CN115561145A

CN115561145A - Detection method and kit for cancer cell specific T cells

Info

Publication number: CN115561145A
Application number: CN202210801405.XA
Authority: CN
Inventors: 刘密
Original assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Current assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date: 2022-07-08
Filing date: 2022-07-08
Publication date: 2023-01-03
Also published as: WO2024007388A1

Abstract

The invention relates to a detection method and a kit of cancer cell specific T cells, which comprises the steps of incubating T cells in peripheral blood, peripheral immune organs or tumor infiltrating lymphocytes and particles prepared by antigen presenting cells activated by particles loaded with whole cell components to activate the cancer cell specific T cells capable of identifying and killing cancer cells, detecting and analyzing the number and proportion of the activated cancer cell specific T cells by using ELISPOT, flow cytometry, ELISA and other technologies, and further evaluating the strength of cancer cell specific immunity in a patient body. The invention overcomes the problem that broad-spectrum and polyclonal specific T cells in peripheral blood, peripheral immune organs or tumor infiltrating lymphocytes can not be effectively screened clinically at present, can detect broad-spectrum effector cell specific T cells with specific killing function from cells, has the characteristics of easy separation and acquisition and high specificity, and can be used as an effective biomarker.

Description

Detection method and kit for cancer cell specific T cells

Technical Field

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

Background

T cells, especially cancer cell-specific T cells, play a major role in cancer resistance. The T cells are the main cells for the body to specifically recognize and kill cancer cells, and the clone of each cancer cell specific T cell can specifically recognize an epitope. Cancer patients, particularly those undergoing immunotherapy or radiotherapy, contain a number of cancer cell-specific T cells. Research shows that the number of cancer cell-specific T cells in a cancer patient treated by immunotherapy is positively correlated with the prognosis of the cancer patient, so that the detection of the number of cancer cell-specific T cells in the cancer patient is particularly important. The inventor has found that the detection of cancer cell-specific T cells is assisted by nanoparticles or microparticles loaded with whole cell components of cancer cells (application No. 202011027741.0, a method for detecting tumor-specific T cells), but the detection system needs to contain Antigen-presenting cells (APCs), so that the detection system contains multiple cells at the same time, and the process of Antigen-activated T cell-assisted detection is an indirect process rather than a direct process. In order to solve the above problems, the applicant has proposed the present invention.

Disclosure of Invention

To solve the above technical problems, the present invention provides a method for detecting killer (effector) cancer cell-specific T cells (T cells) using nanoparticles and/or microparticles prepared from antigen-presenting cells activated by Nanoparticles (NP) or Microparticles (MP) loaded with cancer cell whole cell antigens _eff ) The method of (1), which uses nanoparticles or microparticles prepared from activated antigen-presenting cells to activate cancer cell-specific T cells first, and then uses the activated killer cancer cell-specific T cells (T cells) _eff ) The specifically expressed marker analyzes the content and proportion of the cancer cell specific T cells in the cells to be detected. Effectively solves the problem of how to detect broad-spectrum and polyclonal cancer cell specific T cells with the capacity of identifying and killing cancer cells in peripheral blood, peripheral immune organs or tumor infiltrating lymphocytes. Furthermore, since the nanoparticles or microparticles for detecting cancer cell-specific T cells are loaded with antigen-presenting cell membrane components, the system can be incubated with T cells without the assistance of antigen-presenting cells, which is more directAnd (3) a detection method.

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

s1, incubating the antigen presenting cells and the first particles together to obtain activated antigen presenting cells; wherein the first particle is a nanoparticle or microparticle loaded with tumor tissue and/or cancer cell whole cell components;

s2, preparing the activated cell membrane components of the antigen presenting cells into nano vesicles; or the cell membrane component of the activated antigen presenting cell and the second particle are acted together to make the cell membrane component loaded on the second particle to obtain the second particle loaded with the cell membrane component; wherein the second particle is a nanoparticle or microparticle loaded with tumor tissue and/or cancer cell whole cell components;

and S3, co-incubating the second particles of the nano vesicles and/or the loaded cell membrane components obtained in the step S2 with cells to be detected, activating broad-spectrum cancer cell-specific T cells capable of recognizing antigens, analyzing markers in or on the activated cancer cell-specific T cells by using a proper detection technology, and analyzing the number and the proportion of the T cells containing the markers to obtain the number and the proportion of the cancer cell-specific T cells.

Further, the marker in the cell or on the cell surface includes a protein or a nucleic acid.

Further, where the specific surface marker is a protein, it includes, but is not limited to, interferon- γ, interleukin, granzyme, perforin, CD69, CD25, OX40 (CD 134), CD39, CD103, CD56, CD279, CD278, CD244, CD27, CD154, TCF-1, CD137, CD44, CD28, and the like. Techniques for detecting the number and proportion of cancer cell-specific T cells using surface marker analysis include, but are not limited to, flow cytometry, magnetic bead sorting, enzyme-linked immunospot (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and the like.

