A method and kit for detecting cancer cell-specific t cells

Abstract

The present application relates to a kind of detection method and kit of cancer cell specific T cell, T cell in peripheral blood, peripheral immune organ or tumor infiltrating lymphocyte is co-incubated with the particle activated antigen presenting cell prepared by the particle loaded whole cell component to activate the cancer cell specific T cell that can recognize and kill cancer cell, then using ELISPOT, flow cytometry, ELISA and other techniques detect and analyze the number and proportion of activated cancer cell specific T cell, further evaluate the strength of cancer cell specific immunity in patient body.The present application overcomes the difficulty that currently cannot effectively screen specific T cell of broad spectrum and polyclonal in peripheral blood, peripheral immune organ or tumor infiltrating lymphocyte, can detect broad spectrum of specific T cell of effector cell specific T cell with specific killing function from cell, and with the characteristics of easy separation and high specificity, can be used as effective biomarker.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a method and kit for detecting cancer cell-specific T cells. Background Technology

[0002] T cells, especially cancer cell-specific T cells, play a crucial role in the fight against cancer. T cells are the main cells in the body that specifically recognize and kill cancer cells, and each clone of a cancer cell-specific T cell can specifically recognize an antigenic epitope. Cancer patients, especially those who have undergone immunotherapy or radiotherapy, possess a certain number of cancer cell-specific T cells. Studies have shown that the number of cancer cell-specific T cells in cancer patients treated with immunotherapy and other methods is positively correlated with their prognosis; therefore, detecting the number of cancer cell-specific T cells in cancer patients is particularly important. The inventors previously proposed using nanoparticles or microparticles loaded with whole-cell components of cancer cells to assist in the detection of cancer cell-specific T cells (application number 202011027741.0, Detection Method for Tumor-Specific T Cells). However, the above detection system requires the simultaneous presence of antigen-presenting cells (APCs), resulting in the presence of multiple cell types in the detection system, and the process of antigen-activated T cells assisting in detection is indirect rather than direct. To address these issues, the applicant proposes this invention. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a method for detecting cytotoxic (effective) cancer cell-specific T cells (T cells) using nanoparticles (NPs) or microparticles (MPs) activated by antigen-presenting cells loaded with whole-cell antigens of cancer cells. eff This method utilizes nanoparticles or microparticles prepared from activated antigen-presenting cells to first activate cancer cell-specific T cells, and then uses the activated cytotoxic cancer cell-specific T cells (T cells) to... eff This invention analyzes the content and proportion of cancer cell-specific T cells in test cells using biomarkers specifically expressed in the assay. It effectively solves the problem of how to detect broad-spectrum and polyclonal cancer cell-specific T cells with the ability to recognize and kill cancer cells in peripheral blood, peripheral immune organs, or tumor-infiltrating lymphocytes. Furthermore, because the nanoparticles or microparticles used to detect cancer cell-specific T cells are loaded with antigen-presenting cell membrane components, the system can be co-incubated with T cells without the assistance of antigen-presenting cells, making it a more direct detection method.

[0004] The first objective of this invention is to provide a method for detecting cancer cell-specific T cells using particles prepared from activated antigen-presenting cells, comprising the following steps:

[0005] S1. The antigen-presenting cells are co-incubated with the first particle to obtain activated antigen-presenting cells; wherein, the first particle is a nanoparticle or microparticle loaded with tumor tissue and / or whole cell components of cancer cells;

[0006] S2. Prepare nanovesicles from the cell membrane components of activated antigen-presenting cells; or co-act the cell membrane components of activated antigen-presenting cells with a second particle to load the cell membrane components onto the second particle, thereby obtaining a second particle loaded with cell membrane components; wherein, the second particle is a nanoparticle or microparticle loaded with whole cell components of tumor tissue and / or cancer cells.

[0007] S3. Co-incubate the nanovesicles and / or second particles loaded with cell membrane components from step S2 with the cells to be tested to activate the broad-spectrum cancer cell-specific T cells that can recognize antigens. Then, use appropriate detection techniques to analyze the markers inside or on the surface of the activated cancer cell-specific T cells. The number and proportion of the cancer cell-specific T cells are obtained by analyzing the number and proportion of T cells containing the markers.

[0008] Furthermore, the aforementioned markers inside or on the cell surface include proteins or nucleic acids.

[0009] Furthermore, when the specific surface marker is a protein, it includes, but is not limited to, interferon-γ, interleukin, granzyme, perforin, CD69, CD25, OX40 (CD134), CD39, CD103, CD56, CD279, CD278, CD244, CD27, CD154, TCF-1, CD137, CD44, CD28, etc. Techniques for analyzing and detecting the number and proportion of cancer cell-specific T cells using surface markers include, but are not limited to, flow cytometry, magnetic bead sorting, enzyme-linked immunospot assay (ELISPOT), and enzyme-linked immunosorbent assay (ELISA).

[0010] Furthermore, the cells to be tested can be T cells or a mixture of cells containing T cells, such as T cells or a mixture of cells containing T cells derived from peripheral blood, peripheral immune organs, or tumor-infiltrating lymphocytes.

[0011] Furthermore, before co-incubating the test cells with the S2 product, they can be sorted to separate T cells. Specifically, CD3+ cells can be sorted from peripheral blood, peripheral immune tissues, and tumor-infiltrating lymphocytes using flow cytometry or magnetic bead sorting. + Cells, sorted out CD45 + CD3 + Cells, sorted out CD3 + CD8 +Cells, sorted out CD45 + CD3 + CD8 + Cells, sorted out CD3 + CD4 + Cells or sorted CD45 + CD3 + CD4 + . cells.

[0012] Furthermore, the co-incubation system of the antigen-presenting cells and the first particle in step S1 may contain cytokines or antibodies.

[0013] Furthermore, in step S3, the product of S2 may contain cytokines or antibodies in the co-incubation system with the cells to be tested.

[0014] Preferably, the co-incubation system contains granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-7 and IL-12.

[0015] Furthermore, cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-14 (IL-14), interleukin-4 (IL-4), interleukin-15 (IL-15), interleukin-21 (IL-21), interleukin-17 (IL-17), interleukin-12 (IL-12), interleukin-6 (IL-6), interleukin-33 (IL-33), interferon-gamma (IFN-γ), and TNF-α.

[0016] Furthermore, the antibodies include, but are not limited to, αCD-3 antibodies, αCD-4 antibodies, αCD-8 antibodies, αCD-28 antibodies, αCD-40 antibodies, αOX-40 antibodies, and αOX-40L antibodies.

[0017] Furthermore, in the above preparation method, the first or second particle may also be loaded with bacterial components or bacterial exovesicle components. These bacterial components or bacterial exovesicle components are obtained by lysing the bacteria or bacterial exovesicles with a lysis buffer containing a lysis agent. The lysis agent may be urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein-degrading enzymes, albumin, lecithin, Triton, Tween, amino acids, glycosides, or choline. The bacteria include, but are not limited to, BCG, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus geranioli, Lactobacillus reuteri, and Lactobacillus rhamnosus.

[0018] Furthermore, the activated antigen-presenting cell membrane component can be mixed with cancer cell membrane component or cancer cell extracellular vesicle membrane component to prepare a mixed membrane component, which can then be prepared into nanovesicles or loaded onto the surface of a second particle.

[0019] Furthermore, the first or second particle is also loaded with an immune-enhancing adjuvant, which includes, but is not limited to, pattern recognition receptor agonists, BCG, BCG cell wall cytoskeleton, BCG methanol extract residue, BCG muramyl dipeptide, Mycobacterium tumefaciens, polyantigen A, mineral oil, virus-like particles, immune-enhancing regenerated influenza virus bodies, cholera enterotoxin, saponins and their derivatives, resiquimod, thymosin, newborn bovine liver bioactive peptides, miquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), and immune adjuvant poly ICLC, Corynebacterium spp. vaccine, hemolytic streptococcal preparations, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, cytokines, interleukins, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, squalene, vegetable oils, endotoxins, liposome adjuvants, MF59, double-stranded RNA, double-stranded DNA, CAF01, active ingredients of ginseng and astragalus, etc.

[0020] Preferably, the immune-enhancing adjuvant includes (1) Poly(I:C) or Poly(ICLC); (2) CpG-ODN, wherein the CpG-ODN is at least two of type A CpG-ODN, type B CpG-ODN and type C CpG-ODN, and at least one of them is type B CpG-ODN or type C CpG-ODN. Among them, Class A CpG-ODN is selected from CpG-ODN 2216, CpG-ODN 1585 or CpG-ODN 2336; Class B CpG-ODN is selected from CpG-ODN 1018, CpG-ODN 2006, CpG-ODN 1826, CpG-ODN 1668, CpG-ODN 2007, CpG-ODN BW006 or CpG-ODN SL01; and Class C CpG-ODN is selected from CpG-ODN 2395, CpG-ODN SL03 or CpG-ODNM362.

[0021] Furthermore, the first or second particle is also loaded with positively charged polypeptides (such as KALA polypeptide, RALA polypeptide, bee venom peptide, etc.), arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH4HCO3, protamine, or histone, etc.

[0022] Furthermore, the first or second particle is also loaded with a target that actively targets antigen-presenting cells. The target may be mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc.

[0023] Furthermore, the first or second particle can be prepared from the following materials: organic synthetic polymers including but not limited to PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, synthetic peptides, etc.; natural polymers including but not limited to lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, peptides, etc.; and inorganic materials including but not limited to ferric oxide, ferric oxide, carbonates, phosphates, etc.

[0024] Furthermore, the particle size of the first or second particle is in the nanometer or micrometer range. This ensures that the particles are phagocytosed by the antigen-presenting cells. To improve phagocytosis efficiency, the particle size must be within a suitable range. The particle size of nanoparticles (NP) is 1 nm-1000 nm, more preferably 30 nm-1000 nm, and most preferably 100 nm-600 nm; the particle size of microparticles (MP) is 1 μm-1000 μm, more preferably 1 μm-100 μm, more preferably 1 μm-10 μm, and most preferably 1 μm-5 μm.

[0025] Further, in step S2, the activated antigen-presenting cells are mechanically destroyed, filtered through a membrane, or centrifuged to prepare nanovesicles, or the activated antigen-presenting cells are mechanically destroyed, filtered through a membrane, or centrifuged, and the product is co-treated with the second particle to obtain the second particle loaded with cell membrane components.

[0026] Furthermore, the mechanical destruction method is selected from one or more of ultrasound, homogenization, slurrying, high-speed stirring, high-pressure destruction, high-shear destruction, swelling, chemical substances, and shrinkage. The co-action method is selected from one or more of co-incubation, co-extrusion, ultrasound, stirring, homogenization, and slurrying. After co-action with nanoparticles or microparticles, the antigen-presenting cell components cover the surface of the original nanoparticles or microparticles to form new nanoparticles or microparticles.

[0027] Further, cancer cells or tumor tissue are frozen at -20℃ to -273℃, and then subjected to repeated freeze-thaw lysis after adding water or a solution without a solvent. The resulting supernatant is the water-soluble component, and the portion of the precipitate that becomes soluble after dissolution with a solvent is the water-insoluble component. The water-soluble and water-insoluble components are combined to obtain the whole-cell component of cancer cells. The solvent is selected from at least one of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein-degrading enzymes, albumin, lecithin, inorganic salts (0.1-2000 mg / mL), Triton, Tween, amino acids, glycosides, and choline.

[0028] Furthermore, the antigen-presenting cells include at least one of B cells, dendritic cells (DCs) and macrophages, preferably two or more including DCs, and more preferably a combination of the three types of cells.

[0029] Furthermore, the obtained cancer cell-specific T cells included CD4 + T cells and / or CD8 + T cells, preferably including CD4+ + T cells and CD8 + T cells.

[0030] In this invention, nanoparticles and / or microparticles loaded with cancer cell whole-cell antigens are used to first specifically activate antigen-presenting cells. Then, the antigen-presenting cells are prepared into nanoparticles or microparticles, which are loaded with cancer cell whole-cell antigen epitopes. The nanoparticles or microparticles prepared from the antigen-presenting cells are then used to activate cancer cell-specific T cells in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes. The activated cancer cell-specific T cells are then used to analyze the number and proportion of the most diverse and broad-spectrum cancer-specific T cells with the function of recognizing and killing cancer cells by means of flow cytometry and other methods, taking advantage of the high expression of certain molecules within or on the surface of the activated cancer cell-specific T cells.

[0031] Furthermore, antigen-presenting cells can be derived from the same organism, allogeneic, cell line, or stem cell from cancer cell-specific T cells.

[0032] Furthermore, in the first or second particle, at least one of the cancer cells or tumor tissues used to prepare the antigen has the same disease type as the cancer cell-specific T cell being detected.

[0033] A second object of the present invention is to provide a kit for detecting cancer cell-specific T cells, the kit comprising at least one of the following (1)-(2):

[0034] (1) Nanovesicles prepared from activated antigen-presenting cells;

[0035] (2) Particles loaded with activated antigen-presenting cell membrane components;

[0036] in,

[0037] The nanovesicles are prepared by co-incubating antigen-presenting cells with the first particle to obtain activated antigen-presenting cells, and then extracting the cell membrane components of the activated antigen-presenting cells.

[0038] The particles loaded with activated antigen-presenting cell membrane components are obtained by co-interacting the activated antigen-presenting cell membrane components with second particles, thereby loading the cell membrane components onto the second particles.

[0039] The first or second particle is independently selected from nanoparticles or microparticles loaded with whole-cell components of tumor tissue and / or cancer cells.

[0040] This invention breaks through the limitations of existing detection methods, enabling particles to be loaded with all antigens and activated antigen-presenting cell membranes, thus enabling the detection of a broader and more diverse range of cancer cell-specific T cells with high specificity. It is particularly effective in immunoassays, thereby providing a potential biomarker detection method for immunotherapy.

[0041] By means of the above-described solution, the present invention has at least the following advantages:

[0042] This invention provides a technique for in vitro detection of cancer cell-specific T cells in immune cells using a nanoscale or microscale particle delivery system. The analyzed and detected cancer cell-specific T cells are broad-spectrum and highly specific, encompassing all clones of effector (cytotoxic) cancer cell-specific T cells (T cells). eff Furthermore, compared to particles prepared from unloaded activated antigen-presenting cells, particles prepared from activated antigen-presenting cells exhibit a higher level of specific markers secreted by activated T cells, making them easier to detect and avoiding situations where poor signals prevent detection. These advantages allow the particle detection method of this invention to avoid the situation where some cancer cell-specific T cells cannot be detected due to weak expression of specific markers after activation, thus resulting in higher detection accuracy. Furthermore, this invention optimizes the antigen-presenting cell activation process, the incubation process with T cells, and the loading substances of the first and second particles, enabling the method to detect a more comprehensive range of cancer cell-specific T cells with stronger and more accurate signals.

[0043] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0044] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0045] Figure 1 This is a schematic diagram illustrating the preparation process and application of the cell system of the present invention; wherein, a is a schematic diagram of collecting and preparing nanoparticles or microparticles from water-soluble and insoluble components respectively; b is a schematic diagram of dissolving whole-cell antigens of cancer cells in a dissolving solution containing a solubilizer and preparing nanoparticles or microparticles; c is a schematic diagram of activating antigen-presenting cells using nanoparticles and / or microparticles prepared in a or b, and then preparing the activated antigen-presenting cells into particles for detection of cancer cell-specific T cells.

[0046] Figure 2-14 The figures represent the results of experiments using nanoparticles or microparticles to detect cancer cell-specific T cells in Examples 1-13, respectively; * indicates p≤0.05, indicating a significant difference. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0048] The present invention describes a method for detecting cancer cell-specific T cells in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes. During detection, these cancer cell-specific T cells are first co-incubated with nanoparticles and / or microparticles prepared from activated antigen-presenting cells. Then, flow cytometry, ELISPOT, or enzyme-linked immunosorbent assay (ELISA) are used to analyze the molecules highly expressed by the cancer cell-specific T cells after antigen-specific activation, thus obtaining information on a broad spectrum of cancer cell-specific T cells.

[0049] In this process, antigen-presenting cells used to prepare nanoparticles or microparticles are first activated by nanoparticles and / or microparticles loaded with tumor tissue and / or cancer cell whole-cell antigens or mixtures thereof. The process and applications of detecting cancer cell-specific T cells include... Figure 1 As shown.

