A cancer cell-specific t cell vaccine, and a method of activating cancer cell-specific t cells

Abstract

The present application relates to a kind of cancer cell specific T cell vaccine, and the method for activating cancer cell specific T cell, T cell in peripheral blood, peripheral immune organ or tumor infiltrating lymphocyte is co-incubated with the particle prepared by activated antigen presenting cell to activate cancer cell specific T cell.The present application overcomes the difficulty that the broad-spectrum and polyclonal cancer cell specific T cell in tumor infiltrating lymphocyte cannot be effectively screened in clinic at present, can separate the broad-spectrum of specific tumor killing function of effector cancer cell specific T cell from peripheral blood, peripheral immune organ or tumor infiltrating lymphocyte, and has the characteristics of easy separation and acquisition and high specificity, can be used for the prevention and treatment of cancer.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy, and more particularly to a cancer cell-specific T cell vaccine and a method for activating 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. However, the number of cancer cell-specific T cells in tumor patients is generally insufficient, thus limiting their ability to kill cancer cells. If cancer cell-specific T cells can be isolated, expanded, and re-infused into the patient, the immune microenvironment can be regulated, effectively controlling cancer progression.

[0003] However, it is crucial to identify effector cancer cell-specific T cells with cancer cell recognition and killing functions from millions or even tens of millions of different T cell clones. Currently, there is no particularly efficient method to comprehensively isolate this subset of effector cancer cell-specific T cells with specific tumor-killing functions from numerous T cells. Therefore, this invention is proposed. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for the separation and amplification of 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 broad-spectrum polyclonal cancer cell-specific T cells, and then employs activated cytotoxic cancer cell-specific T cells (T cells). eff This invention utilizes biomarkers to isolate and extract cancer cell-specific T cells, which can then be reinfused into patients for cancer prevention or treatment. This effectively solves the problem of how to specifically isolate and extract broad-spectrum and polyclonal cancer cell-specific T cells with the ability to recognize and kill cancer cells from peripheral blood, peripheral immune organs, or tumor-infiltrating lymphocytes. Furthermore, because the nanoparticles or microparticles used to assist in the isolation and expansion of cancer cell-specific T cells are loaded with antigen-presenting cell membrane components, the system can be incubated with T cells without the assistance of antigen-presenting cells.

[0005] The first objective of this invention is to provide a method for preparing a cancer T-cell vaccine from particle-activated antigen-presenting cells, comprising the following steps:

[0006] S1. The antigen-presenting cells are co-incubated with the first particle to obtain activated antigen-presenting cells; wherein the first particle is loaded with tumor tissue and / or whole cell components of cancer cells;

[0007] 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 particles loaded with cell membrane components; wherein, the second particle loads whole cell components of tumor tissue and / or cancer cells.

[0008] S3. Co-incubate the nanovesicles of S2 and / or particles loaded with cell membrane components with immune cells containing T cells to activate cancer cell-specific T cells that can recognize cancer cell antigens. Then, use specific methods to sort out the activated cancer cell-specific T cells to obtain a cancer T cell vaccine.

[0009] Furthermore, in step S3, after sorting out the activated cancer cell-specific T cells, the step of expanding the cancer cell-specific T cells is also included.

[0010] Furthermore, the above sorting involves screening cancer cell-specific T cells activated by whole-cell components of cancer cells using specific surface markers. Specific surface markers include, but are not limited to, CD69, PD-1, TIM-3, LAG-3, CD25, OX40 (CD134), TCF-1, CD137, CD44, CD39, CD103, CD56, CD279, CD278, CD244, CD27, CD154, and CD28. Techniques for separating cancer cell-specific T cells using surface markers include, but are not limited to, flow cytometry and magnetic bead sorting.

[0011] Furthermore, immune cells containing T cells can be derived from peripheral blood, peripheral immune organs, or tumor-infiltrating lymphocytes. Before co-incubation with the product of S2, these immune cells can be sorted to identify T cells. Specifically, flow cytometry or magnetic bead sorting can be used to sort CD3+ cells from peripheral blood, peripheral immune tissues, and tumor-infiltrating lymphocytes. + 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 above-mentioned amplification is in vitro amplification, that is, co-incubating cancer cell-specific T cells with cytokines and / or antibodies.

[0013] Furthermore, the co-incubation system of antigen-presenting cells and the first particle in step S1, and the co-incubation system of nanovesicles and particles loaded with cell membrane components and immune cells in step S3, may contain cytokines and / or antibodies.

[0014] Preferably, the incubation system contains IL-2 and IL-7.

[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), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-17 (IL-17), IL-12, interleukin-12 (IL-12), interleukin-6 (IL-6), macrophage colony-stimulating factor (M-CSF), 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 step S3, the co-incubation system may also contain unactivated antigen-presenting cells.

[0018] Furthermore, in the above preparation method, the first or second particle may also be loaded with bacterial lysis components and / or bacterial exovesicle lysis components. These antibacterial lysis components and / or bacterial exovesicle lysis components are obtained by lysing bacteria or bacterial exovesicles with a lysis buffer containing a lysis agent. The lysis agent is an aqueous solution of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein-degrading enzymes, albumin, lecithin, Triton, Tween, amino acids, glycosides, choline, etc. The bacteria include, but are not limited to, BCG, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus geranioli, Lactobacillus reuteri, Lactobacillus rhamnosus, etc.

[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 consists of two or more Toll-like receptor agonists, such as (1) Poly(I:C) or Poly(ICLC); (2) CpG-ODN, wherein the CpG-ODN is at least two of class A CpG-ODN, class B CpG-ODN and class C CpG-ODN, and at least one of them is class B CpG-ODN or class C CpG-ODN. Among them, Class A CpG-ODN is selected from CpG-ODN2216, 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-ODN M362.

[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 an appropriate range. The particle size of the nanoparticles is 1 nm-1000 nm, more preferably 30 nm-1000 nm, and most preferably 50 nm-600 nm. The particle size of the microparticles 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 a second particle to obtain particles that encapsulate cell membrane components.

[0026] Furthermore, the mechanical disruption method is selected from one or more of the following: ultrasound, homogenization, slurrying, high-speed stirring, high-pressure disruption, high-shear disruption, swelling, chemical substances, and shrinkage. The co-action method is selected from one or more of the following: co-incubation, co-extrusion, ultrasound, stirring, dialysis, ultrafiltration, 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, the whole-cell component of tumor tissue and / or cancer cells is prepared by the following steps: freezing cancer cells and / or tumor tissue at -20℃ to -273℃, adding water or a solution without a solvent, and then repeatedly freezing and thawing to lyse the tissue. The resulting supernatant is the water-soluble component, and the precipitate, after being dissolved in a solution containing a solvent, becomes the insoluble component. The water-soluble and insoluble components are combined to obtain the whole-cell component of tumor tissue and / or cancer cells; or lysing and dissolving cancer cells and / or tumor tissue in a dissolving solution containing a solvent to obtain the soluble component, which is the whole-cell component of tumor tissue and / or 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, and more preferably a combination of 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 specifically activate antigen-presenting cells. The antigen-presenting cells are then 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 that have been pre-activated in lymph nodes and are stored in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes. The activated cancer cell-specific T cells secrete specific cytokines or highly express certain surface molecules. Cancer cell-specific T cells are then isolated using flow cytometry and other methods. After in vitro expansion, they are reinfused into patients. This method can isolate and expand the most diverse and broad-spectrum cancer cell-specific T cells with the function of recognizing and killing cancer cells.

[0031] A second objective of this invention is to provide the use of the above-mentioned cancer T-cell vaccine in the preparation of medicaments for treating or preventing cancer.

[0032] Furthermore, T cells in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes are derived from the patient's own body or from an allogeneic source.

[0033] Furthermore, antigen-presenting cells can be derived from the same organism, allogeneic, cell line, or stem cell from cancer cell-specific T cells.

[0034] Furthermore, in either the first or second particle, at least one of the cancer cells or tumor tissues used to prepare the antigen is the same type of disease as the target disease treated by the aforementioned drug. The vaccine prepared by this invention is specific.

[0035] A third objective of this invention is to provide a method for activating cancer cell-specific T cells in vitro, the method comprising the following steps:

[0036] S1. The antigen-presenting cells are co-incubated with the first particle to obtain activated antigen-presenting cells; wherein the first particle is loaded with tumor tissue and / or whole cell components of cancer cells;

[0037] 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 particles loaded with cell membrane components; wherein, the second particle loads whole cell components of tumor tissue and / or cancer cells.

[0038] S3. Co-incubate the nanovesicles of S2 and / or particles loaded with cell membrane components with cells containing T cells. The cancer cell-specific T cells that can recognize cancer cell antigens are activated. The activated cancer cell-specific T cells are sorted out using a specific method and then expanded in vitro to obtain the final product.

[0039] This invention breaks through the limitations of existing activation methods, enabling particles to be loaded with all antigens and activated antigen-presenting cell membranes. This facilitates the separation of a broader and more diverse range of cancer cell-specific T cells, which are highly specific and have better effects in immunotherapy, thus providing a more powerful alternative drug for cell therapy.

[0040] By means of the above-described solution, the present invention has at least the following advantages:

[0041] This invention provides a technique for isolating cancer cell-specific T cells from immune cells after in vitro activation using a nanoscale or microscale particle delivery system. The isolated 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 By expanding cancer cell-specific T cells, the resulting cells can be used for cancer prevention and treatment. Based on this, the activation process of antigen-presenting cells, the incubation process with T cells, and the loading substances of the first and second particles were optimized to obtain a cancer vaccine with excellent therapeutic and preventative effects.

[0042] 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

[0043] 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.

[0044] 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 antigens and insoluble antigens 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 with nanoparticles and / or microparticles prepared in a or b, activating cancer cell-specific T cells with particles prepared from the activated antigen-presenting cells, separating and extracting cancer cell-specific T cells based on the characteristics of the activated T cells, then expanding such T cells, and using such cells to prevent or treat cancer;

