator with peripheral immune cells, and then detecting a specific molecule of tumor-specific T cells, thereby achieving the detection of tumor-specific T cells.

[0006] A method for detecting tumor-specific T cell content, comprising the steps of incubating an activator with peripheral immune cells, then detecting specific molecules of tumor-specific T cells, and obtaining the tumor-specific T cell content according to the ratio of the number of tumor-specific T cells to the number of peripheral immune cells.

[0007] In the present invention, the activator includes tumor cells, whole cells of tumor tissue, tumor cell lysate components, whole cell lysate components of tumor tissue, and immune adjuvants, and the lysate components in the tumor cell lysate components and whole cell lysate components of tumor tissue may be water-soluble lysate components or insoluble lysate components, and are preferably water-soluble lysate components and insoluble lysate components.

[0008] In the present invention, the activator may be a free cell or a free lysate component, or a lysate component supported on a micro-nanoparticle, preferably a free lysate component or a lysate component supported on a micro-nanoparticle, and the lysate is supported inside and / or on the surface of the micro-nanoparticle. The method by which the lysate component is supported inside and / or on the surface of the micro-nanoparticle includes, but is not limited to, non-covalent adsorption, electrostatic interaction, hydrophobic interaction, hydrogen bond interaction, covalent bond, etc. The present invention may simultaneously use micro-nanoparticles carrying a water-soluble component and micro-nanoparticles carrying a water-insoluble component, or may simultaneously use micro-nanoparticles carrying both water-soluble and water-insoluble components, or may use micro-nanoparticles carrying only water-soluble components, or may use micro-nanoparticles carrying only water-insoluble components.

[0009] In the present invention, incubation conditions are those under which cells can survive, for example, 4° C. to 60° C., preferably 37° C., and the incubation time is 1 to 100 hours, for example, 5 to 70 hours, preferably 10 to 50 hours.

[0010] In the present invention, the micro-nanoparticles can be organic materials, inorganic materials, or biological materials such as synthetic polymer materials, natural polymer materials, or inorganic materials. The micro-nanoparticles are microparticles or nanoparticles, and the particle size of the nanoparticles is 1 nm to 1000 nm, preferably 30 nm to 800 nm, more preferably 50 nm to 600 nm, and the particle size of the microparticles 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. Specifically, the preparation method of the micro-nanoparticles is the prior art such as solvent evaporation method, dialysis method, extrusion method, hot melt method, etc., and the shape of the micro-nanoparticles can be spherical, elliptical, barrel-shaped, polygonal, linear, worm-shaped, square, triangular, butterfly-shaped, disk-shaped, etc., but is not limited thereto.

[0011] In the present invention, the method of supporting the cell lysate components on the micro-nanoparticles is a solvent volatilization method such as a double emulsion method, and other methods of supporting the cell lysate on the micro-nanoparticles can also be used. Specifically, when the activator is a cell lysate component supported on the micro-nanoparticles, the preparation method is to add the aqueous phase solution to the organic phase solution of the micro-nanoparticle material, add it to the first emulsifier solution after ultrasonic or stirring or homogenization, add it to the second emulsifier solution after ultrasonic or stirring or homogenization, and after stirring, the micro-nanoparticles supporting the cell lysate components are obtained, which are used as the activator. For example, the method includes the following steps: Step (1): Add the aqueous phase solution to the organic phase solution of the polymer material, add it to the first emulsifier solution after ultrasonic or stirring or homogenization, add it to the second emulsifier solution after ultrasonic or stirring or homogenization, and after stirring, centrifuge, resuspend the pellet to obtain the residue, or obtain the residue after ultrafiltration.

[0012] Step (2): The residue from step (1) is freeze-dried and then dispersed in a dispersion liquid, or the residue from step (1) is dispersed in a dispersion liquid and then an aqueous phase solution is added, mixed, and allowed to stand to obtain micro- or nano-particles to be used as activators.

[0013] By adding it to a secondary emulsifier solution, stirring, and then centrifuging or ultrafiltration, micro- and nanoparticles carrying the lysate component or the lysate component / immunoadjuvant inside are obtained. Furthermore, the lysate component or the lysate component / immunoadjuvant is carried on the surface of the micro- and nanoparticles carrying the lysate component or the lysate component / immunoadjuvant inside.

[0014] The aqueous phase solution is a lysate component solution or a lysate component / immune adjuvant solution, the ultrasonic wave is a probe ultrasonic wave or other ultrasonic method, the stirring is mechanical stirring, magnetic stirring, etc., and the homogenization is a high pressure homogenization or high shear homogenization, etc.

[0015] Preferably, when the aqueous phase solution is a lysate component solution, the protein and polypeptide concentrations are higher than 1ng / mL, preferably 1mg / mL to 100mg / mL, and when the aqueous phase solution is a lysate component / immunoadjuvant solution, the protein and polypeptide concentrations are higher than 1ng / mL, preferably 1mg / mL to 100mg / mL, and the immunoadjuvant concentration is higher than 0.01ng / mL, preferably 0.01mg / mL to 20mg / mL. In the polymeric material organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane, and the polymeric material concentration is 0.5mg / mL to 5000mg / mL, preferably 100mg / mL. The primary emulsifier solution is preferably an aqueous polyvinyl alcohol solution having a concentration of 10mg / mL to 50mg / mL, preferably 20mg / mL. The secondary emulsifier solution is preferably an aqueous polyvinyl alcohol solution having a concentration of 1 mg / mL to 20 mg / mL, preferably 5 mg / mL. The dispersion liquid is a PBS buffer solution, a physiological saline solution, or pure water.

[0016] Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is more than 50 rpm, and the stirring time is more than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.5 hours to 5 hours. In the case of ultrasonic treatment, the ultrasonic output is 50W to 500W, and the time is more than 0.1 seconds, for example, 2 to 200 seconds. In the case of homogenization, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used, and the pressure when using a high-pressure / ultra-high-pressure homogenizer is more than 20 psi, and the rotation speed when using a high-shear homogenizer is more than 1000 rpm. Nanometerization is performed by ultrasonic or stirring or homogenization, and the length of ultrasonic time or stirring speed or the pressure and time of homogenization can control the size of the prepared nanoparticles, and if it is too large or too small, it will result in a change in the size of the particle size.