[0050] In preparing nanoparticles or microparticles for activating antigen-presenting cells, water-soluble components and water-insoluble antigens can be collected separately after lysing cells or tissues and then prepared into nanoparticle or microparticle systems. Alternatively, cells or tissues can be directly lysed and cancer cell whole-cell antigens dissolved using a solvent to prepare nanoparticle or microparticle systems. The cancer cell whole-cell antigens described in this invention can be prepared into nanoparticles or microparticles before or after lysis, subject to treatments including but not limited to inactivation or denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease endolysis, or degradation. Alternatively, nanoparticles or microparticles can be prepared directly before or after cell lysis without any inactivation or denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease endolysis, or degradation. In some embodiments of this invention, tumor tissue cells undergo inactivation and / or denaturation treatment before lysis. In actual use, inactivation and / or denaturation treatment can also be performed after cell lysis, or both before and after cell lysis. In some embodiments of this invention, the inactivation and / or denaturation treatment methods before and after cell lysis are ultraviolet irradiation and high-temperature heating. In actual use, methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, protease endocleavage or degradation, and freeze-drying can also be used. Those skilled in the art will understand that appropriate adjustments can be made according to specific circumstances during practical applications.

[0051] When preparing activated antigen-presenting cells into nanoparticles or microparticles, the antigen-presenting cells are first mechanically destroyed, and then filtered using centrifugation and / or a filter membrane with a certain pore size, optionally in conjunction with nanoparticles or microparticles.

[0052] Activated antigen-presenting cells retain a certain cell membrane structure after being mechanically disrupted.

[0053] After being mechanically destroyed, activated antigen-presenting cells interact with nanoparticles or microparticles to form new nanoparticles or microparticles. The antigen-presenting cell component of these new nanoparticles or microparticles is located in the outer layer of the particle.

[0054] Antigen-presenting cells prepared as nanoparticles or microparticles, and antigen-presenting cells used for co-incubation with T cells, can be derived from autologous or allogeneic sources, or from cell lines or stem cells. Antigen-presenting cells can be dendritic cells (DCs), B cells, macrophages, or any mixture of the above, or other cells with antigen-presenting function.

[0055] When using whole-cell components loaded with tumor tissue and / or cancer cells to activate antigen-presenting cells, the system may contain cytokines and / or antibodies to improve activation efficiency.

[0056] When using nanoparticles and / or microparticles prepared from activated antigen-presenting cells to activate cancer cell-specific T cells, the system may contain cytokines and / or antibodies to improve activation efficiency.

[0057] In some implementation schemes, nanoparticles or microparticles loaded with whole-cell antigens of cancer cells are first used to activate antigen-presenting cells, and then the antigen-presenting cells are prepared into nanoparticles or microparticles. The specific method for using nanoparticles or microparticles prepared from antigen-presenting cells to detect cancer cell-specific T cells in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes is as follows:

[0058] Step 1: Add a first predetermined volume of aqueous solution containing a first predetermined concentration to a second predetermined volume of organic phase containing a second predetermined concentration of raw material for preparing particles.

[0059] In some embodiments, the aqueous phase solution may contain components of the cancer cell / tumor tissue lysate and an immunomodulatory adjuvant; the components of the lysate are either water-soluble components or originally water-insoluble components dissolved in a solvent containing urea or guanidine hydrochloride during preparation. The concentration of the water-soluble components or the originally water-insoluble components in the aqueous phase solution, i.e., the first predetermined concentration, requires a protein / peptide concentration greater than 1 ng / mL to load sufficient whole-cell antigens of cancer cells to activate relevant cells. The concentration of the immunomodulatory adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0060] In some embodiments, dichloromethane is selected as the organic solvent. Additionally, in some embodiments, the second predetermined concentration for preparing the particulate raw material ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0061] In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase. In this invention, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1:10. During implementation, the first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0062] 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, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0063] Step 2: The mixture obtained in Step 1 is subjected to ultrasonic treatment for more than 2 seconds, or stirring, homogenization, or microfluidic treatment for more than 1 minute. 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, the stirring speed is 50 rpm to 1500 rpm and the stirring time is 0.1 hours to 24 hours; when ultrasonic treatment is performed, the ultrasonic power is greater than 5W 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 the pressure being greater than 5 psi when using a high-pressure / ultra-high-pressure homogenizer, for example, 20 psi to 100 psi, and the rotation speed being greater than 100 rpm when using a high-shear homogenizer, for example, 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, for example, 0.1 mL / min to 100 mL / min. Nanoparticles and / or microparticles can be prepared by ultrasonication, stirring, homogenization, or microfluidic processing. The duration of ultrasonication, stirring speed, or homogenization pressure and time can control the size of the prepared micro- and nanoparticles. If the particles are too large or too small, the particle size will change.

[0064] Step 3 involves adding the mixture obtained in Step 2 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of emulsifier and subjecting it to ultrasonic treatment for more than 2 seconds, or stirring for more than 1 minute, or homogenization or microfluidic treatment. In this step, the mixture obtained in Step 2 is added to the emulsifier aqueous solution and further ultrasonicated or stirred to achieve nano- or micron-sized structures. In this invention, the ultrasonic time is greater than 0.1 seconds, for example, 2 to 200 seconds; the stirring speed is greater than 50 rpm, for example, 50 to 500 rpm; and the stirring time is greater than 1 minute, for example, 60 to 6000 seconds. 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 50 W to 500 W 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, for example, 20 psi to 100 psi, and a rotation speed greater than 1000 rpm when using a high-shear homogenizer, for example, 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, for example, 0.1 mL / min to 100 mL / min. Nanoparticles or microparticles are produced through ultrasonication, stirring, homogenization, or microfluidic treatment. The duration of ultrasonication, stirring speed, or homogenization pressure and time can control the size of the prepared nanoparticles or microparticles; excessively large or small particles will lead to changes in particle size.

[0065] In some embodiments, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg / mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In this invention, the ratio of the second predetermined volume to the third predetermined volume is set in the range of 1:1.1-1:1000, preferably 2:5. In specific implementations, the ratio of the second predetermined volume to the third predetermined volume can be adjusted to control the size of nanoparticles or microparticles. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution, and the concentration in this step are all determined to obtain nanoparticles or microparticles of suitable size.

[0066] Step 4: Add the liquid obtained after step 3 to a fourth predetermined volume of an emulsifier aqueous solution of a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.

[0067] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0068] The fourth predetermined concentration is 5 mg / mL. The selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size. The selection of the fourth predetermined volume is determined by the ratio of the third predetermined volume to the fourth predetermined volume. In this invention, the ratio of the third predetermined volume to the fourth predetermined volume ranges from 1:1.5 to 1:2000, preferably 1:10. In specific implementations, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the size of the nanoparticles or microparticles.

[0069] In this invention, the predetermined stirring condition for this step is until the organic solvent has completely evaporated, that is, the dichloromethane in step 1 has completely evaporated.

[0070] Step 5: After centrifuging the mixture that has been processed in Step 4 to meet the predetermined stirring conditions for more than 1 minute at a speed greater than 100 RPM, remove the supernatant and resuspend the remaining precipitate in a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of lyophilization protectant or in a sixth predetermined volume of PBS (or physiological saline).

[0071] Step 6: After freeze-drying the suspension containing the freeze-drying protectant obtained in Step 5, the freeze-dried material is ready for use.

[0072] Step 7: Use the nanoparticle-containing suspension obtained in step 5, resuspended in PBS (or physiological saline) in the sixth predetermined volume, or use the freeze-dried material containing nanoparticles or microparticles and a freeze-drying protectant obtained in step 6, resuspended in the sixth predetermined volume of PBS (or physiological saline) directly; or use the above sample after mixing with the water-soluble component or the dissolved original non-water-soluble component in the seventh predetermined volume.

[0073] In this invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.

[0074] Step 8: Incubate the antigen-presenting cells with the prepared nanoparticles and / or microparticles for a certain period of time. The tumor tissue and / or cancer cells from which the nanoparticles and / or microparticles are prepared, along with the antigen-presenting cells, can be derived from the patient's own body or from an allogeneic source.

[0075] Step 9: Collect the co-incubated cells and perform mechanical disruption such as sonication, homogenization, and mechanical stirring.

[0076] Step 10: Centrifuge the ultrasonicated sample and / or filter it using a filter membrane with a certain pore size and / or react it with nanoparticles and / or microparticles loaded with whole cell components of cancer cells to prepare nanoparticles or microparticles based on antigen-presenting cells.

[0077] Step 11: Obtain peripheral blood, peripheral immune tissue, or tumor tissue, and collect T cells or immune cells containing T cells from these tissues. The peripheral blood, peripheral immune tissue, or tumor tissue can be derived from the patient's own body or from an allogeneic source.

[0078] Step 12: Mix the nano and / or micro particles prepared in step 10 with the T cells or immune cells containing T cells obtained in step 11 and incubate them together for a certain period of time.

[0079] Step 13: Analyze the content of antigen-activated cancer cell-specific T cells using flow cytometry, enzyme-linked immunospot assay, enzyme-linked immunosorbent assay, or magnetic bead sorting.

[0080] Example 1: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs

[0081] This embodiment uses mouse melanoma as a cancer model to illustrate how to use nanoparticles prepared from antigen-presenting cells activated by nanoparticles to detect cancer cell-specific T cells in mouse spleen cells. In this embodiment, B16F10 melanoma tumor tissue was lysed to prepare water-soluble and insoluble components of the tumor tissue. Then, using PLGA as the nanoparticle framework material and Polyyinosinic-polycytidylic acid (poly(I:C)) as the immunoadjuvant, a nanoparticle system loaded with the water-soluble and insoluble components of the tumor tissue was prepared by solvent evaporation. The nanoparticles were then used to activate antigen-presenting cells, and the antigen-presenting cells were mechanically destroyed and centrifuged to prepare nanoparticles. These nanoparticles were then used to assist in the detection of cancer cell-specific T cells in peripheral immune organs.

[0082] (1) Lysis of tumor tissue and collection of its components

[0083] 1.5 × 10⁻⁶ mmol / L was subcutaneously injected into the back of each C57BL / 6 mouse. 5 Several B16F10 cells were observed when the tumor reached a volume of approximately 1000 mm². 3 Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and filtered through a cell filter with an appropriate amount of ultrapure water. The mixture was then subjected to five freeze-thaw cycles, accompanied by sonication, to destroy and lyse the cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component in pure water. Adding 8M urea to the resulting precipitate dissolved the insoluble component in pure water, thus converting it into a soluble component in an 8M urea aqueous solution. This constitutes the source of antigen raw materials for preparing the nanoparticle system.

[0084] (2) Preparation of nanoparticles loaded with whole-cell components

[0085] In this embodiment, nanoparticle 1 was prepared using the double emulsion method within the solvent evaporation process. During preparation, nanoparticles loaded with the water-soluble components of whole-cell antigen from cancer cells and nanoparticles loaded with the insoluble components of whole-cell antigen from cancer cells were prepared separately and then used together. The PLGA nanoparticle material used had a molecular weight of 24 kDa-38 kDa, and the immunoadjuvant used was poly(I:C), which was distributed only within the nanoparticles. The preparation method was as described above. First, the cell components and adjuvant were loaded inside the nanoparticles using the double emulsion method. After loading the cell lysis components internally, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, with each mg of PLGA nanoparticles loading approximately 100 μg of protein or polypeptide components, and each mg of PLGA nanoparticles used 0.02 mg of poly(I:C) immunoadjuvant.

[0086] (3) Preparation of bone marrow-derived dendritic cells (BMDCs)

[0087] This example illustrates how to prepare BMDCs using dendritic cells prepared from mouse bone marrow cells. First, a 6-8 week old C57 mouse was euthanized by cervical dislocation. The tibia and femur of the hind leg were surgically removed and placed in PBS. The surrounding muscle tissue was removed using scissors and forceps. Both ends of the bone were cut off with scissors. PBS solution was drawn using a syringe, and the needle was inserted into the bone marrow cavity from both ends of the bone to repeatedly flush the bone marrow into a culture dish. The bone marrow solution was collected, centrifuged at 400g for 3 minutes, and 1 mL of erythrocyte lysis buffer was added to lyse the cells. 3 mL of RPMI 1640 (10% FBS) medium was added to terminate lysis, and the cells were centrifuged at 400g for 3 minutes, discarding the supernatant. The cells were cultured in 10 mm culture dishes using RPMI 1640 (10% FBS) medium, with recombinant mouse GM-CSF (20 ng / mL) added, at 37°C and 5% CO2 for 7 days. On day 3, gently shake the culture flask and add the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL). On day 6, replace half of the medium. On day 7, collect a small number of suspension and semi-adherent cells and analyze them by flow cytometry. When CD86... + CD80 + Cells in CD11c + When the proportion of BMDC in cells is between 15-20%, the induced cultured BMDC can be used for the next step of the experiment.

[0088] (4) Activation of antigen-presenting cells

[0089] Nanoparticles loaded with whole-cell components of cancer cells derived from tumor tissue (250 μg nanoparticles loaded with water-soluble components + 250 μg nanoparticles loaded with insoluble components) and BMDC (10 million units) were co-incubated in 15 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). The incubation system contained either cytokine combination 1: granulocyte-macrophage colony-stimulating factor (GM-CSF, 500 U / mL), IL-2 (500 U / mL), IL-7 (500 U / mL), IL-12 (500 U / mL) or cytokine combination 2: GM-CSF (500 U / mL), IL-4 (500 U / mL), tumor necrosis factor α (TNF-α, 500 U / mL), IL-10 (500 U / mL).

[0090] (5) Preparation of DC-derived nanoparticles

[0091] 10 million dendritic cells (DCs) incubated with cytokine component 1 were collected by centrifugation at 400g for 5 minutes. The cells were then washed twice with physiological saline, resuspended in saline, and sonicated at 4°C and 7.5W for 20 minutes to disrupt the cells and prepare a sample containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes, and the supernatant was collected. The supernatant was then centrifuged at 7000g for 20 minutes, and the supernatant was collected again. Finally, the supernatant was collected after centrifugation at 15000g for 120 minutes, and the supernatant was discarded. The precipitate was resuspended in PBS to obtain nanoparticle 2, with a particle size of 120 nm.

[0092] Alternatively, collect 10 million DCs prepared in step (3) that have not been activated by any nanoparticles or microparticles, wash the cells twice with physiological saline, resuspend the cells in physiological saline, and sonicate them at 4°C and 7.5W for 20 minutes to destroy the cells and prepare a sample containing cell membrane components. Then, centrifuge the sample at 2000g for 20 minutes and collect the supernatant. Centrifuge the supernatant at 7000g for 20 minutes and collect the supernatant. Incubate the supernatant with 40mg of nanoparticles 1 (20mg of nanoparticles loaded with water-soluble components + 20mg of nanoparticles loaded with insoluble components) prepared in step (2) for 10 minutes. Then, repeatedly co-extract the supernatant using a 0.45μm filter membrane. Centrifuge the extrudate at 15000g for 120 minutes and collect the supernatant. Collect the precipitate and resuspend it in PBS to obtain nanoparticles 3 with a particle size of 300nm.

[0093] Alternatively, 10 million DCs (cells) incubated with cytokine component 2 were collected by centrifugation at 400g for 5 minutes. The cells were then washed twice with physiological saline, resuspended in physiological saline, and sonicated at 4°C and 7.5W for 20 minutes to destroy the cells and prepare a sample containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected. The supernatant was then centrifuged at 7000g for 20 minutes and the supernatant was collected again. The supernatant was co-incubated with 40mg of nanoparticles 1 (20mg of nanoparticles loaded with water-soluble components + 20mg of nanoparticles loaded with insoluble components) prepared in step (2) for 10 minutes. The nanoparticles were then repeatedly co-extruded using a 0.45μm filter membrane. The extrudate was collected by centrifugation at 15000g for 120 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS to obtain nanoparticles. Nanoparticles 4, with a particle size of 300nm, were obtained by co-incubating nanoparticles 1 with the membrane components.

[0094] Alternatively, collect 10 million DCs after incubation with cytokine component 1 by centrifuging at 400g for 5 minutes, then wash the cells twice with physiological saline, resuspend the cells in physiological saline, and sonicate at 4°C and 7.5W for 20 minutes to destroy the cells and prepare a sample containing cell membrane components. Then centrifuge the sample at 2000g for 20 minutes and collect the supernatant. Centrifuge the supernatant at 7000g for 20 minutes and collect the supernatant. Co-incubate the supernatant with 40mg of nanoparticles 1 (20mg of nanoparticles loaded with water-soluble components + 20mg of nanoparticles loaded with insoluble components) prepared in step (2) for 10 minutes. Then co-extract the mixture repeatedly using a 0.45μm filter membrane. Centrifuge the extrudate at 15000g for 120 minutes, discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain nanoparticle 5 with a particle size of 300nm.

[0095] (6) Detection of cancer cell-specific T cells

[0096] 0.5 × 10⁻⁶ cells were subcutaneously injected into the back of each C57BL / 6 mouse. 5 Several B16F10 cells were observed when the tumor reached a volume of approximately 1000 mm². 3 Mice were sacrificed and spleen cells were harvested. Single-cell suspensions of the mouse spleen cells were prepared, and CD3+ cells were then sorted from the single-cell suspensions using flow cytometry. + T cells.