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

Further, before the cells to be tested are incubated with the S2 product, they may be sorted to sort out T cells therein, and in particular, CD3 may be sorted from peripheral blood, peripheral immune tissue, tumor-infiltrating lymphocytes using flow cytometry or magnetic bead sorting ⁺ Cell of (2), sorting out CD45 ⁺ CD3 ⁺ Cell of (2), sorting CD3 ⁺ CD8 ⁺ Cell of (2), sorting CD45 ⁺ CD3 ⁺ CD8 ⁺ Cell of (2), sorting CD3 ⁺ CD4 ⁺ Or sorting out CD45 ⁺ CD3 ⁺ CD4 ⁺ The cell of (1).

Further, the co-incubation system of the antigen-presenting cells and the first particles in step S1 may contain a cytokine or an antibody.

Further, in step S3, the product of S2 and the test cell may contain a cytokine or an antibody in the co-incubation system.

Preferably, the co-incubation system comprises 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), interferon gamma (IFN- γ), TNF- α.

Further, antibodies include, but are not limited to, α CD-3 antibodies, α CD-4 antibodies, α CD-8 antibodies, α CD-28 antibodies, α CD-40 antibodies, α OX-40L antibodies.

Further, in the above preparation method, the first particles or the second particles may further carry a bacterial component or a bacterial outer vesicle component, the bacterial component or the bacterial outer vesicle component is obtained by lysing the bacteria or the bacterial outer vesicles with a lysis solution containing a lysing agent, wherein the lysing agent is urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, a protein degrading enzyme, albumin, lecithin, triton, tween, an amino acid, a glycoside, choline, and the bacteria include, but are not limited to, bacillus calmette-guerin, escherichia coli, bifidobacterium longum, bifidobacterium breve, bifidobacterium lactis, lactobacillus acidophilus, lactobacillus formaticus, lactobacillus reuteri, lactobacillus rhamnosus, and the like.

Furthermore, the activated antigen presenting cell membrane component can be mixed with a cancer cell membrane component or a cancer cell extracellular vesicle membrane component to prepare a mixed membrane component and then prepare a nano vesicle or load the nano vesicle on the surface of a second particle.

Further, the first particle or the second particle is loaded with an immune enhancing adjuvant including, but not limited to, a pattern recognition receptor agonist, bcg cell wall skeleton, bcg methanol extraction residue, bcg muramyl dipeptide, mycobacterium phlei, polyclonal antibody, mineral oil, virus-like particles, immune enhanced reconstituted influenza virions, cholera enterotoxin, saponin and its derivatives, resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly (I: C), immune adjuvant poly ICLC, brevibacterium clavatum, hemolytic streptococcal preparation, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, cytokine, interleukin, interferon, polyinosinic acid, polyamphatic acid, alum, aluminum phosphate, lanolin, squalene, vegetable oil, endotoxin, liposome adjuvant, double stranded MF59, double stranded DNA, CAF01, etc., effective components of astragalus, etc.

Preferably, the immunopotentiating adjuvant comprises (1) Poly (I: C) or Poly (ICLC); (2) The CpG-ODN is at least two of A class CpG-ODN, B class CpG-ODN and C class CpG-ODN, and at least one of them is B class CpG-ODN or C class CpG-ODN. Wherein, the A class CpG-ODN is selected from CpG-ODN 2216, cpG-ODN 1585 or CpG-ODN 2336, the B class 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, the C class CpG-ODN is selected from CpG-ODN 2395, cpG-ODN SL03 or CpG-ODN M362.

Further, the first particle or the second particle is loaded with polypeptide (such as KALA polypeptide, RALA polypeptide, melittin, etc.), arginine, and polyarginineLysine, polylysine, histidine, polyhistidine, NH ₄ HCO ₃ Protamine or histone, and the like.

Further, the first particle or the second particle is loaded with a target head for actively targeting the antigen-presenting cell, wherein the target head can be mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody and the like.

Further, the first particles or the second particles may be prepared from the following materials: organic synthetic polymer materials include, but are not limited to, PLGA, PLA, PGA, PEG, PCL, poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, synthetic polypeptides, etc.; natural polymer materials include, but are not limited to, lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, polypeptides, and the like; inorganic materials include, but are not limited to, iron sesquioxide, iron tetroxide, carbonates, phosphates, and the like.