[0017] In the present invention, the volume ratio of the aqueous phase solution to the polymeric material organic phase solution is 1:(1.1-5000), preferably 1:(1.5-500), the volume ratio of the polymeric material organic phase solution to the primary emulsifier solution is 1:(1.1-1000), preferably 1:(1.5-500), the volume ratio of the primary emulsifier solution to the secondary 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, most preferably 1:30-30:1.

[0018] In the present invention, all the cells of the cancer cells or tumor tissue are in a free state. The tumors include blood tumors and solid tumors such as endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, respiratory system tumors, blood cancer, 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, gum cancer, retinoblastoma, and sarcoma.

[0019] In the present invention, the solubilizing agent for dissolving the water-insoluble dissolved component is urea aqueous solution, guanidine hydrochloride solution, sodium deoxycholate aqueous solution, SDS aqueous solution, glycerin aqueous solution, alkaline solution, acidic solution, protease aqueous solution, albumin aqueous solution, lecithin aqueous solution, inorganic salt aqueous solution, polyethylene glycol octylphenyl ether (Triton) aqueous solution, dimethyl sulfoxide (DMSO), acetonitrile, ethanol, methanol, N,N-dimethylformamide (DMF), propanol, isopropanol, Tween, acetic acid, cholesterol, amino acid, glycoside, choline. The water-insoluble dissolved component can be dissolved in a solubilizing agent or an organic solvent, and the organic solvent can be DMSO, glycerol, acetonitrile, ethanol, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc.

[0020] In the present invention, the specific molecule secreted by T cells after the activator activates the T cells is a protein, a polypeptide, a nucleic acid, a sugar (carbohydrate) or a lipid. The specific molecule can be located in the cell membrane, cytoplasm, organelle or nucleus after expression, and the tumor-specific T cells can be detected qualitatively or quantitatively. The detection methods include, but are not limited to, flow cytometry, enzyme-linked immunospot assay, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, gene detection technology, and multi-cytokine detection technology.

[0021] The immune adjuvant of the present invention is an immune enhancing agent or an immune suppressing agent, and the method of adding the immune enhancing agent or the immune suppressing agent includes being encapsulated only inside the micro-nanoparticles, being supported only on the surface of the micro-nanoparticles, and being encapsulated inside the micro-nanoparticles and simultaneously supported on the surface of the micro-nanoparticles. The immune enhancing adjuvant is used to enhance the detection of immune cells capable of secreting or expressing pro-inflammatory cell markers such as IFN-γ and IL-12. The immune suppressing adjuvant is used to enhance the detection of immune cells capable of secreting or expressing anti-inflammatory cell markers such as IL-10. Effect of the Invention

[0022] The present invention provides a method for activating cancer-specific T cells in peripheral tissues by using free whole cell components or loaded whole cell cleavage components, and the content of activated cancer-specific T cells can be detected by conventional detection techniques to obtain information supporting the effectiveness of cancer immunotherapy. In the prior art, when cancer-specific T cells are stimulated and activated by polypeptide antigens, the activated and subsequently detected cancer-specific T cells are inaccurate, which affects the design and therapeutic effect of subsequent immunotherapy. In the present invention, the free state or the whole cell components of cancer cells or tissues are loaded onto nano / microparticles, which are used to activate cancer-specific T cells and detect the content of activated cancer-specific T cells, and the detected cancer-specific T cell content is more extensive and accurate.

[0023] In order to more clearly describe the embodiments of the present invention or the technical means in the prior art, the drawings necessary for depicting the embodiments or the prior art are briefly described below. [Brief description of the drawings]

[0024] [Figure 1] 1 is a flow diagram for the preparation of an activator of the present invention. [Diagram 2] Schematic diagram of micro- and nano-particles carrying water-soluble and water-insoluble cellular components. [Diagram 3] Schematic diagram of micro- and nano-particles carrying water-soluble and water-insoluble cellular components. [Figure 4] Schematic diagram of micro- and nano-particles carrying water-soluble and water-insoluble cellular components. [Diagram 5] Schematic diagram of micro- and nano-particles carrying water-soluble and water-insoluble cellular components. [Figure 6] 1 is a graph showing the experimental results of melanoma in Example 1. [Figure 7] 1 is a graph showing the experimental results of breast cancer in Example 2. [Figure 8] 1 is a graph showing the experimental results of melanoma in Example 3. [Figure 9] 1 is a graph showing the experimental results of lung cancer in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In the following experimental results, each data point in the graph for the tumor growth inhibition experiment is the mean ± standard error (mean ± SEM), and the data points for other experiments are the mean ± standard deviation (mean ± SD). ANOVA was used to analyze the significant difference in the tumor growth inhibition experiment, and the significant difference in other experiments was analyzed by t-test. * indicates that this group is significantly different from the control group at P<0.05. ** indicates that this group is significantly different from the control group at P<0.01. *** indicates that this group is significantly different from the control group at P<0.0001.

[0026] The present invention discloses a method for detecting the content of tumor-specific T cells in peripheral tissues to predict the prognosis of patients and to aid in the diagnosis and treatment of diseases. Those skilled in the art can achieve this by appropriately improving the process parameters with reference to the contents of this specification. It should be noted that all equivalent replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and product of the present invention are described through preferred embodiments, and those skilled in the art can realize and apply the present invention by improving or appropriately modifying and combining the methods described herein without obviously departing from the content, spirit and scope of the present invention.

[0027] The present invention discloses a technique for detecting tumor-specific T cells and their content in peripheral tissues: an activator is incubated with peripheral immune cells, and then the specific molecules of tumor-specific T cells are detected to realize the detection of tumor-specific T cells. An activator is 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.

[0028] When the present invention is used to detect whole cells or tumor tissue, it can be divided into three steps: (1) a step of collecting tumor cells or tumor tissue, (2) a step of co-culturing the whole cells or tumor tissue with a sample containing immune cells such as T cells from peripheral tissue for 10 minutes or more, for example, 16 hours, and (3) a step of detecting a specific molecule that marks the activation of T cells by a method such as flow cytometry, enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), or multi-cytokine detection method. The specific molecule can be secreted outside the T cell, expressed on the surface of the T cell, or located inside the T cell. The specific molecule can be a protein, a nucleic acid, a carbohydrate, or a lipid.