[0097] Nanoparticles 1 (50 μg of nanoparticles loaded with water-soluble components + 50 μg of nanoparticles loaded with insoluble components), or nanoparticles 2 (100 μg), 3 (100 μg), 4 (100 μg), or 5 (100 μg) were incubated with 1 million T cells from spleen cells in 10 mL of RPMI 1649 complete medium for 72 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, the T cells were first treated with Fc blocks to avoid nonspecific loading. Extracellular staining of mouse spleen cells was performed using CD3, CD4, and CD8 antibodies. Cells were then fixed and permeabilized, and intracellular staining of T cells was performed using FN-γ antibody. Flow cytometry was then used to analyze the T cell samples. CD4 values ​​were analyzed separately. + 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. Above CD4 + IFN-γ + T cells and CD8 + IFN-γ + T cells are cancer cell-specific T cells.

[0098] Alternatively, 1 million T cells derived from spleen cells were co-incubated in 10 mL of RPMI 1649 complete medium for 72 hours (37°C, 5% CO2). After cell collection, the cells were centrifuged at 400 g for 5 minutes, resuspended in PBS, and treated with Fcblock to avoid non-specific cell loading. Extracellular staining of mouse spleen cells was then performed using anti-mouse CD3, anti-mouse CD4, and anti-mouse CD8 antibodies linked to specific fluorescent probes. Cells were then fixed and permeabilized, and intracellular staining was performed using anti-mouse IFN-γ antibodies linked to fluorescent probes. Flow cytometry was then used to detect T cells containing IFN-γ antibody-linked fluorescent signals. CD4+ was analyzed separately. + 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. Above CD4 + IFN-γ + T cells and CD8 + IFN-γ+ T cells are cancer cell-specific T cells.

[0099] After nanoparticles / microparticles loaded with whole-cell components of tumor tissue and / or cancer cells are phagocytosed by antigen-presenting cells, the antigens are degraded into polypeptide epitopes and bound to major histocompatibility complex (MHC) molecules, which are then presented to the surface of the antigen-presenting cell membrane. Since whole-cell antigens loaded on nanoparticles and microparticles can be cross-presented, cancer cell antigen epitopes can be presented to the surface of the antigen-presenting cell membrane via both MHC I and MHC II pathways. After the cell membrane of antigen-presenting cells is prepared into nanoparticles or microparticles, the loaded MHC molecules and antigen polypeptide complexes can directly bind to T cell surface receptors that specifically recognize cancer cell antigens. Furthermore, if the T cell is a specific T cell capable of killing cancer cells, the cancer cell will begin to secrete cytotoxic substances such as IFN-γ and granzymes. By analyzing the T cells that secrete cytotoxic substances, the proportion of effector cancer cell-specific T cells with the ability to recognize and kill cancer cells can be obtained.

[0100] (8) Experimental Results

[0101] like Figure 2As shown, the number of cancer cell-specific T cells in the T cell control group and the nanoparticle 1 detection group was very low. However, nanoparticles 2, 3, 4, and 5 could all assist in the detection of a certain amount of cancer cell-specific T cells. Among them, nanoparticle 5 showed the best effect, outperforming nanoparticles 2, 3, and 4. The superiority of nanoparticle 5 over nanoparticle 4 indicates that adding cytokine combination 1 during the activation of antigen-presenting cells is more effective than cytokine component 2; the superiority of nanoparticle 5 over nanoparticle 2 indicates that solid nanoparticles with antigen-presenting cell membrane components on the surface and cell-loaded interiors are more effective than nanovesicle structures with only membrane components on the surface; the superiority of nanoparticles 4 and 5 over nanoparticle 3 indicates that nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell components of cancer cells are more effective than nanoparticles prepared from unactivated antigen-presenting cells. In summary, the particle system described in this invention can be used to detect cancer cell-specific T cells. Antigen-presenting cells (ARTCs) activated by nanoparticles loaded with whole-cell components of cancer cells degrade and present broad-spectrum whole-cell antigens from the cancer cell components loaded with the engulfed nanoparticles. The cancer cell antigenic epitopes presented to the cell membrane surface by the ARTCs have already bound to the major histocompatibility complex (MHC) molecules. After mechanical disruption of the ARTCs, the cell membrane components of the ARTCs contain MHC-bound antigenic epitopes. Through centrifugation and / or filtration using a filter membrane with a specific pore size and / or co-processing with nanoparticles or microparticles, the cell membrane components of the ARTCs will form nanoparticles or microparticles loaded with MHC molecules and degraded and presented cancer cell antigenic epitopes. Therefore, cancer cell-specific T cells can be directly activated for detection without the assistance of ARTCs.

[0102] Example 2: Detection of Cancer Cell-Specific T Cells Based on Antigen-Presenting Cell Particles

[0103] This embodiment uses mouse melanoma as a cancer model to illustrate how to prepare nanoparticle-assisted detection of cancer cell-specific T cells using nanoparticle-activated antigen-presenting cells. In this embodiment, B16F10 melanoma tumor tissue was lysed to prepare water-soluble and insoluble components of the tumor tissue. Then, using PLGA as the nanoparticle framework material and poly(I:C) and CpG1018 as immunoadjuvants, a nanoparticle system loaded with the water-soluble and insoluble components of the tumor tissue was prepared by solvent evaporation. The nanoparticles were then used to activate antigen-presenting cells, and the activated antigen-presenting cells were prepared into nanoparticle-based detection of cancer cell-specific T cells in peripheral blood immune cells.

[0104] (1) Lysis of tumor tissue and collection of its components

[0105] 1.5 × 10⁻⁶ mmol / L was subcutaneously injected into the back of each C57BL / 6 mouse. 5 Several B16F10 cells were observed when the tumor reached a volume of approximately 1000 mm². 3 Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to a freeze-thaw cycle five times, possibly accompanied by sonication, to destroy and lyse the cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. Adding 8M urea to the resulting precipitate dissolved the insoluble component, converting it into a soluble component in an 8M urea aqueous solution. The water-soluble and insoluble components were mixed at a 1:1 mass ratio to obtain the antigen raw material for preparing the nanoparticle system.

[0106] (2) Preparation of nanoparticle systems

[0107] In this embodiment, the nanoparticles were prepared using a solvent evaporation method. The nanoparticle 1 (NP1) loaded with whole-cell components was prepared using PLGA, which has a molecular weight of 7 Da-17 KDa. The adjuvants used were poly(I:C) and CpG1018, with the adjuvants encapsulated within the nanoparticles. The preparation method was as described previously. First, the antigen and adjuvant were loaded internally into the nanoparticles using a double emulsion method. After loading the antigen (lysed component) internally, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm; each mg of PLGA nanoparticles loaded approximately 100 μg of protein and polypeptide components, and each mg of PLGA nanoparticles used 0.02 mg of poly(I:C) and CpG1018 adjuvants. Nanoparticle 1 is also referred to as nanovaccine 1. In this embodiment, peptide nanoparticles 2, loaded with equal masses of four peptide neoantigens—B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ), and TRP2:180-188 (SVYDFFVWL)—were used as control nanoparticles. The preparation materials and methods were the same as for nanoparticle 1. Control nanoparticle 2 had a particle size of approximately 280 nm, loaded with 100 μg of peptide components, and loaded with an equal amount of adjuvant. Blank nanoparticle 3 was prepared using the same materials and methods as nanoparticle 1, with a particle size of approximately 280 nm, but loaded only with an equal amount of immune adjuvant without loading any antigen components.

[0108] (3) Preparation of antigen-presenting cells

[0109] Bone marrow-derived dendritic cells (BMDCs) and B cells were used as antigen-presenting cells. The preparation of BMDCs was the same as in Example 1. The B cell extraction procedure was as follows: After euthanizing the mice, the spleen was removed, and a single-cell suspension of mouse spleen cells was prepared. CD19 cells were then separated from the single-cell suspension using magnetic bead sorting. + B cells. BMDCs and B cells are mixed in a 1:1 ratio and used as mixed antigen-presenting cells.

[0110] (4) Activation of antigen-presenting cells

[0111] Nanoparticle 1 (500 μg) or peptide nanoparticle 2 (500 μg) or blank nanoparticle 3 (500 μg) + free lysis buffer were incubated with 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million B cells) in 15 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2); the incubation system contained a combination of cytokines: IL-15 (500 U / mL), IL-2 (500 U / mL), IL-7 (500 U / mL), and IL-12 (1000 U / mL).

[0112] Alternatively, nanoparticle 1 and 20 million BMDCs were co-incubated in 15 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2); the incubation system contained a combination of cytokines: IL-15 (500 U / mL), IL-2 (500 U / mL), IL-7 (500 U / mL), and IL-12 (1000 U / mL).

[0113] (5) Preparation of nanoparticles based on antigen-presenting cells

[0114] 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million B cells) were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with physiological saline, resuspended in physiological saline, and sonicated at 4°C with low power (7.5W) for 10 minutes to destroy the cells and prepare a sample containing cell membrane components. The sample was then filtered through filter membranes with pore sizes of 50μm, 10μm, 5μm, 1μm, 0.45μm, and 0.22μm. The collected filtrate was co-incubated for 10 minutes with the corresponding nanoparticles 1 (50mg) loaded with whole cell components of cancer cells, or peptide nanoparticles 2 (50mg), or blank nanoparticles 3 (50mg) prepared in step (2). The nanoparticles were then repeatedly co-extruded through a 0.45μm filter membrane. The extrudate was centrifuged at 15000g for 60 minutes, the supernatant was discarded, and the precipitate obtained after resuspending in physiological saline was the nanoparticle. Among them, nanoparticle 4 is prepared by co-processing the mixed antigen-presenting cell membrane component activated by nanoparticle 1 with nanoparticle 1, and has a particle size of 300 nm; nanoparticle 5 is prepared by co-processing the mixed antigen-presenting cell membrane component activated by peptide nanoparticle 2 with peptide nanoparticle 2, and has a particle size of 300 nm; nanoparticle 6 is prepared by co-processing the mixed antigen-presenting cell membrane component activated by blank nanoparticle 3 with blank nanoparticle 3, and has a particle size of 300 nm.

[0115] Alternatively, 20 million BMDCs incubated with nanoparticle 1 can be collected by centrifuging at 400g for 5 minutes. The BMDCs are then washed twice with physiological saline. After resuspending the cells in physiological saline, they are sonicated at 4°C with low power (7.5W) for 10 minutes to destroy the cells and prepare a sample containing cell membrane components. The sample is then filtered through filter membranes with pore sizes of 50μm, 10μm, 5μm, 1μm, 0.45μm, and 0.22μm. The filtrate is collected and co-incubated with nanoparticle 1 (50mg) prepared in step (2) for 10 minutes. Then, the sample is repeatedly co-extruded through a 0.45μm filter membrane. The extrudate is centrifuged at 15000g for 60 minutes, the supernatant is discarded, and the precipitate is resuspended in physiological saline. The precipitate has a particle size of 300nm.

[0116] (6) Detection of cancer cell-specific T cells

[0117] On day 0, 1.5 × 10⁻⁶ mmol / L was subcutaneously injected into the back of each C57BL / 6 mouse. 5B16F10 cells were administered subcutaneously to mice on days 10 and 14, consisting of 100 μL of 1 mg PLGA nanovaccine loaded with whole-cell cancer cells or 100 μL of PBS. Tumor growth rates were monitored in both the PBS and vaccine groups. Mice were sacrificed on day 18, and peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were isolated from the PBMCs using density gradient centrifugation, and CD3+ cells were then isolated from the PBMCs using flow cytometry. + T cells. Among them, the CD3 group treated with nanovaccine 1... + T cells were from the vaccine-treated group, while T cells from the PBS-treated group served as the PBS control group.

[0118] The 100 μg nanoparticles (nanoparticle 4, nanoparticle 5, nanoparticle 6, or nanoparticle 7) based on antigen-presenting cells prepared in step (5) were co-incubated with 5 million B cells and 400,000 T cells derived from tumor-infiltrating lymphocytes (vaccine group or PBS group) in 5 mL of RPMI 1640 complete medium for 24 hours (37°C, 5% CO2). Then, the incubated CD3 cells were sorted by flow cytometry. + IFN-γ + T cells. Simultaneously, CD3 was analyzed. + IFN-γ + IFN-γ in T cells + The mean fluorescence intensity (MFI) of the connected fluorescent probe. The stronger the fluorescence intensity, the more cytotoxic substances are expressed by the cancer cell-specific T cells, resulting in higher sensitivity and better accuracy in detection.

[0119] Alternatively, 100 μg of nanoparticles (nanoparticle 1, nanoparticle 2, or nanoparticle 3) prepared in step (2) can be co-incubated with 5 million B cells and 400,000 T cells derived from tumor-infiltrating lymphocytes (vaccine group or PBS group) in 5 mL of RPMI 1640 complete medium for 24 hours (37°C, 5% CO2), and then CD3+ cells after incubation can be sorted by flow cytometry. + IFN-γ + T cells. Simultaneously, CD3 was analyzed. + IFN-γ + IFN-γ in T cells + The average fluorescence intensity (MFI) of the attached fluorescent probe.

[0120] Alternatively, 100 μg of antigen-presenting cell-based nanoparticles (nanoparticle 4, nanoparticle 5, nanoparticle 6, or nanoparticle 7) prepared in step (5) can be co-incubated with 400,000 T cells derived from tumor-infiltrating lymphocytes (vaccine group or PBS group) in 5 mL of RPMI 1640 complete medium for 24 hours (37°C, 5% CO2), and then CD3+ cells after incubation can be sorted by flow cytometry. + IFN-γ + T cells. Simultaneously, CD3 was analyzed. + IFN-γ + IFN-γ in T cells + The mean fluorescence intensity (MFI) of the connected fluorescent probe.

[0121] Alternatively, 100 μg of nanoparticles (nanoparticle 1) prepared in step (2) can be co-incubated with 400,000 T cells (vaccine group) derived from tumor-infiltrating lymphocytes in 5 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2), and then CD3+ cells after incubation can be sorted by flow cytometry. + IFN-γ + T cells.

[0122] (7) Experimental Results

[0123] like Figure 3 As shown in figures a and b, the tumor growth rate in the PBS group was significantly faster than that in the vaccine group. This indicates that the vaccine group mice had significantly more cancer cell-specific T cells than the PBS group, thus delaying and controlling tumor growth. The PBS group was not induced by the vaccine, therefore it contained fewer cancer cell-specific T cells, while the vaccine group, after induction, had more cancer cell-specific T cells, such as... Figure 3As shown, the PBS control group and the vaccine group showed different results after detection with different nanoparticles. Nanoparticle 4 showed the best performance, detecting the most comprehensive range of cancer cell-specific T cells. Nanoparticle 1 could not detect cancer cell-specific T cells without the assistance of antigen-presenting cells. Nanoparticles 4, 5, and 7 could also detect cancer cell-specific T cells without the assistance of antigen-presenting cells, with nanoparticle 4 showing significantly better performance than the other two nanoparticles. Moreover, nanoparticle 4 detected cancer cell-specific T cells better without the assistance of antigen-presenting cells than nanoparticle 7 detected cancer cell-specific T cells with the assistance of antigen-presenting cells. Furthermore, there was little difference in the number of cancer cell-specific T cells detected by nanoparticles 4, 5, and 7 with and without the assistance of antigen-presenting cells. This indicates that nanoparticles prepared using antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens of cancer cells are beneficial for better detection of cancer cell-specific T cells; and that mixed antigen-presenting cells of DCs and B cells are better than single DCs. Moreover, the method for detecting broad-spectrum cancer cell-specific T cells described in this invention can be performed without relying on antigen-presenting cells. Moreover, in the process of detecting cancer cell-specific T cells, it is not necessary to add antigen-presenting cells to achieve the same effect as adding antigen-presenting cells in detecting cancer cell-specific T cells. This is also an advantage of the nanoparticles or microparticles prepared from activated antigen-presenting cells described in this invention for detecting cancer cell-specific T cells.

[0124] Nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens of cancer cells showed better detection of cancer-specific T cells than those prepared from nanoparticles activated by nanoparticles loaded with four novel antigenic peptides. This indicates that nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with four novel antigenic peptides can detect a limited variety of cancer-specific T cell clones. In contrast, nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens of cancer cells can detect a broader spectrum of cancer-specific T cells, thus resulting in a wider range of T cell clones after expansion.

