WO2024007388A1

WO2024007388A1 - Method and kit for detecting cancer cell-specific t cell

Info

Publication number: WO2024007388A1
Application number: PCT/CN2022/108956
Authority: WO
Inventors: 刘密
Original assignee: 苏州尔生生物医药有限公司
Priority date: 2022-07-08
Filing date: 2022-07-29
Publication date: 2024-01-11
Also published as: CN115561145A

Abstract

A method and kit for detecting a cancer cell-specific T cell. The method comprises: co-incubating T cells in peripheral blood, peripheral immune organs or tumor-infiltrating lymphocytes with a particle prepared from an antigen-presenting cell activated by the particle loaded with whole cell components, so as to activate the cancer cell-specific T cell that can recognize and kill cancer cells, and then detecting and analyzing the number and proportion of the activated cancer cell-specific T cell by means of using techniques, such as ELISPOT, flow cytometry and ELISA, and thereby evaluating the strength of the cancer cell-specific immunity in patient. According to the present invention, the problem that the broad-spectrum polyclonal specific T cell in peripheral blood, peripheral immune organs or tumor-infiltrating lymphocytes cannot be effectively screened clinically at present is overcome, and the broad-spectrum effector cell-specific T cell having a specific killing function can be detected in cells. The particle has the characteristics of easiness to separate and obtain and a high specificity, and can be used as an effective biomarker.

Description

A detection method and kit for cancer cell-specific T cells

Technical field

The present invention relates to the field of detection technology, and in particular, to a detection method and kit for cancer cell-specific T cells.

Background technique

T cells, especially cancer cell-specific T cells, play the main role in the fight against cancer. T cells are the main cells in the body that specifically recognize and kill cancer cells. Each clone of cancer cell-specific T cells can specifically recognize an antigenic epitope. Cancer patients, especially those who have undergone immunotherapy or radiotherapy, contain a certain number of cancer cell-specific T cells. Studies have shown that the number of cancer cell-specific T cells in cancer patients treated with immunotherapy and other methods is positively correlated with the prognosis of cancer patients. Therefore, it is particularly important to detect the number of cancer cell-specific T cells in cancer patients. The inventor has previously proposed using nanoparticles or microparticles loaded with whole cell components of cancer cells to assist in the detection of cancer cell-specific T cells (Application No. 202011027741.0, detection method of tumor-specific T cells), but in the above detection system it is necessary to simultaneously It contains Antigen-presenting cells (APC), which makes the detection system contain a variety of cells at the same time, and the process of antigen-activated T cells to assist detection is an indirect process rather than a direct process. In order to solve the above problems, the applicant proposes the present invention.

Contents of the invention

In order to solve the above technical problems, the present invention provides a method for detecting and killing nanoparticles and/or microparticles prepared by using antigen-presenting cells activated by nanoparticles (NP) or microparticles (MP) loaded with cancer cell whole cell antigens. The method of effective (effector) cancer cell-specific T cells (T _eff ) uses nanoparticles or microparticles prepared by activated antigen-presenting cells to first activate cancer cell-specific T cells, and then uses the activated killing cells Markers specifically expressed by cancer cell-specific T cells (T _eff ) analyze the content and proportion of cancer cell-specific T cells in the cells to be tested. It effectively solves the problem of how to detect broad-spectrum and polyclonal cancer cell-specific T cells with the ability to recognize and kill cancer cells in peripheral blood, peripheral immune organs or tumor-infiltrating lymphocytes. Moreover, since the nanoparticles or microparticles used to detect cancer cell-specific T cells are loaded with antigen-presenting cell membrane components, there may be no antigen-presenting cell membrane components in the system when the nanoparticles or microparticles of the present invention are used to co-incubate with T cells. With the help of cells, it is a more direct detection method.

The first object of the present invention is to provide a method for detecting cancer cell-specific T cells using particles prepared from activated antigen-presenting cells, which includes the following steps:

S1. Co-incubate the antigen-presenting cells with the first particles to obtain activated antigen-presenting cells; wherein the first particles are nanoparticles or microparticles loaded with tumor tissue and/or cancer cell whole cell components;

S2. Preparing the activated cell membrane components of the antigen-presenting cells into nanovesicles; or cooperating the activated cell membrane components of the antigen-presenting cells with the second particles to load the cell membrane components on the second particles. , obtaining second particles loaded with cell membrane components; wherein the second particles are nanoparticles or microparticles loaded with tumor tissue and/or cancer cell whole cell components;

S3. Incubate the nanovesicles and/or second particles loaded with cell membrane components in step S2 with the cells to be tested, activate broad-spectrum cancer cell-specific T cells that can recognize antigens, and then use appropriate detection technology to analyze the cells to be tested. Markers inside or on the surface of activated cancer cell-specific T cells are analyzed to obtain the number and proportion of the cancer cell-specific T cells by analyzing the number and proportion of T cells containing the markers.

Furthermore, the above-mentioned markers inside cells or on cell surfaces include proteins or nucleic acids.

Further, when the specific surface markers are proteins, they include but are not limited to interferon-γ, interleukins, granzymes, perforin, CD69, CD25, OX40 (CD134), CD39, CD103, CD56, CD279, CD278, CD244, CD27, CD154, TCF-1, CD137, CD44, CD28, etc. Technologies that use surface marker analysis to detect the number and proportion of cancer cell-specific T cells include but are not limited to flow cytometry, magnetic bead sorting, enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), etc. .

Further, the cells to be tested may be T cells or a cell mixture containing T cells, such as T cells or a cell mixture containing T cells derived from peripheral blood, peripheral immune organs or tumor-infiltrating lymphocytes.

Furthermore, before the cells to be tested are co-incubated with the products of S2, they can be sorted to sort out the T cells. Specifically, flow cytometry or magnetic bead sorting is used to select cells from peripheral blood and peripheral immune tissues. , sorting CD3 ⁺ cells from tumor infiltrating lymphocytes, sorting CD45 ⁺ CD3 ⁺ cells, sorting CD3 ⁺ CD8 ⁺ cells, sorting CD45 ⁺ CD3 ⁺ CD8 ⁺ cells, sorting CD3 ⁺ CD4 ⁺ cells or sort CD45 ⁺ CD3 ⁺ CD4 ⁺ cells.

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

Further, in step S3, the co-incubation system of the product of S2 and the cells to be tested may contain cytokines or antibodies.

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

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

Further, antibodies include, but are not limited to, αCD-3 antibody, αCD-4 antibody, αCD-8 antibody, αCD-28 antibody, αCD-40 antibody, αOX-40 antibody, and αOX-40L antibody.

Further, in the above preparation method, the first particle or the second particle can also be loaded with a bacterial component or a bacterial outer vesicle component, and the bacterial component or bacterial outer vesicle component is lysed by a lysis solution containing a lysis agent. Obtained from bacteria or bacterial external vesicles, the lysing agent is urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein degrading enzyme, albumin, lecithin, Triton, Tween, amino acids , glycosides, choline, bacteria including but not limited to Bacillus Calmette-Guérin, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus formati, Lactobacillus reuteri, Lactobacillus rhamnosus Bacilli etc.

Furthermore, the activated antigen-presenting cell membrane component can also be mixed with the cancer cell cell membrane component or the cancer cell extracellular vesicle membrane component to prepare a mixed membrane component and then be prepared into nanovesicles or loaded on second particles. surface.

Further, the first particle or the second particle is also loaded with an immune-enhancing adjuvant. The immune-enhancing adjuvant includes but is not limited to pattern recognition receptor agonist, BCG, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl Dipeptides, Mycobacterium phlei, polyantigen, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, Imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium parvum vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycysteine Glycolic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, cytokine, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, squalene, vegetable oil, internal Toxins, liposome adjuvants, MF59, double-stranded RNA, double-stranded DNA, CAF01, ginseng, active ingredients of astragalus, etc.

Preferably, the immune-enhancing adjuvant includes (1) Poly(I:C) or Poly(ICLC); (2) CpG-ODN, wherein CpG-ODN is type A CpG-ODN, type B CpG-ODN and type C At least two types of CpG-ODN, and at least one of them is type B CpG-ODN or type C CpG-ODN. Among them, type A CpG-ODN is selected from CpG-ODN 2216, CpG-ODN 1585 or CpG-ODN 2336, and type 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, Class C CpG-ODN is selected from CpG-ODN 2395, CpG-ODN SL03 or CpG-ODN M362.

Furthermore, the first particle or the second particle is also loaded with positively charged polypeptides (such as KALA polypeptide, RALA polypeptide, melittin, etc.), arginine, polyarginine, lysine, and polylysine. , histidine, polyhistidine, NH ₄ HCO ₃ , protamine or histone, etc.

Furthermore, the first particle or the 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, or CD11c antibody. , CD103 antibody, CD44 antibody, etc.

Further, the first particle or the second particle can be prepared from the following materials: organic synthetic polymer materials 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 polymer materials include but are not limited to lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, peptides, etc.; inorganic materials include But it is not limited to ferric oxide, ferric oxide, carbonate, phosphate, etc.

Furthermore, the particle size of the first particle or the second particle is nanometer or micron, which can ensure that the particles are engulfed by the antigen-presenting cells. In order to improve the phagocytosis efficiency, the particle size should be within an appropriate range. The particle size of nanoparticles (NP) is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 100nm-600nm; the particle size of microparticles (MP) The size is 1 μm-1000 μm, more preferably, the particle size is 1 μm-100 μm, more preferably, the particle size is 1 μm-10 μm, and most preferably, the particle size is 1 μm-5 μm.

Further, in step S2, the activated antigen-presenting cells are subjected to mechanical destruction, membrane filtration or gradient centrifugation to prepare nanovesicles, or the activated antigen-presenting cells are subjected to mechanical destruction, membrane filtration or gradient centrifugation, The product is combined with the second particle to obtain the second particle loaded with cell membrane components.

Further, the mechanical destruction method is selected from one or more of ultrasonic, homogenization, homogenization, high-speed stirring, high-pressure destruction, high-shear force destruction, swelling, chemical substances, and shrinkage. The co-action method is selected from one or more of co-incubation, co-extrusion, ultrasound, stirring, homogenization and homogenization. After co-action with nanoparticles or microparticles, the antigen-presenting cell components cover the original nanoparticles. New nanoparticles or microparticles are formed on the surface of particles or microparticles.

Further, the cancer cells or tumor tissues are frozen at -20°C to -273°C, and water or a solution without a dissolving agent is added, followed by repeated freeze-thaw lysis. The resulting supernatant is a water-soluble component, which is dissolved during the precipitation. After the agent is dissolved, the soluble part becomes the non-water-soluble component. The water-soluble component and the non-water-soluble component are combined to obtain the whole cell component of the cancer cells. The dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerin, protein degrading enzyme, albumin, lecithin, inorganic salts (0.1-2000mg/mL), Triton, and methane. At least one of temperature, amino acid, glycoside and choline.

Further, the antigen-presenting cells include at least one of B cells, dendritic cells (DC) and macrophages, preferably two or more including DC, and more preferably a combination of three types of cells.

Further, the obtained cancer cell-specific T cells include CD4 ⁺ T cells and/or CD8 ⁺ T cells, and preferably include both CD4 ⁺ T cells and CD8 ⁺ T cells.

In the present invention, nanoparticles and/or microparticles loaded with cancer cell whole cell antigens are used to specifically activate antigen-presenting cells, and then the antigen-presenting cells are prepared into nanoparticles or microparticles. The prepared nanoparticles or micron particles are The particles are loaded with whole-cell antigen epitopes of cancer cells, and then nanoparticles or microparticles prepared from antigen-presenting cells are used to activate cancer cell-specific T cells in peripheral blood, peripheral immune tissue or tumor-infiltrating lymphocytes, and then used Characteristics of activated cancer cell-specific T cells that highly express certain molecules within the cell or on the cell surface. Use flow cytometry and other methods to analyze the most diverse and broad-spectrum cancer-specific T cells that have the function of recognizing and killing cancer cells. Quantity and proportion.

Furthermore, the antigen-presenting cells can be derived from the same type as the cancer cell-specific T cells, allogeneic, cell lines, or transformed from stem cells.

Further, in the first particle or the second particle, at least one of the cancer cells or tumor tissues used to prepare the antigen is the same as the disease type corresponding to the detected cancer cell-specific T cells.

The second object of the present invention is to provide a kit for detecting cancer cell-specific T cells, which kit includes at least one of the following (1)-(2):

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

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

in,

The nanovesicles are prepared by co-incubating the antigen-presenting cells with the first particles to obtain activated antigen-presenting cells, and then extracting the cell membrane components of the activated antigen-presenting cells;

The particles loaded with cell membrane components of activated antigen-presenting cells are obtained by cooperating the cell membrane components of activated antigen-presenting cells with second particles, so that the cell membrane components are loaded on the second particles;

The first particles or second particles are independently selected from nanoparticles or microparticles loaded with tumor tissue and/or whole cell components of cancer cells.

The present invention breaks through the limitations of existing detection methods, allowing particles to be loaded with all antigens and activated antigen-presenting cell membranes, capable of detecting a wider spectrum and variety of cancer cell-specific T cells, and is highly specific and effective in immune detection. Better, thus providing a potential biomarker detection method for immunotherapy.

Through the above solutions, the present invention at least has the following advantages:

The present invention provides a technology for in vitro detection of cancer cell-specific T cells in immune cells using a nanoscale or micron-scale particle delivery system. The analyzed and detected cancer cell-specific T cells are broad-spectrum and highly specific, including all clones that can Specific recognition and killing of cancer cell effector (killer) cancer cell-specific T cells (T _eff ); and, compared with particles prepared without loaded activated antigen-presenting cells, particles loaded with activated antigen-presenting cells The T cells activated by the particles secrete a higher content of specific markers, which makes them easier to detect and avoids the situation where the signal is poor and cannot be detected. The above advantages allow the particles of the present invention to detect cancer cell-specific T cells to avoid the situation where some cancer cell-specific T cells cannot be detected because the specific markers expressed after activation are weak. Therefore, the detection method of the present invention The detection accuracy of the method is higher. On this basis, the present invention also optimizes the activation process of antigen-presenting cells, the incubation process with T cells, and the loading materials of the first particle and the second particle, so that the method can detect more comprehensive cancer cell-specific T cells, and The signal is stronger and more accurate during detection.

The above description is only an overview of the technical solutions of the present invention. In order to have a clearer understanding of the technical means of the present invention and implement them according to the contents of the description, the preferred embodiments of the present invention are described below with detailed drawings.

Description of the drawings

In order to make the content of the present invention easier to understand clearly, the present invention will be described in further detail below based on specific embodiments of the present invention and in conjunction with the accompanying drawings.

Figure 1 is a schematic diagram of the preparation process and application of the cell system of the present invention; a is a schematic diagram of collecting and preparing nanoparticles or microparticles from water-soluble components and non-water-soluble components respectively; b is dissolving using a dissolving solution containing a dissolving agent Schematic diagram of cancer cell whole cell antigen and preparation of nanoparticles or microparticles; c is after activating antigen-presenting cells using nanoparticles and/or microparticles prepared in a or b, and preparing the activated antigen-presenting cells into particles. Schematic diagram for detection of cancer cell-specific T cells;

Figures 2-14 respectively show the experimental results of using nanoparticles or microparticles to detect cancer cell-specific T cells in Examples 1-13; * represents p≤0.05, indicating a significant difference.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.

The present invention is used to detect cancer cell-specific T cells in peripheral blood, peripheral immune tissue or tumor infiltrating lymphocytes. The cancer cell-specific T cells are first prepared with activated antigen-presenting cells during detection. Nanoparticles and/or microparticles are co-incubated, and then flow cytometry, enzyme-linked immunospot assay (ELISPOT), or enzyme-linked immunosorbent assay are used to analyze molecules that are highly expressed after cancer cell-specific T cells are specifically activated by antigens. A broad spectrum of cancer cell-specific T cell information can be obtained.