[0029] When the present invention uses nano / microparticles carrying cancer cell lysate components or tumor tissue lysate components for detection, it can be divided into the following four steps: (1) a step of collecting tumor cells or tumor tissues, (2) a step of preparing tumor cell lysate components or whole cell lysate components of tumor tissues and carrying them on micro / nanoparticles, (3) a step of co-culturing the micro / nanoparticles carrying the lysate components or the lysate components with a sample containing immune cells such as T cells from peripheral tissues for 10 minutes or more, for example, 16 hours, and (4) a step of detecting a specific molecule that marks the activation of T cells by a method such as flow cytometry, enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), or multi-cytokine detection method. The specific molecule can be secreted outside the T cells, expressed on the surface of the T cells, or located inside the T cells. The specific molecule can be a protein, a nucleic acid, a carbohydrate, or a lipid.

[0030] In the present invention, tumor cells or whole cells of tumor tissues can be used after being inactivated or denatured before or (and) after lysis, or can be used directly without being inactivated or (and) denatured before or (and) after lysis. The inactivation and (and) denaturation method can be ultraviolet irradiation or high temperature heating, and in the actual use process, inactivation or denaturation methods such as radiation irradiation, high pressure, freeze-drying, and formaldehyde can also be used. Those skilled in the art can understand that in the actual application process, the skilled artisan can make appropriate adjustments according to the specific situation.

[0031] Further, the activator includes an immune adjuvant that is an immunosuppressant or an immune enhancer, and can be encapsulated only within the micro-nanoparticles, can be encapsulated within the micro-nanoparticles and simultaneously supported on the surface of the micro-nanoparticles, or can be encapsulated only on the surface of the micro-nanoparticles.

[0032] FIG. 1 is a flow chart of the preparation of the activator of the present invention. The schematic diagram of the micro-nanoparticles carrying whole cells is shown in FIG. 2 to FIG. 5. In the actual use process, one kind of micro-nanoparticles with a specific structure can be used, or two or more kinds of micro-nanoparticles with different structures can be used simultaneously. In FIG. 2, the surface and the inside of the micro-nanoparticles contain immune adjuvants. In FIG. 3, the immune adjuvants are distributed only inside the micro-nanoparticles. In FIG. 4, the micro-nanoparticles contain immune adjuvants only on the outer surface. In FIG. 5, there is no immune adjuvant inside and on the outer surface of the micro-nanoparticles. When the water-soluble or water-insoluble components in the cell or tissue components carried by the micro-nanoparticles in 2a to 2j in FIG. 2, 6a to 6j in FIG. 3, 10a to 10j in FIG. 4, and 14a to 14j in FIG. 5 are distributed inside the micro-nanoparticles, no obvious inner core is formed. When the water-soluble or water-insoluble components in the cell or tissue components carried by the micro-nanoparticles in 3a to 3j of Figure 2, 7a to 7j of Figure 3, 11a to 11j of Figure 4, and 15a to 15j of Figure 5 are distributed inside the micro-nanoparticles, an inner core part is formed, and the inner core may be generated during the preparation process or formed by a method such as a polymer or an inorganic salt. When the water-soluble or water-insoluble components in the cell or tissue components carried by the micro-nanoparticles in 4a to 4j of Figure 2, 8a to 8j of Figure 3, 12a to 12j of Figure 4, and 16a to 16j of Figure 5 are distributed inside the micro-nanoparticles, a plurality of inner core parts are formed, and the inner core may be generated during the preparation process or formed by a method such as a polymer or an inorganic salt. When the water-soluble or water-insoluble components of the cells or tissue components encapsulated in the micro-nanoparticles in 5a-5j in Figure 2, 9a-9j in Figure 3, 13a-13j in Figure 4, and 17a-17j in Figure 5 are distributed inside the micro-nanoparticles, they are located in the outer layer of the formed inner core. In each figure, 1 represents the water-soluble components of the cells or tissue components, 2 represents the water-insoluble components of the cells or tissue components, 3 represents the immune adjuvant, 4 represents the micro-nanoparticles, and 5 represents the inner core portion of the micro-nanoparticles.a: both the components encapsulated within the micro / nanoparticle and supported on the surface are water-soluble components of the cell or tissue component; b: both the components encapsulated within the micro / nanoparticle and supported on the surface are water-insoluble components of the cell or tissue component; c: the components encapsulated within the micro / nanoparticle are water-insoluble components of the cell or tissue component and supported on the surface of the cell or tissue component; d: the components encapsulated within the micro / nanoparticle are water-soluble components of the cell or tissue component and supported on the surface of the cell or tissue component; e: both the water-soluble and water-insoluble components of the cell or tissue component are encapsulated within the micro / nanoparticle and both the water-soluble and water-insoluble components of the cell or tissue component are supported on the surface of the micro / nanoparticle; f: g represents the water-soluble and water-insoluble components of the cells or tissue component being encapsulated simultaneously inside the particles, and the water-soluble components of the cells or tissue component being carried only on the surface of the micro / nanoparticles; h represents the water-insoluble components of the cells or tissue component being encapsulated only inside the micro / nanoparticles, and the water-soluble components of the cells or tissue component being carried only on the surface of the micro / nanoparticles; i represents the water-soluble components of the cells or tissue component being encapsulated only inside the micro / nanoparticles, and the water-soluble components of the cells or tissue component being carried only on the surface of the micro / nanoparticles.

[0033] In some embodiments, the cell lysate components can be first encapsulated in the micro-nanoparticles together with the immune adjuvant, and then the cell lysate components are loaded onto the surface of the micro-nanoparticles, and the immune adjuvant is loaded onto the surface of the micro-nanoparticles at the same time. In practical applications, the tumor cells or whole cells of the tumor tissue can be directly lysed with a solubilizing agent (8M urea solution or 6M guanidine hydrochloride solution), and then the cell lysate components can be directly dissolved and then loaded onto the micro-nanoparticles.

[0034] The method for loading cell lysate components into micro- and nanoparticles is the solvent volatilization method, and other methods that can load cell lysate components into micro- and nanoparticles can also be used. In some embodiments, the nanoparticles are prepared using the double emulsion method in the solvent volatilization method, and the preparation material for the micro- and nanoparticles used is a polymer material such as an organic polymer polylactic acid-co-glycolic acid (PLGA), whose molecular weight is 24KDa to 38KDa. The PLGA material is a biodegradable material that is approved by the FDA as a dressing material and is suitable as a preparation material for the micro- and nanoparticles. The immune adjuvant used is poly(I:C) or CpG.