[0045] Figure 2-14 The results of experiments on tumor growth rate and survival in mice using isolated and expanded cancer cell-specific T cells to prevent or treat cancer, as described in Examples 1-13, are respectively. Figure 2-14 In the figure, a represents the results of the tumor growth rate experiment during cancer prevention or treatment (n≥8); b represents the results of the mouse survival experiment during cancer prevention or treatment (n≥8). Each data point is the mean ± standard error (mean±SEM). Figure 3 , 4 In Figures 1 and 13, c and d represent the results of flow cytometry analysis of the proportion of cancer cell-specific T cells activated by cancer cell whole-cell antigens to the corresponding T cells; the significant differences in the tumor growth inhibition experiment in Figure a were analyzed using ANOVA, and the significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests; *** indicates a significant difference compared to the PBS blank control group (p < 0.005); ** indicates a significant difference compared to the PBS blank control group (p < 0.01); * indicates a significant difference compared to the PBS blank control group (p < 0.05); ε represents a significant difference compared to the group of cancer cell-specific T cells separated with nanoparticles / microparticles activated by co-incubation with specific cytokine components; ### indicates a significant difference compared to the control group of T cells separated with nanoparticles assisted by antigen-presenting cells prepared by incubation alone without any nanoparticle activation (p < 0.005). The p-value indicates a significant difference compared to the control group of T cells isolated with the assistance of hollow nanoparticles (without loading whole cell components of cancer cells) prepared from antigen-presenting cells activated by nanoparticles / microparticles (p < 0.005); &&& indicates a significant difference compared to the control group of T cells isolated with the assistance of nanoparticles and / or microparticles prepared from antigen-presenting cells activated by blank nanoparticles / microparticles + free lysis buffer (p < 0.005); δδδ represents the difference between the nanoparticles / microparticles prepared from antigen-presenting cells activated by peptide nanoparticles / microparticles (p < 0.005). Compared with the T cell group prepared by particle-assisted separation, p < 0.005, there was a significant difference; ω represents a significant difference compared with the T cell group prepared by nanoparticle / microparticle-activated DC cells or B cells, p < 0.05; χ represents a significant difference compared with the T cell group prepared by nanoparticle / microparticle-activated antigen-presenting cells loaded with whole cancer cell components and bacterial components lysed and dissolved by Tween 80, p < 0.05; θ represents a significant difference compared with the T cell group prepared by using two types of CpG and Poly Compared with the T cell group prepared by nanoparticle / microparticle-assisted isolation from antigen-presenting cells activated by ICLC / Poly(I:C) as a mixed adjuvant, p < 0.05, there was a significant difference; ττ represents a significant difference compared with the T cell group prepared by nanoparticle / microparticle-assisted isolation from antigen-presenting cells activated by nanoparticle / microparticle using a mixed adjuvant of type A CpG and type B CpG, p < 0.01; μμ represents a significant difference compared with the T cell group prepared by nanoparticle / microparticle-assisted isolation from antigen-presenting cells activated by nanoparticle / microparticle using only Poly(I:C) as an adjuvant, p < 0.01; OOO represents a significant difference compared with the T cell group prepared by nanoparticle / microparticle-assisted isolation from antigen-presenting cells activated by nanoparticle / microparticle using nanoparticle / microparticle-activated cells with internally loaded cancer cell whole cell components but without any antigen-presenting cell membrane components loaded on the surface, p < 0.005; φ represents a significant difference compared with the T cell group prepared by nanoparticle / microparticle-activated DCs, p < 0.05. The T cell group prepared by nanoparticle / microparticle-assisted separation, representing antigen-presenting cells activated by nanoparticles / microparticles without adjuvant loading, showed a significant difference (p < 0.005). The group of T cells separated with nanoparticles / microparticles activated by antigen-presenting cells loaded with antigen but not adjuvanted showed a significant difference (p < 0.01). The group of T cells separated with nanoparticles / microparticles loaded with whole-cell components of cancer cells, DC cells, and T cells was significantly different (p < 0.05). The group of T cells separated with nanoparticles / microparticles activated by antigen-presenting cells without the addition of cytokines showed a significant difference (p < 0.05). The group of T cells separated with nanoparticles / microparticles activated by antigen-presenting cells without the addition of cytokines showed a significant difference (p < 0.05). The group of T cells separated with nanoparticles / microparticles activated by antigen-presenting cells without the addition of cytokines showed a significant difference (p < 0.05). The group of T cells separated with nanoparticles / microparticles activated by antigen-presenting cells without the addition of cytokines showed a significant difference (p < 0.05). The group of T cells separated with nanoparticles / microparticles activated by antigen-presenting cells without the addition of lysosomal escape substances ... loaded with only one type of CpG showed a significant difference (p < 0.05). Compared with the group of T cells isolated by nanoparticles / microparticles activated by +Poly(I:C) mixed adjuvant, p < 0.05, indicating a significant difference; υυυ represents the group of T cells isolated by nanoparticles or microparticles with surface loaded with membrane cell components but no internal cancer cell components, indicating a significant difference; ΣΣ represents the group of T cells isolated by nanoparticles / microparticles with nanoparticles / microparticles with nanoparticles / microparticles that are loaded with whole cancer cell components but not adjuvanted internally, and surface loaded with membrane components of antigen-presenting cells activated by nanoparticles without adjuvant loading, indicating a significant difference; λλ represents the group of T cells isolated by nanoparticles / microparticles activated by nanoparticles with nanoparticles only loaded with two types of CpG as adjuvants, indicating a significant difference; π represents the group of T cells isolated by nanoparticles / microparticles activated by nanoparticles with cancer cell specific CD8, indicating a significant difference compared with the group of T cells isolated by nanoparticles / microparticles activated by nanoparticles. + The T cell group showed a significant difference (p < 0.05). Detailed Implementation

[0046] 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.

[0047] The T-cell system for cancer prevention or treatment described in this invention comprises cancer cell-specific T cells specifically isolated and expanded from peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes. These cancer cell-specific T cells are first activated by nanoparticles and / or microparticles prepared from activated antigen-presenting cells during isolation, and then isolated using highly expressed specific molecules after activation. The expanded cancer cell-specific T cells can be derived from allogeneic or allogeneic sources. The antigen-presenting cells used to prepare the 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 preparation process and application areas of the T-cell system for cancer prevention or treatment are as follows. Figure 1 As shown.

[0048] In preparing nanoparticles or microparticles for activating antigen-presenting cells, water-soluble and water-insoluble antigens can be collected separately after lysing cells or tissues and then prepared into nanoparticle or microparticle systems. Alternatively, a dissolving solution containing a solvent can be used to directly lyse cells or tissues and dissolve whole-cell antigens from cancer cells to prepare nanoparticle or microparticle systems. The whole-cell antigens from cancer cells described in this invention can be treated before or after lysis, including but not limited to inactivation or denaturation, solidification, biomineralization, ionization, chemical modification, protease endolysis or degradation, and nuclease treatment, before preparing nanoparticles or microparticles. Alternatively, nanoparticles or microparticles can be prepared directly before or after cell lysis without any inactivation or denaturation, solidification, biomineralization, ionization, chemical modification, protease endolysis or degradation, or nuclease treatment. 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, protease endolysis or degradation, collagenase treatment, 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.

[0049] 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.

[0050] Activated antigen-presenting cells retain a certain cell membrane structure after being mechanically disrupted.

[0051] 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.

[0052] When using nanoparticles and / or microparticles prepared from antigen-presenting cells to activate cancer cell-specific T cells in vitro, antigen-presenting cells can also be added to the system as an adjunct. The antigen-presenting cells prepared as nanoparticles or microparticles, as well as those used to co-incubate with T cells, can be derived from the patient's own cells or allogeneic cells, or from cell lines or stem cells. Antigen-presenting cells can be dendritic cells (DCs), B cells, macrophages, or any mixture of these three, or other cells with antigen-presenting function.

[0053] 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.

[0054] 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.

[0055] In some implementation schemes, nanoparticles or microparticles loaded with cancer cell whole-cell antigens are used to first activate antigen-presenting cells, and then the antigen-presenting cells are prepared into nanoparticles or microparticles. The specific preparation method of using nanoparticles or microparticles prepared from antigen-presenting cells to assist in the separation and expansion of cancer cell-specific T cells from peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes is as follows:

[0056] 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.

[0057] 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 antigens or pre-water-insoluble antigens dissolved in a solvent containing urea or guanidine hydrochloride during preparation. The concentration of the water-soluble antigens or pre-water-insoluble antigens in the aqueous phase solution, i.e., the first predetermined concentration, requires a protein / peptide concentration greater than 1 ng / mL, sufficient to load sufficient whole-cell antigens from cancer cells to activate relevant cells. The concentration of the immunomodulatory adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0066] 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.

[0067] In this invention, the predetermined stirring conditions for this step are until the organic solvent has completely evaporated, that is, the dichloromethane in step 1 has completely evaporated.

[0068] 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).

[0069] Step 6: After freeze-drying the suspension containing the freeze-drying protectant obtained in Step 5, the freeze-dried material is ready for use.

[0070] Step 7: Use the nanoparticle-containing suspension obtained in Step 5, which is 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, which is resuspended in the sixth predetermined volume of PBS (or physiological saline) directly; or use the above sample after mixing it with the seventh predetermined volume of water-soluble antigen or dissolved original non-water-soluble antigen.

[0071] 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.

[0072] 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.

[0073] Step 9: Collect the co-incubated cells and perform mechanical disruption such as sonication, homogenization, and mechanical stirring.

[0074] 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.

[0075] 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.

[0076] Step 12: Mix the nano and / or micro particles prepared in step 10 with the immune cells containing T cells obtained in step 11 and incubate them together for a certain period of time.

[0077] Step 13: Use flow cytometry, magnetic bead sorting, and other methods to separate antigen-activated T cells.

[0078] Step 14: Expand the isolated T cells activated by cancer cell whole-cell antigen in vitro.

[0079] Step 15: The expanded cancer cell-specific T cells are reinfused into the patient to prevent or treat cancer.

[0080] Example 1: Cancer cell-specific T cell isolation and expansion for melanoma prevention.

[0081] This embodiment uses mouse melanoma as a cancer model to illustrate how nanoparticles, prepared from antigen-presenting cells activated by nanoparticles, can be used to assist in the isolation and expansion of cancer cell-specific T cells in tumor-infiltrating lymphocytes for melanoma prevention. In this embodiment, B16F10 melanoma tumor tissue was lysed to prepare water-soluble and insoluble antigens from 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 water-soluble and insoluble antigens from the tumor tissue was prepared by solvent evaporation. The nanoparticles were then used to activate antigen-presenting cells, and the cells were mechanically destroyed and centrifuged to prepare nanoparticles. These nanoparticles were used to assist in the isolation of cancer cell-specific T cells from tumor-infiltrating lymphocytes, and after expansion, they were injected in vivo to prevent melanoma.

[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. 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 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 antigen. Adding 8M urea to the resulting precipitate dissolved the insoluble antigen, converting it into a soluble antigen 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 in solvent evaporation. During preparation, nanoparticles loaded with water-soluble antigens from whole-cell antigens of cancer cells and nanoparticles loaded with non-water-soluble antigens from whole-cell antigens of 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 into the nanoparticles using the 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. 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, collect 10 million DCs after incubation with cytokine component 2 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 the nanoparticles. Among them, nanoparticle 4 was obtained by co-incubating nanoparticle 1 with the membrane component, with a particle size of 300 nm.

[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℃ 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) Isolation and expansion 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 tumor tissue and spleen cells were harvested. The tumor tissue was cut into small pieces, digested with collagenase for 15 minutes, and then a single-cell suspension was prepared by passing through a cell sieve. After centrifugation and washing with PBS, CD3+ was isolated from the tumor tissue single-cell suspension by flow cytometry from live cells (dead cells were removed by labeling with a live-dead cell dye). + T cells. Nanoparticles 2 (100 μg), 3 (100 μg), 4 (100 μg), or 5 (100 μg) were co-incubated with 500,000 T cells derived from tumor-infiltrating lymphocytes in 3 mL of RPMI 1649 complete medium for 96 hours (37°C, 5% CO2). CD3+ was then sorted from the incubated cells using flow cytometry. + CD8 + CD69 + T cells, also known as cancer cell-specific CD8 cells, are... +T cells. The cancer cell-specific T cells obtained above were co-incubated with IL-2 (2000 U / mL), IL-7 (2000 U / mL), IL-12 (200 U / mL), IL-15 (200 U / mL), and αCD-3 antibody (10 ng / mL) for 10 days (with medium changed every two days) to amplify the sorted cancer cell-specific T cells, which constitute the T cell vaccine. Specifically, the cancer cell-specific T cell vaccine obtained with the assistance of nanoparticle 2 for sorting and amplification is T cell vaccine 1; the cancer cell-specific T cell vaccine obtained with the assistance of nanoparticle 3 for sorting and amplification is T cell vaccine 2; the cancer cell-specific T cell vaccine obtained with the assistance of nanoparticle 4 for sorting and amplification is T cell vaccine 3; and the cancer cell-specific T cell vaccine obtained with the assistance of nanoparticle 5 for sorting and amplification is T cell vaccine 4.