[0125] like Figure 3As shown in Figure c, stronger fluorescence intensity indicates that the cancer cell-specific T cells express more cytotoxic substances, resulting in higher sensitivity and accuracy during detection. When activated cancer cell-specific T cells were labeled with anti-mouse IFN-γ antibodies using the same fluorescent probe, the fluorescence signal (mean fluorescence intensity, MFI) of the fluorescent probe linked to the IFN-γ antibody activated by nanoparticle 4, which was loaded with both activated antigen-presenting cell membrane components and whole-cell components of cancer cells, was stronger than that of the fluorescent probe linked to the IFN-γ antibody activated by nanoparticle 1, which was loaded only with whole-cell components of cancer cells. Moreover, regardless of whether antigen-presenting cells were present in the incubation system during detection and activation, the fluorescence signal detected by cancer cell-specific T cells activated by nanoparticle 4 was stronger than that of cancer cell-specific T cells activated by nanoparticle 1. This indicates that cancer cell-specific T cells activated by nanoparticle 4 expressed more specific biomarkers and were therefore easier to detect.

[0126] Therefore, the nanoparticles prepared by antigen-presenting cells activated by nanoparticles loaded with whole-cell components as described in this invention can better detect cancer cell-specific T cells with the ability to recognize and kill cancer cells. The whole-cell antigens of cancer cells loaded on the nanoparticles are degraded into antigen epitopes after being phagocytosed by antigen-presenting cells and presented on the surface of the antigen-presenting cells. Specific T cells that can recognize the whole-cell antigens of cancer cells are activated and express specific surface markers. By analyzing the proportion of T cells highly expressing specific surface markers using flow cytometry, the number and proportion of activated cancer cell-specific T cells with recognition and killing efficacy can be determined.

[0127] Example 3: Detection of cancer cell-specific T cells in mouse peripheral spleen cells

[0128] This embodiment uses mouse melanoma as a cancer model to illustrate how to use nanoparticles prepared from antigen-presenting cells activated by nanoparticles to detect cancer cell-specific T cells. In this embodiment, B16F10 melanoma tumor tissue and cancer cells are first lysed to prepare a mixture of water-soluble components (mass ratio 1:1) and an insoluble component mixture (mass ratio 1:1), and then the water-soluble and insoluble component mixtures are mixed at a 1:1 mass ratio. Then, using PLGA as the nanoparticle framework material and Poly(I:C) and CpG2006 as adjuvants, nanoparticles loaded with the lysate components are prepared. The nanoparticles are then co-incubated with antigen-presenting cells for a period of time to activate the antigen-presenting cells, and the antigen-presenting cells are then used to prepare nanoparticles for detecting cancer cell-specific T cells.

[0129] (1) Lysis of tumor tissue and cancer cells and collection of their components

[0130] When collecting tumor tissue, 1.5 × 10⁻⁶ mmol / L was first subcutaneously injected into the back of each C57BL / 6 mouse. 5 Several B16F10 cells were observed when the tumor reached a volume of approximately 1000 mm². 3 Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and filtered through a cell filter. A suitable amount of pure water was added, and the mixture was subjected to five freeze-thaw cycles, possibly accompanied by sonication, to destroy the lysed sample. When collecting cultured B16F10 cancer cell lines, the culture medium was removed by centrifugation, followed by washing twice with PBS and centrifugation to collect the cancer cells. The cancer cells were resuspended in ultrapure water and subjected to three freeze-thaw cycles, accompanied by sonication, to destroy and lyse the cancer cells. After the tumor tissue or cancer cells were lysed, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. Adding 8M urea to the resulting precipitate dissolved the insoluble component in pure water, converting it into a soluble component in an 8M urea aqueous solution. The water-soluble and insoluble components of the tumor tissue and cancer cells were mixed at a 1:1 mass ratio; the insoluble components of the tumor tissue and cancer cells were also mixed at a 1:1 mass ratio. The mixture of water-soluble and insoluble components at a 1:1 mass ratio provided the antigenic raw material for preparing nanoparticles.

[0131] (2) Preparation of bacterial extracellular vesicles (OMV)

[0132] Bifidobacterium longum was centrifuged at 5000g for 30 minutes, and the supernatant was collected after discarding the precipitate. The supernatant was filtered through a 1μm filter membrane, sonicated at 20W for 5 minutes at 4℃, and then centrifuged at 16000g for 90 minutes. The precipitate was resuspended in PBS to obtain the collected bacterial extravesicular membrane component. The bacterial extravesicular membrane component was then lysed and dissolved using 8M urea aqueous solution.

[0133] Alternatively, centrifuge Bifidobacterium longum at 5000g for 30 minutes, discard the precipitate and collect the supernatant. Filter the supernatant through a 1μm filter membrane, sonicate at 20W for 5 minutes at 4°C, and then centrifuge at 16000g for 90 minutes. Resuspend the precipitate in PBS to obtain the collected bacterial extravesicular membrane component. Then, use Tween 80 aqueous solution to lyse and dissolve the bacterial membrane component.

[0134] (3) Preparation of nanoparticles

[0135] In this embodiment, nanoparticle 1 was prepared using a double emulsion method. The molecular weight of the PLGA material was 7 kDa-17 kDa, and the adjuvants used were poly(I:C) and CpG2006, with the adjuvants encapsulated within the nanoparticles. The preparation method was as described above. First, the lysis buffer components and adjuvants were loaded inside the nanoparticles using a double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h for later use. The average particle size of nanoparticle 1 was approximately 250 nm. Each mg of PLGA nanoparticle 1 loaded approximately 130 μg of protein or polypeptide components, and each mg of PLGA nanoparticle 1 loaded with 0.02 mg each of poly(I:C) and CpG2006 adjuvants. Nanoparticle 1 is also referred to as nanovaccine 1 during injection.

[0136] In this embodiment, the materials and preparation method for nanoparticle 2 are the same as those for nanoparticle 1. Nanoparticle 2 is loaded with both the antigen component prepared in step (1) and the bacterial extravesicular membrane component dissolved in 8M urea prepared in step (2), with a mass ratio of 1:1. The adjuvants used are poly(I:C) and CpG2006, and the adjuvants are encapsulated within the nanoparticles. During the preparation process, a double emulsion method is first used to load the tumor tissue lysis fluid component, bacterial extravesicular membrane component, and adjuvants inside the nanoparticles. Then, 100 mg of nanoparticles are centrifuged at 10000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h for later use. The average particle size of nanoparticle 2 is approximately 250 nm. Each 1 mg of PLGA nanoparticle 2 is loaded with approximately 130 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticle 2 is loaded with 0.02 mg of poly(I:C) and 0.02 mg of CpG2006 adjuvants.

[0137] In this embodiment, the materials and preparation method for nanoparticle 3 are the same as those for nanoparticle 1. Nanoparticle 3 is loaded with both the antigen component prepared in step (1) and the bacterial extravesicular membrane component dissolved in Tween 80 prepared in step (2), with a mass ratio of 1:1. The adjuvants used are poly(I:C) and CpG2006, and the adjuvants are encapsulated within the nanoparticles. During the preparation process, a double emulsion method is first used to load the tumor tissue lysis buffer component, bacterial extravesicular membrane component, and adjuvants inside the nanoparticles. Then, 100 mg of nanoparticles are centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h for later use. The average particle size of nanoparticle 3 is approximately 250 nm. Each 1 mg of PLGA nanoparticle 3 is loaded with approximately 130 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticle 3 is loaded with 0.02 mg of poly(I:C) and CpG2006 adjuvants.

[0138] The materials and preparation method for blank nanoparticles 4 are the same as those for nanoparticles 1, but blank nanoparticles 4 are loaded with only an equal amount of adjuvant and without any tumor tissue lysate components. The particle size of nanoparticles 4 is approximately 250 nm.

[0139] (4) Isolation of B cells

[0140] After euthanizing C57BL / 6 mice, the spleens were harvested, and a single-cell suspension of mouse spleen cells was prepared. CD19 cells were then isolated from the spleen cells using magnetic bead sorting. + B cells.

[0141] (5) Activation of antigen-presenting cells

[0142] 500 μg of nanoparticle 1, or 500 μg of nanoparticle 2, or 500 μg of nanoparticle 3, or 500 μg of nanoparticle 4 were incubated with 10 million B cells in 15 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). The incubation system contained GM-CSF (2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), albumin (50 ng / mL), and CD80 antibody (10 ng / mL).

[0143] (6) Preparation of nanoparticles derived from antigen-presenting cells

[0144] B cells were collected after incubation by centrifugation at 400g for 5 minutes, then washed three times with PBS. After resuspending the cells in PBS, they were sonicated at low power (10W) for 15 minutes. The sample was then centrifuged at 500g for 5 minutes and the supernatant was collected. The supernatant was filtered sequentially through membranes with pore sizes of 30μm, 10μm, 5μm, 0.45μm, and 0.22μm. The resulting filtrate was centrifuged at 18000g for 60 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain nanoparticles. Among them, nanoparticle 5 was prepared using antigen-presenting cells activated by nanoparticle 1, with a particle size of 110 nm; nanoparticle 6 was prepared using antigen-presenting cells activated by nanoparticle 2, with a particle size of 110 nm; nanoparticle 7 was prepared using antigen-presenting cells activated by nanoparticle 3, with a particle size of 110 nm; and nanoparticle 8 was prepared using antigen-presenting cells activated by nanoparticle 4, with a particle size of 110 nm.

[0145] (7) Detection of cancer cell-specific T cells

[0146] On day 0, each C57BL / 6 mouse was subcutaneously injected with 1.5 × 10⁻⁶ mmol / L on its back. 5On days 7, 12, and 17, mice were subcutaneously injected with 0.7 mg of PLGA nanoparticles 1 (nanovaccine 1) prepared in step (3) or 100 μL of PBS. Mice were sacrificed on day 21, and their spleens were harvested and a single-cell suspension of the spleen was prepared. CD3+ cells from the spleen cells were then sorted using magnetic bead sorting. + T cells. The isolated T cells (4 million) were co-incubated with 100 μg of nanoparticles (nanoparticle 5, nanoparticle 6, nanoparticle 7, or nanoparticle 8) prepared in step (6) in 40 mL of high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2). Cells were then collected by centrifugation at 400 g for 5 minutes, and after antibody labeling, CD3+ in the incubated T cells was analyzed by flow cytometry. + IFNγ + T cells are the number and proportion of cancer cell-specific T cells that are specifically activated by cancer cell whole-cell antigens.

[0147] Cancer cell whole-cell antigens loaded on nanoparticles can be degraded into antigenic epitopes after being phagocytosed by antigen-presenting cells and presented on the surface of the antigen-presenting cell membrane. Nanoparticles prepared from antigen-presenting cells, loaded with these degraded and presented antigenic epitopes, can be recognized by cancer cell-specific T cells, activating them and leading to the secretion of cytotoxic cytokines. IFN-γ is the most important cytokine secreted by antigen-specific T cells after recognition of antigens. CD3+ was analyzed using flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells that can recognize and kill cancer cells.

[0148] (5) Experimental Results

[0149] like Figure 4 As shown, the tumors in the PBS control group mice grew rapidly, while those in the nanoparticle-treated group grew more slowly. This indicates that nanoparticles induce cancer cell-specific T cells, which can control tumor growth. Nanoparticle 8 hardly activated cancer cell-specific T cells; while nanoparticles 5, 6, and 7 detected more cancer cell-specific T cells. Moreover, nanoparticle 6 was more effective than nanoparticles 5 and 7, suggesting that antigen-presenting cells activated by loading bacterial exovesicle components, lysed and dissolved using appropriate methods, onto nanoparticles are beneficial for detecting cancer cell-specific T cells.

[0150] Example 4: Detection of cancer cell-specific T cells using nanoparticles or microparticles

[0151] In this embodiment, 6M guanidine hydrochloride was first used to lyse the whole-cell antigen of B16F10 melanoma cancer cells. Then, using PLGA as the microparticle framework material and CpG BW006 (class B), CPG2216 (class A), and Poly ICLC as immunoadjuvants, a microparticle system loaded with the whole-cell antigen of cancer cells was prepared. After activating antigen-presenting cells using the microparticles, the antigen-presenting cells were prepared into nanoparticles or microparticles to detect cancer cell-specific T cells.

[0152] (1) Lysis of cancer cells

[0153] After collecting the cultured B16F10 melanoma cancer cell line, centrifuge at 350g for 5 minutes, discard the supernatant, wash twice with PBS, then resuspend and lyse the cancer cells with 6M guanidine hydrochloride. The whole-cell antigen of the cancer cells was lysed and dissolved in 6M guanidine hydrochloride, which became the source of antigen raw materials for preparing the micron particle system.

[0154] (2) Preparation of micron-sized particle systems

[0155] In this embodiment, the microparticles were prepared using a double emulsion method. The microparticle 1 material used had a molecular weight of PLGA (38 kDa-54 kDa), and the immunoadjuvants used were CpG BW006, CPG2216, and Poly ICLC. Poly ICLC is a Toll-like receptor 3 agonist, while various CpGs are Toll-like receptor 9 agonists. Both Toll-like receptor 3 and Toll-like receptor 9 are located in the endosome membrane structure within cells. First, the lysate components and immunoadjuvants were co-loaded into the microparticles, then centrifuged at 10000g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Before use, the particles were resuspended in 7 mL of PBS, then 3 mL of cancer cell lysate components (protein concentration 50 mg / mL) were added, and the mixture was incubated at room temperature for 10 minutes to obtain microparticle 1 loaded with lysate both internally and externally. The average particle size of these microparticles is about 2.50 μm, and the surface potential is about -2 mV. Each 1 mg of PLGA microparticles is loaded with about 140 μg of protein or polypeptide components, including 0.02 mg each of CpG BW006 (Class B), CPG2216 (Class A), and Poly ICLC.

[0156] The control microparticles 2 were prepared using the same materials and methods, but were loaded with CpG2336 (class A), CPG2216 (class A), and Poly ICLC as immunoadjuvants. The control microparticles 2 had a particle size of approximately 2.50 μm and a surface potential of approximately -2 mV. Each mg of PLGA microparticles loaded approximately 140 μg of protein or peptide components, and each mg of PLGA microparticles contained 0.02 mg each of CpG2336 (class A), CPG2216 (class A), and Poly ICLC immunoadjuvants.

[0157] The control microparticles 3 were prepared using the same materials and methods, but were loaded with CpG BW006 (class B) and CPG2216 (class A) as immunoadjuvants. Each 1 mg of PLGA microparticles in the control microparticles 3 contained 0.02 mg of adjuvant, had a particle size of approximately 2.50 μm, a surface potential of approximately -2 mV, and loaded approximately 140 μg of protein or polypeptide components per 1 mg of PLGA microparticles. Each 1 mg of PLGA microparticles also contained 0.03 mg each of CpG BW006 (class B) and CPG2216 (class A).

[0158] (3) Preparation of antigen-presenting cells

[0159] After euthanizing the mice, the lymph nodes and spleen were collected. The lymph nodes or spleen were chopped and ground, and then filtered through a cell sieve to prepare single-cell suspensions. The lymph node single-cell suspensions and spleen single-cell suspensions were mixed and CD19 cells were isolated by flow cytometry. + B cells and CD11c + DC.

[0160] (4) Activation of antigen-presenting cells

[0161] Microparticles (500 μg) loaded with whole-cell components of cancer cells were co-incubated with 10 million DCs and 10 million B cells in 20 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2). The incubation system contained granulocyte-macrophage colony-stimulating factor (GM-CSF, 2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL) and CD86 antibody (10 ng / mL).

[0162] (5) Preparation of nanoparticles or microparticles derived from antigen-presenting cells

[0163] DCs and B cells were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were sonicated at low power (22.5W) for 1 minute at 4°C. The samples were then centrifuged at 3000g for 15 minutes, and the supernatant was collected. The supernatant was then centrifuged at 8000g for 15 minutes, and the supernatant was collected again. Finally, the supernatant was collected after centrifugation at 16000g for 90 minutes, and the supernatant was discarded. The precipitate was resuspended in PBS to obtain nanoparticles. Nanoparticle 1, with a particle size of 110 nm, was prepared from mixed antigen-presenting cells activated by microparticle 1. Nanoparticle 2, with a particle size of 110 nm, was prepared from mixed antigen-presenting cells activated by microparticle 2. Nanoparticle 3, with a particle size of 110 nm, was prepared from mixed antigen-presenting cells activated by microparticle 3.

[0164] Alternatively, incubated DCs and B cells (activated using microparticles 1) can be collected by centrifuging at 400g for 5 minutes. The cells are then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they are sonicated at low power (22.5W) for 1 minute at 4°C. The sample is then centrifuged at 3000g for 15 minutes, and the supernatant is collected. The supernatant is then centrifuged at 8000g for 15 minutes, and the supernatant is collected again. After centrifugation at 16000g for 90 minutes, the supernatant is discarded, and the precipitate is collected. The precipitate is resuspended in PBS to obtain nanoparticles. 20 mg of nanoparticles are mixed with 100 mg of microparticles 1 and incubated at room temperature for 15 minutes. Then, the mixture is sonicated at 10W for 1 minute, centrifuged at 8000g for 15 minutes, the supernatant is discarded, and the precipitate is collected. The precipitate is resuspended to obtain microparticles 4, with a particle size of approximately 2.55 μm.