[0035] Preferably, the residue from step (1) is freeze-dried after being resuspended in an aqueous solution of a freeze-drying protectant, preferably trehalose or sucrose, at a concentration of 2-8 wt%, preferably 3-6 wt%.

[0036] In the present invention, the nanoparticles are prepared by double emulsion method, and other commonly used micro-nanoparticle preparation methods can be used in actual production. The material for preparing the micro-nanoparticles is PLGA, and other materials that can prepare micro-nanoparticles can be used in actual applications. In some embodiments of the present invention, nanoparticles are used, and in some embodiments, microparticles are used, and those skilled in the art can choose to use micro-nanoparticles in actual applications according to the actual situation. The detection method used in some embodiments of the present invention is flow cytometry, and the detection method used in some embodiments is enzyme-linked immunospot assay (ELISPOT) or enzyme-linked immunosorbent assay (ELISA). In actual applications, other methods such as multi-cytokine detection methods can be used for detection according to the actual situation. In some embodiments of the present invention, the specific molecule of tumor-specific T cells is interferon-γ (IFN-γ), and in actual applications, any other specific molecule can be used, which can be secreted or membrane-bound, and is a protein, nucleic acid, carbohydrate, or lipid. The specific cytokine detected in this embodiment is pro-inflammatory, and in actual applications, anti-inflammatory cytokines such as IL-10 and TGF-β can also be used.

[0037] In the present invention, poly(I:C) and CpG are used as immune adjuvants, and in practical applications, it is not necessary to add immune adjuvants or any other immune adjuvants with immune enhancing / suppressing functions, such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyactin A, mineral oil, virus-like particles, immune enhancing reconstituted influenza virus particles, cholera enterotoxin, saponin and its derivatives, Bacillus Calmette-Guerin (BCG), resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant poly The active ingredients of Chinese herbal medicines such as ICLC, Corynebacterium parvum, hemolytic streptococcus preparations, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM-CSF, MF59, double-stranded RNA, double-stranded DNA, aluminum hydroxide, CAF01, ginseng, and astragalus can be added. In the present invention, the immune adjuvant may or may not be added, but when the immune adjuvant is added, the immune adjuvant is at least one of the following: immune adjuvant derived from microorganisms, products of the immune system of humans or animals, natural immune agonists, acquired immune agonists, chemically synthesized medicines, fungal polysaccharides, and Chinese herbal medicines.

[0038] In order to provide a better understanding of the present invention, the technical means in the embodiments of the present invention will be described in detail with reference to the embodiments of the present invention, but it is obvious that the described embodiments are only some of the embodiments of the present invention and do not include all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without any creative activity are within the scope of protection of the present invention.

[0039] Unless otherwise specified, the specific methods used in the examples of the present invention are conventional methods, and the materials, reagents, etc. used are commercially available. The micro- and nano-particle structures, preparation methods, co-culture methods with T cells in peripheral tissues, and strategies used in detecting activated T cells in the examples of the present invention are merely representative methods, and other micro- and nano-particle structures, preparation methods, co-culture methods with T cells in peripheral tissues, and strategies used in detecting activated T cells can also be used according to the methods described in the present invention. The examples only list the application of the present invention in some cancers, but the present invention can also be used in other cancers. Regarding the specific methods or materials used in the examples, a person skilled in the art can replace the conventional methods with existing technologies based on the technical ideas of the present invention, and are not limited to the specific descriptions of the examples of the present invention. EXAMPLES

[0040] Detection of cancer-specific T cells in peripheral tissues of melanoma-bearing mice This embodiment describes the detection of tumor cell-specific T cells in peripheral tissues using the whole cell lysate of free cancer cells with mouse melanoma as a cancer model, and the detection of the content of tumor cell-specific T cells.Since the peripheral blood of mice is small, the number of peripheral immune cells in peripheral blood is limited, and the blood flow of the spleen contains abundant and sufficient peripheral immune cells, this embodiment uses peripheral immune cells in the spleen of mice to carry out related detection, and the immune cells in the spleen belong to peripheral immune cells, and the immune cells in peripheral blood also belong to peripheral immune cells.In practical clinical applications, peripheral immune cells in human peripheral blood can be used for detection.

[0041] In this example, B16-F10 mouse melanoma cells were used as a tumor cell model. First, B16-F10 cells were lysed to prepare the lysate components of B16-F10 cells, then the free tumor cell lysate components were co-cultured with peripheral immune cells overnight, and finally, the specific molecule gamma interferon of tumor-specific T cells was analyzed by flow cytometry. Specifically, the process is as follows. (1) Lysis of tumor cells and collection of various components: B16-F10 cells were collected, the medium was removed, frozen at -80°C, ultrapure water was added, and the freeze-thawing was repeated three times. At the same time, 150W ultrasonic treatment was also used to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a rotation speed of 12,000 RPM for 5 minutes, the supernatant, which is the water-soluble lysate component of B16-F10, was removed, and the resulting sediment was dissolved by adding an 8M urea aqueous solution to obtain a water-insoluble lysate component of B16-F10.

[0042] (2) Co-culture of free tumor cell lysate components with peripheral immune cells: 6-8 week-old female C57BL / 6 were selected to prepare mice bearing melanoma tumors. On day 0, 150,000 B16-F10 cells were inoculated subcutaneously into the lower right back of each mouse. On days 4, 7, 10, 15, and 20, nanocancer vaccines carrying whole cell components of cancer cells or PBS were injected subcutaneously, respectively. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated by the following formula:

[0043] v=0.52×a×b 2 Wherein v is the tumor volume, a is the tumor length, and b is the tumor width. On days 18 and 24, C57BL / 6 mice in the PBS and vaccine treatment groups were sacrificed and peripheral immune cells in the spleen were collected for parallel experiments.

[0044] The cells were resuspended in DMEM medium containing 10% FBS and diluted to 4x10 6The cells / mL concentration was adjusted to 100 μg / mL, and then 10% volume (based on the medium) of the soluble lysate component (40 mg / mL) and 1% volume (based on the medium) of the insoluble lysate component (30 mg / mL) were added thereto, and the mixture was incubated at 37° C. and 5% CO2 for 20 hours, followed by centrifugation at 400 g, after which the spleen cells of the incubated mice were collected.