Wherein, the antigen-presenting cells used to prepare nanoparticles or microparticles are first activated by nanoparticles and/or microparticles loaded with tumor tissue and/or cancer cell whole cell antigens or mixtures thereof. The process and application fields of detecting cancer cell-specific T cells are shown in Figure 1.

When preparing nanoparticles or microparticles that activate antigen-presenting cells, the cells or tissues can be lysed and the water-soluble components and water-insoluble antigens can be collected separately to prepare nanoparticle or microparticle systems respectively; alternatively, a solution containing a dissolving agent can be directly used. The lysis solution directly lyses cells or tissues and dissolves whole cell antigens of cancer cells to prepare nano or micro particle systems. The cancer cell whole cell antigen of the present invention can be subjected to a process including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease treatment before or (and) after lysis. Nanoparticles or microparticles can be prepared after endolysis or degradation; or without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nucleic acid treatment before or after cell lysis. Nanoparticles or microparticles are directly prepared by enzymatic treatment, proteolytic digestion or degradation. In some embodiments of the present invention, tumor tissue cells are inactivated or/and denatured before lysis. In actual use, they can also be inactivated or/and denatured after cell lysis, or the cells can also be lysed. Inactivation or (and) denaturation treatment is performed before and after lysis; in some embodiments of the present invention, the inactivation or (and) denaturation treatment method before or (and) after cell lysis is ultraviolet irradiation and high-temperature heating. In actual use Treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, protease endolysis or degradation, freeze-drying and other treatment methods can also be used in the process. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific circumstances.

When preparing activated antigen-presenting cells into nanoparticles or micron particles, the antigen-presenting cells are first mechanically destroyed, and then centrifuged and/or filtered with a filter membrane of a certain pore size. Optionally, the antigen-presenting cells are combined with the nanoparticles or micron particles. Particles work together.

Activated antigen-presenting cells contain a certain cell membrane structure after mechanical destruction.

After the activated antigen-presenting cells are mechanically destroyed, they interact with nanoparticles or microparticles to form new nanoparticles or microparticles. The components of the antigen-presenting cells are located in the outer layer of the particles.

The antigen-presenting cells prepared into nanoparticles or microparticles and the antigen-presenting cells used to incubate with T cells can be derived from autologous or allogeneic sources, or from cell lines or stem cells. Antigen-presenting cells can be DC cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen-presenting functions.

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

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

In some embodiments, 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, and the nanoparticles or microparticles prepared by the antigen-presenting cells are used. The specific method of using micron particles to detect cancer cell-specific T cells in peripheral blood, peripheral immune tissue or tumor-infiltrating lymphocytes is as follows:

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

In some embodiments, the aqueous solution may contain each component of the cancer cell/tumor tissue lysate and an immune-enhancing adjuvant; each component of the lysate is a water-soluble component during preparation or is dissolved in a solution containing The original non-water-soluble components in the solution of dissolving agents such as urea or guanidine hydrochloride. The concentration of water-soluble components or the original concentration of non-water-soluble components contained in the aqueous phase solution, that is, the first predetermined concentration requires the concentration of protein polypeptide to be greater than 1ng/mL, and can load enough cancer cell whole cell antigens to activate related cells. . The concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.

In some embodiments, methylene chloride is used as the organic solvent. In addition, in some embodiments, the second predetermined concentration of raw materials for preparing particles ranges from 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.

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 the present invention, the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is It is 1:1.1-1:5000, preferably 1:10. During the specific implementation process, 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.

Preferably, when the aqueous phase solution is a lysate component solution, the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ~ 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL~100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL~20mg/mL. In the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg/mL~ 5000mg/mL, preferably 100mg/mL.

Step 2: subject the mixed solution obtained in Step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute. Preferably, when the stirring is mechanical stirring or magnetic stirring, 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 ~ 1500 rpm, and the stirring time is 0.1 hour ~ 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5W, and the time Greater than 0.1 seconds, such as 2 to 200 seconds; use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer for homogenization processing. When using a high-pressure/ultra-high-pressure homogenizer, the pressure is greater than 5 psi, such as 20 psi to 100 psi. Use a high-pressure homogenizer. The rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used for nanonization and/or micronization. The length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared micro-nano particles. Too large or too small will cause to changes in particle size.

Step 3: Add the mixture obtained after step 2 to a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and perform ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or perform homogenization or microfluidic treatment. deal with. In this step, the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers. In the present invention, the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds, the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm, and the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute. For example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.5 to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W. , the time is greater than 0.1 seconds, such as 2 to 200 seconds; when homogenizing, use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer. When using a high-pressure/ultra-high-pressure homogenizer, the pressure is greater than 20 psi, such as 20 psi to 100 psi. When using a high-shear homogenizer, the rotation speed is greater than 1000rpm, such as 1000rpm ~ 5000rpm; when using microfluidic processing, the flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used to nanonize or micronize the particles. The length of ultrasonic time or stirring speed or homogenization process pressure and time can control the size of the prepared nano or micron particles. Too large or too small will cause Changes in particle size.

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 the present invention, the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5. In order to control the size of nanoparticles or microparticles during specific implementation, the ratio of the second predetermined volume and the third predetermined volume can be adjusted. Similarly, the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.

Step 4: Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.

In this step, the emulsifier aqueous solution is PVA solution or other solutions.

The fourth predetermined concentration is 5 mg/mL, and 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 based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume ranges from 1:1.5 to 1:2000, preferably 1:10. During specific implementation, the ratio between the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.

In the present invention, the predetermined stirring condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of methylene chloride in step 1 is completed.

Step 5: After centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of Five predetermined concentrations of an aqueous solution containing a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).

Step 6: After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.

Step 7: Resuspend a sixth predetermined volume of the nanoparticle-containing suspension obtained in Step 5 in PBS (or physiological saline) or use a sixth predetermined volume of PBS (or physiological saline) to resuspend the nanoparticle-containing suspension obtained in Step 6 The freeze-dried substance containing nanoparticles or microparticles and a lyoprotectant is used directly; or the above sample is mixed with a seventh predetermined volume of water-soluble components or dissolved original non-water-soluble components and used.

In the present 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 .

Step 8: Incubate the antigen-presenting cells with the nanoparticles and/or microparticles prepared above for a certain period of time. The tumor tissue and/or cancer cells and antigen-presenting cells used to prepare nanoparticles and/or microparticles can be from autologous or allogeneic sources.

Step 9: Collect the co-incubated cells and perform mechanical damage such as sonication, homogenization, and mechanical stirring.

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

Step 11: Obtain peripheral blood, peripheral immune tissue or tumor tissue, and collect T cells or immune cells containing T cells in the above tissues. The above peripheral blood, peripheral immune tissue or tumor tissue can be from autologous or allogeneic.

Step 12: Mix the nanoparticles and/or microparticles prepared in step 10 with the T cells or immune cells containing T cells obtained in step 11 and incubate them together for a certain period of time.

Step 13: Use flow cytometry, enzyme-linked immunospot method, enzyme-linked immunosorbent assay, or magnetic bead sorting method to analyze the content of cancer cell-specific T cells activated by the antigen.

Example 1 Nanoparticles are used to detect cancer cell-specific T cells in peripheral immune organs

This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticles prepared by nanoparticle-activated antigen-presenting cells to detect cancer cell-specific T cells in mouse splenocytes. In this example, B16F10 melanoma tumor tissue was lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue. Then, the organic polymer material PLGA was used as the nanoparticle skeleton material, and Polyinosinic-polycytidylic acid (poly(I :C)) For immune adjuvants, a solvent evaporation method is used to prepare a nanoparticle system loaded with water-soluble components and non-water-soluble components of tumor tissue, and then the nanoparticles are used to activate antigen-presenting cells, and the antigen-presenting cells are mechanically Nanoparticles are prepared by centrifugation after destruction, and the nanoparticles are used to assist in the detection of cancer cell-specific T cells in peripheral immune organs.

(1) Lysis of tumor tissue and collection of components

1.5 × 10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm ³ , the mice were sacrificed and the tumor tissue was removed. The tumor tissue was cut into pieces and then ground. An appropriate amount of ultrapure water was added through a cell filter and frozen and thawed 5 times repeatedly, accompanied by ultrasound to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant, which is the water-soluble component that is soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble component from the pure water. The water-insoluble components of water are converted into soluble in 8M urea aqueous solution. The above are the sources of antigen raw materials for preparing nanoparticle systems.

(2) Preparation of nanoparticles loaded with whole cell components

In this embodiment, the nanoparticle 1 is prepared by the double emulsion method in the solvent evaporation method. During preparation, nanoparticles loaded with water-soluble components in whole cell antigens of cancer cells and nanoparticles loaded with non-water-soluble components in whole cell antigens of cancer cells are prepared separately, and then used together. The molecular weight of PLGA, the material used to prepare the nanoparticles, is 24KDa-38KDa. The immune adjuvant used is poly(I:C) and poly(I:C) is only distributed inside the nanoparticles. The preparation method is as described above. During the preparation process, the double emulsion method is first used to load cell components and adjuvants inside the nanoparticles. After loading the cell lysis components inside, 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of nanoparticles containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 h. The average particle size of the nanoparticles 1 is about 280nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components. The poly(I:C) immune adjuvant used per 1 mg of PLGA nanoparticles is 0.02 mg.

(3) Preparation of bone marrow-derived dendritic cells (BMDC)

This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare BMDC. First, a 6-8 week old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The muscle tissue around the bones was removed with scissors and tweezers. Use scissors to cut off both ends of the bone, and then use a syringe to draw the PBS solution. The needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly flushed into the culture dish. Collect the bone marrow solution, centrifuge at 400g for 3 minutes, and then add 1 mL of red blood cell lysis solution to lyse the red blood. Add 3 mL of RPMI 1640 (10% FBS) medium to stop lysis, centrifuge at 400 g for 3 min, and discard the supernatant. The cells were placed in a 10 mm culture dish and cultured in RPMI 1640 (10% FBS) medium with recombinant mouse GM-CSF (20 ng/mL) added at 37 degrees Celsius and 5% CO ₂ for 7 days. On the third day, shake the culture bottle gently and add the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20ng/mL). On the 6th day, half of the culture medium was replaced. On day 7, collect a small amount of suspended and semi-adherent cells. Through flow cytometry, when the proportion of CD86 ⁺ CD80 ⁺ cells in CD11c ⁺ cells is between 15-20%, the induced cultured BMDC can be used for the following One step experiment.

(4) Activation of antigen-presenting cells

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 non-water-soluble components) were completely cultured with BMDC (10 million) in 15 mL RPMI1640. The base was incubated for a total of 48 hours (37°C, 5% CO ₂ ); the incubation system contained cytokine combination 1: granulocyte-macrophage colony-stimulating factor (GM-CSF, 500U/mL), IL-2 (500U/mL) ), IL-7 (500U/mL), IL-12 (500U/mL) or containing cytokine component 2: GM-CSF (500U/mL), IL-4 (500U/mL), tumor necrosis factor α ( TNF-α, 500U/mL), IL10 (500U/mL).

(5) Preparation of DC-derived nanoparticles

Collect the incubated DCs (10 million) with the addition of cytokine component 1 by centrifugation at 400g for 5 minutes, then wash the cells twice with physiological saline, resuspend the cells in physiological saline, and incubate at 4°C and 7.5W. Sonicate for 20 minutes to disrupt cells and prepare samples 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. Then collect the supernatant after centrifugation at 15000g for 120 minutes. Discard the supernatant and collect the pellet. Place the pellet in PBS. After resuspension, nanoparticles 2 are obtained, and the particle size of nanoparticles 2 is 120 nanometers.

Or collect the DCs (10 million) prepared in step (3) without any nanoparticle or microparticle activation, then wash the cells twice with physiological saline, resuspend the cells in physiological saline and incubate at 4°C and 7.5W. Sonicate for 20 minutes to disrupt cells and prepare samples containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected. The supernatant was centrifuged at 7000g for 20 minutes and the supernatant was collected. The supernatant was mixed with 40 mg of nanoparticles loaded with cancer cell whole cell components prepared in step (2). Particle 1 (20 mg of nanoparticles loaded with water-soluble components + 20 mg of nanoparticles loaded with non-water-soluble components) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 μm filter membrane. The extrudate was centrifuged at 15,000g for 120 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain nanoparticle 3 with a particle size of 300 nm.

Alternatively, collect the incubated DCs (10 million) with the addition of cytokine component 2 by centrifugation at 400g for 5 minutes, then wash the cells twice with physiological saline, resuspend the cells in physiological saline, and incubate at 4°C and 7.5W. Sonicate for 20 minutes to disrupt cells and prepare samples containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected. The supernatant was centrifuged at 7000g for 20 minutes and the supernatant was collected. The supernatant was mixed with 40 mg of nanoparticles loaded with cancer cell whole cell components prepared in step (2). Particle 1 (20 mg of nanoparticles loaded with water-soluble components + 20 mg of nanoparticles loaded with non-water-soluble components) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 μm filter membrane. The extrudate was centrifuged at 15,000g for 120 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain nanoparticles. Among them, the nanoparticle 4 obtained by incubating the nanoparticle 1 with the membrane component has a particle size of 300 nm.

Alternatively, collect the incubated DCs (10 million) added with cytokine component 1 by centrifugation at 400 g for 5 minutes, then wash the cells twice with physiological saline, resuspend the cells in physiological saline, and incubate at 4°C and 7.5W. Sonicate for 20 minutes to disrupt cells and prepare samples containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected. The supernatant was centrifuged at 7000g for 20 minutes and the supernatant was collected. The supernatant was mixed with 40 mg of nanoparticles loaded with cancer cell whole cell components prepared in step (2). Particle 1 (20 mg of nanoparticles loaded with water-soluble components + 20 mg of nanoparticles loaded with non-water-soluble components) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 μm filter membrane. The extrudate was centrifuged at 15,000g for 120 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain nanoparticle 5 with a particle size of 300 nm.

(6) Detection of cancer cell-specific T cells

0.5 × 10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm ³ , the mice were sacrificed and splenocytes were harvested. Mouse splenocytes were prepared into a single cell suspension, and then CD3 ⁺ T cells were sorted from the mouse splenocyte single cell suspension using flow cytometry.

Nanoparticle 1 (50 μg nanoparticles loaded with water-soluble components + 50 μg nanoparticles loaded with non-water-soluble components) or nanoparticles 2 (100 μg) or nanoparticles 3 (100 μg) or nanoparticles 4 (100 μg) or nanoparticles 5 (100 μg) was incubated with splenocyte-derived T cells (1 million) in 10 mL of RPMI 1649 complete medium for 72 hours (37°C, 5% _CO2 ). The cells were then collected and centrifuged at 400g for 5 minutes. The cells were resuspended in PBS and the T cells were first treated with Fc block to avoid non-specific loading. Then, CD3 antibody, CD4 antibody, and CD8 antibody were used for extracellular staining of mouse splenocytes, and then the cells were fixed and membrane-broken, and FN-γ antibody was used for intracellular staining of T cells. The sample T cells were then detected using flow cytometry. Analyze the proportion of CD4 ⁺ T cells that can secrete IFN-γ after activation among all CD4 ⁺ T cells and the proportion of CD8 ⁺ T cells that can secrete IFN-γ after activation among all CD8 ⁺ T cells. Proportion of T cells. The above CD4 ⁺ IFN-γ ⁺ T cells and CD8 ⁺ IFN-γ ⁺ T cells are cancer cell-specific T cells.