[0165] (6) Detection of cancer cell-specific T cells

[0166] On day 0, 1.5 × 10⁻⁶ mmol / L was subcutaneously injected into the back of each C57BL / 6 mouse. 5 Mice were treated with B16F10 cells. On days 10, 15, and 20, the tumor sites were irradiated with radiation or injected with 100 μL of PBS. Mice were sacrificed on day 24, and peripheral blood was collected from each group. Peripheral blood mononuclear cells (PBMCs) were then prepared, and CD3+ cells were sorted using magnetic bead sorting. + T cells. The sorted T cells (5 million units) and the nanoparticles 1-3 (100 μg) or microparticles 4 (100 μg) prepared in step (5) were co-incubated in 2 mL of RPMI 1640 complete medium for 24 hours (37℃, 5% CO2). Then, flow cytometry was used to detect CD3+ in the T cells. +IFN-γ + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens. Simultaneously, CD3 was analyzed. + IFN-γ + IFN-γ in T cells + The average fluorescence intensity (MFI) of the attached fluorescent probe.

[0167] (7) Experimental Results

[0168] like Figure 5 As shown in Figure a, nanoparticles prepared from antigen-presenting cells activated by microparticles loaded with a mixture of CpG adjuvant and Poly ICLC adjuvant showed better results in detecting cancer cell-specific T cells than nanoparticles prepared from antigen-presenting cells activated by microparticles loaded with a mixture of two CpG adjuvants. Furthermore, nanoparticles prepared from antigen-presenting cells activated by microparticles loaded with a mixture of one type B CpG, one type A CpG, and Poly ICLC adjuvant showed better results than nanoparticles prepared from antigen-presenting cells activated by microparticles loaded with a mixture of two type A CpGs and Poly ICLC adjuvants; moreover, particles prepared from the membrane of activated antigen-presenting cells that simultaneously internally loaded with whole-cell components of cancer cells showed even better results. CD3 + IFN-γ + IFN-γ in T cells + The analysis results of the mean fluorescence intensity (MFI) of the connected fluorescent probes are consistent with the above trend. Figure 5 (b) This indicates that antigen-presenting cells activated by microparticles loaded with a mixed adjuvant of two different Toll-like receptors can produce nanoparticles with better performance. Moreover, nanoparticles prepared from antigen-presenting cells activated by microparticles containing a mixed adjuvant of class B CpG and a Toll-like receptor 3 agonist are even more effective.

[0169] Example 5: Detection of cancer cell-specific T cells in tumor tissue

[0170] In this embodiment, B16F10 melanoma tumor tissue was first lysed using 8M urea, and the lysate components were dissolved. Then, nanoparticles loaded with cancer cell whole-cell antigens were prepared using PLGA as the nanoparticle framework material and Poly(I:C), CpG2006 (class B), and CpGSL01 (class B) as immunoadjuvants. After activating antigen-presenting cells with the nanoparticles, the nanoparticles were prepared, and then cancer cell-specific T cells in the tumor tissue were detected.

[0171] (1) Collection and lysis of tumor tissue

[0172] 1.5 × 10⁻⁶ mmol / L was subcutaneously injected into the back of each C57BL / 6 mouse. 5Several B16F10 cells were observed when the tumor reached a volume of approximately 1000 mm². 3 Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and then filtered through a cell filter. An appropriate amount of 8M urea was added to lyse the cells and dissolve the cell lysates. This is the source of the antigen raw materials for preparing the nanoparticle system.

[0173] (2) Preparation of nanoparticles

[0174] In this embodiment, the nanoparticles were prepared using a solvent evaporation method. The PLGA material used for nanoparticle 1 had a molecular weight of 7-17 kDa, and the immunoadjuvants used were Poly(I:C), CpG2006, and CpGSL01. The lysate components and adjuvants were encapsulated within the nanoparticles. The preparation method was as described above: after loading the lysate components and adjuvants inside the nanoparticles, 100 mg of nanoparticles were centrifuged at 12000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours to obtain a lyophilized powder for later use. The average particle size of these nanoparticles was approximately 270 nm, and the surface potential was approximately -3 mV. Each 1 mg of PLGA nanoparticles loaded approximately 80 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles used 0.02 mg each of Poly(I:C), CpG2006, and CpGSL01.

[0175] The control nanoparticles 2 were prepared using the same materials and methods as above, with a particle size of approximately 270 nm, loaded with an equal amount of lysate components, and loaded with the immunoadjuvant Poly(I:C), with 0.06 mg of Poly(I:C) loaded per 1 mg PLGA.

[0176] The preparation materials and methods for the control nanoparticles 3 were the same as above, with a particle size of about 270 nm. They were loaded with equal amounts of lysate components and were loaded with immune adjuvants Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A). Each 1 mg of PLGA was loaded with 0.02 mg of each of Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A).

[0177] (3) Preparation of DC and B cells

[0178] After euthanizing C57BL / 6 mice, lymph nodes were harvested, and single-cell suspensions of the lymph nodes were prepared. CD11c cells were then isolated from the lymph node single-cell suspensions using flow cytometry. + DC and CD19 + B cells.

[0179] (4) Activation of antigen-presenting cells

[0180] Nanoparticles 1 (500 μg), 2 (500 μg), or 3 (500 μg) loaded with whole-cell components of cancer cells were co-incubated with DCs (5 million cells) and B cells (5 million cells) in 20 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2). The incubation system contained granulocyte-macrophage colony-stimulating factor (GM-CSF, 2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), and CD86 antibody (10 ng / mL).

[0181] (5) Preparation of nanoparticles based on antigen-presenting cells

[0182] DCs and B cells were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were homogenized at 2000 rpm for 25 minutes at 4°C. The samples were then centrifuged at 3000g for 15 minutes, and the supernatant was collected. This supernatant was then centrifuged at 8000g for 15 minutes, and the supernatant was collected again. Finally, after centrifugation at 15000g for 30 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS to obtain nanoparticles. Nanoparticle 4 (150 nm) was prepared using antigen-presenting cells activated by nanoparticle 1; nanoparticle 5 (150 nm) was prepared using antigen-presenting cells activated by nanoparticle 2; and nanoparticle 6 (150 nm) was prepared using antigen-presenting cells activated by nanoparticle 3.

[0183] (6) Detection of cancer cell-specific T cells

[0184] On day 0, 1.5 × 10⁻⁶ mmol / L was subcutaneously injected into the back of each C57BL / 6 mouse. 5 Mice were injected subcutaneously with 100 μL of αPD-1 antibody (10 mg / kg) or 100 μL of PBS on days 8, 10, 12, 14, and 16, respectively. Mice were sacrificed on day 20, and tumor tissue was collected. The tumor tissue was cut into small pieces and filtered through a cell sieve to prepare a single-cell suspension. CD3+ was then separated from the suspension using magnetic bead sorting. + T cells. The sorted T cells (500,000) were then co-incubated with allogeneic B cells (2.5 million), nanoparticles prepared in step (5) (100 μg), or in 20 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). The mixture was then centrifuged at 400 g for 5 minutes, and the supernatant was collected. The concentration of IFN-γ in the supernatant was analyzed using ELISA.

[0185] In the ELISA detection method, activated cancer cell-specific T cells secrete cytotoxic substances such as IFN-γ. The concentration of these cytotoxic substances indicates the number of activated cancer-specific T cells and the strength of their cancer cell-killing ability.

[0186] (7) Experimental Results

[0187] like Figure 6 As shown, compared with the PBS control group, nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens of cancer cells showed a higher detection rate of IFN-γ after co-incubation. Furthermore, nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with two types of B CpGs and Poly(I:C) as a mixed adjuvant performed better than nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with two types of A CpGs and Poly(I:C) as a mixed adjuvant, or nanoparticles loaded only with Poly(I:C) as an adjuvant.

[0188] Example 6: Detection of cancer cell-specific T cells in colon cancer

[0189] This embodiment uses MC38 mouse colon cancer as a cancer model to illustrate how to use nanoparticles prepared from antigen-presenting cells activated by nanoparticles to detect a broad spectrum of cancer cell-specific T cells. First, colon cancer tumor tissue and lung cancer cells were lysed to prepare water-soluble and insoluble components. The antigen was then degraded into peptides in vitro using a protease. In practical applications, other enzymes or methods can also be used to degrade the proteins in the whole-cell components into peptides first. Then, a mixture of water-soluble and insoluble components (mass ratio 1:1) was prepared and mixed at a mass ratio of 1:1. Next, nanoparticles were prepared using PLA as the nanoparticle framework material and CpGM362, CPG1018, and Poly ICLC as immunoadjuvants. These nanoparticles were then used to detect cancer cell-specific T cells in vitro.

[0190] (1) Lysis of tumor tissue and cancer cells and collection of their components

[0191] 2 × 10⁻⁶ mice were subcutaneously injected into the back of each C57BL / 6 mouse. 6 Each MC38 cell grew to a tumor volume of approximately 1000 mm². 3Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to five freeze-thaw cycles, possibly accompanied by sonication, to destroy and lyse the cells. After cell lysis, the lysate was centrifuged at a speed greater than 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. 8M urea was added to the resulting precipitate to dissolve the insoluble component, converting it into a soluble component in 8M urea solution. Trypsin (0.5 mg / mL) and chymotrypsin (0.5 mg / mL) were added to the water-soluble component (80 mg / mL), and the mixture was incubated for 1 hour. The inactivated proteases were then inactivated by heating at 95°C for 10 minutes.

[0192] After collecting the cultured LLC lung cancer cell line, centrifuge at 350g for 5 minutes, discard the supernatant, wash twice with PBS, resuspend the cells in ultrapure water, and repeat freeze-thaw cycles 5 times, possibly accompanied by sonication to destroy lysed cells. After cell lysis, centrifuge the lysate at 3000g for 6 minutes and collect the supernatant, which is the water-soluble component soluble in pure water. Add 8M urea to the resulting precipitate to dissolve the insoluble component, thus converting it into a soluble component in 8M urea aqueous solution. Add trypsin (0.5mg / mL) and chymotrypsin (0.5mg / mL) to the water-soluble component (80mg / mL) and incubate for 1 hour, then heat at 95℃ for 10 minutes to inactivate the proteases for later use.

[0193] Water-soluble components from colon cancer tumor tissue and lung cancer cells were mixed at a mass ratio of 1:1; insoluble components dissolved in 8M urea were also mixed at a mass ratio of 1:1. The mixture of water-soluble components and the mixture of insoluble components were then mixed at a mass ratio of 1:1; this mixture served as the raw material source for preparing nanoparticles.

[0194] (2) Cleavage and dissolution of BCG

[0195] BCG was collected, and the BCG was pyrolyzed using an 8M urea aqueous solution. The pyrolyzed components were then dissolved for later use.

[0196] (3) Preparation of nanoparticles

[0197] In this embodiment, nanoparticles 1 were prepared using a solvent evaporation method. The PLA material used to prepare nanoparticles 1 has a molecular weight of 20 kDa. The nanoparticles are internally loaded with tumor tissue and cancer cell lysates, bacterial lysates, and an immune adjuvant, while the surface is loaded with components of the tumor tissue and cancer cell lysates. The immune adjuvants used are CpGM362, CPG1018, and poly ICLC, and the adjuvants are loaded internally within the nanoparticles. The mass ratio of tumor tissue and cancer cell lysates to bacterial lysates used in the preparation of the nanoparticles is 1:1. The preparation method is as described above. Firstly, a double emulsion method is used to load the mixture of lysates, bacterial lysate components, and adjuvants internally into the nanoparticles. After internal loading of the lysates and adjuvants, 100 mg of nanoparticles are centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. Before use, 20 mg of nanoparticles are resuspended in 0.9 mL of PBS and mixed with 0.1 mL of a sample containing an equal amount of a mixture of cancer cell and tumor tissue lysate and bacterial lysate (80 mg / mL). The mixture is then incubated at room temperature for 5 minutes before use. Nanoparticle 1 has an average particle size of approximately 290 nm. Each 1 mg of PLGA nanoparticle 1 is loaded with approximately 140 μg of protein or peptide components. Each 1 mg of PLGA nanoparticles contains 0.04 mg each of CpGM362, CPG1018, and Poly ICLC immunoadjuvant.

[0198] (4) Preparation of antigen-presenting cells

[0199] Peripheral blood was collected from mice after euthanasia of C57BL / 6 mice. Peripheral blood mononuclear cells (PBMCs) were isolated from the PBMCs, and CD11c was then sorted from the PBMCs using flow cytometry. + DC and CD19 + B cells. In this example, both BMDCs and BMDMs were used as antigen-presenting cells. The preparation method of BMDCs was the same as in Example 1. The preparation method of BMDMs is as follows:

[0200] C57 mice were anesthetized and euthanized by dislocation. The mice were disinfected with 75% ethanol. A small incision was made in the mouse's back with scissors, and the skin was torn open to the lower leg joint, removing the paw joint and skin. The hind limb was detached along the greater trochanter of the thigh, and the muscle tissue was removed. The limb was then immersed in a culture dish containing 75% ethanol for 5 minutes, and a new culture dish containing 75% ethanol was placed in a clean bench. The ethanol-soaked leg bone was then immersed in cold PBS to wash away the ethanol from the tibia and femur. This process could be repeated three times. The cleaned femur and tibia were separated, and both ends were cut off with scissors. Using a 1mL syringe, cold induction culture medium was drawn to expel bone marrow from the femur and tibia. This process was repeated three times until no obvious red color was visible inside the leg bone. Using a 5 mL pipette, repeatedly pipette the culture medium containing bone marrow cells to disperse cell clumps. Then, filter the cells through a 70 μm cell filter and transfer them to 15 mL centrifuge tubes. Centrifuge at 1500 rpm for 5 min, discard the supernatant, add erythrocyte lysis buffer, let stand for 5 min, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend in cold, prepared bone marrow macrophage induction medium (DMEM high-glucose medium containing 15% L929 medium). Plate the cells. Culture the cells overnight to remove other contaminating cells such as fibroblasts that adhere quickly. Collect non-adherent cells and seed them into dishes or cell culture plates according to the experimental design. Stimulate bone marrow cells with macrophage colony-stimulating factor (M-CSF) at a concentration of 40 ng / mL to induce differentiation into monocytes and macrophages. After 8 days of culture, observe the morphological changes of macrophages under a light microscope. Eight days later, the cells were digested and collected. They were then incubated at 4°C in the dark for 30 minutes with anti-mouse F4 / 80 antibody and anti-mouse CD11b antibody. The proportion of successfully induced macrophages was then determined by flow cytometry.

[0201] (5) Activation of antigen-presenting cells

[0202] Nanoparticle 1 (1000 μg) was co-incubated with peripheral blood-derived DCs (20 million units) and BMDCs (20 million units) in RPMI 1640 complete medium for 72 hours (37°C, 5% CO2); the incubation system contained GM-CSF (500 U / mL), IL-2 (500 U / mL), IL-7 (500 U / mL), IL-12 (500 U / mL) and CD80 antibody (10 ng / mL).

[0203] Alternatively, nanoparticle 1 (1000 μg) was co-incubated with peripheral blood-derived DCs (10 million), BMDCs (10 million), BMDMs (10 million), and B cells (10 million) in 20 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). The incubation system contained GM-CSF (500 U / mL), IL-2 (500 U / mL), IL-7 (500 U / mL), IL-12 (500 U / mL), and CD80 antibody (10 ng / mL).

[0204] (6) Preparation of nanoparticles based on antigen-presenting cells

[0205] Peripheral blood-derived DCs (20 million cells) and BMDCs (20 million cells) were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were homogenized in a high-pressure homogenizer (5000 bar) for 5 minutes. The sample was then centrifuged at 2000g for 15 minutes and the supernatant was collected. The supernatant was then centrifuged at 8000g for 15 minutes and the supernatant was collected. The supernatant was co-incubated with nanoparticles 1 (50 mg) prepared in step (3) at 4°C for 16 hours. Then, the nanoparticles were repeatedly co-extruded through a 0.45 μm filter membrane. After centrifugation at 13000g for 20 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain nanoparticles 2 with a particle size of 310 nm.