Further, the first particles or the second particles have a particle size of nanometer or micrometer order, which ensures phagocytosis of the particles by antigen-presenting cells, and the particle size is within a suitable range for enhancing phagocytosis efficiency. Nanoparticles (NPs) having a particle size of from 1nm to 1000nm, more preferably a particle size of from 30nm to 1000nm, most preferably a particle size of from 100nm to 600nm; the Microparticles (MP) have a particle size of 1 μm to 1000 μm, more preferably a particle size of 1 μm to 100 μm, more preferably a particle size of 1 μm to 10 μm, most preferably a particle size of 1 μm to 5 μm.

Further, in step S2, the activated antigen-presenting cells are subjected to mechanical disruption, membrane filtration or gradient centrifugation to prepare nanovesicles, or the activated antigen-presenting cells are subjected to mechanical disruption, membrane filtration or gradient centrifugation to allow the product to interact with the second particles, thereby obtaining second particles loaded with cell membrane components.

Further, the mechanical disruption means is selected from one or more of ultrasound, homogenization, high speed stirring, high pressure disruption, high shear disruption, swelling, chemicals, and shrinkage. The co-action mode is selected from one or more of co-incubation, co-extrusion, ultrasound, stirring, homogenization and homogenate, and the antigen presenting cell component covers the surface layer of the original nano-particle or micro-particle after the co-action with the nano-particle or micro-particle to form a new nano-particle or micro-particle.

Further, freezing cancer cells or tumor tissues at the temperature of between 20 ℃ below zero and 273 ℃ below zero, adding water or a solution without a dissolving agent, performing repeated freeze-thaw lysis to obtain a supernatant as a water-soluble component, dissolving the precipitate by the dissolving agent, converting the dissolved part into a soluble part as a water-insoluble component, and combining the water-soluble component and the water-insoluble component to obtain the cancer cell whole-cell component. The dissolving agent is at least one selected from urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein degrading enzyme, albumin, lecithin, inorganic salt (0.1-2000 mg/mL), triton, tween, amino acids, glycoside, and choline.

Further, the antigen-presenting cell includes at least one of a B cell, a Dendritic Cell (DC) and a macrophage, preferably a combination of two or more, more preferably three, cells including a DC.

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

According to the invention, nanoparticles and/or microparticles loaded with cancer cell whole cell antigens are used for specifically activating antigen presenting cells, then the antigen presenting cells are prepared into nanoparticles or microparticles, the prepared nanoparticles or microparticles are loaded with cancer cell whole cell epitopes, then the nanoparticles or microparticles prepared from the antigen presenting cells are used for activating cancer cell specific T cells in peripheral blood, peripheral immune tissues or tumor infiltrating lymphocytes, and then the characteristics of high expression of certain molecules in the activated cancer cell specific T cells or on the surfaces of the cells are utilized, and the number and the proportion of the most diverse and broad-spectrum cancer specific T cells with the functions of identifying and killing cancer cells are analyzed by means of flow cytometry and the like.

Further, the antigen-presenting cell may be autologous, allogeneic, cell-line or transformed from stem cells with the cancer cell-specific T cell.

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

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

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

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

wherein the content of the first and second substances,

the nano vesicle is prepared by incubating an antigen presenting cell and a first particle together to obtain an activated antigen presenting cell and extracting a cell membrane component of the activated antigen presenting cell;

the particles loaded with the activated antigen-presenting cell membrane components are obtained by enabling the activated antigen-presenting cell membrane components to act together with second particles so that the cell membrane components are loaded on the second particles;

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

The invention breaks through the limitation of the existing detection mode, enables the particles to load all antigens and activated antigen presenting cell membranes, can detect wider and various cancer cell specific T cells, has high specificity and better effect in immunoassay, and thus provides a potential biomarker detection method for immunotherapy.

By means of the scheme, the invention at least has the following advantages:

the present invention provides a technique for in vitro detection of cancer cell-specific T cells in immune cells using a nano-or micro-sized particle delivery system, the assay being performedThe tested cancer cell specific T cells are broad-spectrum and highly specific, and comprise all cloned T cells (T) which can specifically recognize and kill cancer cell effector (killer) cancer cells _eff ) (ii) a Moreover, compared with particles prepared by not loading activated antigen-presenting cells, the particles prepared by loading activated antigen-presenting cells have higher content of specific markers secreted by activated T cells, so that the particles are easier to detect, and the condition that the T cells cannot be detected due to poor signals is avoided. The above advantages enable the particle of the invention to avoid the situation that some cancer cell-specific T cells cannot be detected due to the fact that the specific markers expressed after the activation are weak when the particle of the invention is used for detecting the cancer cell-specific T cells, so that the detection accuracy of the detection method of the invention is higher. The method also optimizes the activation process of the antigen presenting cells, the incubation process of the antigen presenting cells and the T cells, and the loading substances of the first particles and the second particles on the basis, so that the method can detect more comprehensive cancer cell specific T cells, and the signals are stronger and more accurate during detection.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present invention may be more readily and clearly understood, reference will now be made in detail to the present invention, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a schematic diagram of the preparation process and application of the cell system of the present invention; wherein a is a schematic diagram of collecting and preparing nano particles or micro particles from water-soluble components and water-insoluble components respectively; b is a schematic diagram of dissolving cancer cell whole cell antigen and preparing nano particles or micro particles by using a dissolving solution containing a dissolving agent; c is a schematic diagram of using the nano-particles and/or micro-particles prepared in a or b to activate antigen presenting cells, and preparing the activated antigen presenting cells into particles for detecting cancer cell specific T cells;