Alternatively, only splenocyte-derived T cells (1 million) were incubated in 10 mL of RPMI 1649 complete medium for 72 hours (37°C, 5% CO ₂ ). The cells were then collected and centrifuged at 400g for 5 minutes. The cells were resuspended in PBS and the T cells were first treated with Fc block to avoid non-specific loading. Then use anti-mouse CD3 antibody, anti-mouse CD4 antibody, and anti-mouse CD8 antibody connected with specific fluorescent probes to perform extracellular staining of mouse splenocytes. Then, the cells were fixed and membrane-broken, and the cells were fixed and membrane-broken using the anti-mouse CD4 antibody and anti-mouse CD8 antibody connected with specific fluorescent probes. Intracellular staining of T cells with fluorescently probed anti-mouse IFN-γ antibody. Flow cytometry is then used to detect T cells in the sample that contain fluorescent signals linked to IFN-γ antibodies. Analyze the proportion of CD4 ⁺ T cells that can secrete IFN-γ after activation among all CD4 ⁺ T cells and the proportion of CD8 ⁺ T cells that can secrete IFN-γ after activation among all CD8 ⁺ T cells. Proportion of T cells. The above CD4 ⁺ IFN-γ ⁺ T cells and CD8 ⁺ IFN-γ ⁺ T cells are cancer cell-specific T cells.

After nanoparticles/microparticles loaded with tumor tissue and/or cancer cell whole cell components are engulfed by antigen-presenting cells, the antigen will be degraded into polypeptide epitopes and combined with major histocompatibility complex (MHC) molecules. Presented to the surface of antigen-presenting cell membranes. Since whole-cell antigens loaded with nanoparticles and microparticles can be cross-presented and released, cancer cell antigen epitopes can be presented to the surface of antigen-presenting cell membranes through two pathways, MHC I and MCH II. After the antigen-presenting cell membrane is prepared into nanoparticles or microparticles, the MHC molecules and antigen peptide complexes loaded therein can directly bind to T cell surface receptors on the surface of T cells that can specifically recognize cancer cell antigens. Moreover, if the T cells are specific T cells that can kill cancer cells, the cancer cells will begin to secrete killer substances such as IFN-γ and granzymes. By analyzing the T cells secreting killer substances, we can obtain the characteristics of cancer cells. Proportion of effector cancer cell-specific T cells with recognition and killing capabilities.

(8)Experimental results

As shown in Figure 2, the cancer cell-specific T cells in the T cell alone control group and the nanoparticle 1 detection group were very low. Nanoparticle 2, nanoparticle 3, nanoparticle 4 and nanoparticle 5 can all assist in detecting a certain amount of cancer cell-specific T cells. Among them, nanoparticle 5 has the best effect, and nanoparticle 5 has a better effect than nanoparticle 2, nanoparticle 3 and nanoparticle 4. Nanoparticle 5 is better than nanoparticle 4, indicating that the effect of adding cytokine combination 1 during the activation of antigen-presenting cells is better than cytokine component 2; nanoparticle 5 is better than nanoparticle 2, indicating that the surface of nanoparticles that assist in the detection of T cells is loaded with antigens. Presenting cell membrane components, the effect of solid nanoparticles loaded with cell components inside is better than that of nanovesicle structures that only load membrane components on the surface;

Nanoparticles

4 and 5 are better than Nanoparticles 3, indicating that whole cells of cancer cells are loaded The nanoparticles prepared by the antigen-presenting cells activated by the nanoparticles of the component are more effective than the nanoparticles prepared by the unactivated antigen-presenting cells. In summary, the particle system of the present invention can be used to detect cancer cell-specific T cells. The antigen-presenting cells activated by the nanoparticles loaded with cancer cell whole cell components will degrade and present the broad-spectrum whole-cell antigens in the cancer cell whole cell components loaded with the phagocytosed nanoparticles, which are presented to Cancer cell antigen epitopes on the cell membrane surface have been bound to major histocompatibility complex (MHC) molecules. After the above-mentioned antigen-presenting cells are mechanically destroyed, the cell membrane components of the antigen-presenting cells contain antigen epitopes that bind to MHC. Through centrifugation and/or filtration using a filter membrane with a certain pore size and/or co-operation with nanoparticles or microparticles, the cell membrane components in the above antigen-presenting cells will form nanoparticles or microparticles, and be loaded with MHC molecules and degraded. Therefore, it can directly activate cancer cell-specific T cells for detecting this type of cells without the assistance of antigen-presenting cells.

Example 2 Antigen-presenting cell-based particles are used for the detection of cancer cell-specific T cells

This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticle-activated antigen-presenting cells to prepare nanoparticles to assist in the detection of cancer cell-specific T cells. In this example, B16F10 melanoma tumor tissue was lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue. Then, PLGA was used as the nanoparticle framework material, and poly(I:C) and CpG1018 were used as immune adjuvants. The agent uses a solvent evaporation method to prepare a nanoparticle system loaded with water-soluble components and non-water-soluble components of tumor tissue, then uses nanoparticles to activate antigen-presenting cells, and prepares the activated antigen-presenting cells into nanoparticle detection Cancer cell-specific T cells among peripheral blood immune cells.

(1) Lysis of tumor tissue and collection of components

1.5 × 10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm ³ , the mice were sacrificed and the tumor tissue was removed. Cut the tumor tissue into pieces and then grind it. Add an appropriate amount of pure water through a cell filter and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant, which is the water-soluble component that is soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble component from the pure water. The water-insoluble components of water are converted into soluble in 8M urea aqueous solution. Mixing water-soluble components and non-water-soluble components at a mass ratio of 1:1 is the source of antigen raw materials for preparing nanoparticle systems.

(2) Preparation of nanoparticle system

In this example, nanoparticles were prepared by solvent evaporation method. The molecular weight of PLGA, the preparation material for nanoparticle 1 (NP1) loaded with whole cell components, is 7Da-17KDa. The immune adjuvants used are poly(I:C) and CpG1018, and the adjuvants are contained inside the nanoparticles. The preparation method is as described above. During the preparation process, the double emulsion method is first used to load the antigen and adjuvant inside the nanoparticles. After loading the antigen (cleavage component) inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 h. The average particle size of the nanoparticles 1 is about 280nm; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein and peptide components, and each 1 mg of PLGA nanoparticles uses 0.02 mg of poly(I:C) and CpG1018 immune adjuvant; nanoparticles 1 Also called nanovaccine1. In this example, four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were loaded with equal mass. Peptide nanoparticle 2 is used as a control nanoparticle. Its preparation materials and preparation methods are the same as nanoparticle 1. The particle size of control nanoparticle 2 is about 280 nm, and it is loaded with 100 μg of peptide component and an equal amount of adjuvant. The preparation materials and methods of blank nanoparticle 3 are the same as those of nanoparticle 1. The particle size is about 280 nm. It only carries the same amount of immune adjuvant but does not load any antigen component.

(3) Preparation of antigen-presenting cells

Bone marrow-derived dendritic cells (BMDC) and B cells were used as antigen-presenting cells. The preparation of BMDC is the same as in Example 1. The B cell extraction process is as follows: kill the mouse and remove the mouse spleen, then prepare a mouse splenocyte single cell suspension, and then use magnetic bead sorting to sort CD19 ⁺ B cells from the splenocyte single cell suspension. Mix BMDC and B cells at a ratio of 1:1 and use them as mixed antigen-presenting cells.

(4) Activation of antigen-presenting cells

Mix nanoparticle 1 (500 μg) or peptide nanoparticle 2 (500 μg) or blank nanoparticle 3 (500 μg) + free lysate with 20 million mixed antigen-presenting cells (10 million BMDC + 10 million B cells) in 15 mL Incubate in RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ); the incubation system contains a combination of cytokines: IL-15 (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL) mL), IL-12 (1000U/mL).

Or

nanoparticle

1 and 20 million BMDCs were incubated in 15mL RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ); the incubation system contained a combination of cytokines: IL-15 (500U/mL), IL-2 ( 500U/mL), IL-7 (500U/mL), IL-12 (1000U/mL).

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

Collect the incubated 20 million mixed antigen-presenting cells (10 million BMDC + 10 million B cells) by centrifugation at 400g for 5 minutes, then wash the cells twice with physiological saline, resuspend the cells in physiological saline, and Use low-power 7.5W ultrasonic for 10 minutes at 4°C to disrupt cells and prepare samples containing cell membrane components. Then, the sample is filtered once through a filter membrane with pore sizes of 50 μm, 10 μm, 5 μm, 1 μm, 0.45 μm, and 0.22 μm. The obtained filtrate is collected and combined with the nanometer nanoparticles loaded with whole cell components of cancer cells prepared in the corresponding step (2). Particle 1 (50 mg) or peptide nanoparticle 2 (50 mg) or blank nanoparticle 3 (50 mg) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 μm filter. The extruded liquid was centrifuged at 15000g for 60 minutes and discarded. After removing the supernatant, resuspend in physiological saline and the resulting precipitate is nanoparticles. Among them, the nanoparticles prepared by using the mixed antigen-presenting cell membrane components activated by nanoparticle 1 and nanoparticle 1 are nanoparticles 4, with a particle size of 300 nm; the mixed antigen-presenting cell membrane activated by peptide nanoparticles 2 The nanoparticles prepared by the interaction between the components and the polypeptide nanoparticles 2 are nanoparticles 5, with a particle size of 300 nm; they are prepared by the interaction between the mixed antigen-presenting cell membrane components activated by the blank nanoparticles 3 and the blank nanoparticles 3. The obtained nanoparticles were nanoparticle 6, with a particle size of 300 nm.

Or collect 20 million BMDCs after incubation with nanoparticles 1 by centrifugation at 400g for 5 minutes, then wash the BMDCs twice with physiological saline, resuspend the cells in physiological saline and use low power 7.5W ultrasonic for 10 minutes at 4°C. to disrupt cells and prepare samples containing cell membrane components. Then filter the sample through a filter membrane with pore sizes of 50 μm, 10 μm, 5 μm, 1 μm, 0.45 μm, and 0.22 μm. Collect the filtrate and incubate it with the nanoparticle 1 (50 mg) prepared in step (2) for 10 minutes, and then Repeated co-extrusion using a 0.45 μm filter membrane, centrifuge the extrudate at 15,000 g for 60 minutes, discard the supernatant and resuspend in physiological saline, and the resulting precipitate is Nanoparticle 7, with a particle size of 300 nm.

(6) Detection of cancer cell-specific T cells

On day 0, 1.5×10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. On days 10 and 14, the mice were subcutaneously injected with 100 μL of 1 mg PLGA nanovaccine loaded with whole cell components of cancer cells1. Or 100 μL PBS; monitor the tumor growth rate of mice in the PBS group and vaccine group during the process. The mice were sacrificed on day 18, and their peripheral blood was collected. Peripheral blood mononuclear cells (PBMC) were isolated from the mouse peripheral blood using density gradient centrifugation, and then CD3 ⁺ T cells were isolated from the PBMC using flow cytometry. . Among them, the CD3 ⁺ T cells in the group treated with Nano Vaccine 1 are the vaccine treatment group, and the T cells in the group treated with PBS are the PBS control group.

Combine 100 μg of antigen-presenting cell-based nanoparticles prepared in step (5) (nanoparticle 4, or nanoparticle 5, or nanoparticle 6, or nanoparticle 7) with B cells (5 million) and 400,000 from tumors Lymphocyte-infiltrating T cells (vaccine group or PBS group) were incubated in 5 mL RPMI1640 complete medium for 24 hours (37°C, 5% CO ₂ ), and then flow cytometry was used to sort the incubated CD3 ⁺ IFN-γ ⁺ T cells. At the same time, the mean fluorescence intensity (MFI) of the fluorescent probe connected to IFN-γ ⁺ in CD3 ⁺ IFN-γ ⁺ T cells was analyzed. The stronger the fluorescence intensity, the more killer substances expressed by the cancer cell-specific T cells, and the detection sensitivity will be higher and the accuracy will be better.

Or combine 100 μg of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3) prepared in step (2) with B cells (5 million) and 400,000 T cells from tumor-infiltrating lymphocytes (vaccine group or PBS group) were incubated in 5 mL RPMI1640 complete medium for a total of 24 hours (37°C, 5% CO ₂ ), and then flow cytometry was used to sort the incubated CD3 ⁺ IFN-γ ⁺ T cells. At the same time, the mean fluorescence intensity (MFI) of the IFN-γ ⁺ -linked fluorescent probe in CD3 ⁺ IFN-γ ⁺ T cells was analyzed.

Or combine 100 μg of antigen-presenting cell-based nanoparticles prepared in step (5) (nanoparticle 4, or nanoparticle 5, or nanoparticle 6, or nanoparticle 7) with 400,000 T cells derived from tumor-infiltrating lymphocytes ( Vaccine group or PBS group) were incubated in 5 mL RPMI1640 complete medium for a total of 24 hours (37°C, 5% CO ₂ ), and then flow cytometry was used to sort the incubated CD3 ⁺ IFN-γ ⁺ T cells. At the same time, the mean fluorescence intensity (MFI) of the fluorescent probe connected to IFN-γ ⁺ in CD3 ⁺ IFN-γ ⁺ T cells was analyzed.

Or incubate 100 μg of nanoparticles (nanoparticle 1) prepared in step (2) with 400,000 T cells (vaccine group) derived from tumor infiltrating lymphocytes in 5 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ), and then use flow cytometry to sort the incubated CD3 ⁺ IFN-γ ⁺ T cells.

(7)Experimental results

As shown in a and b in Figure 3, the tumor growth rate of mice in the PBS group was significantly faster than that of the vaccine group. This shows that there are significantly more cancer cell-specific T cells in the mice in the vaccine group than in the PBS group, and thus the mouse tumors can be delayed and controlled. Volume growth. The PBS group has not been induced by vaccines, so it contains fewer cancer cell-specific T cells, while the vaccine group will have more cancer cell-specific T cells after vaccine induction. As shown in Figure 3, the PBS control group and vaccine group There are differences in the results after testing different nanoparticles. Nanoparticle 4 has the best effect and can detect the most comprehensive cancer cell-specific T cells. Nanoparticle 1 cannot detect cancer cell-specific T cells without the assistance of antigen-presenting cells. Nanoparticle 4, nanoparticle 5 and nanoparticle 7 can also detect cancer cell-specific T cells without the assistance of antigen-presenting cells, and the effect of nanoparticle 4 is significantly better than the other two nanoparticles. Moreover, the effect of nanoparticle 4 on detecting cancer cell-specific T cells without the assistance of antigen-presenting cells was better than that of nanoparticle 7 on detecting cancer cell-specific T cells with the assistance of antigen-presenting cells. Moreover, there is little difference in the cancer cell-specific T cells that can be detected by Nanoparticle 4, Nanoparticle 5 and Nanoparticle 7 with or without the assistance of antigen-presenting cells. This shows that nanoparticles prepared by using antigen-presenting cells activated by nanoparticles loaded with cancer cell whole-cell antigens are conducive to better detection of cancer cell-specific T cells; and the effect of mixed antigen-presenting cells of DC and B cells is better than Single DC. Furthermore, the method of detecting broad-spectrum cancer cell-specific T cells described in the present invention may not rely on antigen-presenting cells. Moreover, in the process of detecting cancer cell-specific T cells, it is not necessary to add antigen-presenting cells to obtain the same effect as adding antigen-presenting cells to detect cancer cell-specific T cells. This is also the method of the present invention. Nanoparticles or microparticles prepared by activated antigen-presenting cells have an advantage in detecting cancer cell-specific T cells.

Nanoparticles loaded with cancer cell whole cell antigens and activated by antigen-presenting cells are more effective in detecting cancer cell-specific T cells than nanoparticles loaded with four antigen peptides. Nanoparticles prepared by activated antigen-presenting cells are better at detecting cancer cells. Cell-specific T cells. This shows that the types of cancer cell-specific T cell clones that can be detected by nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with four neoantigen peptides are limited. Nanoparticles prepared by activated antigen-presenting cells loaded with nanoparticles loaded with cancer cell whole cell antigens can detect a wider spectrum of cancer cell-specific T cells, so the number of T cell clones that can be obtained after expansion is also broader. .