[0097] (7) Cancer cell-specific T cells for cancer prevention

[0098] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to eliminate immune cells in the recipient mice. Then, 4 million cancer cell-specific T cells (T cell vaccine 1, or T cell vaccine 2, or T cell vaccine 3, or T cell vaccine 4) prepared in step (6) or 100 μL PBS were intravenously injected into the recipient mice. The next day, 1.5 × 10⁻⁶ cells were subcutaneously injected into the lower right back of each recipient mouse. 5 B16F10 cells were used to monitor tumor growth rate and mouse survival in mice. Tumor volume was recorded every 3 days starting from day 3. Tumor volume was calculated using the formula v = 0.52 × a × b. 2 The calculation is performed, where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to animal testing ethics, in mouse survival trials, tumor volumes exceeding 2000 mm² were not permitted. 3 This means that the mouse is considered dead and is euthanized.

[0099] (8) Experimental Results

[0100] like Figure 2As shown, mice in the PBS control group exhibited rapid tumor growth and short survival. Mice treated with several T-cell vaccines showed significantly slower tumor growth and longer survival. T-cell vaccine 4 demonstrated the best efficacy, outperforming T-cell vaccines 1, 2, and 3. This indicates that: adding cytokine combination 1 during antigen-presenting cell activation is more effective than cytokine component 2; solid nanoparticles with antigen-presenting cell membrane components loaded on the surface and containing cell-loaded internal components are more effective than nanovesicles with only membrane components on the surface; and nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell components of cancer cells are far more effective than those prepared from unactivated antigen-presenting cells. In conclusion, the cancer cell-specific T cells described in this invention have a good preventive effect against melanoma. Antigen-presenting cells (ATCs) activated by nanoparticles loaded with whole-cell components of cancer cells degrade and present whole-cell antigens from the cancer cell components loaded by the engulfed nanoparticles. The cancer cell antigenic epitopes presented to the cell membrane surface by the ATCs have already bound to the major histocompatibility complex (MHC) molecules. After mechanical disruption of the ATCs, the cell membrane components of the ATCs 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 ATCs 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 without the assistance of ATCs.

[0101] Example 2: Cancer cell-specific T cell isolation and expansion for melanoma prevention.

[0102] This embodiment uses mouse melanoma as a cancer model to illustrate how to use nanoparticle-activated antigen-presenting cells to prepare nanoparticle-assisted separation and expansion of cancer cell-specific T cells for melanoma prevention. In this embodiment, B16F10 melanoma tumor tissue was lysed to prepare water-soluble and insoluble antigens from the tumor tissue. Then, using PLGA as the nanoparticle framework material and poly(I:C) and CpG1018 as immunoadjuvants, a nanoparticle system loaded with water-soluble and insoluble antigens from 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 cancer cell-specific T cells from tumor-infiltrating lymphocytes using nanoparticle assistance. After expansion, these T cells were injected in vivo to prevent melanoma.

[0103] (1) Lysis of tumor tissue and collection of its components

[0104] 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 five freeze-thaw cycles, 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 antigen. Adding 8M urea to the resulting precipitate dissolved the insoluble antigen, converting it into a soluble antigen 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.

[0105] (2) Preparation of nanoparticles

[0106] In this embodiment, the nanoparticles, as well as the blank nanoparticles and peptide nanoparticles used as controls, were prepared by solvent evaporation. The nanoparticles 1 loaded with whole-cell components were prepared using PLGA, a material with a molecular weight of 7 Da-17 KDa. The immunoadjuvants used were poly(I:C) and CpG1018, with the adjuvants encapsulated within the nanoparticles. The preparation method was as described above. First, the antigen and adjuvant were loaded into 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. The average particle size of these nanoparticles was approximately 280 nm. Each 1 mg of PLGA nanoparticles loaded approximately 100 μg of protein and peptide components, and each 1 mg of PLGA nanoparticles used 0.02 mg of poly(I:C) and CpG1018 immunoadjuvants. 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.

[0107] (3) Preparation of antigen-presenting cells

[0108] 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.

[0109] (4) Activation of antigen-presenting cells

[0110] 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).

[0111] 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).

[0112] (5) Preparation of nanoparticles based on antigen-presenting cells

[0113] 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 was prepared by co-processing the mixed antigen-presenting cell membrane component activated by blank nanoparticle 3 with blank nanoparticle 3, and the nanoparticle had a particle size of 300 nm; nanoparticle 5 was prepared by co-processing the mixed antigen-presenting cell membrane component activated by peptide nanoparticle 2 with peptide nanoparticle 2, and the nanoparticle 6 was prepared by co-processing the mixed antigen-presenting cell membrane component activated by nanoparticle 1 with nanoparticle 1, and the nanoparticle had a particle size of 300 nm.

[0114] 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.

[0115] (6) Isolation and expansion of cancer cell-specific T cells

[0116] On day 0, 5 × 10⁵ cells were subcutaneously injected into the back of each C57BL / 6 mouse. 5Mice were injected subcutaneously with 100 μL of PLGA nanoparticles loaded with whole cancer cell components on days 7, 14, 21, and 28. Mice were sacrificed on day 32, and their spleens and tumor tissues were collected. The tumor tissues were cut into small pieces and passed through a cell sieve to prepare a single-cell suspension. After centrifugation and washing with PBS, CD3+ was isolated from the tumor tissue single-cell suspension by flow cytometry from live cells (dead cells were removed by labeling with a live-dead cell dye). + T cells. Simultaneously, mouse spleens were processed through a cell sieve and lysed with erythrocytes to prepare a single-cell suspension of spleen cells. Flow cytometry was used to sort live cells (dead cells were removed by labeling them with a live-dead cell dye) from the spleen cell suspension containing CD19. + B cells. 100 μg nanoparticles (nanoparticle 4, or nanoparticle 5, or 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 in 5 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). Then, CD3+ cells after incubation were sorted by flow cytometry. + CD134 + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens; among them, cancer cell-specific T cells obtained by nanoparticle 4-assisted sorting are T cell vaccine 1; among them, cancer cell-specific T cells obtained by nanoparticle 5-assisted sorting are T cell vaccine 2; among them, cancer cell-specific T cells obtained by nanoparticle 6-assisted sorting are T cell vaccine 3; and among them, cancer cell-specific T cells obtained by nanoparticle 7-assisted sorting are T cell vaccine 4. Alternatively, 100 μg of nanoparticle 6 based on antigen-presenting cells prepared in step (5) and 400,000 T cells from tumor-infiltrating lymphocytes are co-incubated in 5 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2), and then the incubated CD3 cells are sorted by flow cytometry. + CD134 + T cells, which are cancer cell-specific T cells activated by cancer cell whole-cell antigens, are T cell vaccines.

[0117] Meanwhile, T cells incubated with anti-mouse CD3 antibody, anti-mouse CD4 antibody, anti-mouse CD8 antibody, and anti-mouse CD134 antibody were labeled with these antibodies, respectively. Flow cytometry was then used to analyze the CD8+ levels after co-incubation of different nanoparticles with T cells and antigen-presenting cells. + T cell subtypes and CD4 + CD134 in T cell subtypes +The proportion of T cells. The whole-cell antigen of cancer cells loaded on nanoparticles can be degraded into antigenic epitopes after being phagocytosed by antigen-presenting cells and presented on the surface of antigen-presenting cells. Specific T cells that can recognize the whole-cell antigen of cancer cells are activated and express specific surface markers. By analyzing the proportion of T cells that highly express specific surface markers by flow cytometry, the number of activated and sortable cancer cell-specific T cells that can recognize and kill cancer cells can be determined.

[0118] The sorted T-cell vaccines were incubated with IL-2 (2000 U / mL) and αCD-3 antibody (20 ng / mL) for 14 days (with the medium changed every two days) to amplify the sorted cancer cell-specific T-cell vaccines.

[0119] (7) Cancer cell-specific T cells for cancer prevention

[0120] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to eliminate immune cells in the recipient mice. Then, 1 million cancer cell-specific T-cell vaccines (T-cell vaccine 1, or T-cell vaccine 2, or T-cell vaccine 3, or T-cell vaccine 4, or T-cell vaccine 5) prepared in step (6) or 100 μL of PBS were intravenously injected into the recipient mice. The next day, 1.5 × 10⁻⁶ cells were subcutaneously injected into the lower right back of each recipient mouse. 5 B16F10 cells were used to monitor tumor growth rate and mouse survival in mice. Tumor volume was recorded every 3 days starting from day 3. Tumor volume was calculated using the formula v = 0.52 × a × b. 2 The calculation is performed, where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to animal testing ethics, in mouse survival trials, tumor volumes exceeding 2000 mm² were not permitted. 3 This means that the mouse is considered dead and is euthanized.

[0121] (8) Experimental Results

[0122] like Figure 3As shown, the tumors in mice in the PBS control group and T-cell vaccine group 1 grew rapidly, and the mice had short survival times. Compared with the two control groups, the tumor growth rates in other T-cell vaccine groups were significantly slower, and some mice even experienced tumor disappearance and complete healing. T-cell vaccines 3 and 5 showed the best effects, significantly outperforming T-cell vaccines 2 and 4; moreover, T-cell vaccines 3 and 5 were comparable in efficacy. This indicates that nanoparticles prepared using antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens of cancer cells are beneficial for isolating better cancer cell-specific T cells; and that mixed antigen-presenting cells of DCs and B cells are more effective than single DCs. The superiority of T-cell vaccine 5 over T-cell vaccine 4 demonstrates that the method for assisting the isolation of 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 assisting the isolation of cancer cell-specific T cells, the same effect as with the addition of antigen-presenting cells can be achieved without the addition of antigen-presenting cells, which is also an advantage of the nanoparticles or microparticles prepared from activated antigen-presenting cells used in this invention for assisting the sorting and amplification of cancer cell-specific T cells.

[0123] The cancer-specific T cells prepared by nanoparticle-assisted separation and expansion using antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens of cancer cells showed superior cancer-specific T cell prevention efficacy compared to those prepared by nanoparticle-assisted separation and expansion using nanoparticles loaded with four novel antigenic peptides. This indicates that the types of cancer-specific T cells isolated by nanoparticle-assisted separation using nanoparticles loaded with four novel antigenic peptides are limited, resulting in a smaller number of T cell clones in the expanded T cell system and thus a smaller number of cancer cells that can be recognized and killed. In contrast, nanoparticles prepared by nanoparticle-assisted separation using antigen-presenting cells loaded with whole-cell antigens of cancer cells can assist in the separation of a broader spectrum of cancer-specific T cells, resulting in a wider range of T cell clones after expansion, enabling the recognition and killing of more cancer cells and leading to better therapeutic or preventative effects against cancer.

[0124] like Figure 3 Figures c and d show the in vitro activation of cancer cell-specific T cells by five T-cell vaccines. The nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell components can activate CD8+. + CD134 + T cells and CD4 + CD134 + T cells account for a significant portion of CD8. + T cells and CD4 +The proportion of T cells was significantly higher than that in the control group. This demonstrates that the nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell components, as described in this invention, can better assist in the separation of cancer cell-specific T cells capable of recognizing and killing cancer cells.

[0125] Example 3: Sorted and expanded cancer cell-specific T cells used for melanoma treatment.

[0126] This embodiment uses mouse melanoma as a cancer model to illustrate how to use nanoparticle-activated antigen-presenting cells to prepare nanoparticle-assisted isolation and expansion of cancer cell-specific T cells in mouse peripheral immune cells for the treatment of melanoma. In this embodiment, B16F10 melanoma tumor tissue and cancer cells are first lysed to prepare a water-soluble antigen mixture (mass ratio 1:1) and an insoluble antigen mixture (mass ratio 1:1), and then the water-soluble and insoluble antigen mixtures are mixed at a mass ratio of 1:1. 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. These antigen-presenting cells are then used to prepare nanoparticle-assisted isolation and activation of cancer cell-specific T cells, which are then expanded and used for the treatment of melanoma.