FIGS. 2 to 14 are the results of experiments for detecting cancer cell-specific T cells using nanoparticles or microparticles in examples 1 to 13, respectively; * Representing that p is less than or equal to 0.05, and has significant difference.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The cancer cell specific T cells are incubated with nano particles and/or micro particles prepared by activated antigen presenting cells during detection, and then the high-expression molecules of the cancer cell specific T cells after being activated by antigen specificity are analyzed by using flow cytometry, enzyme-linked immunosorbent assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA) or the like, so that the broad-spectrum information of the cancer cell specific T cells can be obtained.

Wherein the antigen presenting cells used to prepare the nanoparticles or microparticles are first activated by the nanoparticles and/or microparticles loaded with the 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 fig. 1.

When preparing nano particles or micro particles for activating antigen presenting cells, water-soluble components and water-insoluble antigens are respectively collected and nano or micro particle systems are respectively prepared after cells or tissues are cracked; or directly using a dissolving solution containing a dissolving agent to directly crack cells or tissues and dissolve cancer cell whole cell antigens and prepare a nano or micro particle system. The cancer cell whole cell antigen can be subjected to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease incision or degradation and other treatments before or (and) after lysis, and then nano particles or micro particles are prepared; the nanoparticles or microparticles can also be prepared directly before cell lysis or (and) after cell lysis without any inactivation or (and) denaturation, immobilization, biomineralization, ionization, chemical modification, nuclease treatment, protease inscribation or degradation. In some embodiments of the present invention, the tumor tissue cells are inactivated or (and) denatured before being lysed, or inactivated or (and) denatured after being lysed during the actual use process; in some embodiments of the present invention, the inactivation or/and denaturation treatment before or/and after cell lysis is uv irradiation and high temperature heating, and during the actual use, treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, protease incision or degradation, freeze drying, etc. may also be used. Those skilled in the art can understand that in the practical application process, the skilled person can make appropriate adjustments according to specific situations.

In preparing the activated antigen-presenting cells into nanoparticles or microparticles, the antigen-presenting cells are first mechanically disrupted and then filtered using centrifugation and/or a filter of a certain pore size, optionally in conjunction with nanoparticles or microparticles.

The activated antigen-presenting cells contain a certain cell membrane structure after mechanical disruption.

The activated antigen-presenting cells, after mechanical disruption, interact with the nanoparticles or microparticles to form a new nanoparticle or microparticle in which the antigen-presenting cell component is located on the outer layer of the particle.

The antigen-presenting cells prepared as nanoparticles or microparticles and used to add the antigen-presenting cells for co-incubation with T cells may be autologous or allogeneic, or may be derived from cell lines or stem cells. The antigen presenting cell can be DC cell, B cell, macrophage or any mixture of the three, and can also be other cells with antigen presenting function.

In activating antigen-presenting cells using whole cell fractions loaded with tumor tissue and/or cancer cells, cytokines and/or antibodies may be included in the system to increase the efficiency of activation.

When nanoparticles and/or microparticles prepared from activated antigen-presenting cells are used to activate cancer cell-specific T cells, cytokines and/or antibodies may be included in the system to increase the efficiency of activation.

In some embodiments, the specific method for detecting cancer cell-specific T cells in peripheral blood, peripheral immune tissue or tumor infiltrating lymphocytes using nanoparticles or microparticles prepared from antigen-presenting cells, which are prepared by activating antigen-presenting cells using cancer cell whole-cell antigen-loaded nanoparticles or microparticles, is as follows:

step 1, adding 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 prepared particle raw material of a second predetermined concentration.

In some embodiments, the aqueous phase solution may contain components of the cancer cell/tumor tissue lysate and an immune enhancing adjuvant; the components in the lysate are water-soluble components or original water-insoluble components dissolved in a dissolving solution containing a dissolving agent such as urea or guanidine hydrochloride. The aqueous solution contains a concentration of water soluble components or a concentration of primarily water insoluble components, i.e., a first predetermined concentration, that requires a protein polypeptide concentration of greater than 1ng/mL to be loaded with sufficient cancer cell whole cell antigen to activate the relevant cells. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01ng/mL.