As shown in c in Figure 3, the stronger the fluorescence intensity, the more killer substances expressed by the cancer cell-specific T cells, and the detection sensitivity will be higher and the accuracy will be better. When using the same fluorescent probe-labeled anti-mouse IFN-γ antibody to label activated cancer cell-specific T cells, nanoparticles loaded with activated antigen-presenting cell membrane components and whole cell components of cancer cells 4 were used. The fluorescence signal (mean fluorescence intensity, MFI) of the fluorescent probe connected to the IFN-γ antibody that can be detected by activated cancer cell-specific T cells is stronger than that of nanoparticles simply loaded with whole cell components of cancer cells. 1. The activated cancer cell-specific T cells can detect the fluorescent signal of the fluorescent probe connected to the IFN-γ antibody. Moreover, regardless of whether there are antigen-presenting cells in the incubation system during the detection and activation process, the fluorescence signal detected by the cancer cell-specific T cells activated by nanoparticle 4 is stronger than that of the cancer cell-specific T cells activated by nanoparticle 1. , which shows that the cancer cell-specific T cells activated by nanoparticle 4 express more specific markers and are therefore easier to detect during detection.

It can be seen that the nanoparticles prepared by the antigen-presenting cells activated by the nanoparticles loaded with whole cell components according to the present invention can better detect cancer cell-specific T cells with the ability to recognize and kill cancer cells. The cancer cell whole cell antigens loaded by the nanoparticles can be degraded into antigen epitopes after being phagocytosed by the antigen presenting cells and presented to the surface of the antigen presenting cells. Specific T cells that can recognize the cancer cell whole cell antigens can recognize them. Cancer cells are activated after whole-cell antigen epitopes and express specific surface markers. By analyzing the proportion of T cells that highly express specific surface markers through flow cytometry, we can know that the activated T cells can recognize and have killing efficacy. Number and proportion of cancer cell-specific T cells.

Example 3 Detection of cancer cell-specific T cells in mouse peripheral spleen cells

This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticles prepared by nanoparticle-activated antigen-presenting cells to detect cancer cell-specific T cells. In this example, B16F10 melanoma tumor tissue and cancer cells are first lysed to prepare a mixture of water-soluble components (mass ratio 1:1) and a mixture of non-water-soluble components (mass ratio 1:1) of tumor tissue and cancer cells, And mix the water-soluble component mixture and the water-insoluble component mixture at a mass ratio of 1:1. Then, PLGA is used as the nanoparticle skeleton material, Poly(I:C) and CpG2006 are used as adjuvants to prepare nanoparticles loaded with lysate components, and then the nanoparticles are incubated with antigen-presenting cells for a period of time to activate antigen presentation. cells, and prepare antigen-presenting cells into nanoparticles to detect cancer cell-specific T cells.

(1) Lysis of tumor tissue and cancer cells and collection of components

When collecting tumor tissue, 1.5 × 10 ⁵ B16F10 cells were first subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm ³ , the mice were sacrificed and the tumor tissue was removed. The tumor tissue was cut into sections. Grind, add an appropriate amount of pure water through a cell strainer and freeze and thaw repeatedly 5 times, and can be accompanied by ultrasound to destroy the lysed sample; when collecting the cultured B16F10 cancer cell line, first centrifuge to remove the medium, then wash twice with PBS and centrifuge Cancer cells were collected, resuspended in ultrapure water, frozen and thawed three times, and destroyed and lysed by ultrasound. After the tumor tissue or cancer cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant to obtain the water-soluble component that is soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate. The non-water-soluble components that are insoluble in pure water are converted into soluble in 8M urea aqueous solution. The water-soluble components of the tumor tissue and the water-soluble components of the cancer cells are mixed at a mass ratio of 1:1; the water-insoluble components of the tumor tissue and the non-water-soluble components of the cancer cells are mixed at a mass ratio of 1:1. Mixing the water-soluble component mixture and the water-insoluble component mixture at a mass ratio of 1:1 is the source of the antigen raw material for preparing nanoparticles.

(2) Preparation of bacterial extracellular vesicles (OMV)

Centrifuge Bifidobacterium longum at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 μm filter, ultrasonicate at 4°C for 5 minutes at 20W, and then centrifuge at 16000g for 90 minutes. Resuspend the pellet in PBS to form the collected bacterial outer vesicle membrane components, and then use 8M urea aqueous solution to lyse and dissolve the bacterial outer vesicle membrane components.

Or centrifuge Bifidobacterium longum at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 μm filter, ultrasonicate at 4°C for 5 minutes at 20W, and then centrifuge at 16000g for 90 minutes. , resuspend the pellet in PBS to form the collected bacterial outer vesicle membrane components, and then use Tween 80 aqueous solution to lyse and dissolve the bacterial membrane components.

(3) Preparation of nanoparticles

In this example, nanoparticle 1 is prepared by the double emulsion method. The molecular weight of the preparation material PLGA is 7KDa-17KDa. The immune adjuvants used are poly(I:C) and CpG2006 and the adjuvants are encapsulated in the nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysis solution components and adjuvants inside the nanoparticles. Then 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure solution containing 4% trehalose is used. Resuspend in water and freeze-dry for 48 hours before use. The average particle size of nanoparticles 1 is about 250nm. Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 130 μg of protein or peptide components. Each 1 mg of PLGA nanoparticles 1 carries 0.02 mg of poly(I:C) and CpG2006 immune adjuvant. Nano Particle 1 is also called nanovaccine 1 when injected.

In this embodiment, the preparation materials and preparation method of nanoparticle 2 are the same as nanoparticle 1. The nanoparticle 2 is internally loaded with the antigen component prepared in step (1) and the 8M urea-dissolved bacterial outer vesicle membrane component prepared in step (2) at the same time, and the mass ratio of the two is 1:1. The immune adjuvants used were poly(I:C) and CpG2006, and the adjuvants were encapsulated in nanoparticles. During the preparation process, the double emulsion method was first used to load the tumor tissue inside the nanoparticles with lysate components, bacterial external vesicle components and adjuvants. Then 100 mg of the nanoparticles were centrifuged at 10,000g for 20 minutes, and 10 mL containing 4% trehalose was used. Resuspend in ultrapure water and freeze-dry for 48 hours before use. The average particle size of nanoparticles 2 is about 250nm. Each 1 mg of PLGA nanoparticles 2 is loaded with approximately 130 μg of protein or peptide components. Each 1 mg of PLGA nanoparticles 2 carries 0.02 mg of poly(I:C) and CpG2006 immune adjuvants each.

In this embodiment, the preparation materials and preparation methods of nanoparticle 3 are the same as nanoparticle 1. The nanoparticle 3 is simultaneously loaded with the antigen component prepared in step (1) and the bacterial outer vesicle membrane component dissolved in Tween 80 prepared in step (2), and the mass ratio of the two is 1:1. The immune adjuvants used were poly(I:C) and CpG2006, and the adjuvants were encapsulated in nanoparticles. During the preparation process, the double emulsion method was first used to load the tumor tissue inside the nanoparticles with lysate components, bacterial external vesicle components and adjuvants. Then 100 mg of the nanoparticles were centrifuged at 10,000g for 20 minutes, and 10 mL containing 4% trehalose was used. Resuspend in ultrapure water and freeze-dry for 48 hours before use. The average particle size of nanoparticles 3 is about 250nm. Each 1 mg of PLGA nanoparticles 3 is loaded with approximately 130 μg of protein or peptide components. Each 1 mg of PLGA nanoparticles 3 is loaded with 0.02 mg of poly(I:C) and CpG2006 immune adjuvant.

The preparation materials and methods of blank nanoparticle 4 are the same as nanoparticle 1, but blank nanoparticle 4 only carries the same amount of adjuvant and does not load any tumor tissue lysate components. The particle size of the nanoparticles 4 is approximately 250 nm.

(4) Isolation of B cells

After the C57BL/6 mice were sacrificed, the spleens of the mice were removed, a single cell suspension of mouse splenocytes was prepared, and CD19 ⁺ B cells in the splenocytes were isolated using magnetic bead sorting.

(5)Activation of antigen-presenting cells

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 B cells (10 million cells) in 15 mL of RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ), the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), albumin (50ng /mL), and CD80 antibody (10ng/mL).

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

The incubated B cells were collected by centrifugation at 400g for 5 minutes, then washed with PBS three times, resuspended in PBS water and sonicated at low power (10W) for 15 minutes. Then centrifuge the sample at 500g for 5 minutes and collect the supernatant. The supernatant is filtered through membranes with pore diameters of 30 μm, 10 μm, 5 μm, 0.45 μm, and 0.22 μm. The resulting filtrate sample is centrifuged at 18000g for 60 minutes and discarded. The supernatant and the pellet were resuspended in PBS to obtain nanoparticles. Among them, the nanoparticles prepared by using antigen-presenting cells activated by nanoparticle 1 are nanoparticles 5, with a particle size of 110 nanometers; the nanoparticles prepared by using antigen-presenting cells activated by nanoparticles 2 are nanoparticles 6, with a particle size of 110 nanometers. Nanoparticles; the nanoparticles prepared using antigen-presenting cells activated by nanoparticle 3 are nanoparticles 7, with a particle size of 110 nanometers; the nanoparticles prepared using antigen-presenting cells activated with nanoparticles 4 are nanoparticles 8, with a particle size of 110 nanometers nanometer.

(7) Detection of cancer cell-specific T cells

Inoculate 1.5×10 ⁵ B16F10 cells subcutaneously on the back of each C57BL/6 mouse on day 0, and subcutaneously inject 0.7 mg of PLGA prepared in step (3) into the mice on day 7, day 12, and day 17 respectively. Nanoparticle 1 (Nanovaccine 1) or 100μL PBS. The mice were sacrificed on the 21st day, the mouse spleens were removed and mouse spleen single cell suspension was prepared, and the CD3 ⁺ T cells in the spleen cells were sorted using magnetic bead sorting method. Put the isolated T cells (4 million) and 100 μg of nanoparticles prepared in step (6) (nanoparticle 5, or nanoparticle 6, or nanoparticle 7, or nanoparticle 8) in 40 mL of high-glucose DMEM complete medium The CCP was incubated for 48 hours (37°C, 5% CO ₂ ), and then centrifuged at 400 g for 5 minutes to collect the cells. After antibody labeling, flow cytometry was used to analyze the CD3 ⁺ IFNγ ⁺ T cells in the incubated T cells, which were the The number and proportion of cancer cell-specific T cells specifically activated by cancer cell whole-cell antigens.

The cancer cell whole cell antigens loaded by the nanoparticles can be degraded into antigen epitopes after being phagocytosed by the antigen-presenting cells and presented to the surface of the antigen-presenting cell membrane. The nanoparticles prepared by the antigen-presenting cells are loaded with the above-mentioned degraded and presented products. The antigenic epitope can be recognized by cancer cell-specific T cells and activate cancer cell-specific T cells. After activation, they secrete killer cytokines. IFN-γ is the most important cytokine secreted by antigen-specific T cells that are activated after recognizing antigens. CD3 ⁺ IFN-γ ⁺ T cells analyzed using flow cytometry are cancer cell-specific T cells that can recognize and kill cancer cells.

(5)Experimental results

As shown in Figure 4, the tumor growth rate of mice in the PBS control group was very fast, while the tumor growth rate of the nanoparticle-treated group was slower, which shows that the nanoparticles induce cancer cell-specific T cells and can control the growth of tumors. Nanoparticle 8 could hardly activate cancer cell-specific T cells; while nanoparticle 5, nanoparticle 6, and nanoparticle 7 could detect more cancer cell-specific T cells. Moreover, the effect of Nanoparticle 6 is better than that of Nanoparticle 5 and Nanoparticle 7, which shows that the activated antigen-presenting cells after loading the bacterial outer vesicle components lysed and dissolved using appropriate methods into the nanoparticles are beneficial to the specific detection of cancer cells. Sexual T cells.

Example 4 Nanoparticles or microparticles detect cancer cell-specific T cells

In this example, 6M guanidine hydrochloride was first used to cleave the whole cell antigen of B16F10 melanoma cancer cells. Then, a micron particle system loaded with cancer cell whole cell antigens was prepared using PLGA as the micron particle skeleton material, CpG BW006 (B type), CPG2216 (A type) and Poly ICLC as immune adjuvants. After using micron particles to activate antigen-presenting cells, the antigen-presenting cells are prepared into nanoparticles or micron particles to detect cancer cell-specific T cells.

(1) Lysis of cancer cells

The cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, and then the cancer cells were resuspended and lysed with 6M guanidine hydrochloride. The whole cell antigen of the cancer cells was lysed and dissolved in 6M Guanidine hydrochloride is the source of antigen raw materials for preparing micron particle systems.

(2) Preparation of micron particle system

In this embodiment, the micron particles are prepared by the double emulsion method. The molecular weight of PLGA, the material used to prepare micron particles 1, is 38KDa-54KDa, and the immune adjuvants used are CpG BW006, CPG2216 and Poly ICLC. Poly ICLC is a toll-like receptor 3 agonist, while various CpGs are Toll-like receptor 9 agonists, and both Toll-like receptor 3 and Toll-like receptor 9 are located in the endosome membrane structure within the cell. First, the lysate components and immune adjuvant were loaded into the micron particles, and then centrifuged at 10,000g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours; before use, the particles were Resuspend in 7 mL of PBS and then add 3 mL of cancer cell lysate component (protein concentration 50 mg/mL) and incubate at room temperature for 10 min to obtain micron particles 1 loaded with lysate both inside and outside. The average particle size of the microparticles is about 2.50μm, and the surface potential is about -2mV; each 1 mg of PLGA microparticles is loaded with approximately 140 μg of protein or peptide components, and the loaded CpG BW006 (Class B), CPG2216 (Class A) and Poly ICLC 0.02mg each.

The preparation materials and preparation methods of control microparticle 2 are the same, and the loaded immune adjuvants are CpG2336 (Class A), CPG2216 (Class A) and Poly ICLC. The diameter of the control micron particles 2 is about 2.50 μm, and the surface potential is about -2mV. Each 1 mg PLGA micron particle is loaded with approximately 140 μg of protein or peptide components. Each 1 mg PLGA micron particle is loaded with CpG2336 (Class A) and CPG2216 (Class A). and Poly ICLC immune adjuvant are 0.02mg each.

The preparation materials and preparation methods of control microparticle 3 are the same, and the loaded immune adjuvants are CpG BW006 (category B) and CPG2216 (category A). The adjuvant used in control microparticle 3 is 0.02 mg per 1 mg PLGA microparticle, the particle size is about 2.50 μm, and the surface potential is about -2mV. Each 1 mg PLGA microparticle is loaded with approximately 140 μg of protein or peptide components. Each 1 mg PLGA microparticle contains The loaded CpG BW006 (Category B) and CPG2216 (Category A) are 0.03mg each.

(3) Preparation of antigen-presenting cells

After the mice were killed, mouse lymph nodes and spleens were collected. The mouse lymph nodes or spleens were minced and ground, and filtered through a cell mesh to prepare a single cell suspension. After mixing the lymph node single cell suspension and the spleen single cell suspension, flow cytometry was used. CD19 ⁺ B cells and CD11c ⁺ DC were sorted by cytometry.

(4) Activation of antigen-presenting cells

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

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

The incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors. The cells were resuspended in PBS water and incubated at 4°C at low power (PBS). 22.5W) Ultrasonic for 1 minute. Then centrifuge the sample at 3000g for 15 minutes and collect the supernatant. Centrifuge the supernatant at 8000g for 15 minutes and collect the supernatant. Then collect the supernatant after centrifugation at 16000g for 90 minutes. Discard the supernatant and collect the pellet. Place the pellet in PBS. After resuspension, nanoparticles are obtained. Among them, the nanoparticles prepared by the mixed antigen-presenting cells activated by micron particles 1 are nanoparticles 1, and the particle size is 110 nanometers; among them, the nanoparticles prepared by the mixed antigen-presenting cells activated by micron particles 2 are nanoparticles 2, and the particle size is 110 nanometers. Nano; the nanoparticles prepared by mixed antigen-presenting cells activated by micron particles 3 are nanoparticles 3, with a particle size of 110 nanometers.