[0045] (3) Detection of activated cancer-specific T cells by flow cytometry: To avoid non-specific binding, first treat the collected mouse spleen cells with Fc block. Then, stain mouse spleen cells extracellularly with CD3, CD4, and CD8 antibodies, fix the cells, permeabilize the cells, and stain mouse spleen cells intracellularly with FN-γ antibody. Then, detect mouse spleen cells with FACS AriaTMIII system, and analyze the results with FlowJo10 software. CD4, which can secrete IFN-γ after activation, + All CD4 T cells + The proportion of T cells and total CD8 T cells that can secrete IFN-γ after activation + The ratios to T cells were analyzed.

[0046] (4) Experimental results: Figure 6 shows the experimental results described above. Figure a shows the experimental results (n≧8) of the inhibitory effect of cancer vaccine treatment on tumor growth rate. Figure b shows the activated cancer-specific CD8+ cells in peripheral immune cells of the spleen after incubation with tumor tissue lysate, analyzed by flow cytometry. + CD8 in the spleen of T cells + Figure c shows the ratio of activated cancer-specific CD4+ cells in peripheral immune cells of the spleen after incubation with tumor tissue lysates, as analyzed by flow cytometry. + CD4 in splenic peripheral immune cells of T cells +6 shows the ratio of activated T cells to the total cells. As shown in FIG. 6, compared with the PBS blank control group, the peripheral immune cells of the mice in the vaccine treatment group had significantly more activated T cells after co-culture with tumor cell lysate, indicating that the cancer-specific T cells in the peripheral tissues of the mice treated with the cancer vaccine were significantly increased. It can be seen that the free whole cells described in the present invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients. EXAMPLES

[0047] Detection of cancer-specific T cells in peripheral tissues of mice bearing breast cancer tumors This example describes the detection of tumor cell-specific T cells in peripheral tissues using the whole cell lysate of free cancer cells with mouse breast cancer as a cancer model, and the detection of the content of tumor cell-specific T cells.Since the peripheral blood of mice is small, the number of peripheral immune cells in peripheral blood is limited, and the blood flow of the spleen contains abundant and sufficient peripheral immune cells, this example uses peripheral immune cells from the mouse spleen to carry out related detection.In practical clinical applications, peripheral immune cells in human peripheral blood can be used for detection.

[0048] In this example, 4T1 mouse mammary tumor cells were used as a tumor cell model. To prepare the soluble and insoluble components, the whole cells of the tumor tissue were first lysed, then the free tumor whole cell lysate components were co-cultured with peripheral immune cells overnight, and finally, the specific molecule gamma interferon of tumor-specific T cells was analyzed by flow cytometry.

[0049] (1) Tumor tissue dissolution and collection of various components: 400,000 4T1 mammary tumor cells were inoculated subcutaneously into the back of each BALB / c mouse, and the inoculated tumors were 200 mm 3 ~1500mm 3When the tumor tissue had grown to a volume of 1000 mg / kg, the mice were sacrificed and the tumor tissue was excised. The tumor tissue was cut into pieces, mashed, filtered through a cell strainer, and frozen and thawed five times. After the cells in the tumor tissue were lysed, the cell lysate was centrifuged at 3000 g for 20 minutes to remove the supernatant, which is the water-soluble component of all the cells in the tumor tissue. An 8M urea solution was added to the resulting sediment to dissolve the sediment, obtaining the water-insoluble components.

[0050] (2) Co-culture of free whole cell components with peripheral immune cells: 6-8 week-old female BALB / c mice bearing mammary tumors were selected. On day 0, 400,000 B16-F10 cells were inoculated subcutaneously into the lower right back of each mouse. On days 4, 7, 10, 15, and 20, nanocancer vaccines carrying whole cell components of cancer cells or PBS were injected subcutaneously, respectively. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated by the following formula:

[0051] v=0.52×a×b 2 Wherein v is the tumor volume, a is the tumor length, and b is the tumor width. On day 24, the mice in the vaccine-treated group and the PBS group were sacrificed, and peripheral immune cells in the spleen were collected.

[0052] The cells were resuspended in RPMI 1640 medium containing 10% FBS and diluted to 4X10 6 The cells / mL concentration was adjusted to 500 μg / mL, and then 5% volume (based on the medium) of water-soluble components (35 mg / mL) and 1% volume (based on the medium) of water-insoluble components dissolved in 8 M urea (35 mg / mL) were added thereto, followed by incubation at 37° C. and 5% CO2 for 18 hours, followed by centrifugation at 400 g, after which the spleen cells of the incubated mice were collected.

[0053] (3) Detection of activated cancer-specific T cells by flow cytometry: To avoid non-specific binding, mouse spleen cells were first treated with Fc block. Then, mouse spleen cells were stained extracellularly with CD3, CD4, and CD8 antibodies, and the cells were fixed and permeabilized, after which mouse spleen cells were stained intracellularly with FN-γ antibody. Mouse spleen cells were then detected with a FACS AriaTMIII system, and the results were analyzed with FlowJo10 software. CD4 T cells, which can secrete IFN-γ after activation, were stained extracellularly with CD3, CD4, and CD8 antibodies. + All CD4 T cells + The proportion of T cells and total CD8 T cells that can secrete IFN-γ after activation + The ratios to T cells were analyzed.

[0054] (4) Experimental results: Figure 7 shows the experimental results of breast cancer described above. Figure a shows the experimental results of the inhibitory effect of cancer vaccine treatment on tumor growth rate (n≧9). Figure b shows the activated cancer-specific CD8+ in peripheral immune cells of the spleen after incubation with tumor tissue lysate. + CD8 in splenic peripheral immune cells of T cells + Figure c shows the ratio of activated cancer-specific CD4+ cells in peripheral immune cells of the spleen after incubation with tumor tissue lysates. + CD4 in splenic peripheral immune cells of T cells + The ratio to cells is shown.

[0055] As shown in Figure 7, compared with the PBS blank control group, the peripheral immune cells of the mice in the vaccine treatment group had significantly more activated T cells after co-culture with the whole cell lysate of tumor cells, indicating that the cancer-specific T cells in the peripheral tissues of the mice treated with the cancer vaccine were significantly increased. It can be seen that the free whole cells described in the present invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients. EXAMPLES

[0056] (Activation of cancer-specific T cells in peripheral tissues by nanoparticles loaded with melanoma tumor tissue lysate components) In this example, a mouse melanoma model is used to prepare nanoparticles encapsulating all cellular components of melanoma tumor tissue, and a method is described for activating cancer-specific T cells in peripheral tissues using the nanoparticles. After T cells are activated, the content of activated cancer-specific T cells is detected by enzyme-linked immunosorbent assay (ELISA).