In some embodiments, the organic solvent is dichloromethane. Additionally, in some embodiments, the second predetermined concentration of the starting material for preparing the particles ranges from 0.5mg/mL to 5000mg/mL, preferably 100mg/mL.

In practice, the second predetermined volume of organic phase is set according to its ratio to the first predetermined volume of aqueous phase, and in the present invention, the ratio between the first predetermined volume of aqueous phase and the second predetermined volume of organic phase ranges from 1.1 to 5000, preferably from 1. 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 nanoparticles or microparticles produced during the implementation process.

Preferably, when the aqueous phase solution is a lysate component solution, the concentration of the protein and the polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL; when the aqueous phase solution is lysate component/immunoadjuvant solution, the concentration of protein and polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL, and the concentration of immunoadjuvant is more than 0.01ng/mL, preferably 0.01 mg/mL-20 mg/mL. In the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5mg/mL to 5000mg/mL, preferably 100mg/mL.

And 2, carrying out ultrasonic treatment for more than 2 seconds or stirring or homogenizing treatment for more than 1 minute or micro-fluidic treatment on the mixed solution obtained in the step 1. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 second, such as 2-200 seconds; a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer is used during homogenization treatment, the pressure is more than 5psi, such as 20-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 100rpm, such as 1000-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-grade and/or micron-grade particles are subjected to ultrasonic treatment, stirring treatment, homogenizing treatment or microfluidic treatment, the size of the prepared micro-nano particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the particle size can be changed when the particles are too large or too small.

And 3, adding the mixture obtained after the treatment in the step 2 into a third predetermined volume of aqueous solution containing a third emulsifier with a predetermined concentration, and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or microfluidic treatment. Adding the mixture obtained in the step 2 into an emulsifier aqueous solution, and continuing to carry out ultrasonic treatment or stirring for nano-crystallization or micro-crystallization. In the present invention, the sonication time is greater than 0.1 seconds, such as from 2 to 200 seconds, the agitation speed is greater than 50rpm, such as from 50rpm to 500rpm, and the agitation time is greater than 1 minute, such as from 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 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W-500W, and the time is more than 0.1 second, such as 2-200 seconds; a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer is used during homogenization treatment, the pressure is more than 20psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-or micrometer-sized particles are subjected to ultrasonic treatment, stirring treatment, homogenizing treatment or microfluidic treatment, the size of the prepared nano-or micrometer-sized particles can be controlled by the length of ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the change of the size of the particles can be caused by over-size or under-size.

In some embodiments, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20mg/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 1.1-1, and preferably may be 2. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during implementation to control the size of the nanoparticles or microparticles. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution and the concentration of the emulsifier aqueous solution are all taken according to the values to obtain the nano-particles or micro-particles with proper size.

And 4, adding the liquid obtained after the treatment in the step 3 into a fourth preset volume of emulsifier aqueous solution with a fourth preset concentration, and stirring until preset stirring conditions are met.

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

The fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nanoparticles or microparticles of a suitable size. The fourth predetermined volume is selected based on a 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 is in the range 1.5-1, preferably 1. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during implementation to control the size of the nanoparticles or microparticles.

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

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition for more than 1 minute at the rotating speed of more than 100RPM, removing the supernatant, and resuspending the rest precipitate in a fifth preset volume of aqueous solution containing the lyoprotectant at a fifth preset concentration or in a sixth preset volume of PBS (or physiological saline).

And 6, freeze-drying the suspension containing the freeze-drying protective agent obtained in the step 5, and then keeping the freeze-dried substance for later use.

Step 7, directly using the suspension containing the nanoparticles, which is obtained in the step 5 and is resuspended in PBS (or normal saline) in a sixth predetermined volume, or using PBS (or normal saline) in a sixth predetermined volume to resuspend the freeze-dried substance containing the nanoparticles or microparticles and the freeze-drying protective agent obtained in the step 6; or the sample is mixed with a seventh predetermined volume of water-soluble component or dissolved original water-insoluble component for use.

In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1 10000 to 10000, the preferential volume ratio is 1.

And 8, incubating the antigen presenting cells and the prepared nano particles and/or micro particles for a certain time. The tumor tissue and/or cancer cells from which the nanoparticles and/or microparticles are prepared and the antigen-presenting cells may be autologous or allogeneic.

And 9, collecting the cells after co-incubation, and performing mechanical disruption such as ultrasonic disruption, homogenization disruption, mechanical agitation and the like.

And step 10, centrifuging the sample subjected to ultrasonic treatment and/or filtering the sample by using a filter membrane with a certain pore size and/or performing combined action with the nano particles and/or the micro particles loaded with the cancer cell whole cell components to prepare the nano particles or the micro particles based on the antigen presenting cells.