Or collect the incubated DC and B cells (activated with micron particles 1) by centrifugation at 400g for 5 minutes, then wash the cells twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, and resuspend the cells in PBS Then ultrasonicate at 4°C for 1 minute at low power (22.5W) in water. Then centrifuge the sample at 3000g for 15 minutes and collect the supernatant. Centrifuge the supernatant at 8000g for 15 minutes and collect the supernatant. Then collect the supernatant after centrifugation at 16000g for 90 minutes. Discard the supernatant and collect the pellet. Place the pellet in PBS. After resuspension, nanoparticles are obtained. Mix 20 mg nanoparticles and 100 mg micron particles 1 and incubate at room temperature for 15 minutes, then ultrasonicate at 10W for 1 minute, then centrifuge at 8000g for 15 minutes, discard the supernatant and collect the precipitate, and resuspend the precipitate. Micron particles 4, the particle size is about 2.55μm.

(6) Detection of cancer cell-specific T cells

On day 0, 1.5×10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. On

days

10, 15, and 20, the mice were subjected to radiotherapy or injected with 100 μL PBS at the tumor site. The mice were sacrificed on the 24th day, and the peripheral blood of mice in each group was collected. Peripheral blood mononuclear cells (PBMC) were then prepared, and CD3 ⁺ T cells were sorted using magnetic bead sorting. The sorted T cells (5 million), nanoparticles 1-3 (100 μg) or microparticles 4 (100 μg) prepared in step (5) were incubated in 2 mL RPMI1640 complete medium for 24 hours (37°C, 5% CO ₂ ), and then flow cytometry was used to detect CD3 ⁺ IFN-γ ⁺ T cells in T cells, which are cancer cell-specific T cells activated by cancer cell whole cell antigens. At the same time, the mean fluorescence intensity (MFI) of the IFN-γ ⁺ -linked fluorescent probe in CD3 ⁺ IFN-γ ⁺ T cells was analyzed.

(7)Experimental results

As shown in Figure 5a, nanoparticles prepared from antigen-presenting cells activated by microparticles loaded with CpG adjuvant and Poly ICLC mixed adjuvant were better at detecting cancer cell-specific T cells than microparticles loaded with two CpG mixed adjuvants. Nanoparticles prepared from activated antigen-presenting cells. Moreover, nanoparticles prepared by activating antigen-presenting cells using microparticles loaded with a type B CpG, a type A CpG, and PolyICLC mixed adjuvant were better than those prepared using micron particles loaded with two type A CpG and PolyICLC mixed adjuvants. Nanoparticles prepared from particle-activated antigen-presenting cells; moreover, particles prepared from activated antigen-presenting cell membranes are more effective at loading whole cell components of cancer cells internally. The analysis results of the mean fluorescence intensity (MFI) of the fluorescent probe connected to IFN-γ ⁺ in CD3 ⁺ IFN-γ ⁺ T cells were consistent with the above trend (Figure 5b). This shows that the nanoparticles activated by antigen-presenting cells loaded with mixed adjuvants of two different toll-like receptors can produce better nanoparticles, and that nanoparticles containing class B CpG and Toll-like receptor 3 agonists are used as mixed adjuvants. Nanoparticles prepared by activating antigen-presenting cells with micron particles are more effective.

Example 5 Detection of cancer cell-specific T cells in tumor tissue

In this example, 8M urea was first used to lyse B16F10 melanoma tumor tissue and dissolve the tumor tissue lysate components. Then, PLGA was used as the nanoparticle skeleton material, and Poly(I:C), CpG2006 (B type) and CpGSL01 (B type) were used as immune adjuvants to prepare nanoparticles loaded with cancer cell whole cell antigens, and the nanoparticles were used to activate the antigens. After presenting the cells, the nanoparticles are prepared and then detected for cancer cell-specific T cells in the tumor tissue.

(1) Collection and lysis of tumor tissue

1.5 × 10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm ³ , the mice were sacrificed and the tumor tissue was removed. Cut the tumor tissue into pieces and grind it. Add an appropriate amount of 8M urea through a cell filter to lyse the cells and dissolve the cell lysate. The above are the sources of antigen raw materials for preparing nanoparticle systems.

(2) Preparation of nanoparticles

In this example, nanoparticles were prepared by solvent evaporation method. The molecular weight of PLGA, the material used to prepare nanoparticle 1, is 7KDa-17KDa. The immune adjuvants used are Poly(I:C), CpG2006 and CpGSL01, and the lysate components and adjuvants are encapsulated inside the nanoparticles. The preparation method is as described above. After loading the lysate components and adjuvants inside the nanoparticles, 100 mg of the nanoparticles are centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h to obtain Freeze-dry the powder for later use. The average particle size of the nanoparticles is about 270nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 80 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles uses Poly(I:C), CpG2006 and CpGSL01 is 0.02 mg each.

The preparation materials and preparation method of control nanoparticle 2 are the same as above. The particle size is about 270nm. It is loaded with an equal amount of lysate components. The loaded immune adjuvant is Poly(I:C). Each 1mg of PLGA is loaded with 0.06mg of Poly(I:C). .

The preparation materials and preparation method of control nanoparticle 3 are the same as above. The particle size is about 270nm. It is loaded with equal amounts of lysate components. The loaded immune adjuvants are Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A). Each 1mg of PLGA loads 0.02mg each of Poly (I:C), CpG1585 (Class A) and CpG2216 (Class A).

(3)Preparation of DCs and B cells

After C57BL/6 were sacrificed, mouse lymph nodes were removed to prepare mouse lymph node single cell suspension, and then flow cytometry was used to sort CD11c ⁺ DC and CD19 ⁺ B cells from the lymph node cell single cell suspension.

(4) Activation of antigen-presenting cells

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

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

Collect the incubated DC and B cells by centrifugation at 400g for 5 minutes, then wash the cells twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, resuspend the cells in PBS water and homogenize at 4°C. The machine was stirred and destroyed at 2000rpm for 25 minutes. Then the sample was centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected. Then the supernatant was discarded after centrifugation at 15000g for 30 minutes to collect the pellet. The pellet was resuspended in PBS. After suspension, nanometer samples are obtained. Among them, nanoparticles 4 were prepared using antigen-presenting cells activated by nanoparticles 1, with a particle size of 150 nanometers; nanoparticles 5 were prepared using antigen-presenting cells activated with nanoparticles 2, and the particle size was 150 nanometers; The antigen-presenting cells activated by particle 3 were prepared as nanoparticles 6, with a particle size of 150 nanometers.

(6) Detection of cancer cell-specific T cells

days

8, 10, 12, 14, and 16, mice were injected subcutaneously with 100 μL. αPD-1 antibody (10 mg/kg) or 100 μL PBS. The mice were sacrificed on the 20th day, and the tumor tissues of the mice were collected respectively. The tumor tissues were cut into small pieces and filtered through a cell mesh to prepare a single cell suspension of tumor tissue, and then CD3 was sorted from it using a magnetic bead sorting method. ⁺ T cells. Then, the sorted T cells (500,000) were co-incubated with allogeneic B cells (2.5 million), the nanoparticles prepared in step (5) (100 μg), or in 20 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ). Then centrifuge at 400g for 5 minutes, collect the supernatant, and analyze the concentration of IFN-γ in the supernatant using ELISA.

In the ELISA detection method, cancer cell-specific T cells secrete killer substances such as IFN-γ after being activated. The concentration of this type of killer substance represents the amount of activated cancer-specific T cells and the strength of the killing ability of cancer cells.

(7)Experimental results

As shown in Figure 6, compared with the PBS control group, more IFN-γ could be detected after co-incubation with nanoparticles prepared by antigen-presenting cells activated by nanoparticles loaded with cancer cell whole cell antigens. Moreover, the effect of nanoparticles loaded with two types of B CpG and Poly(I:C) as mixed adjuvants to activate antigen-presenting cells is better than that of two types of type A CpG with Poly(I:C) as a mixed adjuvant. Nanoparticles prepared by mixing adjuvant nanoparticles or nanoparticles loading only Poly(I:C) as an adjuvant to activate antigen-presenting cells.

Example 6 Detection of cancer cell-specific T cells in colon cancer

This example uses MC38 mouse colon cancer as a cancer model to illustrate how to use nanoparticles prepared from nanoparticle-activated antigen-presenting cells to detect a broad spectrum of cancer cell-specific T cells. Colon cancer tumor tissue and lung cancer cancer cells are first lysed to prepare water-soluble components and water-insoluble components, and the antigen is first degraded into polypeptides using protease in vitro. In practical applications, other enzymes or other methods can also be used to first degrade the proteins in the whole cell fraction into peptides. Then prepare a mixture of water-soluble components (mass ratio 1:1) and non-water-soluble components (mass ratio 1:1), and mix the water-soluble component mixture and the non-water-soluble component mixture in a mass ratio of 1:1 mix. Then, PLA was used as the nanoparticle skeleton material, and CpGM362, CPG1018 and Poly ICLC were used as immune adjuvants to prepare nanoparticles, and the nanoparticles were used to detect cancer cell-specific T cells in vitro.

(1) Lysis of tumor tissue and cancer cells and collection of components

2 × 10 ⁶ MC38 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm ³ , the mice were sacrificed and the tumor tissue was removed. Cut the tumor tissue into pieces and then grind it. Add an appropriate amount of pure water through a cell filter and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at a speed greater than 5000g for 5 minutes and take the supernatant, which is the water-soluble component soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble components. The water-insoluble components of pure water are converted into soluble in 8M urea aqueous solution. Add trypsin (0.5 mg/mL) and chymotrypsin (Chymotrypsin, 0.5 mg/mL) to the water-soluble component (80 mg/mL), incubate for 1 hour, and then heat at 95°C for 10 minutes to inactivate the proteases for later use.

Collect the cultured LLC lung cancer cell lines and centrifuge them at 350g for 5 minutes. Then discard the supernatant and wash twice with PBS. Then resuspend the cells in ultrapure water and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells. . After the cells are lysed, centrifuge the lysate at 3000g for 6 minutes and take the supernatant to obtain the water-soluble components that are soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble components in pure water. The water-insoluble components of water are converted into soluble in 8M urea aqueous solution. Add trypsin (0.5 mg/mL) and chymotrypsin (Chymotrypsin, 0.5 mg/mL) to the water-soluble component (80 mg/mL) and incubate for 1 hour, then heat at 95°C for 10 minutes to inactivate the proteases for later use.

The water-soluble components from colon cancer tumor tissue and lung cancer cancer cells were mixed at a mass ratio of 1:1; the water-insoluble components dissolved in 8M urea were also mixed at a mass ratio of 1:1. Then, the water-soluble component mixture and the water-insoluble component mixture are mixed at a mass ratio of 1:1, and this mixture is the source of raw materials for preparing nanoparticles.

(2) Lysis and dissolution of Bacillus Calmette-Guérin (BCG)

Collect BCG, use 8M urea aqueous solution to cleave BCG, and then dissolve the cleavage components for later use.

(3) Preparation of nanoparticles

In this embodiment, nanoparticle 1 is prepared by solvent evaporation method. The molecular weight of PLA, the material for preparing nanoparticle 1, is 20KDa. The nanoparticles are loaded with tumor tissue and cancer cell lysate, bacterial lysate and immune adjuvant inside, and the tumor tissue and cancer cell lysate components are loaded on the surface. The immune adjuvants used were CpGM362, CPG1018 and poly ICLC, and the adjuvants were loaded inside the nanoparticles. The mass ratio of tumor tissue and cancer cell lysate to bacterial lysate used in preparing the nanoparticles was 1:1. The preparation method is as mentioned above. During the preparation process, the lysate mixture, bacterial lysate components and adjuvants are first loaded inside the nanoparticles using the double emulsion method. After loading the lysates and adjuvants inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h. Before use, resuspend 20 mg of nanoparticles in 0.9 mL of PBS and mix and incubate at room temperature for 5 minutes with 0.1 mL of samples containing equal amounts of cancer cell and tumor tissue lysate mixture and bacterial lysate components (80 mg/mL). use. The average particle size of nanoparticles 1 is about 290nm. Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 140 μg of protein or peptide components. Each 1 mg of PLGA nanoparticles contains 0.04 mg each of CpGM362, CPG1018 and Poly ICLC immune adjuvant.

(4) Preparation of antigen-presenting cells

Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of mice after killing C57BL/6, and then CD11c ⁺ DC and CD19 ⁺ B cells were sorted from PBMC using flow cytometry. In this example, BMDC and BMDM were used simultaneously as antigen-presenting cells. The preparation method of BMDC is the same as in Example 1. The preparation method of BMDM is as follows:

C57 mice were anesthetized and sacrificed by dislocation. The mice were disinfected with 75% ethanol. Then, a small opening was made on the back of the mouse with scissors. The skin was directly torn open to the calf joint of the mouse by hand, and the foot joint and foot joint of the mouse were removed. skin. Use scissors to remove the hind limbs along the greater trochanter at the root of the mouse's thigh, remove the muscle tissue and place it in a petri dish containing 75% ethanol to soak for 5 minutes. Replace the petri dish with a new one with 75% ethanol and move it to a clean bench. Move the leg bones soaked in ethanol into cold PBS and soak them to wash off the ethanol on the surfaces of the tibia and femur. This process can be repeated three times. Separate the cleaned femur and tibia, and use scissors to cut off both ends of the femur and tibia respectively. Use a 1mL syringe to draw cold induction medium and blow out the bone marrow from the femur and tibia. Repeat the blowing 3 times until the inside of the leg bone is no longer visible. to a distinct red color. Use a 5mL pipette to pipe the culture medium containing bone marrow cells repeatedly to disperse the cell clumps, then use a 70μm cell strainer to sieve the cells, transfer to a 15mL centrifuge tube, centrifuge at 1500rpm/min for 5 minutes, discard the supernatant, and add red blood cells to lyse Resuspend the solution and let stand for 5 minutes, then centrifuge at 1500 rpm/min for 5 minutes. Discard the supernatant and resuspend in cold prepared bone marrow macrophage induction medium (DMEM high-glucose medium containing 15% L929 medium), and plate. Culture the cells overnight to remove other miscellaneous cells that adhere quickly, such as fibroblasts, etc. Collect non-adherent cells and seed them into dishes or cell culture plates according to the experimental design. Macrophage colony-stimulating factor (M-CSF) stimulates bone marrow cells to differentiate into mononuclear macrophages at a concentration of 40ng/mL. After culturing for 8 days, the morphological changes of macrophages were observed under a light microscope. Digest and collect the cells after 8 days, use anti-mouse F4/80 antibody and anti-mouse CD11b antibody, incubate at 4°C for 30 minutes in the dark, and use flow cytometry to identify the proportion of successfully induced macrophages.

(5)Activation of antigen-presenting cells

Nanoparticle 1 (1000 μg) was incubated with peripheral blood-derived DC (20 million) and BMDC (20 million) in RPMI1640 complete medium for 72 hours (37°C, 5% CO ₂ ); the incubation system contained GM- CSF (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL), IL-12 (500U/mL) and CD80 antibody (10ng/mL).

Or incubate nanoparticle 1 (1000 μg) with peripheral blood-derived DCs (10 million), BMDC (10 million), BMDM (10 million), and B cells (10 million) in 20 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ), the incubation system contains GM-CSF (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL), and IL-12 (500U/mL) and CD80 antibody (10ng/mL).

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

The incubated peripheral blood-derived DCs (20 million) and BMDCs (20 million) were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors. The cells were resuspended in PBS water and treated 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 centrifuged at 8000g for 15 minutes and the supernatant was collected. The supernatant was mixed with the nanoparticle 1 (50mg) prepared in step (3) at 4°C. Incubate for a total of 16 hours, and then use a 0.45 μm filter to repeatedly co-extrude. Centrifuge the extruded liquid at 13000g for 20 minutes, discard the supernatant and collect the precipitate. Resuspend the precipitate in PBS to obtain nanoparticle 2. diameter is 310 nm.