[0206] Alternatively, peripheral blood-derived DCs (10 million), BMDCs (10 million), B cells (10 million), and BMDMs (10 million) can be collected after incubation by centrifugation at 400g for 5 minutes. The cells are then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they are homogenized in a high-pressure homogenizer (5000 bar) for 5 minutes. The sample is then centrifuged at 2000g for 15 minutes and the supernatant is collected. The supernatant is then centrifuged at 8000g for 15 minutes and the supernatant is collected again. The supernatant is then co-incubated with nanoparticles 1 (50 mg) prepared in step (3) at 4°C for 16 hours. The sample is then repeatedly co-extruded using a 0.45 μm filter membrane. The extrudate is centrifuged at 13000g for 20 minutes, the supernatant is discarded, and the precipitate is collected. The precipitate is resuspended in PBS to obtain nanoparticles 3 with a particle size of 310 nm.

[0207] (7) Detection of cancer cell-specific T cells

[0208] On day 0, each C57BL / 6 mouse was subcutaneously injected with 1.5 × 10⁻⁶ mmol / L on its back. 5Mice were injected subcutaneously with 100 μL of 1 mg PLGA nanoparticles on days 10, 15, and 21 to activate cancer cell-specific T cells. Mice were sacrificed on day 24, and draining lymph nodes and spleens were collected. Single-cell suspensions of the draining lymph node and spleen cells were prepared, and T cells were sorted from them using a magnetic bead method.

[0209] The obtained T cells (4 million), 200 μg of nanoparticles (nanoparticle 1, nanoparticle 2, or nanoparticle 3), IL-2 (500 U / mL), IL-7 (500 U / mL), and IL-15 (5000 U / mL) were co-incubated in 5 mL L DMEM complete medium for 24 hours. Then, the incubated CD3 cells were sorted by flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens. Simultaneously, CD3 was analyzed. + IFN-γ + IFN-γ in T cells + The average fluorescence intensity (MFI) of the attached fluorescent probe. The stronger the fluorescence intensity, the more cytotoxic substances are expressed by the cancer cell-specific T cells, resulting in higher sensitivity and better accuracy during detection.

[0210] Alternatively, the obtained T cells (4 million), peripheral blood-derived DCs (1 million), BMDCs (1 million), B cells (1 million), BMDMs (1 million), and 200 μg of nanoparticles (nanoparticle 1 or nanoparticle 3), IL-2 (500 U / mL), IL-7 (500 U / mL), and IL-15 (5000 U / mL) were co-incubated in 5 mL L DMEM complete medium for 24 hours, and then the incubated CD3 cells were sorted by flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens. Simultaneously, CD3 was analyzed. + IFN-γ + IFN-γ in T cells + The average fluorescence intensity (MFI) of the attached fluorescent probe.

[0211] (8) Experimental Results

[0212] like Figure 7As shown, nanoparticle 3 is superior to nanoparticles 1 and 2. When detecting T cells without antigen-presenting cells, nanoparticle 1 is undetectable, while nanoparticles 2 and 3 are detectable. When detecting T cells with antigen-presenting cells, nanoparticles 1, 2, and 3 can all detect T cells, with nanoparticles 2 and 3 performing better than nanoparticle 1, and nanoparticle 3 performing better than nanoparticle 2. Moreover, even when detecting T cells without antigen-presenting cells, nanoparticle 2 is more effective than nanoparticles 1 and 2 in detecting T cells with antigen-presenting cells. The analysis results of the average fluorescence intensity are consistent with the above trends. This indicates that loading activated antigen-presenting cell membrane components onto the particle surface and using mixed antigen-presenting cell membrane components can improve the effectiveness of nanoparticles or microparticles in detecting cancer cell-specific T cells.

[0213] Example 7: Detection of cancer cell-specific T cells in breast cancer mice

[0214] This embodiment uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how microparticles loaded with cancer cell whole-cell antigens activate antigen-presenting cells and are then prepared into microparticles for detecting cancer cell-specific T cells from peripheral immune organs.

[0215] (1) Lysis of cancer cells

[0216] Cultured 4T1 cells were centrifuged at 400g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. The cells were then subjected to five freeze-thaw cycles accompanied by sonication to lyse the cancer cells. 1 mg / mL of nuclease was added to the lysed cells to degrade the nucleic acids in the lysate. The nuclease was then inactivated by heating at 95°C for 10 minutes. The supernatant was collected after centrifugation at 5000g for 5 minutes; this was the water-soluble component. The precipitate was dissolved in 10% sodium deoxycholate (containing 10M arginine) to obtain the water-insoluble component. The water-soluble and water-insoluble components were mixed at a mass ratio of 3:1 to obtain the raw material for preparing the particle system.

[0217] (2) Preparation of micron-sized particle systems

[0218] In this embodiment, the microparticles were prepared using a double emulsion method. The PLGA backbone material for microparticle 1 had a molecular weight of 38 kDa-54 kDa, and the immunoadjuvants used were CpG2395 (class C), CpGM362 (class C), and Poly(I:C). During preparation, a double emulsion method was used to prepare microparticles internally loaded with lysate components and adjuvants. Then, 100 mg of microparticles were centrifuged at 9000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 h before use. The average particle size of this microparticle system was approximately 2.5 μm, and the surface potential was approximately -6 mV. Each 1 mg of PLGA microparticles loaded approximately 110 μg of protein or peptide components, and 0.02 mg each of CpG2395, CpGM362, and Poly(I:C). The same materials and preparation methods were used for the control microparticles 2, with a particle size of about 2.5 μm and a surface potential of about -6 mV. Each 1 mg of PLGA microparticles was loaded with about 110 μg of protein or polypeptide components, and each 1 mg of PLGA was loaded with 0.02 mg each of CpG1585 (Class A), CpG2336 (Class A) and Poly(I:C).

[0219] (3) Preparation of B cells

[0220] B cells derived from peripheral spleen cells were used. After euthanizing mice, the spleen was harvested, and a single-cell suspension of mouse spleen cells was prepared. CD19 cells in the single-cell suspension were then sorted using magnetic bead sorting. + B cells.

[0221] (4) Activation of antigen-presenting cells

[0222] Microparticles (800 μg) loaded with whole-cell antigen components of cancer cells were co-incubated with 10 million B cells prepared in step (3) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained GM-CSF (2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL) and CD86 antibody (10 ng / mL).

[0223] (5) Preparation of micron-sized particles based on antigen-presenting cells

[0224] 10 million B cells were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were sonicated at low power (20W) at 4°C for 1 minute and then homogenized at 1000rpm for 3 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was then centrifuged at 8000g for 15 minutes and the supernatant was collected. The supernatant was then sonicated at 50W for 2 minutes with the microparticles (60mg) prepared in step (2) and DSPE-PEG-mannose (1mg). After centrifugation at 8000g for 20 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain the microparticles. Microparticle 3 was prepared by co-processing the antigen-presenting cell membrane component activated by microparticle 1 with microparticle 1, and the particle size was 2.6 μm; microparticle 4 was prepared by co-processing the antigen-presenting cell membrane component activated by microparticle 2 with microparticle 2, and the particle size was 2.6 μm.

[0225] (6) Detection of cancer cell-specific T cells

[0226] On day 0, each BALB / c mouse was subcutaneously injected with 1×10⁻⁶ mmol / L on its back. 6 4T1 cells were administered subcutaneously to mice on days 10, 14, and 18, with either 100 μL of 1 mg PLGA microparticles 1 (microparticle vaccine 1) or 100 μL of PBS. Mice were sacrificed on day 22, and spleens were collected to prepare single-cell suspensions of spleen cells. The single-cell suspension of spleen cells (6 million cells), DC2.4 (2 million cells), and microparticles (50 μg) were incubated in 2 mL of DMEM complete medium for 72 hours (37°C, 5% CO2). CD3+ cells were then isolated by flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens.

[0227] (7) Experimental Results

[0228] like Figure 8As shown, compared with the control group, microparticles prepared from antigen-presenting cells activated by microparticles could effectively detect more cancer cell-specific T cells in the treated mice. Furthermore, microparticles 3 prepared from antigen-presenting cells activated by microparticle 1 using two types of CpG and Poly(I:C) as a mixed adjuvant were more effective than microparticles 4 prepared from antigen-presenting cells activated by microparticle 2 using two types of A CpG and Poly(I:C) as a mixed adjuvant. In this embodiment, mannose was used as the active targeting head in the micron vaccine. In practical applications, any target head capable of targeting cells, such as CD32 antibody, mannan, CD205 antibody, or CD19 antibody, can also be used.

[0229] Example 8: Detection of cancer cell-specific T cells in pancreatic cancer

[0230] This embodiment uses mannose as a target to illustrate how to use actively targeted nanoparticles to activate antigen-presenting cells to detect cancer cell-specific T cells in peripheral blood.

[0231] (1) Lysis of cancer cells

[0232] After collecting cultured Pan02 pancreatic cancer cells, the cancer cells were lysed with 10% octyl glucoside and the whole-cell antigens derived from the cancer cells were dissolved.

[0233] (2) Preparation of nanoparticles

[0234] In this embodiment, the nanoparticle system was prepared using a double emulsion method. The nanoparticle materials were PLGA and mannose-modified PLGA, both with molecular weights ranging from 7 kDa to 17 kDa. When preparing nanoparticles with targets, the mass ratio of the two materials used together was 4:1. The immunoadjuvants used were Poly(I:C) and CpGSL03. The preparation method was as described above, using a double emulsion method to co-load the lysate components and adjuvants into the nanoparticles. Then, 100 mg of nanoparticles were centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h before use. The average particle size of the target-tipped nanoparticles 1 was approximately 270 nm, and each 1 mg of PLGA nanoparticles loaded approximately 80 μg of protein and peptide components, containing 0.04 mg each of Poly(I:C) and CpGSL03. Nanoparticles 2 without adjuvant loading but with mannose targets also have a particle size of about 270 nm. They are prepared using an equal amount of cell lysis components but without any immune adjuvants. Each 1 mg of PLGA nanoparticles is loaded with about 80 μg of protein and peptide components.

[0235] (3) Preparation of antigen-presenting cells

[0236] In this embodiment, BMDCs and BMDMs are used as antigen-presenting cells. The preparation methods for BMDCs and BMDMs are the same as above.

[0237] (4) Activation of antigen-presenting cells

[0238] Nanoparticle 1 (1000 μg) or nanoparticle 2 (1000 μg) was incubated with BMDC (10 million units), BMDM (10 million units) and IL-7 (500 U / mL) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2).

[0239] Alternatively, BMDC (10 million units), BMDM (10 million units), and IL-7 (500 U / mL) can be co-incubated in 15 mL of high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2). Both incubation systems contain IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), IFN-γ (500 U / mL), and CD80 antibody (10 ng / mL).

[0240] (5) Preparation of nanoparticles derived from antigen-presenting cells

[0241] DCs and macrophages were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were sonicated at low power (10W) for 20 minutes at 4°C. The samples were then centrifuged at 3000g for 15 minutes, and the supernatant was collected. The supernatant was filtered sequentially through membranes with pore sizes of 30μm, 10μm, 5μm, 2μm, 1μm, 0.45μm, and 0.22μm. The filtrate was collected, centrifuged at 18000g for 50 minutes, and the supernatant was discarded. The precipitate was collected, resuspended in 4% trehalose aqueous solution, and then freeze-dried for 48 hours to obtain nanoparticles. Among them, nanoparticle 3 was prepared using mixed antigen-presenting cells that were not activated by nanoparticles, with a particle size of 110 nanometers; nanoparticle 4 was prepared using mixed antigen-presenting cells activated by nanoparticle 2, with a particle size of 110 nanometers; and nanoparticle 5 was prepared using mixed antigen-presenting cells activated by nanoparticle 1, with a particle size of 110 nanometers.

[0242] (6) Detection of cancer cell-specific T cells

[0243] On day 0, each C57BL / 6 mouse was subcutaneously injected with 1×10⁻⁶ dredges on its back. 6Mice were injected subcutaneously with 100 μL of 1 mg PLGA nanoparticles at the injection sites of Pan02 pancreatic cancer cells on days 10, 15, 20, and 27. Mice were sacrificed on day 24, and peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were prepared from the PBMCs, and CD3+ cells were then isolated from the PBMCs using flow cytometry. + T cells. Five million T cells were incubated with 100 μg of nanoparticles (nanoparticle 1, nanoparticle 3, nanoparticle 4, or nanoparticle 5) in DMEM high-glucose medium for 72 hours (37°C, 5% CO2). The incubation system contained IL-2 (500 U / mL) and IL-7 (500 U / mL). CD3+ cells were then isolated from the incubated cells using flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells.

[0244] (7) Experimental Results

[0245] like Figure 9 As shown, nanoparticles 4 and 5 are more effective than nanoparticles 1 and 3. Furthermore, nanoparticle 5 is more effective than nanoparticle 4. In conclusion, regardless of whether the antigen-presenting cells are activated by adjuvanted nanoparticles, the nanoparticles prepared from them can effectively detect cancer cell-specific T cells; however, nanoparticles prepared from antigen-presenting cells activated by adjuvanted nanoparticles are more effective.

[0246] Example 9: Detection of specific T cells in lung cancer cells using nanoparticles

[0247] This embodiment illustrates the detection of cancer cell-specific T cells in mouse spleen cells using calcified nanoparticles. In practical applications, other biomineralization techniques, cross-linking, gelation, and other particle modifications can also be used. In this embodiment, mouse lung cancer tumor tissue was lysed with 8M urea (containing 200mM sodium chloride), dissolved, and loaded onto a nanoparticle system. After activating antigen-presenting cells using these particles, the antigen-presenting cells were prepared into cancer cell-specific T cells for nanoparticle detection.

[0248] (1) Lysis of tumor tissue and cancer cells

[0249] Female C57BL / 6 mice aged 6-8 weeks were injected with 1×10⁻⁶ spores on their backs. 6 LLC mouse lung cancer cells, until the tumor volume reaches 1000 mm. 3 Mice were sacrificed and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and filtered through a cell sieve to prepare a single-cell suspension. After being irradiated with ultraviolet light for 5 minutes, the suspension was heated at 80°C for 10 minutes. Then, the tumor tissue single-cell suspension was lysed and dissolved with 8M urea (containing 200mM sodium chloride) to obtain cancer cell whole-cell antigen.

[0250] (2) Preparation of nanoparticles

[0251] This embodiment describes the biocalcification of nanoparticles by loading whole-cell antigens from cancer cells onto the interior and surface of nanoparticles. The nanoparticles were prepared using a solvent evaporation method. The PLGA nanoparticle material used had a molecular weight of 7-17 kDa, and the immunoadjuvants CpG2006 and Poly(I:C) were loaded into the nanoparticles. The preparation method is as follows: First, the antigen was loaded into the nanoparticles using a double emulsion method. After loading the lysate components, 100 mg of PLGA nanoparticles were centrifuged at 13000 g for 20 min and resuspended in 18 mL of PBS. Then, 2 mL of tumor tissue and cancer cell lysate (60 mg / mL) dissolved in 8 M urea was added. After incubation at room temperature for 10 min, the mixture was centrifuged at 12000 g for 20 min, and the precipitate was collected. The 100 mg of PLGA nanoparticles were then resuspended in 20 mL of DMEM medium, and 200 μL of CaCl2 (1 mM) was added. The mixture was reacted at 37 °C for two hours. The precipitate was then collected after centrifugation at 10000g for 20 minutes, resuspended in ultrapure water, and washed twice by centrifugation to obtain nanoparticle 1, with an average particle size of approximately 290nm. Each 1mg of PLGA nanoparticle 1 is loaded with approximately 140μg of protein or peptide components, 0.03mg each of CpG2006 and Poly(I:C).

[0252] (3) Preparation of antigen-presenting cells

[0253] In this embodiment, BMDCs and B cells are used as antigen-presenting cells. The preparation method of BMDCs is the same as in Example 1. B cells are obtained from mouse peripheral blood PBMCs and are prepared in the same way. BMDCs and B cells are mixed in a 1:1 ratio to obtain mixed antigen-presenting cells.

[0254] (4) Activation of antigen-presenting cells

[0255] Nanoparticles (1000 μg) loaded with whole cell components of cancer cells were co-incubated with BMDCs (5 million cells) and B cells (5 million cells) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2); the incubation system contained cytokine component 1: IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), and IFN-γ (500 U / mL).

[0256] Alternatively, as a control, nanoparticles (1000 μg) loaded with whole-cell components of cancer cells were co-incubated with BMDCs (5 million units) and B cells (5 million units) in 15 mL of high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2); the incubation system contained no cytokines or antibodies.

[0257] Nanoparticles (1000 μg) loaded with whole-cell components of cancer cells were co-incubated with BMDCs (5 million cells) and B cells (5 million cells) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2); the incubation system contained cytokine combination 2: IL-4 (500 U / mL), IL-10 (200 U / mL), IL-37 (200 U / mL), and TGF-β (500 U / mL).