Step 11, peripheral blood, peripheral immune tissue or tumor tissue is obtained, and T cells or T cell-containing immune cells in the tissue are collected. The above peripheral blood, peripheral immune tissue or tumor tissue may be from autologous or allogeneic sources.

And 12, mixing the nano and/or micro particles prepared in the step 10 with the T cells or the immune cells containing the T cells obtained in the step 11, and then incubating for a certain time.

And step 13, analyzing the content of the cancer cell-specific T cells activated by the antigen by adopting a flow cytometry, an enzyme-linked immunosorbent assay or a magnetic bead sorting method and the like.

Example 1 use of nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs

This example illustrates how nanoparticles prepared using nanoparticle-activated antigen-presenting cells can be used to detect cancer cell-specific T cells in mouse splenocytes using a mouse melanoma cancer model. In this embodiment, a B16F10 melanoma tumor tissue is lysed to prepare a water-soluble component and a water-insoluble component of the tumor tissue, then a nanoparticle system loaded with the water-soluble component and the water-insoluble component of the tumor tissue is prepared by a solvent evaporation method using organic polymer material PLGA as a nanoparticle framework material and Polyinosinic-polycytidyclic acid (poly (I: C)) as an immune adjuvant, then an antigen-presenting cell is activated by the nanoparticle, the antigen-presenting cell is mechanically destroyed and then centrifuged to prepare the nanoparticle, and the nanoparticle is used to assist in detecting cancer cell-specific T cells in peripheral immune organs.

(1) Lysis of tumor tissue and Collection of fractions

Subcutaneous inoculation of 1.5X 10 in the back of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissue was harvested. Cutting tumor tissue into pieces, grinding, adding appropriate amount of ultrapure water through cell filter screen, and freeze thawing repeatedly for 5 timesWith sonication to disrupt lysed cells. After the cells are lysed, centrifuging the lysate for 5 minutes at the rotating speed of 5000g, and taking supernatant fluid which is a water-soluble component soluble in pure water; the addition of 8M urea to the resulting precipitate portion dissolves the precipitate portion, thereby converting the water-insoluble components insoluble in pure water to soluble in an 8M aqueous urea solution. The above is the source of antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticles loaded with Whole cell Components

In this example, the nanoparticles 1 were prepared by a multiple emulsion method in a solvent evaporation method. The nanoparticles carrying the water-soluble component in the cancer cell whole cell antigen and the nanoparticles carrying the water-insoluble component in the cancer cell whole cell antigen are prepared separately and then used together at the time of use. The molecular weight of PLGA used as a nanoparticle preparation material is 24-38 KDa, and the adopted immunologic adjuvant is poly (I: C) which is only distributed in the nanoparticle. As mentioned above, in the preparation process, the cell component and adjuvant are loaded inside the nanoparticles by using a multiple emulsion method, after the cell lysis component is loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are freeze-dried for 48 hours. The average particle diameter of the nano-particle 1 is about 280nm, each 1mg PLGA nano-particle is loaded with about 100 mug protein or polypeptide component, and each 1mg PLGA nano-particle uses 0.02mg poly (I: C) immunologic adjuvant.

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

This example illustrates how to prepare BMDCs by taking dendritic cells prepared from mouse bone marrow cells as an example. Firstly, 1C 57 mouse of 6-8 weeks old is taken out and killed by dislocation of cervical vertebrae, tibia and femur of hind leg are taken out by operation and put into PBS, and muscle tissue around the bone is removed by scissors and forceps. The two ends of the bone are cut off by scissors, the PBS solution is extracted by a syringe, the needles are respectively inserted into the marrow cavity from the two ends of the bone, and the marrow is repeatedly washed into a culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and then 1mL of red blood cell lysate was added to lyse red blood. Lysis was stopped by adding 3mL of RPMI1640 (10% FBS), centrifuging at 400g for 3min, and discarding the supernatant. Placing the cells in a 10mm culture dish for cultureCulturing in RPMI1640 (10% FBS) medium, while adding recombinant mouse GM-CSF (20 ng/mL), 37%, 5% CO ₂ The culture was carried out for 7 days. The flask was gently shaken on day 3 and supplemented with the same volume of medium containing GM-CSF (20 ng/mL) RPMI1640 (10% FBS). On day 6, medium was subjected to half-volume change. On day 7, a small number of suspended and semi-adherent cells were collected and tested by flow assay as CD86 ⁺ CD80 ⁺ Cells in CD11c ⁺ The proportion of cells is between 15 and 20 percent, and the BMDCs cultured by induction can be used for the next experiment.