Or collect the incubated peripheral blood-derived DC (10 million), BMDC (10 million), B cells (10 million), and BMDM (10 million) by centrifugation at 400g for 5 minutes, and then use protease inhibitor-containing Wash the cells twice with 4°C phosphate buffer solution (PBS), resuspend the cells in PBS water and treat them in a high-pressure homogenizer (5000bar) for 5 minutes. The sample was then centrifuged at 2000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected. The supernatant was mixed with the nanoparticle 1 (50mg) prepared in step (3) at 4°C. Incubate for a total of 16 hours, and then use a 0.45 μm filter to repeatedly co-extrude. Centrifuge the extruded liquid at 13000g for 20 minutes, discard the supernatant and collect the precipitate. Resuspend the precipitate in PBS to obtain nanoparticle 3. diameter is 310 nm.

(7) Detection of cancer cell-specific T cells

On day 0, each C57BL/6 mouse was subcutaneously inoculated with 1.5 × 10 ⁵ MC38 cells on the back. On

days

10, 15 and 21, the mice were subcutaneously injected with 100 μL of 1 mg PLGA nanoparticles 1 to activate the mice. Cancer cell-specific T cells in the body. The mice were sacrificed on day 24, and the draining lymph nodes and spleens of the mice were collected. Single-cell suspensions of draining lymph nodes and spleen cells were prepared and T cells were isolated from them using the magnetic bead method.

The obtained T cells (4 million), 200 μg of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3), IL-2 (500 U/mL), IL-7 (500 U/mL) and IL- 15 (5000U/mL) was incubated in 5mL DMEM complete medium for 24 hours, and then flow cytometry was used to sort the incubated CD3 ⁺ IFN-γ ⁺ T cells, which are cancer cell-specific cells activated by cancer cell whole cell antigens. T cells. At the same time, the mean fluorescence intensity (MFI) of the IFN-γ ⁺ -linked fluorescent probe in CD3 ⁺ IFN-γ ⁺ T cells was analyzed. The stronger the fluorescence intensity, the more killer substances expressed by the cancer cell-specific T cells, and the detection sensitivity will be higher and the accuracy will be better.

Alternatively, the obtained T cells (4 million), peripheral blood-derived DCs (1 million), BMDC (1 million), B cells (1 million), BMDM (1 million), and 200 μg of nanoparticles (nano Particle 1, or nanoparticle 3), IL-2 (500U/mL), IL-7 (500U/mL) and IL-15 (5000U/mL) were incubated in 5mL DMEM complete medium for 24 hours, and then flow cytometry was used CD3 ⁺ IFN-γ ⁺ T cells after surgical sorting and incubation are cancer cell-specific T cells activated by cancer cell whole cell antigens. At the same time, the mean fluorescence intensity (MFI) of the IFN-γ ⁺ -linked fluorescent probe in CD3 ⁺ IFN-γ ⁺ T cells was analyzed.

(8)Experimental results

As shown in Figure 7, Nanoparticle 3 outperforms Nanoparticle 1 and Nanoparticle 2. When detecting T cells without antigen-presenting cells, nanoparticle 1 cannot be detected, but both nanoparticle 2 and nanoparticle 3 can be detected. When detecting T cells containing antigen-presenting cells, nanoparticle 1, nanoparticle 2 and nanoparticle 3 can all detect T cells. Furthermore, nanoparticle 2 and nanoparticle 3 are better than nanoparticle 1, and nanoparticle 3 is better than nanoparticle 3. Particle 2. Moreover, even when detecting T cells without antigen-presenting cells, the effect of nanoparticle 2 is better than that of

nanoparticles

1 and 2 when detecting T cells when they contain antigen-presenting cells. The analysis results of average fluorescence intensity are consistent with the above trend. This shows that loading activated antigen-presenting cell membrane components on the particle surface and using mixed antigen-presenting cell membrane components can improve the effectiveness of nanoparticles or microparticles in detecting cancer cell-specific T cells.

Example 7 Detection of cancer cell-specific T cells in breast cancer mice

This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how micron particles loaded with cancer cell whole cell antigens activate antigen-presenting cells and then are prepared into micron particles to detect cancer cell-specific T cells from peripheral immune organs.

(1) Lysis of cancer cells

The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water, and then repeatedly frozen and thawed 5 times with ultrasound to lyse cancer cells. Add 1mg/mL nuclease to the lysed cells to degrade the nucleic acid in the lysate, then heat at 95°C for 10 minutes to inactivate the nuclease, and then centrifuge at 5000g for 5 minutes to collect the supernatant, which is the water-soluble component. Precipitate using 10% sodium deoxycholate (containing 10M arginine) to dissolve the non-water-soluble components. Mix the water-soluble components and the non-water-soluble components at a mass ratio of 3:1 to prepare the particle system. sources of raw materials.

(2) Preparation of micron particle system

In this embodiment, the double emulsion method is used to prepare micron particles. The molecular weight of the micron particle 1 framework material PLGA is 38KDa-54KDa, and the immune adjuvants used are CpG2395 (Category C), CpGM362 (Category C) and Poly (I:C). During preparation, the double emulsion method is used to prepare micron particles loaded with lysate components and adjuvants internally, and then 100 mg of micron particles are centrifuged at 9000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use. The average particle size of this microparticle system is about 2.5 μm, and the surface potential is about -6mV; each 1 mg of PLGA micron particles is loaded with approximately 110 μg of protein or polypeptide components, and each of CpG2395, CpGM362 and Poly(I:C) is loaded with 0.02 mg. The preparation materials and preparation method of control micron particles 2 are the same as above, the particle size is about 2.5 μm, and the surface potential is about -6mV; every 1 mg of PLGA micron particles is loaded with approximately 110 μg of protein or peptide components, and every 1 mg of PLGA is loaded with CpG1585 (Class A), CpG2336 ( Class A) and Poly(I:C) 0.02mg each.

(3) Preparation of B cells

B cells from peripheral splenocytes were used. After the mice were sacrificed, the spleens were removed, and then a single cell suspension of mouse spleen cells was prepared, and the CD19 ⁺ B cells in the single cell suspension were sorted using magnetic bead sorting.

(4) Activation of antigen-presenting cells

The micron particles (800 μg) loaded with cancer cell whole cell antigen components were incubated with the B cells (10 million) prepared in step (3) in 15 mL high-sugar DMEM complete medium for 48 hours (37°C, 5% CO ₂ ) , the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) and CD86 antibody (10ng/mL).

(5) Preparation of microparticles based on antigen-presenting cells

The incubated B cells (10 million) were collected by centrifugation at 400g for 5 minutes, then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, and resuspended in PBS water at 4°C. Ultrasonicate at low power (20W) for 1 minute and then use a homogenizer at 1000rpm for 3 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected. The supernatant was mixed with the microparticles (60mg) prepared in step (2) and DSPE-PEG. -Mannose (1mg) was sonicated at 50W for 2 minutes, then centrifuged at 8000g for 20 minutes and then collected. The supernatant was discarded to collect the precipitate. The precipitate was resuspended in PBS to obtain micron particles. The microparticles prepared by using the antigen-presenting cell membrane component activated by micron particle 1 and micron particle 1 are micron particles 3, with a particle size of 2.6 μm; using the antigen-presenting cell membrane component activated by micron particle 2 and micron particle The micron particles prepared by the combined action of 2 are micron particles 4, with a particle size of 2.6 μm.

(6) Detection of cancer cell-specific T cells

On day 0, each BALB/c mouse was subcutaneously inoculated with 1 × 10 ⁶ 4T1 cells on the back. On

days

10, 14 and 18, the mice were subcutaneously injected with 100 μL of 1 mg PLGA micron particles 1 (micron vaccine 1). ) or 100 μL of PBS. The mice were sacrificed on day 22, their spleens were collected, and a single cell suspension of splenocytes was prepared. Spleen cell single cell suspension (6 million cells), DC2.4 (2 million cells) and micron particles (50 μg) were incubated in 2 mL DMEM complete medium for 72 hours (37°C, 5% CO ₂ ), and then used flow CD3 ⁺ IFN-γ ⁺ T cells, which are cancer cell-specific T cells activated by whole-cell antigens of cancer cells, are sorted out by cytometry.

(7)Experimental results

As shown in Figure 8, compared with the control group, micron particles prepared by micron particle-activated antigen-presenting cells can effectively detect more cancer cell-specific T cells in mice in the treatment group. Moreover, the effect of microparticles 3 prepared by using two types of CpG and Poly(I:C) as mixed adjuvants was better than that of two types of A CpG and Poly(I:C). The micron particles 4 were prepared by mixing the micron particles 2 with the adjuvant to activate the antigen-presenting cells. Mannose is used as an active target in the micron vaccine of this embodiment. In practical applications, any target with the ability to target target cells, such as CD32 antibodies, mannans, CD205 antibodies, and CD19 antibodies, can also be used.

Example 8 Detection of cancer cell-specific T cells in pancreatic cancer

This example uses mannose as the target to illustrate how to detect cancer cell-specific T cells in peripheral blood using nanoparticles prepared by activating antigen-presenting cells using active targeting nanoparticles.

(1) Lysis of cancer cells

The cultured Pan02 pancreatic cancer cells were collected and 10% octylglucoside was used to lyse the cancer cells and dissolve the whole cell antigens derived from the cancer cells.

(2) Preparation of nanoparticles

In this example, the nanoparticle system was prepared using the double emulsion method. The nanoparticle preparation materials are PLGA and mannose-modified PLGA, both of which have molecular weights of 7KDa-17KDa. When the two are used together to prepare nanoparticles with a target, the mass ratio is 4:1. The immune adjuvants used were Poly(I:C) and CpG SL03. The preparation method is as described above. The lysate components and adjuvants are loaded into the nanoparticles using the double emulsion method. Then 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes and resuspended in 10 mL of ultrapure water containing 4% trehalose. Then freeze-dry for 48 hours before use. The average particle size of the nanoparticles 1 with the target is about 270nm. Each 1 mg of PLGA nanoparticles is loaded with approximately 80 μg of protein and peptide components, including 0.04 mg each of Poly(I:C) and CpGSL03. Nanoparticles 2 that are not loaded with adjuvants but have a mannose target are also about 270nm in size. They are prepared using the same amount of cell lysis components but do not contain any immune adjuvants. Each 1 mg of PLGA nanoparticles is loaded with approximately 80 μg of protein and peptides. components.

(3) Preparation of antigen-presenting cells

This example uses BMDC and BMDM as antigen-presenting cells. The preparation methods of BMDC and BMDM are the same as above.

(4) Activation of antigen-presenting cells

Nanoparticle 1 (1000 μg) or nanoparticle 2 (1000 μg) were incubated with BMDC (10 million pcs), BMDM (10 million pcs) and IL-7 (500 U/mL) respectively in 15 mL high-glucose DMEM complete medium for 48 hours. (37°C, 5% CO ₂ ).

Or incubate BMDC (10 million pcs), BMDM (10 million pcs) and IL-7 (500U/mL) in 15mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2). Both of the above incubation systems contain IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN-γ (500U/mL) and CD80 antibody (10ng/mL ).

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

Collect the incubated DCs and macrophages by centrifugation at 400g for 5 minutes, then wash the cells twice using 4°C phosphate buffer solution (PBS) containing protease inhibitors, resuspend the cells in PBS water and incubate at 4°C on low power. (10W) Ultrasound for 20 minutes. Then centrifuge the sample at 3000g for 15 minutes and collect the supernatant. The supernatant is filtered through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, 1 μm, 0.45 μm, and 0.22 μm. The filtrate is collected, and then the filtrate is filtered at 18000 g. After centrifugation for 50 minutes, collect and discard the supernatant to collect the precipitate, resuspend the precipitate in 4% trehalose aqueous solution, and then freeze-dry for 48 hours to obtain nanoparticles. Among them, the one prepared using mixed antigen-presenting cells that have not been activated by nanoparticles is nanoparticle 3, with a particle size of 110 nanometers; the one prepared using mixed antigen-presenting cells activated with nanoparticles 2 is nanoparticle 4, with a particle size of 110 nanometers. ; Nanoparticle 5 was prepared using mixed antigen-presenting cells activated by nanoparticle 1, with a particle size of 110 nanometers.

(6) Detection of cancer cell-specific T cells

On day 0, each C57BL/6 mouse was subcutaneously inoculated with 1 × 10 ⁶ Pan02 pancreatic cancer cells on the back. On day 10, day 15, day 20, and day 27, mice were injected subcutaneously with 100 μL of 1 mg PLGA. Nanoparticles. The mice were sacrificed on day 24 and peripheral blood was collected. Peripheral blood mononuclear cells (PBMC) were prepared from the peripheral blood, and then CD3 ⁺ T cells were isolated from the PBMC using flow cytometry. T cells (5 million) were incubated with 100 μg of nanoparticles (nanoparticle 1, or nanoparticle 3, or nanoparticle 4, or nanoparticle 5) in DMEM high-glucose medium for 72 hours (37°C, 5% CO ₂ ), the incubation system contains IL-2 (500U/mL) and IL-7 (500U/mL). Then flow cytometry is used to sort out CD3 ⁺ IFN-γ ⁺ T cells from the incubated cells, which are cancer cell-specific T cells.

(7)Experimental results

As shown in Figure 9,

nanoparticles

4 and 5 are better than

nanoparticles

1 and 3. And Nanoparticle 5 is better than Nanoparticle 4. In summary, regardless of whether the antigen-presenting cells are activated by nanoparticles with adjuvants, the prepared nanoparticles can effectively detect cancer cell-specific T cells, but the antigens activated by nanoparticles with adjuvants Nanoparticles prepared by presenting cells are more effective.

Example 9 Nanoparticles detect lung cancer cancer cell-specific T cells

This example illustrates the detection of cancer cell-specific T cells in mouse splenocytes using calcified nanoparticles. In actual use, other biomineralization technologies, cross-linking, gelation and other modified particles can also be used. In this example, mouse lung cancer tumor tissue was lysed with 8M urea (containing 200mM sodium chloride), dissolved and loaded into a nanoparticle system. After using the particles to activate antigen-presenting cells, the antigen-presenting cells were prepared into nanoparticles. Detection of cancer cell-specific T cells.

(1) Lysis of tumor tissue and cancer cells

Inoculate 1× ¹⁰ LLC mouse lung cancer cells on the back of female C57BL/6 mice aged 6 to 8 weeks. When the tumor volume reaches 1000 ^mm3 , the mice will be sacrificed. The mouse tumor tissue will be removed, cut into pieces and ground. Then filter it through a cell mesh to prepare a single cell suspension, use ultraviolet irradiation for 5 minutes and then heat at 80°C for 10 minutes, and then use 8M urea (containing 200mM sodium chloride) to lyse and dissolve the tumor tissue single cell suspension to obtain Cancer cell whole cell antigen.

(2) Preparation of nanoparticles

In this embodiment, the nanoparticles are loaded with cancer cell whole cell antigens inside and on the surface, and then the nanoparticles are biocalcified. In this example, the nanoparticles are prepared by a solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA is 7KDa-17KDa. The immune adjuvants CpG2006 and Poly(I:C) are loaded inside the nanoparticles. The preparation method is as follows. During the preparation process, the double emulsion method is first used to load the antigen inside the nanoparticles. After loading the lysis components inside, 100mg PLGA nanoparticles are centrifuged at 13000g for 20 minutes and resuspended in 18mL PBS. Then 2mL is added to dissolve Act in 8M urea tumor tissue and cancer cell lysate (60mg/mL) at room temperature for 10 minutes, then centrifuge at 12000g for 20 minutes to collect the precipitate. The 100 mg PLGA nanoparticles were then resuspended in 20 mL DMEM medium, and then 200 μL CaCl ₂ (1 mM) was added and reacted at 37°C for two hours. Then, collect the precipitate after centrifugation at 10,000 g for 20 minutes, resuspend in ultrapure water, and centrifuge and wash twice to obtain nanoparticles 1, with an average particle size of about 290 nm. Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 140 μg of protein or peptide components, including 0.03 mg of CpG2006 and Poly(I:C).