[0127] (1) Lysis of tumor tissue and cancer cells and collection of their components

[0128] 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 antigen soluble in pure water. Adding 8M urea to the resulting precipitate dissolved the insoluble antigen in pure water, converting it into an soluble antigen in an 8M urea aqueous solution. The water-soluble antigen from the tumor tissue and the water-soluble antigen from the cancer cells were mixed at a 1:1 mass ratio; the insoluble antigen from the tumor tissue and the insoluble antigen from the cancer cells were mixed at a 1:1 mass ratio. The mixture of water-soluble and insoluble antigens at a 1:1 mass ratio provided the antigen raw material for preparing nanoparticles.

[0129] (2) Preparation of bacterial extracellular vesicles (OMV) and cancer cell extracellular vesicles

[0130] 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.

[0131] 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.

[0132] (3) Preparation of nanoparticles

[0133] 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 immunoadjuvants 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 peptide components, and each mg of PLGA nanoparticle 1 loaded with 0.02 mg each of poly(I:C) and CpG2006 immunoadjuvants.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] (4) Isolation of B cells

[0138] 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.

[0139] (5) Activation of antigen-presenting cells

[0140] 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).

[0141] (6) Preparation of nanoparticles derived from antigen-presenting cells

[0142] B cells were collected after incubation by centrifugation at 400g for 5 minutes, then washed three times with PBS, resuspended in PBS, and 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. Nanoparticle 5, with a particle size of 110 nm, was prepared using antigen-presenting cells activated by nanoparticle 1; nanoparticle 6, with a particle size of 110 nm, was prepared using antigen-presenting cells activated by nanoparticle 2; nanoparticle 7, with a particle size of 110 nm, was prepared using antigen-presenting cells activated by nanoparticle 3; and nanoparticle 8, with a particle size of 110 nm, was prepared using antigen-presenting cells activated by nanoparticle 4.

[0143] (7) Isolation and expansion of cancer cell-specific T cells

[0144] On day 0, each C57BL / 6 mouse was subcutaneously injected with 5 × 10⁵ cells on its back. 5 On days 10, 17, and 24, mice were subcutaneously injected with 0.5 mg of PLGA nanoparticles prepared in step (3). Mice were sacrificed on day 31, and their spleens were harvested and a single-cell suspension was prepared. CD45+ in live cells of the spleen cells was sorted using magnetic bead sorting (dead cells were labeled with a live-dead cell dye to remove dead cells). + CD3 + T cells. The isolated T cells (4 million units) 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 72 hours (37°C, 5% CO2). Then, the incubated CD3+ cells were sorted by flow cytometry.+ CD134 + T cells are cancer cell-specific T cells that are specifically activated by the whole-cell antigen of cancer cells. Among them, cancer cell-specific T cells obtained by nanoparticle 5-assisted sorting are T cell vaccine 1; cancer cell-specific T cells obtained by nanoparticle 6-assisted sorting are T cell vaccine 2; cancer cell-specific T cells obtained by nanoparticle 7-assisted sorting are T cell vaccine 3; and cancer cell-specific T cells obtained by nanoparticle 8-assisted sorting are T cell vaccine 4.

[0145] Simultaneously, while sorting T cells by flow cytometry, the CD3+ levels in spleen T cells were analyzed after co-incubating with different nanoparticles in DMEM high-glucose complete medium for 48 hours. + CD134 + The proportion of T cells.

[0146] Meanwhile, T cells from unsorted spleen cells were co-incubated with different nanoparticles prepared in step (6) in DMEM high-glucose complete medium for 48 hours. The incubated cells were then collected and labeled with IFN-γ antibody containing a fluorescent probe. Flow cytometry was then used to analyze the IFN-γ levels in the T cells. + The proportion of T cells. 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 are loaded with these degraded and presented antigenic epitopes, which can be recognized by cancer cell-specific T cells and activate them, 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 levels were analyzed using flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells that can recognize and kill cancer cells.

[0147] The cancer cell-specific T-cell vaccine obtained above was incubated with IL-2 (2000 U / mL) and IL-7 (2000 U / mL) in DMEM high glucose complete medium for 7 days (37℃, 5% CO2, medium changed every two days) to amplify the cancer cell-specific T-cell vaccine.

[0148] (8) Cancer cell-specific T cells for cancer treatment

[0149] Female C57BL / 6 mice aged 6-8 weeks were selected as melanoma-bearing mice. On day 0, each mouse was subcutaneously injected with 1.5 × 10⁻⁶ mmol / L of iodine solution into the lower right back. 5Two B16F10 cells were injected intravenously on days 4, 7, 10, 15, 20, and 25 after melanoma inoculation with either 2 million cancer cell-specific T-cell vaccines (T-cell vaccine 1, T-cell vaccine 2, T-cell vaccine 3, or T-cell vaccine 4) or 100 μL of PBS. Tumor volume was recorded every 3 days starting from day 3. Tumor volume was calculated using the formula v = 0.52 × a × b. 2 The calculation is performed, where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to animal testing ethics, in mouse survival trials, tumor volumes exceeding 2000 mm² were not permitted. 3 This means that the mouse is considered dead and is euthanized.

[0150] (9) Experimental Results

[0151] like Figure 4 As shown in Figures a and b, the tumors in mice treated with T-cell vaccine 4 (prepared from antigen-presenting cells activated by blank nanoparticles) grew rapidly and had short survival times. In contrast, the tumor growth rates in mice treated with T-cell vaccines 1, 2, and 3 were significantly slower, and some mice even experienced tumor disappearance and complete remission. Furthermore, T-cell vaccine 2 was more effective than T-cell vaccines 1 and 3, indicating that antigen-presenting cells activated by loading bacterial exovesicle components onto nanoparticles using appropriate methods are beneficial for assisting in the isolation of cancer cell-specific T cells. In conclusion, the cell system described in this invention exhibits excellent therapeutic effects against cancer.

[0152] like Figure 4 Figures c and d show the in vitro activation of cancer cell-specific T cells by the T-cell vaccine. The proportion of cancer cell-specific T cells activated by different particle-assisted sorting methods is related to the therapeutic effects shown in figures a and b. Furthermore, the proportion of activated cancer cell-specific T cells obtained by sorting using CD134 as an activation surface marker is consistent with the proportion of T cells that can release the cytotoxic cytokine IFN-γ upon encountering cancer cell antigens. This indicates that the T cells sorted using CD134 as an activation surface marker are cancer cell-specific T cells capable of specifically recognizing and killing cancer cells. Therefore, the separation method described in this invention can effectively sort cancer cell-specific T cells in tumor tissue that have the ability to recognize and kill cancer cells.

[0153] Example 4: Nanoparticle-assisted isolation and expansion of cancer cell-specific T cells for cancer prevention.

[0154] 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 nanoparticle-assisted separation of cancer cell-specific T cells, which were then expanded for cancer prevention.

[0155] (1) Lysis of cancer cells

[0156] 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.

[0157] (2) Preparation of micron-sized particles

[0158] 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.

[0159] 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.

[0160] 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).

[0161] (3) Preparation of antigen-presenting cells

[0162] 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.

[0163] (4) Activation of antigen-presenting cells

[0164] 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).

[0165] (5) Preparation of nanoparticles derived from antigen-presenting cells

[0166] 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 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 with a particle size of 110 nm.

[0167] (6) Isolation and expansion of cancer cell-specific T cells

[0168] 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 radiation therapy by irradiating the tumor sites with B16F10 cells on days 10, 15, and 20. Mice were sacrificed on day 25, and tumor tissues from each group were collected. The tumor tissues were cut into small pieces and passed through a cell sieve to prepare single-cell suspensions. Then, a magnetic bead sorting method was used to separate the CD3+ levels in the live cells from the tumor tissue single-cell suspensions (dead cells were removed by labeling them with a live-dead cell dye). + T cells. The sorted T cells (5 million units) and the nanoparticles prepared in step (5) (100 μg) were co-incubated in 2 mL of RPMI 1640 complete medium for 24 hours (37℃, 5% CO2). Then, CD69 groups in the T cells were sorted using a magnetic bead sorting method. + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens. The cancer cell-specific T cells obtained above were incubated with IL-2 (2000 U / mL), αCD-3 antibody (20 ng / mL), and αCD-28 antibody (20 ng / mL) in RPMI 1640 complete medium for 7 days (with medium changes every two days) to expand the sorted cancer cell-specific T cells.

[0169] (7) Cancer-specific T cell expansion for cancer prevention

[0170] Female C57BL / 6 mice aged 6-8 weeks were selected as melanoma-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to deplete immune cells in the recipient mice. On day 0, mice were intravenously injected with 100 μL of cancer cell-specific T cells containing 3 million cells. Simultaneously, on day 0, each mouse was subcutaneously injected with 1.5 × 10⁻⁶ cells. 5 Using B16F10 cells, the tumor volume of mice was recorded every 3 days starting from day 3. Tumor volume was calculated using the formula v = 0.52 × a × b.2 The calculation is performed, where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to animal testing ethics, in mouse survival trials, tumor volumes exceeding 2000 mm² were not permitted. 3 This means that the mouse is considered dead and is euthanized.

[0171] (8) Experimental Results

[0172] like Figure 5 As shown, tumors in the control group mice all grew, while the tumor growth rate in mice treated with cancer cell-specific T cells was significantly slowed and survival was significantly prolonged. Furthermore, T cells prepared from antigen-presenting cells activated by microparticles loaded with a mixture of CpG adjuvant and Poly ICLC adjuvant showed better preventative effects against melanoma than those prepared from microparticles activated by antigen-presenting cells loaded with a mixture of two CpG adjuvants. Moreover, 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 were more effective than those prepared from microparticles activated by microparticles loaded with a mixture of two type A CpGs and Poly ICLC adjuvants. This indicates that nanoparticles prepared from antigen-presenting cells activated by microparticles loaded with a mixture of two different Toll-like receptor adjuvants are more effective, and that nanoparticles prepared from microparticles activated by antigen-presenting cells containing a mixture of type B CpG and a Toll-like receptor 3 agonist are even more effective.

[0173] Example 5: Cancer Cell-Specific T Cells for Cancer Prevention

[0174] 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 were isolated from tumor-infiltrating lymphocytes with the assistance of this method. The cells were then expanded and used for cancer prevention.

[0175] (1) Collection and lysis of tumor tissue

[0176] 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. 3Mice 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.

[0177] (2) Preparation of nanoparticles

[0178] 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 of Poly(I:C), CpG2006, and CpGSL01.

[0179] 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.

[0180] The control nanoparticles 3 have a particle size of about 270 nm and are loaded with an equal amount of lysate components. The loaded immunoadjuvants are Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A). Each 1 mg of PLGA is loaded with 0.02 mg of each of Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A).

[0181] (3) Preparation of DC and B cells

[0182] 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.

[0183] (4) Activation of antigen-presenting cells

[0184] 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).

[0185] (5) Preparation of nanoparticles based on antigen-presenting cells

[0186] 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 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. The 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 with a particle size of 150 nm.