[0258] (5) Preparation of nanoparticles based on antigen-presenting cells

[0259] DC and B cells were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were sonicated at 4°C at low power (20W) for 2 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was then centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45μm membrane and then ultrafiltered and concentrated using an ultrafiltration membrane (molecular weight cutoff of 50KDa). The filtered and concentrated sample was mixed with the nanoparticles 1 prepared in step (2) and homogenized using a high-pressure homogenizer (10000bar) for 3 minutes. After centrifugation at 13000g for 30 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain the nanoparticles. Nanoparticle 2, with a particle size of 300 nm, was prepared from antigen-presenting cells containing cytokine combination 1 in the system when using particle-activated antigen-presenting cells. Nanoparticle 3, with a particle size of 300 nm, was prepared from antigen-presenting cells without cytokines in the system when using particle-activated antigen-presenting cells. Nanoparticle 4, with a particle size of 300 nm, was prepared from antigen-presenting cells containing cytokine combination 2 in the system when using particle-activated antigen-presenting cells.

[0260] (6) Detection of cancer cell-specific T cells

[0261] On day 0, each C57BL / 6 mouse was subcutaneously injected with 0.5 × 10⁻⁶ nitroglycerin on its back. 6 Mice were injected subcutaneously with 100 μL of 1 mg PLGA nanoparticles at three different sites (LLC lung cancer cells) on days 10, 15, and 20. Mice were sacrificed on day 24, and spleen cells were harvested and prepared into single-cell suspensions. CD45 groups were then isolated using flow cytometry.+ CD3 + T cells.

[0262] T cells (5 million) and nanoparticles 2 (100 μg), nanoparticles 3 (100 μg), or nanoparticles 4 (100 μg) prepared from antigen-presenting cells were co-incubated in DMEM high glucose medium for 12 hours (37°C, 5% CO2). During incubation, the system contained IL-2 (500 U / mL) and IL-7 (500 U / mL).

[0263] Alternatively, 5 million T cells and 100 μg of nanoparticles prepared from antigen-presenting cells can be co-incubated in DMEM high glucose medium for 12 hours (37°C, 5% CO2), and the incubation system must not contain any cytokines or antibodies.

[0264] Alternatively, 5 million T cells, 10 million mixed antigen-presenting cells prepared in step (3), and nanoparticle 1 (100 μg) can be co-incubated in DMEM high glucose medium for 12 hours (37°C, 5% CO2). During incubation, the system contains IL-2 (500 U / mL) and IL-7 (500 U / mL).

[0265] The cells were then collected after incubation, centrifuged at 400g for 5 minutes, and labeled with appropriate flow cytometry antibodies. CD3+ was then detected by flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells.

[0266] (7) Experimental Results

[0267] like Figure 10 As shown, compared with the control group, nanoparticles prepared from antigen-presenting cells activated by calcified nanoparticles can detect more cancer cell-specific T cells. Furthermore, when nanoparticles loaded with whole-cell antigens of cancer cells activate antigen-presenting cells, the system containing cytokine combination 1 is superior to the system without cytokines and / or antibodies; the effect of nanoparticles activating antigen-presenting cells in a system containing cytokine combination 1 or a system without cytokines is superior to the system containing cytokine combination 2; moreover, when nanoparticles prepared from antigen-presenting cells are co-incubated with T cells, the system containing cytokine combination 2 is superior to the system without cytokines.

[0268] Example 10: Detection of cancer cell-specific T cells in melanoma using nanoparticles

[0269] (1) Lysis of tumor tissue and cancer cells and collection of their components

[0270] When collecting tumor tissue, 1.5 × 10⁻⁶ mmol / L was first subcutaneously injected into the back of each C57BL / 6 mouse. 5 Several B16F10 cells were observed when the tumor reached a volume of approximately 1000 mm². 3 Mice were euthanized and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and passed through a cell filter to prepare a single-cell suspension. Ultrapure water was added, and the cells were repeatedly freeze-thawed and sonicated to lyse them. Nuclease was then added and inactivated for 5 minutes, followed by inactivation at 95°C for 10 minutes. The suspension was then centrifuged at 8000g for 3 minutes. The supernatant was the water-soluble component; the precipitate was dissolved in a 10% sodium deoxycholate aqueous solution to remove the water-insoluble component. The water-soluble component and the sodium deoxycholate-dissolved water-insoluble component were mixed at a 1:1 mass ratio to obtain the antigen raw material for preparing the nanoparticle system.

[0271] (2) Preparation of nanoparticle systems

[0272] In this embodiment, the nanoparticles were prepared using a double emulsion method and possess the ability to target dendritic cells. The nanoparticles used were prepared from PLGA and mannan-modified PLGA, both with molecular weights ranging from 24 kDa to 38 kDa. The mass ratio of unmodified PLGA to mannan-modified PLGA was 9:1. The immunoadjuvants used were poly(I:C), CpG1018, and CpG2216. The substance that increases lysosomal immune escape was the KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and both the adjuvant and the KALA polypeptide were encapsulated within the nanoparticles. The preparation method was as described above. First, a double emulsion method was used to load the lysis buffer components, adjuvant, and KALA polypeptide inside the nanoparticles. After loading these components, 100 mg of nanoparticles were centrifuged at 12000 g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. The nanoparticles have an average diameter of approximately 250 nm and a surface potential of approximately -5 mV. Each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components. Each 1 mg of PLGA nanoparticles contains 0.02 mg each of poly(I:C), CpG1018, and CpG2216 immunoadjuvants, and 0.05 mg of KALA peptide. Nanoparticle 2 was prepared using the same materials and methods, with a diameter of approximately 250 nm and a surface potential of approximately -5 mV. It does not contain KALA peptide but is loaded with an equal amount of adjuvant and cell lysis components. Nanoparticle 3 was prepared using the same materials and methods, with a diameter of approximately 250 nm and a surface potential of approximately -5 mV. Each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein and peptide components. Each 1 mg of PLGA nanoparticles contains 0.02 mg of poly(I:C), 0.04 mg of CpG1018, and 0.05 mg of KALA peptide.

[0273] (3) Preparation of antigen-presenting cells

[0274] In this embodiment, BMDCs and BMDMs are used as antigen-presenting cells. The preparation methods for BMDCs and BMDMs are the same as above.

[0275] (4) Activation of antigen-presenting cells

[0276] Nanoparticles (1000 μg) loaded with whole-cell components of cancer cells were co-incubated with BMDC (10 million units) and BMDM (10 million units) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained GM-CSF (2000 U / mL), M-CSF (2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), IFN-γ (500 U / mL) and CD80 antibody (10 ng / mL).

[0277] (5) Preparation of nanoparticles based on antigen-presenting cells

[0278] BMDCs and BMDMs were collected after incubation by centrifugation at 400g for 5 minutes. They were then washed three times by centrifugation at 1200 rpm for 3 minutes in 30 mM pH 7.0 Tris-HCl buffer containing 0.0759 M sucrose and 0.225 M mannitol. The antigen-presenting cells were then mechanically destroyed by sonication in the presence of phosphatase and protease inhibitors. The resulting cell membranes were washed with a solution of 10 mM pH 7.5 Tris-HCl and 1 mM EDTA. Then, the sample was filtered through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm and 0.45 μm in sequence. The filtrate was centrifuged at 16000g for 35 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS and co-incubated with the nanoparticles prepared in step (2) for 10 minutes. Then, it was repeatedly co-extruded through a 0.45 μm filter membrane. The extrudate was centrifuged at 12000g for 25 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in an aqueous solution containing 4% trehalose and then freeze-dried to obtain the nanoparticles. Nanoparticle 4, with a particle size of 260 nm, was prepared by co-processing the antigen-presenting cell membrane component activated by nanoparticle 1 with nanoparticle 1; nanoparticle 5, with a particle size of 260 nm, was prepared by co-processing the antigen-presenting cell membrane component activated by nanoparticle 2 with nanoparticle 2; and nanoparticle 6, with a particle size of 260 nm, was prepared by co-processing the antigen-presenting cell membrane component activated by nanoparticle 3 with nanoparticle 3.

[0279] (6) Detection of cancer cell-specific T cells

[0280] Female C57BL / 6 mice aged 6-8 weeks were selected and subcutaneously injected with 0.5 mg of PLGA nanoparticles (loaded with lysate components, Poly(I:C), two CpG adjuvants, and KALA peptide) on days 0, 7, 14, 21, and 28. Mice were sacrificed on day 32, and peripheral blood and lymph nodes were collected. PBMCs in the peripheral blood were separated using gradient centrifugation. Lymph nodes were cut into small pieces, ground, and passed through a cell sieve to prepare a single-cell suspension. The PBMC and lymph node single-cell suspensions were then mixed. CD45 cells were then sorted using magnetic bead sorting. + CD3 + T cells. The sorted CD3... + T cells (5 million), nanoparticles (40 μg), and IL-7 (10 ng / mL) were co-incubated in 2 mL of RPMI 1640 complete medium for 96 hours. CD3+ ions from the incubated T cells were then sorted by flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells that can recognize full-cell antigens of cancer cells.

[0281] (7) Experimental Results

[0282] like Figure 11 As shown, compared with the control group, nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell components showed a higher detection rate of cancer cell-specific T cells. Furthermore, nanoparticles 4 prepared from antigen-presenting cells activated by nanoparticles containing substances that increase lysosomal escape were more effective than nanoparticles 5 prepared from antigen-presenting cells activated by nanoparticles without lysosomal escape; and nanoparticles 4 prepared from antigen-presenting cells activated by nanoparticles using a mixture of two CpG and Poly(I:C) adjuvants were more effective than nanoparticles 6 prepared from antigen-presenting cells activated by nanoparticles using only one CpG and Poly(I:C) adjuvant.

[0283] Example 11 Detection of cancer cell-specific T cells in breast cancer

[0284] This embodiment uses 4T1 triple-negative breast cancer in mice as a cancer model to illustrate how to use microparticles loaded with cancer cell whole-cell antigens to activate antigen-presenting cell membrane components and cancer cell membrane components to detect cancer cell-specific T cells. In this embodiment, breast cancer cells are first inactivated and denatured, then lysed, and the insoluble components in the lysed cancer cells are dissolved with octyl glucoside. Then, using PLGA as the microparticle framework material, CpG2007, CpG1018, and Poly ICLC as immunoadjuvants, and polyarginine and polylysine as substances to enhance lysosomal escape, microparticles loaded with cancer cell whole-cell antigens are prepared. These particles are then used to activate antigen-presenting cells to prepare microparticles based on a mixture of antigen-presenting cell membrane components and cancer cell membrane components. Finally, these microparticles are used to detect cancer cell-specific T cells.

[0285] (1) Lysis of cancer cells

[0286] Cultured 4T1 cells were centrifuged at 400g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. The resulting cancer cells were inactivated and denatured by ultraviolet light and heating at 60°C for 5 minutes, respectively. Then, ultrapure water was added, and the cells were repeatedly frozen and thawed 5 times, followed by sonication to lyse the cancer cells. The cell lysate was centrifuged at 5000g for 10 minutes, and the supernatant was the water-soluble component. The precipitate was dissolved with 10% octyl glucoside to obtain the dissolved original insoluble component. The water-soluble and insoluble components were mixed at a mass ratio of 2:1 to obtain the lysate component required for preparing micron-sized particles.

[0287] (2) Preparation of micron-sized particles

[0288] In this embodiment, microparticle 1 was prepared using a double emulsion method. The PLGA backbone material for the microparticles had a molecular weight of 38 kDa-54 kDa. The immunoadjuvants used were CpG2007, CpG1018, and Poly ICLC. The lysosomal escape-enhancing substances used were polyarginine and polylysine. The preparation process involved first preparing microparticles internally loaded with lysate components, adjuvants, and KALA peptides using a double emulsion method. After internal loading of lysate and adjuvants, 100 mg of microparticles were centrifuged at 9000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use. The average particle size of this microparticle 1 system was approximately 3.1 μm, and the surface potential of the microparticle system was approximately -7 mV. Each 1 mg of PLGA microparticles loaded approximately 110 μg of protein or peptide components, containing 0.01 mg each of CpG2007, CpG1018, and Poly ICLC, and 0.02 mg each of polyarginine and polylysine.

[0289] (3) Preparation of antigen-presenting cells

[0290] In this embodiment, BMDC and DC2.4 were used as antigen-presenting cells. The BMDC preparation method was the same as in Example 1.

[0291] (4) Activation of antigen-presenting cells

[0292] Microparticles (1000 μg) loaded with whole-cell components of cancer cells were co-incubated with BMDC (5 million cells) and DC2.4 cells (5 million cells) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained GM-CSF (2000 U / mL), M-CSF (2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), IFN-γ (500 U / mL) and CD80 antibody (10 ng / mL).

[0293] (5) Preparation of micron-sized particles based on a mixed membrane composition of presenting cells and cancer cells

[0294] 10 million DCs were collected after incubation by centrifugation at 400g for 5 minutes. These were mixed with 10 million 4T1 cells and washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. The cells were then resuspended in PBS and sonicated at 4°C at low power (20W) for 2 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was then centrifuged at 5000g for 10 minutes and the supernatant was collected again. The supernatant was repeatedly co-extruded through a 0.22μm membrane. 30mg of microparticles 1 prepared in step (2) was added and the mixture was sonicated at 10W for 10 seconds and co-incubated for 10 minutes. The mixture was then repeatedly co-extruded through a 5μm membrane. The extrudate was collected by centrifugation at 9000g for 120 minutes, and the supernatant was discarded. The precipitate was collected and resuspended in PBS to obtain microparticles 2 with a particle size of 3.15μm.

[0295] Alternatively, collect 20 million DCs after incubation by centrifuging at 400g for 5 minutes, wash the cells twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C, resuspend the cells in PBS, and sonicate at low power (20W) for 2 minutes at 4°C. Then centrifuge the sample at 3000g for 15 minutes and collect the supernatant. Centrifuge the supernatant at 5000g for 10 minutes and collect the supernatant. Repeatedly co-extrude the supernatant through a 0.22μm membrane, add 30mg of micron particles 1 prepared in step (2), sonicate at 10W for 10 seconds, and co-incubate for 10 minutes. Then repeatedly co-extrude through a 5μm membrane. Centrifuge the extrudate at 9000g for 120 minutes, discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain micron particles 3 with a particle size of 3.15μm.

[0296] (6) Detection of cancer cell-specific T cells

[0297] Female BALB / c mice aged 6-8 weeks were selected, and 2×10⁻⁶ mice were subcutaneously injected into the back of the mice on day 0. 6 4T1 breast cancer cells were subcutaneously injected with 0.3 mg of PLGA microparticles on days 7, 14, and 21. Mice were sacrificed on day 25, and peripheral blood was collected. PBMCs were isolated from the peripheral blood, and CD3+ was sorted from the PBMCs. + T cells. CD3 + One million T cells and 100 μg of either microparticle 2 or microparticle 3 were co-incubated in 10 mL of RPMI 1640 complete medium for 24 hours (37°C, 5% CO2). Cell membrane staining with different fluorescent probe-modified anti-mouse CD4 and anti-mouse CD8 antibodies was then performed. After cell fixation, intracellular staining with fluorescent probe-modified anti-mouse granzyme B antibody was performed. Flow cytometry was then used to detect CD8 levels in the incubated cells. + Granzyme B + T cells and CD4 + Granzyme B + T cells are cancer cell-specific T cells that can recognize whole-cell antigens of cancer cells. After being activated by antigens, cancer cell-specific T cells begin to synthesize and express cytotoxic substances; granzyme B is one such cytotoxic substance, and it has the strongest apoptosis-inducing activity. Simultaneously, CD8+ analysis... + Granzyme B + The mean fluorescence intensity (MFI) of the fluorescent probes attached to Granzyme B in T cells.

[0298] (7) Experimental Results

[0299] like Figure 12 As shown, micron particles 2, prepared by mixing antigen-presenting cell membrane components activated by micron particles with cancer cell membrane components, can better detect cancer cell-specific T cells than micron particles 3, and the activated cancer cell-specific T cells can synthesize more cytotoxic substances. Furthermore, during detection, the cancer cell-specific T cells activated by micron particles 2 can synthesize more cytotoxic substances, making them easier to detect and thus leading to more accurate detection.

[0300] Example 12 Detection of cancer cell-specific T cells in breast cancer

[0301] (1) Lysis of cancer cells and bacterial vesicles

[0302] The cultured 4T1 cells were centrifuged at 400g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. The resulting cancer cells were inactivated and denatured by ultraviolet light and high-temperature heating, respectively. Then, the cancer cells were lysed with an 8M urea aqueous solution (containing 500mM sodium chloride) and the lysate components were dissolved to obtain the antigen components for the preparation of the micron particle system.