(3) Preparation of antigen-presenting cells

This example uses BMDC and B as antigen-presenting cells. The preparation method of BMDC is the same as in Example 1. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above. Mix BMDC and B cells at a ratio of 1:1 to obtain mixed antigen-presenting cells.

(4) Activation of antigen-presenting cells

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

Or as a control, nanoparticles (1000 μg) loaded with cancer cell whole cell components were incubated with BMDC (5 million) and B cells (5 million) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ); does not contain any cytokines or antibodies in the incubation system.

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

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

The incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors. The cells were resuspended in PBS water and incubated at 4°C at low power (PBS). 20W) Ultrasonic for 2 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45μm membrane and ultrafiltrated using an ultrafiltration membrane (molecular weight cutoff 50KDa). Filter, centrifuge, filter and concentrate. Mix the filtered and concentrated sample with the nanoparticles 1 prepared in step (2) and treat it with a high-pressure homogenizer (10000bar) for 3 minutes. Then centrifuge at 13000g for 30 minutes and discard the supernatant for collection. Precipitate and resuspend the precipitate in PBS to obtain nanoparticles. Among them, when particles are used to activate antigen-presenting cells, the antigen-presenting cells containing cytokine combination 1 in the system are prepared as nanoparticles 2, with a particle size of 300 nanometers; when particles are used to activate antigen-presenting cells, the system does not contain cytokines. The nanoparticles 3 prepared by the antigen-presenting cells have a particle size of 300 nanometers; the nanoparticles 4 prepared by the antigen-presenting cells containing the cytokine combination 2 in the system when activating the antigen-presenting cells using particles are nanoparticles 4 with a particle size of 300 nanometers. 300 nm.

(6) Detection of cancer cell-specific T cells

On day 0, each C57BL/6 mouse was subcutaneously inoculated with 0.5 × 10 ⁶ LLC lung cancer cells on the back. On

days

10, 15, and 20, the mice were subcutaneously injected with 100 μL of 1 mg PLGA nanoparticles. On day 24, the mice were sacrificed and spleen cells were harvested and prepared into single cell suspension, and then flow cytometry was used to isolate CD45 ⁺ CD3 ⁺ T cells.

T cells (5 million) were incubated with nanoparticles 2 (100 μg) or nanoparticles 3 (100 μg) or nanoparticles 4 (100 μg) prepared by antigen-presenting cells in DMEM high-glucose medium for 12 hours (37°C, 5 %CO ₂ ), the system contains IL-2 (500U/mL) and IL-7 (500U/mL) during the incubation process;

Or T cells (5 million) and nanoparticles 2 (100 μg) prepared from antigen-presenting cells were incubated in DMEM high-glucose medium for 12 hours (37°C, 5% CO2), and the incubation system did not contain any cytokines or Antibody;

Or incubate T cells (5 million), 10 million mixed antigen-presenting cells prepared in step (3) and nanoparticle 1 (100 μg) in DMEM high-glucose medium for 12 hours (37°C, 5% CO ₂ ) , the system contained IL-2 (500U/mL) and IL-7 (500U/mL) during the incubation process.

Then collect the above-incubated cells, centrifuge at 400g for 5 minutes and use the corresponding flow cytometry antibody to label the incubated cells, and then use flow cytometry to detect CD3 ⁺ IFN-γ ⁺ T cells, which are cancer cell-specific T cells. .

(7)Experimental results

As shown in Figure 10, compared with the control group, nanoparticles prepared from antigen-presenting cells activated by calcified nanoparticles could detect more cancer cell-specific T cells. Moreover, when nanoparticles loaded with whole cell antigens of cancer cells activate antigen-presenting cells, the system containing cytokine combination 1 is better than the system without cytokines and/or antibodies; when nanoparticles activate antigen-presenting cells, the system contains cytokines. The effect of combination 1 or the system without cytokines is better than that of the system with cytokine combination 2; moreover, when the nanoparticles prepared by antigen-presenting cells are co-incubated with T cells, the effect of the system with cytokine combination 2 is better than that of the system without cytokine combination 2. Contains cytokines.

Example 10 Nanoparticles detect cancer cell-specific T cells in melanoma

(1) Lysis of tumor tissue and cancer cells and collection of components

When collecting tumor tissue, 1.5 × 10 ⁵ B16F10 cells were first subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm ³ , the mice were sacrificed and the tumor tissue was removed. The tumor tissue was cut into sections. Grind and pass through the cell strainer to prepare a single cell suspension. Add ultrapure water and freeze and thaw repeatedly with ultrasound to lyse the above cells. Then add nuclease and incubate for 5 minutes, and then inactivate the nuclease at 95°C for 10 minutes. Then centrifuge at 8000g for 3 minutes, the supernatant part is the water-soluble component; the precipitate part uses 10% sodium deoxycholate aqueous solution to dissolve the non-water-soluble component. The water-soluble component and the non-water-soluble component after dissolving sodium deoxycholate are miscible at a mass ratio of 1:1, which is the source of the antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle system

In this embodiment, the nanoparticles are prepared by the double emulsion method and have the ability to target dendritic cells. The nanoparticle preparation materials used are PLGA and mannan-modified PLGA, both of which have molecular weights of 24KDa-38KDa. When used, the mass ratio of unmodified PLGA to mannan-modified PLGA is 9:1. The immune adjuvants used are poly(I:C), CpG1018 and CpG2216. The substance that increases lysosomal immune escape is KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and the adjuvants and KALA polypeptide are encapsulated in nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysis solution components, adjuvants, and KALA polypeptide inside the nanoparticles. After loading the above components inside, 100 mg of the nanoparticles are centrifuged at 12,000g for 25 minutes. And resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h. The average particle size of the nanoparticles is about 250nm, and the surface potential is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C), CpG1018 and CpG2216 immune The adjuvants are 0.02mg each, and the loaded KALA polypeptide is 0.05mg. The preparation materials and methods of Nanoparticle 2 are the same. Its particle size is about 250nm, its surface potential is about -5mV, it does not load KALA polypeptide, and it loads equal amounts of adjuvants and cell lysis components. The preparation materials and preparation methods of nanoparticle 3 are the same, about 250nm, and the surface potential is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) 0.02mg, the loaded CpG1018 is 0.04mg, and the loaded KALA polypeptide is 0.05mg.

(3) Preparation of antigen-presenting cells

(4) Activation of antigen-presenting cells

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

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

Collect the incubated BMDC and BMDM by centrifugation at 400g for 5 minutes, then wash three times using 1200rpm for 3min in 30mM pH 7.0 Tris-HCl buffer containing 0.0759M sucrose and 0.225M mannitol, and then incubate with phosphatase inhibitors and protease inhibitors. Ultrasound mechanically destroys antigen-presenting cells in the presence of the agent. After centrifugation, the cell membrane obtained was washed with a solution of 10mM Tris-HCl pH 7.5 and 1mM EDTA. Then filter the sample through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, and 0.45 μm in sequence. Centrifuge the filtrate at 16,000g for 35 minutes and discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS and proceed with step (2) The prepared nanoparticles were incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 μm filter membrane. The extruded liquid was centrifuged at 12,000g for 25 minutes and the supernatant was discarded to collect the precipitate. The precipitate was placed in a 4% trehalose aqueous solution. Resuspend and freeze-dry to obtain nanoparticles. Among them, the antigen-presenting cell membrane component activated by nanoparticle 1 and nanoparticle 1 were used to prepare nanoparticle 4, with a particle size of 260 nanometers; the antigen-presenting cell membrane component activated by nanoparticle 2 was used together with nanoparticle 2. The nanoparticle 5 prepared by the interaction has a particle size of 260 nanometers; the nanoparticle 6 is prepared by using the antigen-presenting cell membrane component activated by the nanoparticle 3 and the nanoparticle 3 to interact with the nanoparticle 3, and the particle size is 260 nanometers.

(6) Detection of cancer cell-specific T cells

Female C57BL/6 mice aged 6-8 weeks were selected and injected subcutaneously with 0.5 mg of PLGA nanoparticles (lysate-loaded group) on

days

0, 7, 14, 21 and 28. points, Poly(I:C) and two CpG adjuvants and KALA polypeptide). The mice were sacrificed on day 32, and their peripheral blood and lymph nodes were collected. Gradient centrifugation was used to isolate PBMCs from the peripheral blood of mice. The lymph nodes were cut into small pieces and ground through a cell mesh to prepare a single cell suspension. The PBMCs were then mixed with the single cell suspension of lymph node cells. CD45 ⁺ CD3 ⁺ T cells were then sorted using magnetic bead sorting. The sorted CD3 ⁺ T cells (5 million cells) were incubated with nanoparticles (40 μg) and IL-7 (10 ng/mL) in 2 mL RPMI1640 complete medium for 96 hours. Then flow cytometry is used to sort the CD3 ⁺ IFN-γ ⁺ T cells in the incubated T cells, which are cancer cell-specific T cells that can recognize cancer cell whole cell antigens.

(7)Experimental results

As shown in Figure 11, compared with the control group, nanoparticles prepared by activated antigen-presenting cells loaded with nanoparticles loaded with whole cell components can detect more cancer cell-specific T cells. Moreover, the effect of nanoparticles 4 prepared by adding antigen-presenting cells activated by nanoparticles that increase lysosome escape substances is better than that of nanoparticles prepared by adding nanoparticles activated by antigen-presenting cells without adding lysosome escape substances 5; using two Nanoparticles prepared with CpG and Poly(I:C) as mixed adjuvants activated antigen-presenting cells. Nanoparticles 4 are more effective than nanoparticles activated with only one CpG and Poly(I:C) mixed adjuvant. Presentation of Cell-Prepared Nanoparticles 6.

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

This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use microparticles loaded with cancer cell whole cell antigens to activate the antigen-presenting cell membrane component and microparticles prepared from cancer cell membrane components to detect cancer cell-specific T cell. In this embodiment, breast cancer cells are first inactivated and denatured, and then the cells are lysed, and octylglucoside is used to dissolve and lyse the non-water-soluble components in the cancer cells. Then, PLGA was used as the micron particle skeleton material, CpG2007, CpG1018, and Poly ICLC were used as immune adjuvants, and polyarginine and polylysine were used as substances that enhance lysosomal escape to prepare whole cell antigens loaded with cancer cells. Micron particles are used to activate antigen-presenting cells to prepare mixed micron particles based on antigen-presenting cell membrane components and cancer cell membrane components, and then the micron particles are used to detect cancer cell-specific T cells.

(1) Lysis of cancer cells

The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells were inactivated and denatured using ultraviolet light and high-temperature heating at 60°C for 5 minutes, and then ultrapure water was added and repeatedly frozen and thawed 5 times, supplemented by ultrasound to lyse the cancer cells. The cell lysate was centrifuged at 5000g for 10 minutes, and the supernatant The liquid is the water-soluble component. Dissolve the precipitate with 10% octyl glucoside to become the original dissolved non-water-soluble component. Mix the water-soluble component and the non-water-soluble component at a mass ratio of 2:1 , which is the lysate component required to prepare micron particles.

(2) Preparation of micron particles

In this example, the micron particles 1 are prepared using the double emulsion method. The molecular weight of the micron particle skeleton material PLGA is 38KDa-54KDa. The immune adjuvants used are CpG2007, CpG1018 and Poly ICLC. The lysosomal escape increasing substance used is poly. Arginine and polylysine. During preparation, first use the double emulsion method to prepare microparticles internally loaded with lysate components, adjuvants and KALA polypeptides. After loading lysate and adjuvants internally, centrifuge 100mg of microparticles at 9000g for 20 minutes, and use 10mL containing 4% trehalose. Resuspend in ultrapure water and dry for 48 hours before use. The average particle size of the micron particle 1 system is about 3.1 μm, and the surface potential of the micron particle system is about -7mV; every 1 mg of PLGA micron particles is loaded with approximately 110 μg of protein or peptide components, including 0.01 mg each of CpG2007, CpG1018 and Poly ICLC. 0.02mg each of arginine and polylysine.

(3) Preparation of antigen-presenting cells

This example uses BMDC and DC2.4 as antigen-presenting cells. The preparation method of BMDC is the same as in Example 1.

(4) Activation of antigen-presenting cells

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

(5) Preparation of microparticles based on mixed membrane components of presenting cells and cancer cells

Collect 10 million DCs after incubation by centrifugation at 400 g for 5 minutes, mix with 10 million 4T1 cells, wash the cells twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, resuspend the cells in PBS water, and Ultrasound at 4°C for 2 minutes at low power (20W). The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was collected after centrifugation at 5000g for 10 minutes. The supernatant was repeatedly co-extruded through a 0.22μm membrane, and then 30mg of step (2) was added. The prepared micron particles 1 were ultrasonicated at 10W for 10 seconds and incubated for 10 minutes, and then repeatedly co-extruded using a 5 μm membrane. The extruded liquid was collected after centrifugation at 9000g for 120 minutes. The supernatant was discarded to collect the precipitate, and the precipitate was placed in PBS. After medium resuspension, micron particles 2 were obtained, with a particle size of 3.15 μm.

Alternatively, collect 20 million DCs after incubation by centrifugation at 400 g for 5 minutes, wash the cells twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, resuspend the cells in PBS water and incubate at 4°C with low power (20W ) Ultrasound for 2 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was collected after centrifugation at 5000g for 10 minutes. The supernatant was repeatedly co-extruded through a 0.22μm membrane, and then 30mg of step (2) was added. The prepared micron particles 1 were ultrasonicated at 10W for 10 seconds and incubated for 10 minutes, and then repeatedly co-extruded using a 5 μm membrane. The extruded liquid was collected after centrifugation at 9000g for 120 minutes. The supernatant was discarded to collect the precipitate, and the precipitate was placed in PBS. After medium resuspension, micron particles 3 were obtained, with a particle size of 3.15 μm.

(6) Detection of cancer cell-specific T cells

Select 6-8 week old female BALB/c mice and inoculate 2×10 ⁶ 4T1 breast cancer cells subcutaneously on the back of the mice on day 0; subcutaneously inject 0.3mg on days 7, 14 and 21 respectively. PLGA micron particles1. On the 25th day, the mice were sacrificed, peripheral blood was collected, PBMC were isolated from the peripheral blood, and CD3 ⁺ T cells were sorted from the PBMC. CD3 ⁺ T cells (1 million), 100 μg of microparticles 2 or microparticles 3 were incubated in 10 mL of RPMI1640 complete medium for 24 hours (37°C, 5% CO ₂ ). Then, the cell membrane was stained with anti-mouse CD4 antibodies and anti-mouse CD8 antibodies modified with different fluorescent probes. After the cells were fixed, intracellular staining with anti-mouse granzyme B (Granzyme B) antibodies modified with fluorescent probes was performed. , and then use flow cytometry to detect CD8 ⁺ Granzyme B ⁺ T cells and CD4 ⁺ Granzyme B ⁺ T cells in the incubated cells, which are cancer cell-specific T cells that can recognize cancer cell whole cell antigens. Cancer cell-specific T cells will begin to synthesize and express killer substances after being activated by antigens. Granzyme B is one of these killer substances and has the strongest activity in inducing cell apoptosis. At the same time, the mean fluorescence intensity (MFI) of the fluorescent probe connected to Granzyme B in CD8 ⁺ Granzyme B ⁺ T cells was analyzed.

(7)Experimental results

As shown in Figure 12, micron particles 2 prepared by mixing the antigen-presenting cell membrane components activated by micron particles and the cancer cell membrane components can better detect cancer cell-specific T cells than micron particles 3, and the cancer cells activated by them are better than those of micron particles 3. Cell-specific T cells can synthesize more killer substances. Moreover, during detection, cancer cell-specific T cells activated by micron particles 2 can synthesize more killer substances, because they are easier to detect and the detection will be more accurate.