[0187] (6) Isolation and expansion of cancer cell-specific T cells

[0188] 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) on ​​days 8, 10, 12, 14, 16, 18, and 20 of B16F10 cells. Mice were sacrificed on day 24, and tumor tissues were collected from each group. Tumor tissue single-cell suspensions were prepared, and CD3+ was extracted from live cells (dead cells were removed by labeling with a live-dead cell dye) using magnetic bead sorting. + T cells. Then, the sorted T cells (500,000) were co-incubated with allogeneic B cells (2.5 million) and nanoparticles prepared in step (5) (100 μg) in 10 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). The incubated CD3+ cells were then sorted by flow cytometry. + CD8 + CD69 + T cells and CD3 + CD4 + CD69 +T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens. The cancer cell-specific T cells obtained above were incubated with IL-2 (2000 U / mL), αCD-3 antibody (20 ng / mL), and αCD-28 antibody (20 ng / mL) in RPMI 1640 complete medium for 11 days (with medium changed every two days) to expand the sorted cancer cell-specific T cells.

[0189] (7) Cancer cell-specific T cells for cancer prevention

[0190] Female C57BL / 6 mice aged 6-8 weeks were selected as melanoma-bearing mice. One day before transplantation of cancer cell-specific T cells, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to deplete immune cells in the recipient mice. On day 0, mice were subcutaneously injected with 100 μL of 800,000 amplified cancer cell-specific CD8+ cells. + T cells and 200,000 expanded cancer cell-specific CD4 cells + T cells were also administered subcutaneously to each mouse on day 0. 5 Using B16F10 cells, the tumor volume of mice was recorded every 3 days starting from day 3. Tumor volume was calculated using the formula v = 0.52 × a × b. 2 The calculation is performed, where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to animal testing ethics, in mouse survival trials, tumor volumes exceeding 2000 mm² were not permitted. 3 This means that the mouse is considered dead and is euthanized.

[0191] (8) Experimental Results

[0192] like Figure 6 As shown, tumors in the control group mice all grew, while tumor-specific T cells obtained by transplantation from antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens of cancer cells showed significantly slower tumor growth, and most mouse cancer cells disappeared after inoculation. Furthermore, T cells obtained by nanoparticle-assisted separation and expansion of antigen-presenting cells activated by nanoparticles loaded with two types of B CpG and Poly(I:C) as a mixed adjuvant were more effective than T cells obtained by nanoparticle-assisted separation and expansion of antigen-presenting cells activated by nanoparticles loaded with two types of A CpG and Poly(I:C) as a mixed adjuvant, or by nanoparticles loaded only with Poly(I:C) as an adjuvant.

[0193] Example 6: Cancer Cell-Specific T Cells for the Treatment of Colon Cancer

[0194] This embodiment uses MC38 mouse colon cancer as a cancer model to illustrate how to use nanoparticles to assist in the isolation of broad-spectrum cancer cell-specific T cells for the treatment of colon cancer. First, colon cancer tumor tissue and lung cancer cells are lysed to prepare water-soluble antigens, which are then degraded into peptides in vitro using a protease. In practical applications, other enzymes or methods can be used to degrade proteins in whole-cell components into peptides first. Then, a mixture of water-soluble antigens (1:1 mass ratio) and an insoluble antigen mixture (1:1 mass ratio) are prepared and mixed at a 1:1 mass ratio. Next, nanoparticles are prepared using PLA as the nanoparticle framework material and CpGM362, CPG1018, and Poly ICLC as immunoadjuvants. These nanoparticles are then used to activate cancer cell-specific T cells in vitro, and the expanded cancer cell-specific T cells are then isolated, extracted, and used for the treatment of colon cancer.

[0195] (1) Lysis of tumor tissue and cancer cells and collection of their components

[0196] 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. 3 Mice were sacrificed 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 antigen. Adding 8M urea to the resulting precipitate dissolved the insoluble antigen, converting it into a soluble antigen in 8M urea solution. Trypsin (0.5 mg / mL) and chymotrypsin (0.5 mg / mL) were added to the water-soluble antigen (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.

[0197] 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 antigen soluble in pure water. Add 8M urea to the resulting precipitate to dissolve the precipitate, thus converting the insoluble antigen into one soluble in 8M urea aqueous solution. Add trypsin (0.5mg / mL) and chymotrypsin (0.5mg / mL) to the water-soluble antigen (80mg / mL) and incubate for 1 hour, then heat at 95℃ for 10 minutes to inactivate the proteases for later use.

[0198] Water-soluble antigens from colon cancer tumor tissue and lung cancer cells were mixed at a mass ratio of 1:1; insoluble antigens dissolved in 8M urea were also mixed at a mass ratio of 1:1. The mixture of water-soluble and insoluble antigens was then mixed at a mass ratio of 1:1; this mixture served as the raw material source for preparing nanoparticles.

[0199] (2) Cleavage and dissolution of BCG

[0200] BCG was collected, and the BCG was pyrolyzed using an 8M urea aqueous solution. The pyrolyzed components were then dissolved for later use.

[0201] (3) Preparation of nanoparticles

[0202] In this embodiment, nanoparticle 1 was prepared using a solvent evaporation method. The PLA material used to prepare nanoparticle 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. First, a double emulsion method is used to load the mixture of lysates, bacterial lysates, and adjuvants internally into 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. 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 the mixture of cancer cell and tumor tissue lysates and bacterial lysates (80 mg / mL). The mixture is then incubated at room temperature for 5 minutes before use. The average particle size of nanoparticle 1 is about 290 nm. Each 1 mg of PLGA nanoparticle 1 is loaded with about 140 μg of protein or peptide components. Each 1 mg of PLGA nanoparticle contains 0.04 mg each of CpGM362, CPG1018 and Poly ICLC immune adjuvant.

[0203] (4) Preparation of antigen-presenting cells

[0204] 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 BMDC and BMDM were used as antigen-presenting cells. The preparation method of BMDC was the same as in Example 2. The preparation method of BMDM was as follows: C57 mice were anesthetized and euthanized by dislocation. The mice were disinfected with 75% ethanol. Then, a small incision was made on the back of the mouse with scissors, and the skin was torn open directly to the lower leg joint. The mouse paw joint and skin were removed. The hind limb was removed along the greater trochanter of the thigh with scissors. After removing the muscle tissue, the limb was placed in a culture dish containing 75% ethanol and soaked for 5 minutes. A new culture dish containing 75% ethanol was then placed in a clean bench. The ethanol-soaked leg bone was transferred to cold PBS and soaked to wash away the ethanol on the surface of the tibia and femur. This process could be repeated 3 times. The cleaned femur and tibia were separated, and the two ends of the femur and tibia were cut off with scissors. The bone marrow was blown out of the femur and tibia using a 1mL syringe with cold induction culture medium. This was repeated 3 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.

[0205] (5) Activation of antigen-presenting cells

[0206] 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).

[0207] 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).

[0208] (6) Preparation of nanoparticles based on antigen-presenting cells

[0209] 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 prepared in step (3) at 4°C for 16 hours. Then, the nanoparticles were repeatedly co-extruded through a 0.45μm 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.

[0210] 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.

[0211] (7) Isolation and expansion of cancer cell-specific T cells

[0212] On day 0, each C57BL / 6 mouse was subcutaneously injected with 1.5 × 10⁻⁶ mmol / L on its back. 5MC38 cells were subcutaneously injected into mice with 100 μL of 1 mg PLGA nanoparticles on days 10, 15, and 21. Mice were sacrificed on day 24, and draining lymph nodes and spleens were collected. Single-cell suspensions of the draining lymph nodes and spleen cells were prepared, and T cells were sorted from them using magnetic beads. The resulting T cells (4 million cells), 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 incubated in 5 mL L DMEM complete medium for 96 hours. CD3+ cells were then sorted by flow cytometry. + CD8 + CD69 + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens. The cancer cell-specific T cells obtained above were incubated with IL-2 (1000 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), and αCD-3 antibody (10 ng / mL) in DMEM complete medium for 8 days (with medium changed every two days) to amplify the sorted cancer cell-specific CD8+. + T cells. Among them, cancer cell-specific T cells sorted and expanded with the assistance of nanoparticle 1 are designated as T cell vaccine 1; cancer cell-specific T cells sorted and expanded with the assistance of nanoparticle 2 are designated as T cell vaccine 2; and cancer cell-specific T cells sorted and expanded with the assistance of nanoparticle 3 are designated as T cell vaccine 3.

[0213] (8) Cancer cell-specific CD8 + T-cell therapy for cancer

[0214] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer-bearing mice. On day 0, each mouse was subcutaneously injected with 2×10⁻⁶ g of [unspecified substance]. 6 Mice were injected with 100 μL of MC38 cells containing 2 million cancer cell-specific CD8+ on days 4, 7, 10, 15, and 20. + T cells. The methods for monitoring tumor volume and survival in mice are the same as above.

[0215] (9) Experimental Results

[0216] like Figure 7As shown, the tumors in mice in the PBS control group and the T-cell vaccine group 1 grew rapidly, resulting in short survival times. Compared to these two groups, the tumor growth rate in mice treated with T-cell vaccines 2 and 3 was significantly slower, and some mice even experienced tumor disappearance and complete remission. Furthermore, T-cell vaccine 3 was more effective than T-cell vaccine 2. 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 effect of nanoparticles or microparticles in assisting the separation and expansion of cancer cell-specific T cells. In conclusion, the T-cell vaccine of this invention has a good therapeutic effect on colon cancer.

[0217] Example 7: Cancer Cell-Specific T Cells for Breast Cancer Prevention

[0218] 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 used to prepare microparticle-assisted separation of specific T cells from peripheral blood cancer cells for breast cancer prevention.

[0219] (1) Lysis of cancer cells

[0220] 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 antigen component. The precipitate was dissolved in 10% sodium deoxycholate (containing 10M arginine) to obtain the water-insoluble antigen component. The water-soluble and water-insoluble antigen components were mixed at a mass ratio of 3:1 to obtain the raw material for preparing the particle system.

[0221] (2) Preparation of micron-sized particle systems

[0222] 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).

[0223] (3) Preparation of B cells

[0224] 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.

[0225] (4) Activation of antigen-presenting cells

[0226] 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).

[0227] (5) Preparation of micron-sized particles based on antigen-presenting cells

[0228] 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 100W for 2 minutes with the micronized particles (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 micronized particles with a particle size of 2.6μm.

[0229] (6) Isolation and expansion of cancer cell-specific T cells

[0230] 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, 17, and 24, with 100 μL of 1 mg PLGA microparticles. Mice were sacrificed on day 30, and peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were isolated from the PBMCs, and CD3+ cells were then isolated from the PBMCs using flow cytometry. + T cells. One million T cells, two million DC2.4 cells, and 50 μg of microparticles were incubated in 2 mL of DMEM complete medium for 72 hours (37°C, 5% CO2). CD3+ cells were then isolated by flow cytometry. + CD69 + T cells are cancer cell-specific T cells activated by cancer cell whole-cell antigens. The cancer cell-specific T cells obtained above were incubated with IL-2 (4000 U / mL), IL-7 (2000 U / mL), and αCD-3 antibody (20 ng / mL) in DMEM complete medium for 12 days (with medium changed every two days) to expand the sorted cancer cell-specific T cells.

[0231] (4) Cancer cell-specific T cells for cancer prevention

[0232] Female BALB / c mice aged 6-8 weeks were selected as breast cancer tumor-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to deplete immune cells in the recipient mice. On day 0, mice were subcutaneously injected with 100 μL of 1.5 million expanded cancer cell-specific T cells. Simultaneously, on day 0, each mouse was subcutaneously injected with 1 × 10⁻⁶ cells. 6Four T1 cells were used, and the size of the mouse tumor was recorded every three days starting from day 3. The methods for monitoring mouse tumor growth and survival were the same as above.