[0057] In this embodiment, IFN-γ secreted from activated T cells is detected by enzyme-linked immunosorbent assay (ELISA), but in practical applications, other substances secreted from activated T cells or other substances expressed on the surface of the cell membrane can also be detected by other methods such as enzyme-linked immunospot assay and flow cytometry.

[0058] In this example, mouse B16-F10 melanoma cells were transplanted into C57BL / 6 mice, and the tumor tissue was then excised. The water-soluble components in the tumor tissue lysate and the original water-insoluble components dissolved in 8M urea were simultaneously loaded onto the interior and surface of nanoparticles. Next, nanoparticles loaded with the water-soluble and water-insoluble components of the tumor tissue lysate were prepared by solvent evaporation using PLGA as the nanoparticle framework material, and the nanoparticles were used to activate cancer-specific tumor cells in peripheral tissues.

[0059] (1) Dissolution of tumor tissue and collection of various components: 150,000 B16-F10 melanoma cells were inoculated subcutaneously into the back of each C57BL / 6 mouse, and the inoculated tumors were 200 mm 3 ~1500mm 3When the tumor tissue grew to a volume of 1000 μg, the mice were sacrificed and the tumor tissue was removed. The tumor tissue was cut into pieces, ground, passed through a cell strainer, and added with pure water, and then frozen and thawed five times, thawed with 150 W ultrasonic waves for 2 minutes each time. After lysing the cells at the tumor tissue site, the cell lysate of the tumor tissue was centrifuged at a rotation speed of more than 100 g for 5 minutes, the supernatant, which is the water-soluble component of all the cells of the tumor tissue, was removed, and the resulting sediment was added with an 8M urea aqueous solution to dissolve the sediment, so that the water-insoluble components that are originally insoluble in pure water can be solubilized in the 8M urea aqueous solution. The water-soluble components of the tumor tissue lysate obtained above and the original water-insoluble components dissolved in 8M urea are used as raw materials for nanoparticle preparation.

[0060] (2) Preparation of nanoparticles carrying all cellular components: In this example, nanoparticles carrying cellular components and blank control nanoparticles were prepared by the double emulsion method in the solvent evaporation method, and the molecular weight of the nanoparticle preparation material PLGA used was 24KDa to 38KDa, and the preparation method was as described above. In this example, nanoparticles carrying four types of melanoma polypeptide antigens were also prepared, and the four types of 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 when preparing the nanoparticles was 5mg / mL. Specifically, it is as follows. 200 μL of the above water-soluble component solution (30 mg / mL) and water-insoluble component solution (30 mg / mL) were added to 1 mL of PLGA (100 mg) dichloromethane solution, and then ultrasonicated for 30 seconds, and mixed with 2.5 mL of polyvinyl alcohol aqueous solution (20 mg / mL). After ultrasonicated for 30 seconds, the mixture was mixed with 50 mL of polyvinyl alcohol aqueous solution (5 mg / mL). The mixture was stirred as usual until the organic solvent (dichloromethane) was evaporated and disappeared. The mixture was then centrifuged at 12,000 rpm for 10 minutes, the supernatant was removed, and the sediment was resuspended in 20 mL of trehalose aqueous solution (4 wt%), freeze-dried at -80°C, and resuspended in 10 mL of physiological saline. The mixture was mixed with 0.5 mL of the above water-soluble component solution (30 mg / mL) and 0.5 mL of water-insoluble component solution (30 mg / mL), and allowed to stand for 60 seconds to obtain an activator carrying the dissolved component inside and on the surface of the nanoparticles.

[0061] Preparations of blank nanoparticles, nanoparticles loaded with the four melanoma polypeptide antigens are prepared as above, but without the addition of lysate components or simply replacing the lysate components.

[0062] Before the lysate components are loaded on the surface of the nanoparticles, the average particle size of the nanoparticles is about 280 nm, and after the lysate components are loaded on the surface of the nanoparticles, the particle size of the nanoparticles obtained is about 300 nm, and 150 μg of the lysate components are loaded on each mg of PLGA nanoparticles. The particle size of the blank control nanoparticles is about 250 nm. The particle size of the nanoparticles loaded with the polypeptide antigen is about 290 nm, and the total amount of polypeptide loaded on each mg of PLGA nanoparticles is 50 μg of the polypeptide antigen.

[0063] (3) Activation of cancer-specific T cells by nanoparticles: 6-8 week-old female C57BL / 6 mice bearing melanoma tumors were selected. On day 0, 150,000 B16-F10 melanoma cells were inoculated subcutaneously into the lower right back of each mouse. On days 4, 7, 10, 15, and 20, nano cancer vaccines carrying whole cell components of cancer cells or PBS were injected subcutaneously, respectively. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated by the following formula:

[0064] v=0.52×a×b 2 Wherein v is the tumor volume, a is the tumor length, and b is the tumor width. On days 18 and 24, the mice in the PBS and vaccine treatment groups were sacrificed, and peripheral immune cells in the spleen were collected.

[0065] The cells were resuspended in RPMI 1640 medium containing 10% FBS and diluted to 5x10 6 The concentration of cells / mL is adjusted. Nanoparticles loaded with water-soluble and water-insoluble components at a final concentration of 300 μg / mL or nanoparticles loaded with polypeptide antigens at a final concentration of 300 μg / mL, or an equal amount of blank nanoparticles, or an equal amount of free whole cell lysate components, or an equal amount of free polypeptide antigens are added to the sample. Then, the sample is incubated at 37°C (5% CO 2 The cells were then cultured in a 350-well plate in a 400-well incubator for 72 hours, after which they were centrifuged at 400 g for 5 minutes, the supernatant was removed, and the IFN-γ concentration in the supernatant was analyzed by ELISA.

[0066] In the ELISA detection method, tumor-specific T cells secrete specific cell secretions such as IFN-γ after activation, and the higher concentration of the specific cell secretions indicates the higher content of activated cancer-specific T cells.