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

(1) Cleavage of cancer cell and bacterial external vesicles

The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells were inactivated and denatured using ultraviolet light and high-temperature heating respectively, and then an 8M urea aqueous solution (containing 500mM sodium chloride) was used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing the micron particle system.

Centrifuge Lactobacillus acidophilus at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 μm filter membrane, and then centrifuge at 16000g for 90 minutes. The precipitate is treated with 8M urea aqueous solution (containing 500mM chloride). Sodium) cleaves and solubilizes bacterial outer vesicle components.

(2) Preparation of micron particles

In this embodiment, the double emulsion method is used to prepare micron particles. The skeleton material of micron particle 1 is unmodified PLA and mannose-modified PLA, both with a molecular weight of 40KDa. The ratio of unmodified PLA to mannose-modified PLA is 4:1. The immune adjuvants used were CpG2006, CpG2216 and Poly ICLC, and the lysosomal escape-increasing substances used were arginine and histidine. The mass ratio of cancer cell lysate components and bacterial external vesicle components used in the preparation of micron particles is 1:1. During preparation, the double emulsion method is first used to prepare micron particles that are internally loaded with cancer cell lysate components, bacterial external vesicle components, adjuvants, arginine and histidine. Then, 100mg of micron particles are centrifuged at 9000g for 20 minutes. Use 10 mL of ultrapure water containing 4% trehalose to resuspend and dry for 48 hours to obtain micron particles 1. The average particle size is about 1.5 μm. Each 1 mg of PLGA micron particles 1 is loaded with approximately 100 μg of protein or peptide components, including CpG2006 and CpG2216. and Poly ICLC 0.02mg each, containing 0.05mg each of arginine and histidine. The preparation materials and preparation methods of control micron particle 2 are the same as those of micron particle 1. The particle size is about 1.5 μm, and it is loaded with equal amounts of arginine, histidine and equal amounts of cancer cell lysate components and bacterial outer vesicle components. However, Does not contain any adjuvants.

(3) Preparation of antigen-presenting cells

This example uses BMDC, B cells and BMDM as antigen-presenting cells. The preparation methods of BMDC and BMDM are the same as above. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above. Mix BMDC, B cells and BMDM in a quantity ratio of 2:1:1 to form mixed antigen-presenting cells.

(4) Activation of antigen-presenting cells

1000 μg of

microparticles

1 or 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% CO ₂ ); the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN-γ (500U/mL), CD80 antibody (10ng/mL) and CD40 antibody (20mg/mL).

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

Collect the incubated 40 million mixed antigen-presenting cells by centrifugation at 400g for 5 minutes, then wash the cells twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, resuspend the cells in PBS water and incubate at 4 ℃ low power (20W) ultrasonic for 2 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45μm membrane and ultrafiltrated using an ultrafiltration membrane (molecular weight cutoff 50KDa). Filter, centrifuge, filter and concentrate. Use a high-pressure homogenizer (10,000 bar) to process the filtered and concentrated sample for 3 minutes. Then, centrifuge at 13,000g for 30 minutes. Discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS. Nanoparticles. Among them, the mixed antigen-presenting cells activated using micron particles 1 were prepared as nanoparticles 1, with a particle size of 250 nanometers; the mixed antigen-presenting cells activated using micron particles 2 were prepared as nanoparticles 2, with a particle size of 250 nanometers.

Or collect 40 million mixed antigen-presenting cells after incubation with

microparticles

1 or 2 by centrifugation at 400g for 5 minutes, and then wash the cells twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors. The cells were resuspended in PBS water and sonicated at 4°C for 2 minutes at low power (20W). The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45μm membrane and ultrafiltrated using an ultrafiltration membrane (molecular weight cutoff 50KDa). Filter, centrifuge, and concentrate. Use a high-pressure homogenizer (10,000 bar) to treat the filtered and concentrated sample for 3 minutes, and then react it with 60 mg of

micron particles

1 or 2 prepared in step (2) for 10 minutes before use. The 2 μm filter membrane was repeatedly co-extruded, and then the extruded liquid was centrifuged at 10,000 g for 20 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS to obtain micron particles. Among them, the mixed antigen-presenting cell membrane component activated by micron particle 1 and the micron particle 1 are combined to prepare micron particle 3, with a particle size of 1.6 μm; the mixed antigen-presenting cell membrane component activated by micron particle 2 and micron particle 2 are prepared by the combined action of micron particles 4, with a particle size of 1.6 μm.

(6) Analysis of cancer cell-specific T cells

Select female BALB/c mice that are 6-8 weeks old and subcutaneously inject 100 μL of micron particles containing 0.4 mg of PLGA prepared in step (2) on

days

0, 7, 14, 21, and 28. 1. The mice were sacrificed on day 32, peripheral blood and spleen were collected, and then a single cell suspension of PBMC and spleen cells was prepared and mixed, and then CD3 ⁺ T cells were sorted out using magnetic bead sorting method. The sorted CD3 ⁺ T cells (2 million), 100 μg nanoparticles or microparticles (nanoparticle 1, or nanoparticle 2, or microparticle 3, or microparticle 4), DC2.4 cells (1 million) Incubate in 10mL RPMI1640 complete medium for a total of 48 hours (37°C, 5% CO ₂ ). The incubation system contains IL-2 (200U/mL), IL-7 (200U/mL), and IL-15 (200U/mL). and CD80 antibody (10ng/mL). Then flow cytometry was used to analyze the CD3 ⁺ IFN-γ ⁺ T cells in the incubated CD3 ⁺ T cells, which are cancer cell-specific T cells that can recognize cancer cell whole cell antigens. At the same time, the mean fluorescence intensity (MFI) of the fluorescent probe connected to IFN-γ in CD3 ⁺ IFN-γ ⁺ T cells was analyzed.

The cancer cell whole cell antigens loaded by the nanoparticles can be degraded into antigen epitopes after being phagocytosed by the antigen-presenting cells and presented to the surface of the antigen-presenting cell membrane. The nanoparticles prepared by the antigen-presenting cells are loaded with the above-mentioned degraded and presented products. The antigenic epitope can be recognized by cancer cell-specific T cells and activate cancer cell-specific T cells. After activation, they secrete killer cytokines. IFN-γ is the most important cytokine secreted by antigen-specific T cells that are activated after recognizing antigens. CD3 ⁺ IFN-γ ⁺ T cells analyzed using flow cytometry are cancer cell-specific T cells that can recognize and kill cancer cells. At the same time, the mean fluorescence intensity (MFI) of the fluorescent probe connected to IFN-γ in CD3+IFN-γ+T cells was analyzed.

(7)Experimental results

As shown in Figure 13, the proportion of cancer cell-specific T cells is related to the efficacy in the figure, which shows that the T cells detected using the particles of the present invention are cancer cell-specific T cells that can specifically recognize and kill cancer cells. Moreover, nanoparticle 1 is better than nanoparticle 2; microparticle 3 is better than microparticle 4. This shows that the particles prepared by activated antigen-presenting cells containing micron particles containing substances that increase lysosomal escape function and mixed adjuvants are more effective in detecting cancer cell-specific T cells than those containing only substances with lysosomal escape function without mixed adjuvants. Adjuvant micron particles are prepared by activating antigen-presenting cells. Moreover, micron particles 3 are better than nanoparticles 1, and microparticles 4 are better than nanoparticles 2, which shows that solid particles loaded with cancer cell lysis components inside and activated antigen-presenting cell components on the surface detect cancer cell-specific T The cellular effect is better than vesicle particles simply loaded with activated antigen-presenting cell components. Moreover, the analysis results of the mean fluorescence intensity (MFI) of the fluorescent probe connected to IFN-γ were consistent with the above results. It can be seen that the use of mixed adjuvants and the internal loading of cancer cell whole cell components are helpful in detecting cancer cell-specific T cells.

Example 13 Detection of cancer cell-specific T in colon cancer

This example uses mouse colon cancer as a cancer model to illustrate how to detect cancer cell-specific T cells using nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with colon cancer whole-cell antigens. In this example, 8M urea aqueous solution is first used to lyse colon cancer tumor tissue and dissolve the lysed components. Then, PLGA is used as the skeleton material, Poly(I:C), CpG2336 and CpG2006 are used as adjuvants, and NH ₄ HCO ₃ is used as the adjuvant. Add lysosomal escape substances to prepare nanoparticles, use the nanoparticles to activate antigen-presenting cells, prepare the antigen-presenting cells into nanoparticles, and then use the nanoparticles to detect cancer cell-specific T cells.

(1) Lysis of tumor tissue and collection of components

When collecting tumor tissue, 2 × ¹⁰ MC38 colon cancer cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm, the mice were ^sacrificed and the tumor tissue was removed and cut into sections. After the block was ground, an 8M urea aqueous solution was added through a cell strainer to digest the tumor tissue and dissolve the lysed components. The above are the sources of antigen raw materials for preparing nanoparticle systems.

(2) Preparation of nanoparticle system

In this example, the nanoparticles were prepared using the double emulsion method. The preparation material of nanoparticles is PLGA with a molecular weight of 7KDa-17KDa, Poly(I:C) and CpG as adjuvants, NH ₄ HCO ₃ as a substance that increases lysosomal escape, and the adjuvants and NH ₄ HCO ₃ are loaded on the nanoparticles Within; the preparation method is as described above. During the preparation process, the lysis solution components and adjuvants are first loaded inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used to rehydrate the nanoparticles. Suspend and freeze-dry for 48 hours before use; the average particle size of the nanoparticles is about 260nm, and the surface potential is about -7mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I :C), CpG2336 and CpG2006 immune adjuvant 0.02mg each, loaded with NH ₄ HCO ₃ 0.01mg.

The preparation materials and methods of nanoparticle 2 are the same as nanoparticle 1. The particle size is about 260nm and the surface potential is about -7mV. Each 1 mg PLGA nanoparticle is loaded with approximately 90 μg of protein and peptide components. Each 1 mg PLGA nanoparticle is loaded with NH ₄ HCO. ₃ 0.01mg, loaded with 0.03mg each of CpG2336 and CpG2006.

(3) Preparation of antigen-presenting cells

This example uses BMDC and B as antigen-presenting cells. The preparation method of BMDC is the same as in Example 1. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.

(4) Activation of antigen-presenting cells

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

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

The incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors. The cells were resuspended in PBS water and incubated at 4°C at low power (PBS). 20W) Ultrasonic for 2 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45μm membrane and ultrafiltrated using an ultrafiltration membrane (molecular weight cutoff 50KDa). Filter, centrifuge, filter and concentrate. Mix the filtered and concentrated sample with the nanoparticles prepared in step (2) and treat it with a high-pressure homogenizer (10000bar) for 3 minutes. Then centrifuge at 13000g for 30 minutes and then discard the supernatant to collect the precipitate. , resuspend the precipitate in PBS to obtain nanoparticles. Among them, the one prepared by using the antigen-presenting cells activated by the nanoparticle 1 and the nanoparticle 1 is nanoparticle 3, with a particle size of 300 nanometers; the one prepared by using the antigen-presenting cells activated by the nanoparticle 2 and the nanoparticle 2. is nanoparticle 4, with a particle size of 300 nanometers.

(6) Detection of cancer cell-specific T cells

Female C57BL/6 mice aged 6-8 weeks were selected and subcutaneously injected with 100 μL of nanoparticles containing 0.5 mg PLGA (loaded lysate component, mixed adjuvant) on

days

0, 7, 14, and 28. and substances that increase lysosomal escape) or 100 μL PBS. Mice were sacrificed on day 32, peripheral blood was collected and peripheral blood mononuclear cells (PBM C) were isolated, and then flow cytometry was used to sort CD3 ⁺ T cells from PBMC.

This example uses enzyme-linked immunospot method (ELISPOT) to detect cancer cell-specific T cells. First, anti-mouse IFN-γ antibody a (capture antibody, coating antibody) was coated in a 96-well plate for 12 hours, then the medium was added for blocking treatment for 1 hour, and then washed with PBS, and then 100 μL of RPMI 1640 complete medium was added. T cells (5000) obtained by the above sorting were added to each well and 50 μg of

nanoparticles

3 or 4 prepared from antigen-presenting cells were added, and incubated at 37°C (5% CO ₂ ) for 24 hours. Afterwards, the mixture of cells and particles was discarded, and the 96-well plate was washed and cultured in a 37° C. (5% CO ₂ ) incubator for more than 2 hours after adding anti-mouse IFN-γ antibody b (detection antibody). Discard the solution containing anti-mouse IFN-γ antibody b, wash the 96-well plate, and use the corresponding method to develop color to form spots on the surface of the 96-well plate. Use an ELISPOT analyzer to read the data and analyze the number of spots formed in each well, which are cancer cell-specific T cells that can recognize cancer cell whole-cell antigens.

(7)Experimental results

As shown in Figure 14, compared with the PBS control group, the mice injected with particles loaded with whole cell components contained more cancer cell-specific T cells, and the antigen-presenting cells activated by the nanoparticles of the present invention The prepared nanoparticles can effectively detect cancer cell-specific T cells. Moreover, nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with mixed adjuvants, lysate components and lysosomal escape substances are more effective in assisting in the isolation of cancer cell-specific T cells than those loaded with lysate components and both. Nanoparticles of CpG adjuvants and lysosomal escape substances.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications may be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

A method for detecting cancer cell-specific T cells using particles prepared from activated antigen-presenting cells, which is characterized by including the following steps:

S1. Incubate the antigen-presenting cells with the first particles to obtain activated antigen-presenting cells; wherein the first particles are nanoparticles or microparticles loaded with tissue and/or whole cell components;

S2. Preparing the activated cell membrane components of the antigen-presenting cells into nanovesicles; or cooperating the activated cell membrane components of the antigen-presenting cells with second particles to obtain second particles loaded with cell membrane components; Wherein, the second particles are nanoparticles or microparticles loaded with whole cell components of tissues and/or cells;

S3. Incubate the nanovesicles and/or the second particles loaded with cell membrane components described in step S2 with the cells to be tested, detect the markers inside or on the surface of the cells to be tested, and achieve the desired results by analyzing the T cells containing the markers. Detection of cancer cell-specific T cells.
The method of claim 1, wherein the marker includes protein or nucleic acid.
The method according to claim 1, characterized in that: before the nanovesicles and/or the second particles loaded with cell membrane components are co-incubated with the cells to be tested, the method further includes the step of sorting the T cells in the cells to be tested. step.
The method according to claim 1, characterized in that in step S1 or step S3, the co-incubation system contains cytokines and/or antibodies.
The method according to claim 1, wherein the first particle or the second particle is loaded with bacterial components or bacterial outer vesicle components.
The method according to claim 5, characterized in that: the bacterial component or bacterial outer vesicle component is obtained by lysing the bacteria or bacterial outer vesicles with a lysis solution containing a lysing agent; the lytic agent is urea, guanidine hydrochloride , one or more of deoxycholate, lauryl sulfate, glycerol, protein degrading enzymes, albumin, lecithin, Triton, Tween, amino acids, glycosides and choline.
The method according to claim 1, characterized in that: in step S2, it also includes mixing the cell membrane components of cancer cells or extracellular vesicle membrane components with the cell membrane components of activated antigen-presenting cells to prepare Nanovesicles or the step of loading onto a second particle.
The method of claim 1, wherein the first particle or the second particle is loaded with an immune-enhancing adjuvant.
The method of claim 8, wherein the immune-enhancing adjuvant includes two or more Toll-like receptor agonists.
A kit for detecting cancer cell-specific T cells, characterized in that the kit includes at least one of the following (1)-(2):

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

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

in,

The nanovesicles are prepared by co-incubating the antigen-presenting cells with the first particles to obtain activated antigen-presenting cells, and then extracting the cell membrane components of the activated antigen-presenting cells;

The particles loaded with cell membrane components of activated antigen-presenting cells are obtained by cooperating the cell membrane components of activated antigen-presenting cells with second particles, so that the cell membrane components are loaded on the second particles;

The first particles or second particles are independently selected from nanoparticles or microparticles loaded with tumor tissue and/or whole cell components of cancer cells.