[0233] (5) Experimental Results

[0234] like Figure 8 As shown, compared with the control group, mice treated with cancer cell-specific T cells obtained by microparticle-assisted isolation from antigen-presenting cells activated by microparticles exhibited significantly slower tumor growth and prolonged survival. Furthermore, microparticles prepared from antigen-presenting cells activated by microparticles using a mixture of two types of CpG and Poly(I:C) as adjuvants showed better efficacy than those prepared from microparticles activated by microparticles using a mixture of two types of A CpG and Poly(I:C) as adjuvants. 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.

[0235] Example 8: Cancer Cell-Specific T Cells for Cancer Prevention

[0236] This embodiment uses mannose as a target to illustrate how to use actively targeted nanoparticles to activate antigen-presenting cells to assist in the isolation of cancer cell-specific T cells for cancer prevention. In practical applications, the specific dosage form, adjuvant, administration time, frequency of administration, and administration regimen can be adjusted according to the circumstances. Actively targeted nanoparticles can be taken up by mannose receptors on the surface of dendritic cells and enter the cells.

[0237] (1) Lysis of cancer cells

[0238] 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.

[0239] (2) Preparation of nanoparticles

[0240] 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.

[0241] (3) Preparation of antigen-presenting cells

[0242] This embodiment uses BMDC and BMDM as antigen-presenting cells. The preparation methods for BMDC and BMDM are the same as above.

[0243] (4) Activation of antigen-presenting cells

[0244] 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). Alternatively, BMDC (10 million units), BMDM (10 million units), and IL-7 (500 U / mL) were 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). Both incubation systems contained 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).

[0245] (5) Preparation of nanoparticles derived from antigen-presenting cells

[0246] 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.

[0247] (6) Isolation and expansion of cancer cell-specific T cells

[0248] On day 0, each C57BL / 6 mouse was subcutaneously injected with 1×10⁻⁶ dredges on its back. 6 Pan02 pancreatic cancer cells were injected subcutaneously into mice on days 10, 15, 20, and 27 with 100 μL of 1 mg PLGA nanoparticles. Mice were sacrificed on day 24, and tumor tissue and lymph nodes were harvested. Single-cell suspensions were prepared from the tumor tissue and lymph nodes, respectively. CD45 groups were then isolated from the tumor tissue and lymph node single-cell suspensions using flow cytometry. + CD3 + T cells were obtained by mixing T cells from tumor tissue and lymph nodes. The T cells (5 million units) were then co-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), IL-7 (500 U / mL), and IFN-γ (500 U / mL). CD3+ was then isolated from the incubated cells using flow cytometry. + CD69 +T cells, specifically cancer cell-specific T cells, were obtained by incubating the sorted T cells with IL-2 (2000 U / mL), IL-7 (2000 U / mL), IL-15 (1000 U / mL), and αCD-3 antibody (50 ng / mL) in DMEM high-glucose medium for 12 days (with medium changed every two days) to expand the resulting cancer cell-specific T cells. Specifically, cancer cell-specific T cells sorted and expanded using nanoparticle 1 were designated as T cell vaccine 1; those sorted and expanded using nanoparticle 3 were designated as T cell vaccine 2; those sorted and expanded using nanoparticle 4 were designated as T cell vaccine 3; and those sorted and expanded using nanoparticle 5 were designated as T cell vaccine 4.

[0249] (7) Cancer cell-specific T cells for cancer prevention

[0250] Female C57BL / 6 mice aged 6-8 weeks were selected as pancreatic cancer-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to deplete immune cells in the recipient mice. Then, 5 million cancer cell-specific T cells (T cell vaccine 1, T cell vaccine 2, T cell vaccine 3, or T cell vaccine 4) or 100 μL of PBS were intravenously injected into the recipient mice. The following day, each recipient mouse was subcutaneously inoculated with 1 × 10⁻⁶ cells on the lower right back. 6 One Pan02 pancreatic cancer cell line was used. Tumor growth rate and survival time in mice were monitored. The methods for monitoring tumor growth and survival time were the same as above.

[0251] (8) Experimental Results

[0252] like Figure 9 As shown, the tumors in mice treated with PBS, T-cell vaccine 1, and T-cell vaccine 2 grew rapidly, leading to rapid mortality. Compared to the aforementioned groups, the tumor growth rate in mice treated with T-cell vaccine 3 and T-cell vaccine 4 was significantly slower. Furthermore, T-cell vaccine 4 was more effective than T-cell vaccine 3. In conclusion, regardless of whether the antigen-presenting cells are activated by adjuvanted nanoparticles, the nanoparticles prepared from them can effectively assist in the separation of cancer cell-specific T cells from tumor-infiltrating lymphocytes. However, nanoparticles prepared from antigen-presenting cells activated by adjuvanted nanoparticles are more effective. This indicates that the cancer cell-specific T cells described in this invention can effectively prevent cancer.

[0253] Example 9: Nanoparticle-assisted separation of cancer cell-specific T cells for lung cancer prevention

[0254] This embodiment illustrates how calcified nanoparticles aid in the separation of cancer cell-specific T cells from tumor-infiltrating lymphocytes. 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 nanoparticles to aid in the separation of tumor-infiltrating lymphocytes and cancer cell-specific T cells from peripheral blood. These nanoparticles were then expanded for lung cancer prevention.

[0255] (1) Lysis of tumor tissue and cancer cells

[0256] 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.

[0257] (2) Preparation of nanoparticles

[0258] 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. Then, 100 mg of PLGA nanoparticles were centrifuged at 13000 g for 20 min and resuspended in 18 mL of PBS. Next, 2 mL of tumor tissue and cancer cell lysis buffer (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 PLGA nanoparticles were then resuspended in 20 mL of DMEM culture 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. The average particle size of the nanoparticles was approximately 290 nm; each 1 mg of PLGA nanoparticles was loaded with approximately 140 μg of protein or peptide components, 0.03 mg each of CpG2006 and Poly(I:C).

[0259] (3) Preparation of antigen-presenting cells

[0260] In this embodiment, BMDCs and B cells were used as antigen-presenting cells. The BMDC preparation method was the same as in Example 1. B cells were obtained from mouse peripheral blood PBMCs and prepared in the same way.

[0261] (4) Activation of antigen-presenting cells

[0262] Nanoparticles (1000 μg) loaded with whole-cell components of cancer cells were co-incubated with 5 million BMDCs and 5 million B 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), or as a control, the incubation system did not contain any cytokines or antibodies.

[0263] (5) Preparation of nanoparticles based on antigen-presenting cells

[0264] 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 nanoparticles with a particle size of 300 nm.

[0265] (6) Isolation and expansion of cancer cell-specific T cells

[0266] On day 0, each C57BL / 6 mouse was subcutaneously injected with 1×10⁻⁶ dredges on its back. 6 LLC lung cancer cells were administered to mice via subcutaneous injection of 100 μL of 1 mg PLGA nanoparticles on days 10, 15, 20, and 27. Mice were sacrificed on day 24, and tumor tissue and peripheral blood were collected. Single-cell suspensions were prepared from the tumor tissue, and CD45+ cells were isolated from these suspensions using flow cytometry. + CD3 + T cells; PBMCs were isolated from peripheral blood, and CD45 was isolated from PBMCs using flow cytometry. +CD3 + T cells were prepared by mixing T cells from tumor tissue and peripheral blood. Then, 5 million T cells were co-incubated with 100 μg of antigen-presenting cell nanoparticles in DMEM high-glucose medium for 72 hours (37°C, 5% CO2), with IL-2 (500 U / mL) and IL-7 (500 U / mL) present during incubation; or 5 million T cells were co-incubated with 100 μg of antigen-presenting cell nanoparticles in DMEM high-glucose medium for 72 hours (37°C, 5% CO2), with no cytokines or antibodies present in the incubation system. CD3+ was then isolated from the incubated cells using flow cytometry. + CD69 + T cells are cancer cell-specific T cells. The sorted T cells were incubated with IL-2 (2000 U / mL), IL-7 (2000 U / mL), IL-15 (1000 U / mL) and αCD-3 antibody (50 ng / mL) in DMEM high glucose medium for 12 days (with medium changed every two days) to expand the T cells.

[0267] (7) Cancer cell-specific T cells for cancer prevention

[0268] Female C57BL / 6 mice aged 6-8 weeks were selected as lung cancer tumor-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to deplete immune cells in the recipient mice. Then, 5 million cancer cell-specific T cells were intravenously injected into the recipient mice. The following day, each recipient mouse was subcutaneously inoculated with 1 × 10⁻⁶ cells on the lower right back. 6 Several LLC lung cancer cells were used. Tumor growth rate and mouse survival were monitored. The methods for monitoring tumor growth and survival were the same as above.

[0269] (8) Experimental Results

[0270] like Figure 10 As shown, compared with the control group, cancer cell-specific T cells prepared from antigen-presenting cells activated by calcified nanoparticles can prolong the survival of mice and effectively prevent cancer. Furthermore, when antigen-presenting cells are activated by nanoparticles loaded with whole-cell antigens of cancer cells, the system containing cytokines and / or antibodies is superior to the system without cytokines and / or antibodies; moreover, when nanoparticles prepared from antigen-presenting cells are co-incubated with T cells, the system containing cytokines and / or antibodies is superior to the system without cytokines and / or antibodies.

[0271] Example 10: Cancer-specific T cell therapy for melanoma treatment

[0272] (1) Lysis of tumor tissue and cancer cells and collection of their components

[0273] 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. Then, nuclease (0.5 mg / mL), trypsin (0.5 mg / mL), and chymotrypsin (0.5 mg / mL) were added and incubated for 15 minutes, followed by inactivation of the nuclease at 95°C for 10 minutes. The mixture was then centrifuged at 8000g for 3 minutes. The supernatant was the water-soluble antigen; the precipitate was dissolved in a 10% sodium deoxycholate aqueous solution to remove the water-insoluble antigen. The water-soluble antigen and the sodium deoxycholate-dissolved water-insoluble antigen were mixed at a 1:1 mass ratio to obtain the antigen raw material for preparing the nanoparticle system.

[0274] (2) Preparation of nanoparticle systems

[0275] 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 peptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and both the adjuvant and the KALA peptide were encapsulated within the nanoparticles. The preparation method was as described above. First, the lysis buffer components, adjuvant, and KALA peptide were loaded inside the nanoparticles using a double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 12000 g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. Nanoparticle 1 has an average particle size 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 is loaded with 0.02 mg each of poly(I:C), CpG1018, and CpG2216 immune adjuvants, and 0.05 mg of KALA peptide. Nanoparticle 2 is prepared using the same materials and methods as nanoparticle 1, with a particle size of approximately 250 nm and a surface potential of approximately -5 mV. Nanoparticle 2 does not load KALA peptide, but it is loaded with an equal amount of adjuvant and cell lysis components. The materials and preparation method of nanoparticle 3 are the same as those of nanoparticle 1. It is about 250 nm in size and has a surface potential of about -5 mV. Each 1 mg of PLGA nanoparticles is loaded with about 100 μg of protein and peptide components. Each 1 mg of PLGA nanoparticles is loaded with 0.02 mg of poly(I:C), 0.04 mg of CpG1018, and 0.05 mg of KALA peptide.

[0276] (3) Preparation of antigen-presenting cells

[0277] In this embodiment, BMDC and BMDM are used as mixed antigen-presenting cells. The preparation methods of BMDC and BMDM are the same as above.

[0278] (4) Activation of antigen-presenting cells

[0279] 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).