[0067] (4) Experimental results: Figure 8 shows the experimental results of melanoma mentioned above. Figure a shows the experimental results (n≧8) of the inhibitory effect of cancer vaccine treatment on tumor growth rate. Figure b shows the content of activated cancer T cells in peripheral immune cells of spleen after incubation with free tumor tissue lysate or nanoparticles carrying tumor tissue lysate by ELISA.

[0068] As shown in FIG. 8, compared with the PBS blank control group and the blank nanoparticle action group in the vaccine treatment group, the peripheral immune cells of the mice in the vaccine treatment group have significantly more activated T cells after co-culture with the total cellular components of free tumor tissue, or nanoparticles carrying the total cellular components of tumor tissue, or free polypeptide antigen, or nanoparticles carrying polypeptide antigen. This indicates that the content of cancer-specific T cells in the peripheral tissues of mice undergoing cancer vaccine treatment is significantly increased. As can be seen, the total cellular components of the free tumor tissue of the present invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients. When the total cellular components of free tumor tissue and the free polypeptide antigen are used to stimulate and activate T cells, the free total cellular components can stimulate and activate more T cells. When the nanoparticles carrying the total cellular lysate components and the nanoparticles carrying the polypeptide antigen are used to stimulate and activate T cells, the nanoparticles carrying the total cellular lysate components can stimulate and activate more T cells, and the T cells stimulated and activated by the nanoparticles carrying the total cellular lysate components are more than the T cells stimulated and activated by the free total cellular lysate components. EXAMPLES

[0069] (Lung cancer tumor tissue lysate components and immune adjuvants are loaded onto macroparticles to activate cancer-specific T cells in peripheral tissues) This example describes the preparation of microparticles carrying lung cancer tumor tissue lysate components and immune adjuvants and microparticles carrying only lung cancer tumor tissue lysate components in mouse lung cancer, activating tumor-specific T cells in peripheral tissues, and detecting the content of tumor-specific T cells by enzyme-linked immunospot assay (ELISPOT). This example examines the effect of CpG or poly I:C as immune adjuvants, and the effect of adding immune adjuvants.

[0070] In this embodiment, the specific molecule IFN-γ of activated tumor-specific T cells is detected by enzyme-linked immunospot assay (ELISPOT), and in practical applications, the specific molecules of other tumor-specific T cells can also be detected by other methods such as flow cytometry and ELISA.

[0071] In this example, LLC lung tumor cells from mice were transplanted into C57BL / 6 mice, and the tumor tissue was then excised to obtain the water-soluble components in the tumor tissue lysate and the water-insoluble components dissolved in 6M guanidine hydrochloride. Using PLGA as a microparticle framework material, microparticles carrying the water-soluble or water-insoluble components of the tumor tissue lysate were prepared by a solvent volatilization method. The water-soluble and water-insoluble components in the tumor tissue lysate were carried inside and on the surface of the microparticles, respectively, and in the microparticles containing the immune adjuvant, the immune adjuvant was encapsulated only inside the microparticles. The microparticles were used to detect tumor-specific T cells in the peripheral tissues of mice.

[0072] (1) Dissolution of tumor tissue and collection of various components: 2,000,000 LLC lung tumor cells were inoculated subcutaneously into the back of each C57BL / 6 mouse, and the inoculated tumor was 200 mm 3 ~1500mm 3When the tumor tissues reached a volume of 1000 mg / kg, the mice were sacrificed and the tumor tissues were excised. The tumor tissues were cut into equal sizes, ground, passed through a cell strainer, and pure water was added. All cells in the tumor tissues were inactivated and denatured by conventional UV irradiation and heating, and then frozen and thawed five times, thawed with 250W ultrasound for 1 minute each time. After all the cells in the tumor tissues were lysed, the cell lysate was centrifuged at 5000 rpm for 15 minutes, the supernatant, which is the water-soluble component of all the cells in the tumor tissue, was removed, and the resulting sediment was added with 6M guanidine hydrochloride solution to dissolve the sediment, obtaining the water-insoluble components.

[0073] (2) Preparation of microparticles carrying all cellular components: In this example, microparticles carrying cellular components and blank control microparticles were prepared by the double emulsion method in the solvent evaporation method, and the molecular weight of the microparticle preparation material PLGA used was 24KDa to 38KDa, and the preparation method was as described above. Specifically, it is as follows. 150 μL of the above water-soluble component solution (60 mg / mL) or 200 μL of the water-insoluble component solution (10 mg / mL) was added to 2 mL of PLGA (100 mg) dichloromethane solution, and then mixed with 10 mL of polyvinyl alcohol aqueous solution (15 mg / mL), and then ultrasonicated for 50 seconds, and then mixed with 300 mL of polyvinyl alcohol aqueous solution (8 mg / mL). The mixture was stirred as usual until the organic solvent (dichloromethane) was evaporated and disappeared, and then centrifuged at 10,000 rpm for 30 minutes, the supernatant was removed, and the sediment was resuspended in 20 mL of sucrose aqueous solution (5 wt%), freeze-dried at -80 ° C, and then resuspended in 5 mL of physiological saline, mixed with 3 mL of the above water-soluble component solution (10 mg / mL) and 0.5 mL of the water-insoluble component solution (40 mg / mL), and left to stand for 20 minutes to obtain an activator carrying a dissolved component inside and on the surface of the microparticles.

[0074] Add 150 μL of the above water-soluble component solution (60 mg / mL) or 200 μL of the water-insoluble component solution (10 mg / mL) to 100 μL of immune adjuvant (CpG or poly I:C) solution (0.25 mg / mL) After mixing with the above, the mixture was added to 2 mL of a PLGA (500 mg) dichloromethane solution, and then subjected to normal stirring for 150 seconds, mixed with 10 mL of an aqueous polyvinyl alcohol solution (15 mg / mL), and then subjected to normal ultrasonic treatment for 50 seconds, and then mixed with 300 mL of an aqueous polyvinyl alcohol solution (8 mg / mL). The mixture was stirred as usual until the organic solvent (dichloromethane) was evaporated and disappeared, and then centrifuged at 10,000 rpm for 30 minutes, the supernatant was removed, and the sediment was resuspended in 20 mL of an aqueous sucrose solution (5 wt%), freeze-dried at -80°C, and then resuspended in 5 mL of physiological saline. The mixture was mixed with 2 mL of the above water-soluble component solution (10 mg / mL) and 0.5 mL of the water-insoluble component solution (40 mg / mL), and allowed to stand for 20 minutes to obtain an activator carrying the dissolved component inside and on the surface of the microparticles.