(4) Activation of antigen presenting cells

Incubating the nanoparticles loaded with the whole cell fraction of cancer cells derived from tumor tissue (250. Mu.g of nanoparticles loaded with a water-soluble fraction + 250. Mu.g of nanoparticles loaded with a water-insoluble fraction) with BMDCs (1000 ten thousand) in 15mL of RPMI1640 complete medium (37 ℃,5% ₂ ) (ii) a 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 a mixture comprising cytokine component 2: GM-CSF (500U/mL), IL-4 (500U/mL), tumor necrosis factor alpha (TNF-alpha, 500U/mL), IL10 (500U/mL).

(5) Preparation of DC-derived nanoparticles

The incubated DCs (1000 ten thousand) to which the cytokine fraction 1 was added were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice using physiological saline, and after resuspending the cells in physiological saline, the cells were sonicated at 4 ℃ and 7.5W for 20 minutes to disrupt the cells and prepare a sample containing the cell membrane fraction. And centrifuging the sample at 2000g for 20 minutes, collecting supernatant, centrifuging the supernatant at 7000g for 20 minutes, collecting supernatant, centrifuging at 15000g for 120 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nanoparticles 2, wherein the particle size of the nanoparticles 2 is 120 nanometers.

Or collecting the DCs (1000 ten thousand) prepared in step (3) without any nanoparticle or microparticle activation, then washing the cells twice using physiological saline, and sonicating the cells at 4 ℃ and 7.5W for 20 minutes after resuspending the cells in physiological saline to disrupt the cells and prepare a sample containing cell membrane components. Centrifuging the sample at 2000g for 20 minutes and collecting supernatant, centrifuging the supernatant at 7000g for 20 minutes and collecting supernatant, co-incubating the supernatant with 40mg of the cancer cell whole cell component-loaded nanoparticles 1 prepared in step (2) (water-soluble component-loaded nanoparticles 20mg + non-water-soluble component-loaded nanoparticles 20 mg) for 10 minutes, repeatedly co-extruding through a 0.45-micrometer filter membrane, centrifuging the extruded solution at 15000g for 120 minutes, collecting the supernatant, discarding the precipitate, and re-suspending the precipitate in PBS to obtain nanoparticles 3 with the particle size of 300nm.

Or the incubated DCs (1000 ten thousand) to which the cytokine fraction 2 was added were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice using physiological saline, and after resuspending the cells in physiological saline, the cells were sonicated at 4 ℃ and 7.5W for 20 minutes to disrupt the cells and prepare a sample containing the cell membrane fraction. And (3) centrifuging the sample at 2000g for 20 minutes, collecting supernatant, centrifuging the supernatant at 7000g for 20 minutes, collecting supernatant, co-incubating the supernatant with 40mg of the cancer cell whole cell component-loaded nanoparticles 1 (water-soluble component-loaded nanoparticles 20mg + non-water-soluble component-loaded nanoparticles 20 mg) prepared in the step (2) for 10 minutes, repeatedly co-extruding the mixture by using a 0.45-micrometer filter membrane, centrifuging the extruded solution at 15000g for 120 minutes, collecting the supernatant, discarding the precipitate, and re-suspending the precipitate in PBS to obtain the nanoparticles. Wherein, the nano particle 4 is obtained by using the co-incubation of the nano particle 1 and the membrane component, and the particle size is 300nm.

Or the incubated DCs (1000 ten thousand) to which the cytokine fraction 1 was added were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice using physiological saline, and after resuspending the cells in physiological saline, the cells were sonicated at 4 ℃ and 7.5W for 20 minutes to disrupt the cells and prepare a sample containing the cell membrane fraction. And (3) centrifuging the sample at 2000g for 20 minutes, collecting supernatant, centrifuging the supernatant at 7000g for 20 minutes, collecting supernatant, co-incubating the supernatant with 40mg of the cancer cell whole cell component-loaded nanoparticles 1 (water-soluble component-loaded nanoparticles 20mg + non-water-soluble component-loaded nanoparticles 20 mg) prepared in the step (2) for 10 minutes, repeatedly co-extruding the mixture by using a 0.45-micrometer filter membrane, centrifuging the extruded solution at 15000g for 120 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nanoparticles 5 with the particle size of 300nm.

(6) Detection of cancer cell-specific T cells

Subcutaneous inoculation of 0.5X 10 in the back of each C57BL/6 mouse ⁵ B16F10 cells, each about 1000mm in tumor growth to volume ³ Mice were sacrificed and splenocytes harvested. Preparing mouse spleen cells into single cell suspension, and then separating CD3 from the mouse spleen cell single cell suspension by using flow cytometry ⁺ T cells.