[0280] (5) Preparation of nanoparticles based on antigen-presenting cells

[0281] 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 for 3 minutes (25 W) in the presence of phosphatase and protease inhibitors. The resulting cell membranes were washed with 10 mM pH 7.5 Tris-HCl and 1 mM EDTA solution. The samples were then filtered sequentially through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, and 0.45 μm. The filtrate was centrifuged at 12000g for 45 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in physiological saline containing 4% mannitol and then freeze-dried to obtain nanoparticles. Nanoparticle 4 was prepared using antigen-presenting cells activated by nanoparticle 1, with a particle size of 260 nm; nanoparticle 5 was prepared using antigen-presenting cells activated by nanoparticle 2, with a particle size of 260 nm; and nanoparticle 6 was prepared using antigen-presenting cells activated by nanoparticle 3, with a particle size of 260 nm.

[0282] (6) Preparation of cancer cell-specific T cells

[0283] 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 peptides) 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 separated using magnetic bead sorting. + CD3 + T cells. The sorted CD3...+ 5 million T cells, 40 μg of nanoparticles (nanoparticle 4, 5, or 6), and IL-7 (10 ng / mL) were co-incubated in 2 mL of RPMI 1640 complete medium for 96 hours. CD3+ was then sorted from the incubated T cells using flow cytometry. + OX40 + T cells are cancer cell-specific T cells that can recognize full-cell antigens of cancer cells. The CD3 cells obtained above... + OX40 + T cells were incubated with IL-2 (1000 U / mL), IL-15 (1000 U / mL), IL-21 (1000 U / mL), and αCD-3 antibody (20 ng / mL) in RPMI 1640 complete medium for 14 days (with medium changes every two days) to expand cancer cell-specific T cells. Specifically, cancer cell-specific T cells expanded with the assistance of nanoparticle 4 were designated as T cell vaccine 1; those expanded with the assistance of nanoparticle 5 were designated as T cell vaccine 2; and those expanded with the assistance of nanoparticle 6 were designated as T cell vaccine 3.

[0284] (7) Expanded cancer cell-specific T cells are used to treat cancer.

[0285] Female C57BL / 6 mice aged 6-8 weeks were selected as melanoma-bearing mice. On day 0, each mouse was subcutaneously injected with 1.5 × 10⁻⁶ mmol / L of iodine solution into the lower right back. 5 1.5 million expanded cancer cell-specific T cells were intravenously injected on days 4, 7, 10, 15, and 20 after melanoma inoculation. Tumor volume and survival in mice were monitored using the same methods as above.

[0286] (8) Experimental Results

[0287] like Figure 11As shown, tumors in the PBS control group grew rapidly. Compared to the control group, mice treated with nanoparticle-assisted separation and expansion of cancer cell-specific T cells prepared from antigen-presenting cells loaded with whole-cell components exhibited significantly slower tumor growth and prolonged survival. Furthermore, the cancer cell-specific T cell vaccine 1 prepared from antigen-presenting cells activated by nanoparticle 1 (with added lysosomal escape substances) was superior to the cancer cell-specific T cell vaccine 2 prepared from antigen-presenting cells activated by nanoparticle 5 (without added lysosomal escape nanoparticle 2). The cancer cell-specific T cell vaccine 1 prepared from antigen-presenting cells activated by nanoparticle 1 (using two CpG and Poly(I:C) as a mixed adjuvant) was more effective than the T cell vaccine 3 prepared from antigen-presenting cells activated by nanoparticle 3 (using only one CpG and Poly(I:C) as a mixed adjuvant). In conclusion, the T cells described in this invention have good therapeutic effects on cancer.

[0288] Example 11: Cancer Cell-Specific T Cells for Breast Cancer Prevention

[0289] This embodiment uses 4T1 triple-negative breast cancer in mice as a cancer model to illustrate how nanoparticles, prepared by activating antigen-presenting cells with microparticles loaded with cancer cell whole-cell antigens, can be used to assist in the sorting of cancer cell-specific T cells for breast cancer prevention. In this embodiment, breast cancer cells are first inactivated and denatured, then lysed, and the insoluble antigens 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 nanoparticles. These nanoparticles are then used to assist in the separation and expansion of cancer cell-specific T cells for cancer prevention.

[0290] (1) Lysis of cancer cells

[0291] 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 antigen. The precipitate was dissolved with 10% octyl glucoside to obtain the dissolved original insoluble antigen. The water-soluble antigen and the insoluble antigen were mixed at a mass ratio of 2:1 to obtain the lysate component required for the preparation of micron-sized particles.

[0292] (2) Preparation of micron-sized particle systems

[0293] In this embodiment, the microparticles were prepared using a double emulsion method. The PLGA backbone material of microparticle 1 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 using a double emulsion method to prepare microparticles internally loaded with lysate components, adjuvants, and KALA peptides. 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 hours before use. The average particle size of microparticle 1 was approximately 1.5 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.

[0294] (3) Preparation of antigen-presenting cells

[0295] In this embodiment, BMDCs and mouse spleen cells-derived B cells are used as antigen-presenting cells. The preparation methods for BMDCs and B cells are the same as above. BMDCs and B cells are mixed in a 1:1 ratio to obtain mixed antigen-presenting cells.

[0296] (4) Activation of mixed antigen-presenting cells

[0297] 1000 μg of microparticles loaded with whole-cell components of cancer cells were co-incubated with 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million DC2.4 cells) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained GM-CSF (500 U / mL), M-CSF (500 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).

[0298] (5) Preparation of micron-sized particles derived from antigen-presenting cells

[0299] Two hundred 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 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 co-incubated with 40mg of the nanoparticles prepared in step (2) for 10 minutes and then repeatedly squeezed out through a 2μm filter membrane. The squeezed liquid was then centrifuged at 10000g for 20 minutes and the supernatant was discarded. The precipitate was collected and resuspended in PBS to obtain micron-sized particles 2 with a particle size of 1.6μm.

[0300] (6) Preparation of cancer cell-specific T cells

[0301] 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 administered subcutaneously on days 7, 14, 21, and 28. Microparticles of PLGA (loaded with lysate components, adjuvants, and substances to increase lysosomal escape) were injected subcutaneously. Mice were sacrificed on day 32, and tumor tissue was collected. The tumor tissue was cut into small pieces and passed through a cell sieve to prepare a single-cell suspension. CD3+ cells were isolated from the tumor tissue single-cell suspension using flow cytometry. + Tumor-infiltrating T cells. The sorted CD3... + T cells (1 million), 100 μg microparticles, and IL-7 (500 U / mL) were co-incubated in 5 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2); or the sorted CD3... + T cells (1 million) and 100 μg of micronized particles were co-incubated in 5 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2); or the sorted CD3+ cells were incubated in the medium. + 1 million T cells, 1 million BMDCs prepared in step (3), 100 μg microparticles, and IL-7 (500 U / mL) were co-incubated in 5 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). Then, flow cytometry was used to sort the incubated CD3+ cells. + CD8 in T cells + CD69 + T cells and CD4 + CD69 +T cells are cancer cell-specific T cells that can recognize full-cell antigens of cancer cells. The CD8 cells obtained above... + CD69 + T cells or CD4 + CD69 + T cells were co-incubated for 14 days in RPMI 1640 complete medium with IL-2 (1000 U / mL), IL-6 (1000 U / mL), IL-12 (1000 U / mL), and αCD28 antibody (10 ng / mL) to expand cancer cell-specific T cells. CD3+ was used in this incubation. + Cancer cell-specific T cells obtained by co-incubation and sorting of T cells, microparticles 2, and IL-7 were used as T cell vaccine 1; CD3 was used. + Cancer cell-specific T cells obtained by co-incubation and sorting of T cells and microparticles 2 were used as T cell vaccine 2; CD3 was used. + Cancer cell-specific T cells obtained by co-incubation and sorting of T cells, DCs, microparticles 1, and IL-7 are T cell vaccines 3.

[0302] (7) Cancer cell-specific T cells for cancer prevention

[0303] Female BALB / c mice aged 6-8 weeks were selected as model mice to prepare breast cancer-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to deplete immune cells in the recipient mice. On day 0, mice were subcutaneously injected with 100 μL of T-cell vaccine (containing 1 million amplified CD8+ cells). + T cells and 200,000 expanded CD4 cells + Alternatively, administer 100 μL of PBS. Simultaneously, on day 0, each mouse should be subcutaneously injected with 1 × 10⁻⁶ PBS. 6 4T1 cells. The methods for monitoring mouse tumor growth and survival were the same as above.

[0304] (8) Experimental Results

[0305] like Figure 12As shown, compared with the PBS control group, the tumor growth rate and mouse survival time were significantly slower in the group treated with the cancer cell-specific T cell vaccine prepared from antigen-presenting cells activated by microparticles and assisted in sorting. Furthermore, the cancer cell-specific T cell vaccine 1, obtained by adding IL-7 during co-incubation of nanoparticles prepared from antigen-presenting cells with immune cells containing T cells, was more effective than the cancer cell-specific T cell vaccine 2 obtained without IL-7 assistance during co-incubation. Moreover, T cell vaccine 1 was more effective than T cell vaccine 3. This indicates that adding the cytokine IL-7 during co-incubation is beneficial for assisting the isolation of cancer cell-specific T cells. Furthermore, the cancer cell-specific T cells obtained by using microparticles prepared from activated mixed antigen-presenting cells without antigen-presenting cells were more effective than those obtained by co-incubating nanoparticles loaded with whole-cell components with DC+T cells. Therefore, the cancer cell-specific T cell vaccine described in this invention has a preventive effect against breast cancer.

[0306] Example 12: Cancer Cell-Specific T Cells for Breast Cancer Prevention

[0307] (1) Lysis of cancer cells and bacterial vesicles

[0308] 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.

[0309] 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 dissolved using an 8M urea aqueous solution (containing 500mM sodium chloride) to dissolve the bacterial exovesicle components.

[0310] (2) Preparation of micron-sized particles

[0311] 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.

[0312] (3) Preparation of antigen-presenting cells

[0313] 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. The mixed antigen-presenting cells are obtained by mixing BMDCs, B cells, and BMDMs in a ratio of 2:1:1.

[0314] (4) Activation of antigen-presenting cells

[0315] 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).

[0316] (5) Preparation of particles derived from antigen-presenting cells

[0317] 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.

[0318] 40 million mixed antigen-presenting cells incubated with either microparticle 1 or microparticle 2 were collected 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. 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 co-treated with 60mg of the microparticle 1 or microparticle 2 prepared in step (2) for 10 minutes. The sample was then repeatedly co-extruded using a 2μm filter membrane. The extrudate was centrifuged at 10000g for 20 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended 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.

[0319] (6) Preparation and analysis of cancer cell-specific T cells

[0320] Female BALB / c mice aged 6-8 weeks were selected and subcutaneously injected with 100 μL of micronized particles containing 0.2 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. Then, nanoparticles 1, 2, 3, or 4 are used to assist in the sorting and expansion of cancer cell-specific T cells. The sorted CD3+ cells... + T cells (2 million), nanoparticles or microparticles (100 μg), 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). Then, flow cytometry was used to sort the incubated CD3 cells. + CD3 in T cells + CD8 + CD69 + T cells and CD4 + CD4 in T cells + CD69 + T cells are cancer cell-specific T cells that can recognize full-cell antigens of cancer cells. The CD8 cells obtained above... + CD69 + T cells or CD4 + CD69 + T cells were mixed at a 2:1 ratio and incubated with IL-2 (1000 U / mL), IL-7 (1000 U / mL), and αCD-3 antibody (10 ng / mL) in RPMI 1640 complete medium for 14 days to expand cancer cell-specific T cells, with the medium changed every two days. The cancer cell-specific T cells isolated and expanded using nanoparticle 1 were designated as T cell vaccine 1; those isolated and expanded using nanoparticle 2 were designated as T cell vaccine 2; those isolated and expanded using microparticle 3 were designated as T cell vaccine 3; and those isolated and expanded using microparticle 4 were designated as T cell vaccine 4.