[0075] Before the soluble component is supported on the surface of the microparticle, the average particle size of the microparticle is about 2.0 μm, and after the soluble component is supported on the surface of the microparticle, the particle size of the microparticle obtained is about 2.1 μm, and 160 μg of the soluble component is supported per mg of PLGA microparticle.

[0076] (3) Activation of cancer-specific T cells by microparticles: 6-8 week-old female C57BL / 6 mice bearing lung cancer tumor aneurysms were selected. On day 0, 2,000,000 LLC lung tumor cells were inoculated subcutaneously into the lower right back of each mouse. On days 4, 7, 10, 15, and 20, nano cancer vaccines carrying whole cell components of cancer cells or PBS were injected subcutaneously, respectively. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated by the following formula:

[0077] v=0.52×a×b 2 Wherein v is the tumor volume, a is the tumor length, and b is the tumor width. On day 24, the mice in the vaccine-treated group and the PBS group were sacrificed, and the immune cells in the spleen were collected.

[0078] The cells were resuspended in RPMI 1640 medium containing 10% FBS and diluted to 5x10 6 The concentration of cells / mL was adjusted. 100 μL of the above spleen cells was added to a 96-well plate precoated with IFN-γ antibody a (capture antibody, coating antibody) and blocked with medium for more than 1 hour. 25 μg of microparticles carrying water-soluble components and 25 μg of microparticles carrying water-insoluble components were added to it and incubated at 37°C and 5% CO2 for 72 hours. After that, the mixture of cells and microparticles was discarded, the 96-well plate was washed, and then IFN-γ antibody b (detection antibody) was added and cultured in an incubator at 37°C (5% CO2) for more than 2 hours. The solution containing IFN-γ antibody b was discarded, the 96-well plate was washed, and the color was developed in the corresponding manner to form spots on the surface of the 96-well plate. The data was read with an ELISPOT analyzer and the experimental results were analyzed.

[0079] In the ELISPOT detection method, tumor-specific T cells are activated and secreted IFN-γ and other cell secretions bind to antibody A contained in a 96-well plate, and after antibody B is added, a double antibody sandwich structure is formed, and an enzyme that helps color development is bound to the detection antibody. When a substrate is added to develop color, spots are formed at the position of each activated cell. The formation of one spot represents one activated tumor-specific T cell, so the number of spots formed by color development in each well of the 96-well plate can be measured to determine the number of tumor-specific T cells in the sample to be detected.

[0080] (4) Experimental results: Figure 9 shows the experimental results of lung cancer described above. Figure a shows the experiment (n≧9) of the inhibitory effect of cancer vaccine treatment on tumor growth rate. Figure b shows the content of activated cancer T cells in peripheral immune cells of the spleen after incubation with microparticles carrying tumor tissue lysate by ELISPOT method.

[0081] As shown in FIG. 9, compared with the PBS blank control group, the peripheral immune cells of mice in the vaccine treatment group have significantly more activated T cells after co-culture with microparticles carrying whole cell components of tumor tissue or with microparticles carrying whole cell components of tumor and immune adjuvant. Regardless of CpG as immune adjuvant or Poly(I:C) as immune adjuvant, after co-culture with peripheral immune cells, the microparticles carrying whole cell components of tumor and immune adjuvant activate more T cells than the microparticles carrying whole cell components of tumor. The above results indicate that the addition of immune adjuvant can activate more cancer antigen-specific T cells. It can be seen from this that the free whole cells of the present invention can be used to detect the content of cancer-specific T cells in the peripheral blood of cancer patients.

[0082] The content of cancer-specific T cells in peripheral tissues such as peripheral blood of cancer patients is positively correlated with the prognosis of the patient. The present invention can confirm the content of cancer-specific T cells in peripheral tissues such as peripheral blood by collecting tumor cells or whole cells of tumor tissue, using free whole cells or carrying cell lysates on nano / microparticles, co-culturing with peripheral immune cells from the periphery, activating the cancer-specific T cells, and detecting the specific molecules of the tumor-specific T cells. The inventive step of the present invention is to detect tumor-specific T cells in peripheral immune cells using cancer cells, whole cells of tumor tissue, components of cancer cell lysates, or components of whole cell lysates of tumor tissue as activators, and the particle carrying, cell incubation, specific secretion detection, etc. involved are all prior art in the art.

Claims

1. A method for detecting tumor-specific T cells, comprising the steps of incubating an activator containing either tumor cells or whole cells of tumor tissue, a tumor cell lysate component, or a whole cell lysate component of tumor tissue with peripheral immune cells, and then detecting a specific molecule of the tumor-specific T cells to realize detection of the tumor-specific T cells.

2. A method for detecting tumor-specific T cell content, comprising the steps of incubating an activator containing either tumor cells or whole cells of tumor tissue, a tumor cell lysate component, or a whole cell lysate component of tumor tissue with peripheral immune cells, then detecting a specific molecule of tumor-specific T cells, and further obtaining the tumor-specific T cell content according to the ratio of the number of tumor-specific T cells to the number of peripheral immune cells.

3. The detection method according to claim 1 or 2, characterized in that the lysate components are water-soluble lysate components and / or water-insoluble lysate components.

4. 3. The method according to claim 1 or 2, characterized in that the lysate components are free lysate components or lysate components supported on micro- or nanoparticles.

5. The detection method according to claim 4, characterized in that the lysate components are supported inside and / or on the surface of micro-nanoparticles.

6. The detection method according to claim 4, characterized in that the preparation material of the micro-nano particles is an organic material, an inorganic material or a biological material, and the micro-nano particles are microparticles or nanoparticles.

7. 3. The detection method according to claim 1, wherein the incubation conditions are conditions in which the cells can survive.

8. The method according to claim 1 or 2, characterized in that the tumors include blood tumors and solid tumors, and the specific molecules are proteins, polypeptides, nucleic acids, carbohydrates or lipids.

9. An activator composition for use in the detection of tumor-specific T cells in peripheral immune cells, characterized in that the activator composition contains either tumor cells, whole cells of tumor tissue, a tumor cell lysate component, or a whole cell lysate component of tumor tissue.

10. 10. The activator composition according to claim 9, characterized in that the lysate component is a free lysate component or a lysate component supported on micro-nano particles.

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