[0303] The Lactobacillus acidophilus was centrifuged at 5000g for 30 minutes, and the supernatant was collected after discarding the precipitate. The supernatant was filtered through a 1μm filter membrane and then centrifuged at 16000g for 90 minutes. The precipitate was lysed and the bacterial extravesicular components were dissolved using an 8M urea aqueous solution (containing 500mM sodium chloride).

[0304] (2) Preparation of micron-sized particles

[0305] In this embodiment, the micron-sized particles were prepared using a double emulsion method. The backbone materials of micron-sized particles 1 were unmodified PLA and mannose-modified PLA, both with a molecular weight of 40 kDa, and the ratio of unmodified PLA to mannose-modified PLA was 4:1. The immunoadjuvants used were CpG2006, CpG2216, and Poly ICLC, and the lysosomal escape-enhancing substances used were arginine and histidine. The mass ratio of cancer cell lysate components to bacterial extravesicle components used in the preparation of micron-sized particles was 1:1. In the preparation process, microparticles loaded with cancer cell lysate components, bacterial extravesicular components, adjuvants, arginine, and histidine were first prepared using a double emulsion method. Then, 100 mg of microparticles were centrifuged at 9000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 h to obtain microparticle 1. The average particle size was approximately 1.5 μm. Each mg of PLGA microparticle 1 was loaded with approximately 100 μg of protein or polypeptide components, containing 0.02 mg each of CpG2006, CpG2216, and Poly ICLC, and 0.05 mg each of arginine and histidine. Control microparticle 2 was prepared using the same materials and method as microparticle 1, with a particle size of approximately 1.5 μm, loaded with equal amounts of arginine, histidine, and equal amounts of cancer cell lysate components and bacterial extravesicular components, but without any adjuvants.

[0306] (3) Preparation of antigen-presenting cells

[0307] This embodiment uses BMDCs, B cells, and BMDMs as antigen-presenting cells. The preparation methods for BMDCs and BMDMs are the same as above. B cells are obtained from mouse peripheral blood PBMCs and are prepared using the same method as above. The mixed antigen-presenting cells are obtained by mixing BMDCs, B cells, and BMDMs in a ratio of 2:1:1.

[0308] (4) Activation of antigen-presenting cells

[0309] 1000 μg of microparticle 1 or microparticle 2 were incubated with 40 million mixed antigen-presenting cells (containing 20 million BMDCs, 10 million B cells and 10 million BMDMs) in 15 mL of high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2). The incubation system contained GM-CSF (2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), IFN-γ (500 U / mL), CD80 antibody (10 ng / mL) and CD40 antibody (20 mg / mL).

[0310] (5) Preparation of particles derived from antigen-presenting cells

[0311] 40 million mixed antigen-presenting cells were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were sonicated at low power (20W) for 2 minutes at 4°C. The sample was then centrifuged at 3000g for 15 minutes, and the supernatant was collected. The supernatant was then centrifuged at 5000g for 10 minutes, and the supernatant was collected again. The supernatant was filtered through a 0.45μm membrane and then ultrafiltered and concentrated using an ultrafiltration membrane (molecular weight cutoff of 50kDa). The filtered and concentrated sample was homogenized using a high-pressure homogenizer (10000bar) for 3 minutes, and then centrifuged at 13000g for 30 minutes. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS to obtain nanoparticles. Among them, nanoparticle 1 was prepared using mixed antigen-presenting cells activated by micron-particle 1, with a particle size of 250 nanometers; nanoparticle 2 was prepared using mixed antigen-presenting cells activated by micron-particle 2, with a particle size of 250 nanometers.

[0312] Alternatively, collect 40 million mixed antigen-presenting cells incubated with either microparticle 1 or microparticle 2 after centrifugation at 400g for 5 minutes. Wash the cells twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. Resuspend the cells in PBS and sonicate at low power (20W) for 2 minutes at 4°C. Then centrifuge the sample at 3000g for 15 minutes and collect the supernatant. Centrifuge the supernatant at 5000g for 10 minutes and collect the supernatant. Filter the supernatant through a 0.45μm membrane and then use an ultrafiltration membrane (molecular weight cutoff 50KDa) for ultrafiltration centrifugation and concentration. Process the filtered and concentrated sample using a high-pressure homogenizer (10000bar) for 3 minutes. Then co-process it with 60mg of the microparticle 1 or microparticle 2 prepared in step (2) for 10 minutes and repeatedly co-extract it through a 2μm filter membrane. Centrifuge the extrudate at 10000g for 20 minutes, discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain the microparticles. Among them, microparticle 3 was prepared by co-processing microparticle 1 with mixed antigen-presenting cell membrane components activated by microparticle 1, with a particle size of 1.6 μm; microparticle 4 was prepared by co-processing microparticle 2 with mixed antigen-presenting cell membrane components activated by microparticle 2, with a particle size of 1.6 μm.

[0313] (6) Analysis of cancer cell-specific T cells

[0314] Female BALB / c mice aged 6-8 weeks were selected and subcutaneously injected with 100 μL of micronized particles containing 0.4 mg of PLGA prepared in step (2) on days 0, 7, 14, 21, and 28. Mice were sacrificed on day 32, and peripheral blood and spleen were collected. Single-cell suspensions of PBMCs and spleen cells were prepared and mixed. CD3+ cells were then separated from the mixture using magnetic bead sorting. + T cells. The sorted CD3... + T cells (2 million), 100 μg nanoparticles or microparticles (nanoparticle 1, nanoparticle 2, microparticle 3, or microparticle 4), and DC2.4 cells (1 million) were co-incubated in 10 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). The incubation system contained IL-2 (200 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), and CD80 antibody (10 ng / mL). Flow cytometry was then used to analyze the CD3+ levels after incubation. + CD3 in T cells + IFN-γ + T cells are cancer cell-specific T cells that can recognize full-cell antigens of cancer cells. Simultaneously, CD3 analysis was performed. + IFN-γ +The mean fluorescence intensity (MFI) of the fluorescent probes linked to IFN-γ in T cells.

[0315] Cancer cell whole-cell antigens loaded on nanoparticles can be degraded into antigenic epitopes after being phagocytosed by antigen-presenting cells and presented on the surface of the antigen-presenting cell membrane. Nanoparticles prepared from antigen-presenting cells, loaded with these degraded and presented antigenic epitopes, can be recognized by cancer cell-specific T cells, activating them and leading to the secretion of cytotoxic cytokines. IFN-γ is the most important cytokine secreted by antigen-specific T cells after recognition of antigens. CD3+ was analyzed using flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells that can recognize and kill cancer cells. Simultaneously, the mean fluorescence intensity (MFI) of the fluorescent probes linked to IFN-γ in CD3+IFN-γ+ T cells was analyzed.

[0316] (7) Experimental Results

[0317] like Figure 13 As shown, the proportion of cancer cell-specific T cells is correlated with the therapeutic effect in the figure, indicating that the T cells detected using the particles described in this invention are cancer cell-specific T cells that can specifically recognize and kill cancer cells. Furthermore, nanoparticle 1 is better than nanoparticle 2; microparticle 3 is better than microparticle 4. This indicates that particles prepared from antigen-presenting cells activated by microparticles containing substances that enhance lysosomal escape function and mixed adjuvants are more effective in detecting cancer cell-specific T cells than particles prepared from antigen-presenting cells activated by microparticles containing only substances that enhance lysosomal escape function without mixed adjuvants. Moreover, microparticle 3 is better than nanoparticle 1, and microparticle 4 is better than nanoparticle 2, indicating that solid particles internally loaded with cancer cell lysis components and surface loaded with activated antigen-presenting cell components are more effective in detecting cancer cell-specific T cells than vesicle particles only loaded with activated antigen-presenting cell components. Furthermore, the analysis results of the mean fluorescence intensity (MFI) of the fluorescent probe connected to IFN-γ are consistent with the above results. Therefore, the use of mixed adjuvants and internal loading of whole-cell cancer cell components both contribute to the detection of cancer cell-specific T cells.

[0318] Example 13: Detection of cancer cell-specific T cells in colon cancer

[0319] This embodiment uses mouse colon cancer as a cancer model to illustrate how to use nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with colon cancer whole-cell antigens to detect cancer cell-specific T cells. In this embodiment, colon cancer tumor tissue was first lysed and the lysed components were dissolved using an 8M urea aqueous solution. Then, nanoparticles were prepared using PLGA as the backbone material, Poly(I:C), CpG2336, and CpG2006 as adjuvants, and NH4HCO3 as a substance to increase lysosomal escape. After activating antigen-presenting cells using these nanoparticles, the antigen-presenting cells were prepared into nanoparticles, and then the nanoparticles were used to detect cancer cell-specific T cells.

[0320] (1) Lysis of tumor tissue and collection of its components

[0321] When collecting tumor tissue, 2×10⁻⁶ cells were first subcutaneously injected into the back of each C57BL / 6 mouse. 6 MC38 colon cancer cells, when the tumor grew to a volume of approximately 1000 mm... 3 Mice were sacrificed and tumor tissue was harvested. The tumor tissue was cut into pieces, ground, and then filtered through a cell filter with an 8M urea solution to dissolve and lyse the tumor tissue. This is the source of the antigen raw materials for preparing the nanoparticle system.

[0322] (2) Preparation of nanoparticle systems

[0323] In this embodiment, the nanoparticles were prepared using a double emulsion method. The nanoparticles were prepared using PLGA with a molecular weight of 7-17 kDa, with Poly(I:C) and CpG as adjuvants, and NH4HCO3 as a substance to increase lysosomal escape. The adjuvants and NH4HCO3 were loaded inside the nanoparticles. The preparation method was as described above. In the preparation process, the lysis buffer components and adjuvants were first loaded inside the nanoparticles. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h for later use. The average particle size of the nanoparticles was about 260 nm, and the surface potential was about -7 mV. Each 1 mg of PLGA nanoparticles loaded approximately 90 μg of protein and polypeptide components. Each 1 mg of PLGA nanoparticles loaded 0.02 mg each of poly(I:C), CpG2336, and CpG2006 immune adjuvants, and 0.01 mg of NH4HCO3.

[0324] The materials and preparation method of nanoparticle 2 are the same as those of nanoparticle 1. The particle size is about 260 nm and the surface potential is about -7 mV. Each 1 mg of PLGA nanoparticles is loaded with about 90 μg of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.01 mg of NH4HCO3, 0.03 mg of CpG2336 and 0.03 mg of CpG2006.

[0325] (3) Preparation of antigen-presenting cells

[0326] In this embodiment, BMDCs and B cells were used as antigen-presenting cells. The BMDCs were prepared using the same method as in Example 1. B cells were derived from mouse peripheral blood PBMCs and were prepared using the same method.

[0327] (4) Activation of antigen-presenting cells

[0328] Nanoparticles (1000 μg) loaded with whole-cell components of cancer cells were co-incubated with BMDCs (5 million cells) and B cells (5 million cells) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained GM-CSF (2000 U / mL), IL-2 (500 U / mL), IL-7 (200 U / mL), IL-12 (200 U / mL), IFN-γ (500 U / mL), CD80 antibody (10 ng / mL), and CD40 antibody (20 mg / mL).

[0329] (5) Preparation of nanoparticles based on antigen-presenting cells

[0330] DC and B cells were collected after incubation by centrifugation at 400g for 5 minutes. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. After resuspending the cells in PBS, they were sonicated at 4°C at low power (20W) for 2 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was then centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45μm membrane and then ultrafiltered and concentrated using an ultrafiltration membrane (molecular weight cutoff of 50KDa). The filtered and concentrated sample was mixed with the nanoparticles prepared in step (2) and homogenized using a high-pressure homogenizer (10000bar) for 3 minutes. After centrifugation at 13000g for 30 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain the nanoparticles. Among them, nanoparticle 3 was prepared by co-processing antigen-presenting cells activated by nanoparticle 1 with nanoparticle 1, and the particle size was 300 nanometers; nanoparticle 4 was prepared by co-processing antigen-presenting cells activated by nanoparticle 2 with nanoparticle 2, and the particle size was 300 nanometers.

[0331] (6) Detection of cancer cell-specific T cells

[0332] Female C57BL / 6 mice aged 6-8 weeks were selected and subcutaneously injected with 100 μL of nanoparticles containing 0.5 mg PLGA (loaded with lysate components, mixed adjuvants, and substances that increase lysosomal escape) or 100 μL of PBS on days 0, 7, 14, and 28, respectively. Mice were sacrificed on day 32, peripheral blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated. CD3+ cells were then sorted from the PBMCs using flow cytometry. + T cells.

[0333] This embodiment uses enzyme-linked immunospot assay (ELISPOT) to detect cancer cell-specific T cells. First, anti-mouse IFN-γ antibody a (capture antibody) was coated in 96-well plates for 12 hours. After blocking with culture medium for 1 hour, the plates were washed with PBS. Then, 5000 T cells (selected above) were added to each well in 100 μL of RPMI 1640 complete culture medium, along with 50 μg of antigen-presenting cell nanoparticles 3 or 4. The plates were incubated at 37°C (5% CO2) for 24 hours. Afterward, the mixture of cells and nanoparticles was discarded, the 96-well plates were washed, and anti-mouse IFN-γ antibody b (detection antibody) was added. The plates were then incubated at 37°C (5% CO2) for at least 2 hours. The solution containing anti-mouse IFN-γ antibody b was discarded, the 96-well plates were washed, and appropriate color development methods were used to form spots on the surface of the 96-well plates. The data was read and analyzed using the ELISPOT analyzer, and the number of spots formed in each well was identified as cancer cell-specific T cells that can recognize cancer cell whole-cell antigens.

[0334] (7) Experimental Results

[0335] like Figure 14 As shown, compared with the PBS control group, mice injected with particles loaded with whole-cell components contained more cancer cell-specific T cells. Furthermore, the nanoparticles prepared from antigen-presenting cells activated by the nanoparticles described in this invention can effectively detect cancer cell-specific T cells. Moreover, the nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with a mixture of adjuvants, lysate components, and lysosomal escape substances showed better results in assisted separation of cancer cell-specific T cells than nanoparticles loaded with lysate components, two CpG adjuvants, and lysosomal escape substances.

[0336] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method of detecting cancer cell-specific T cells prepared from activated antigen presenting cells, characterized in that, The method comprises the following steps: S1, co-incubating antigen-presenting cells with first particles to obtain activated antigen-presenting cells; wherein the first particles are nano-particles or micro-particles loaded with tumor tissue and / or whole cell components of cancer cells; S2, preparing cell membrane components of the activated antigen-presenting cells into nano-vesicles; or co-acting the cell membrane components of the activated antigen-presenting cells with second particles to obtain second particles loaded with the cell membrane components; wherein the second particles are nano-particles or micro-particles loaded with tumor tissue and / or whole cell components of cancer cells; S3, co-incubating the nano-vesicles and / or the second particles loaded with the cell membrane components obtained in step S2 with test cells, detecting markers on the surface or inside of the test cells, and analyzing T cells containing the markers to detect the cancer cell-specific T cells; The first particles and the second particles are internally loaded with bacterial outer vesicle components, and the bacterial outer vesicle components are obtained by lysing bacterial outer vesicles with a lysis solution containing a lysis agent; the lysis agent is urea or Tween.

2. The method of claim 1, wherein: The markers include proteins or nucleic acids.

3. The method of claim 1, wherein: Before the nano-vesicles and / or the second particles loaded with the cell membrane components are co-incubated with the test cells, a step of sorting T cells in the test cells is further included.

4. The method of claim 1, wherein: In step S1 or step S3, the co-incubation system contains cytokines and / or antibodies.

5. The method of claim 1, wherein: The first particles or the second particles are further internally loaded with an immune adjuvant.

6. The method of claim 5, wherein: The immune adjuvant includes two or more than two Toll-like receptor agonists.

7. A kit for detecting cancer cell specific T cells, characterized in that, The kit includes at least one of the following (1)-(2): (1) nano-vesicles prepared from the activated antigen-presenting cells; (2) particles loaded with cell membrane components of the activated antigen-presenting cells; The nano-vesicles are prepared by co-incubating antigen-presenting cells with first particles to obtain activated antigen-presenting cells, and then extracting cell membrane components of the activated antigen-presenting cells, The particles loaded with the cell membrane components of the activated antigen-presenting cells are obtained by co-acting the cell membrane components of the activated antigen-presenting cells with second particles, so that the cell membrane components are loaded on the second particles, The first particles and the second particles are nano-particles or micro-particles loaded with tumor tissue and / or whole cell components of cancer cells, and the first particles and the second particles are internally loaded with bacterial outer vesicle components, which are obtained by lysing bacterial outer vesicles with a lysis solution containing a lysis agent; the lysis agent is urea or Tween. ​

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