[0321] Meanwhile, T cells incubated with anti-mouse CD3 antibody, anti-mouse CD3 antibody, and anti-mouse CD69 antibody were labeled, respectively. Then, flow cytometry was used to analyze the CD69 levels in T cells after co-incubation with different nanoparticles and antigen-presenting cells. + The proportion of T cells.

[0322] Meanwhile, T cells from unsorted spleen cells were co-incubated with different nanoparticles prepared in step (6) in DMEM high-glucose complete medium for 48 hours. The incubated cells were then collected and labeled with IFN-γ antibody containing a fluorescent probe. Flow cytometry was then used to analyze the IFN-γ levels in the T cells. + The proportion of T cells. 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 are loaded with these degraded and presented antigenic epitopes, which can be recognized by cancer cell-specific T cells and activate them, 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 levels were analyzed using flow cytometry. + IFN-γ + T cells are cancer cell-specific T cells that can recognize and kill cancer cells.

[0323] (7) Cancer cell-specific T cells for cancer prevention

[0324] Female BALB / c mice aged 6-8 weeks were selected as model mice to prepare breast cancer-bearing mice. One day before adoptive cell transfer, recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to deplete immune cells in the recipient mice. On day 0, mice were subcutaneously injected with a T-cell vaccine (containing 600,000 amplified CD8+ cells). + T cells and 300,000 expanded CD4 cells + T cells or 100 μL PBS. Simultaneously, on day 0, each mouse was subcutaneously injected with 1 × 10⁻⁶ T cells. 6 4T1 cells were used, and the tumor volume and survival of mice were monitored using the same methods as above.

[0325] (8) Experimental Results

[0326] like Figure 13As shown in Figures a and b, compared with the control group, mice treated with T-cell vaccines exhibited significantly slower tumor growth and prolonged survival. Furthermore, T-cell vaccine 1 was superior to T-cell vaccine 2, and T-cell vaccine 3 was superior to T-cell vaccine 4. This indicates that cancer cell-specific T cells isolated with the assistance of particles prepared from antigen-presenting cells activated by micron-sized particles containing substances that enhance lysosomal escape function and a mixed adjuvant are more effective than cancer cell-specific T cells isolated with the assistance of particles prepared from antigen-presenting cells activated by micron-sized particles containing only substances that enhance lysosomal escape function without a mixed adjuvant. Moreover, T-cell vaccine 3 was superior to T-cell vaccine 1, and T-cell vaccine 4 was superior to T-cell vaccine 2. This indicates that cancer cell-specific T cells isolated with the assistance of solid particles internally loaded with cancer cell lysis components and surface loaded with activated antigen-presenting cell components are superior to cancer cell-specific T cells isolated with the assistance of vesicular particles only loaded with activated antigen-presenting cell components. Therefore, the cancer cell-specific T cells described in this invention have the ability to kill breast cancer and can be used for the prevention or treatment of cancer. Furthermore, the use of mixed adjuvants and the internal loading of whole-cell components of cancer cells both help to isolate cancer cell-specific T cells.

[0327] like Figure 13 As shown in Figures c and d, the proportion of cancer cell-specific T cells that can be activated when using different particle-assisted sorting methods is related to the therapeutic effects shown in Figures a and b. This indicates that the T cells obtained by using the particle-assisted sorting method described in this invention are cancer cell-specific T cells that can specifically recognize and kill cancer cells.

[0328] Example 13: Cancer Cell-Specific T Cells for the Treatment of Colon Cancer

[0329] This embodiment uses mouse colon cancer as a cancer model to illustrate how to use nanoparticles loaded with colon cancer whole-cell antigens to activate antigen-presenting cells, prepare nanoparticles to assist in the sorting of cancer cell-specific T cells, and use them for the treatment of colon cancer. In this embodiment, colon cancer tumor tissue is first lysed and the lysed components are dissolved using an 8M urea aqueous solution. Then, nanoparticles are 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 are prepared into nanoparticles. Then, the nanoparticles are used to assist in the sorting of cancer cell-specific T cells. The cancer cell-specific T cells obtained after two steps of sorting are expanded and used for cancer treatment.

[0330] (1) Lysis of tumor tissue and collection of its components

[0331] When collecting tumor tissue, 2×10⁻⁶ cells were first subcutaneously injected into the back of each C57BL / 6 mouse. 6MC38 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.

[0332] (2) Preparation of nanoparticle systems

[0333] 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. 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.

[0334] (3) Preparation of antigen-presenting cells

[0335] In this embodiment, BMDCs and B cells were used as antigen-presenting cells. The BMDC preparation method was the same as in Example 1. B cells were obtained from mouse peripheral blood PBMCs and prepared in the same way.

[0336] (4) Activation of antigen-presenting cells

[0337] 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).

[0338] (5) Preparation of nanoparticles based on antigen-presenting cells

[0339] 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 with a particle size of 300 nm.

[0340] (6) Preparation of cancer cell-specific T cells

[0341] Female C57BL / 6 mice aged 6-8 weeks were selected, and 2×10⁻⁶ mice were subcutaneously injected into their backs on day 0. 6 Mice were injected subcutaneously with 100 μL of nanoparticles containing 0.4 mg PLGA (loaded with lysate components, adjuvant mixture, and substances to increase lysosomal escape) on days 14 and 28. Mice were sacrificed on day 32, and tumor tissue and peripheral blood were collected. Tumor tissue was prepared into a single-cell suspension; peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood. The tumor tissue single-cell suspension and PBMCs were then mixed, and CD3+ cells were isolated from the mixed cells using flow cytometry. + CD8 + T cells and sorted CD3 + CD4 + T cells. The sorted CD8 cells... + T cells (200,000), CD4 +100,000 T cells, 50 μg of antigen-presenting cell nanoparticles, 1 million B cells, and IL-7 (10 ng / mL) were co-incubated in 2 mL of RPMI 1640 complete medium for 48 hours (37°C, 5% CO2). Then, flow cytometry was used to sort the incubated CD8+ cells. + CD8 in T cells + CD69 + T cells and CD4 + CD4 in T cells + CD69 + T cells are cancer cell-specific T cells that can recognize full-cell antigens of cancer cells. The CD8 cells obtained above... + CD69 + T cells or CD4 + CD69 + T cells were incubated with IL-2 (1000 U / mL), IL-12 (1000 U / mL), IL-15 (1000 U / mL), and αCD-3 antibody (10 ng / mL) in RPMI 1640 complete medium for 14 days (with medium changed every two days) to expand cancer cell-specific T cells.

[0342] (7) Cancer cell-specific T cells are used to treat cancer.

[0343] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer mice. On day 0, each mouse was subcutaneously injected with 2×10⁻⁶ cells on the lower right back. 6 MC38 cells. 800,000 CD8+ cells were intravenously injected on days 6, 9, 12, 15, 20, and 25 after inoculation with colon cancer cells. + Cancer-specific T cells and 400,000 CD4+ cells + Cancer-specific T cells; or inject 1.2 million CD8 cells within the above-mentioned number of days. + Cancer cell-specific T cells. The methods for monitoring tumor growth and survival in mice are the same as above.

[0344] (8) Experimental Results

[0345] like Figure 14 As shown, compared with the control group, the tumor growth rate of mice treated with nanoparticle-assisted separation and amplification of cancer cell-specific T cells prepared from antigen-presenting cells activated by nanoparticles was significantly slowed and the survival time of mice was significantly prolonged. Furthermore, CD8+ cells prepared from antigen-presenting cells and simultaneously separated and amplified using nanoparticles showed significantly improved tumor growth. + T cells and CD4 + T cells were superior to CD8 cells obtained solely through nanoparticle-assisted isolation and amplification. +T cells. Furthermore, the nanoparticle-assisted separation of cancer cell-specific T cells prepared from antigen-presenting cells activated by nanoparticles loaded with a mixed adjuvant, lysate components, and lysosomal escape substances showed better efficacy than nanoparticles loaded with lysate components, two CpG adjuvants, and lysosomal escape substances. Therefore, the cancer cell-specific T cells described in this invention have excellent therapeutic effects on cancer.

[0346] 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 cancer T cell vaccine, characterized in that, The preparation method of the cancer T cell vaccine comprises the following steps: S1, co-incubating antigen presenting cells with first particles to obtain activated antigen presenting cells; wherein the first particles are nanoparticles and / or microparticles loaded with tumor tissue and / or whole cell components of cancer cells, immune-enhancing adjuvants and lysosome escape substances; S2, co-acting cell membrane components of the activated antigen presenting cells with second particles to load the cell membrane components on the second particles to obtain particles loaded with cell membrane components; wherein the second particles are nanoparticles and / or microparticles loaded with tumor tissue and / or whole cell components of cancer cells, immune-enhancing adjuvants and lysosome escape substances; S3, co-incubating the particles loaded with cell membrane components in S2 with immune cells containing T cells, activating cancer cell-specific T cells that can recognize cancer cell antigens, sorting out the activated cancer cell-specific T cells, and in vitro expanding the activated cancer cell-specific T cells to obtain the cancer T cell vaccine; The immune-enhancing adjuvant is (1) a combination of Poly(I:C) and CpG-ODN or (2) a combination of Poly(ICLC) and CpG-ODN, wherein the CpG-ODN is at least two of A-class CpG-ODN, B-class CpG-ODN and C-class CpG-ODN, and at least one of them is B-class CpG-ODN or C-class CpG-ODN; In step S3, the sorting is screening the cancer cell-specific T cells by using the markers highly expressed on the surface of the activated T cells; the T cell surface-specific surface markers used for screening are at least one of CD69, PD-1, TIM-3, LAG-3, CD25, OX40, CD137, CD44, CD39, CD103, CD56, CD278, CD244, CD27, CD154, and CD28; The co-incubation system contains one or more of antigen presenting cells, cytokines and antibodies.

2. The cancer T cell vaccine of claim 1, wherein: The cytokine is one or more of GM-CSF, IL-2, IL-7, IL-12, IL-4, TNF-α, IL-10, IL-15, IFN-γ, and M-CSF.

3. The cancer T-cell vaccine of claim 1, wherein: In step S3, after sorting out the activated cancer cell-specific T cells, the step of in vitro expanding the cancer cell-specific T cells is further included; the in vitro expansion is co-incubating the cancer cell-specific T cells with cytokines and / or antibodies.

4. The cancer T-cell vaccine of claim 1, wherein: In step S1, the co-incubation system contains cytokines and / or antibodies.

5. The cancer T-cell vaccine of claim 1, wherein: The antigen presenting cells are selected from one or more of dendritic cells, B cells and macrophages.

6. The cancer T cell vaccine of claim 1, wherein: The first particles or the second particles are further loaded with bacterial lysis components obtained by lysing bacteria with a lysis solution containing a lysis agent; the lysis agent is selected from one or more of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein-degrading enzyme, albumin, lecithin, Triton, Tween, amino acid, glycoside and choline in aqueous solution.

7. Use of the cancer T cell vaccine according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment or prevention of cancer, characterized in that, The cancer is melanoma, colon cancer, breast cancer, pancreatic cancer, or lung cancer. The cancer is melanoma, colon cancer, breast cancer, pancreatic cancer, or lung cancer. The cancer is melanoma, colon cancer, breast cancer, pancreatic cancer, or lung cancer. The cancer is melanoma, colon cancer, breast cancer, pancreatic cancer, or lung

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