Nanoparticle 1 (50. Mu.g of water-soluble component-supporting nanoparticle + 50. Mu.g of water-insoluble component-supporting nanoparticle) or nanoparticle 2 (100. Mu.g) or nanoparticle 3 (100. Mu.g) or nanoparticle 4 (100. Mu.g) or nanoparticle 5 (100. Mu.g) was incubated with splenocyte-derived T cells (100 ten thousand) in 10mL of RPMI 1649 complete medium for 72 hours (37 ℃,5% CO ₂ ). Cells were then harvested and centrifuged at 400g for 5 minutes, and cells resuspended in PBS before treatment of T cells with Fc block to avoid non-specific loading. The mouse splenocytes were then stained extracellularly with CD3, CD4, and CD8 antibodies, followed by cell fixation and rupture, and T cells were stained intracellularly with FN-gamma antibodies. The sample T cells are then detected using a flow cytometer. Separate analysis of CD4 ⁺ T cells activated to secrete IFN-gamma are all CD4 ⁺ Ratio of T cells and CD8 ⁺ T cells that can secrete IFN-gamma after being activated in T cells are all CD8 ⁺ The proportion of T cells. Above CD4 ⁺ IFN-γ ⁺ T cells and CD8 ⁺ IFN-γ ⁺ The T cells are cancer cell specific T cells.

Or incubating only splenocyte-derived T cells (100 ten thousand) in 10mL of RPMI 1649 complete medium for 72 hours (37 ℃,5% CO) ₂ ). Cells were then harvested and centrifuged at 400g for 5 minutes, and cells resuspended in PBS before treatment of T cells with Fc block to avoid non-specific loading. Then adopting anti-mouse CD3 antibody, anti-mouse CD4 antibody and anti-mouse CD8 antibody connected with specific fluorescent probe to make them be used for curing the diseases of spleen and kidneyThe cells were stained extracellularly, then fixed and the cells were disrupted, and T cells were stained intracellularly with an anti-mouse IFN-. Gamma.antibody linked to a fluorescent probe. The sample is then examined by flow cytometry for T cells containing the fluorescent signal linked to the IFN- γ antibody. Separate analysis of CD4 ⁺ T cells that can secrete IFN-gamma after being activated in T cells are all CD4 ⁺ Ratio of T cells and CD8 ⁺ T cells activated to secrete IFN-gamma are found on all CD8 ⁺ The proportion of T cells. The above CD4 ⁺ IFN-γ ⁺ T cells and CD8 ⁺ IFN-γ ⁺ The T cell is cancer cell specific T cell.

After the nano/micro particles loaded with tumor tissues and/or cancer cell whole cell components are phagocytized by antigen presenting cells, the antigen can be degraded into polypeptide epitope and presented on the surface of an antigen presenting cell membrane after being combined with Major Histocompatibility Complex (MHC) molecules. As the nanoparticle and microparticle loaded whole cell antigen can be presented and released in a cross way, the cancer cell epitope can be presented to the surface of an antigen presenting cell membrane through two pathways of MHC I and MCH II. After the cell membrane of the antigen presenting cell is prepared into nano particles or micro particles, the loaded MHC molecules and antigen polypeptide complexes can be directly combined with a T cell surface receptor which can specifically recognize cancer cell antigens on the surface of a T cell. Furthermore, if the T cell is a specific T cell capable of killing cancer cells, the cancer cells begin to secrete a killer substance such as IFN-. Gamma.and granzyme, and the ratio of T cells specific to effector cancer cells having the ability to recognize and kill cancer cells can be obtained by analyzing T cells that secrete the killer substance.

(8) Results of the experiment

As shown in fig. 2, cancer cell-specific T cells were low in the T cell-alone control group and the nanoparticle 1 detection group. And the

nanoparticles

2, 3, 4 and 5 can assist in detecting a certain content of cancer cell-specific T cells. Among them, the nanoparticles 5 are the most effective, and the nanoparticles 5 are more effective than the

nanoparticles

2, 3 and 4. The nano particle 5 is superior to the nano particle 4, which shows that the effect of adding the cytokine combination 1 in the process of activating antigen presenting cells is superior to that of the cytokine component 2; the nano particle 5 is superior to the nano particle 2, which indicates that the surface of the nano particle for assisting in detecting T cells is loaded with antigen presenting cell membrane components, and the effect of the solid nano particle with the internal loaded cell components is superior to that of the nano vesicle structure only with the surface loaded with the membrane components; the

nanoparticles

4 and 5 are superior to the nanoparticles 3, which shows that the effect of the nanoparticles prepared by the antigen presenting cells activated by the nanoparticles loaded with the cancer cell whole cell components is better than that of the nanoparticles prepared by the antigen presenting cells which are not activated. In conclusion, the particle system of the present invention can be used for detecting cancer cell-specific T cells. Antigen-presenting cells activated by nanoparticles loaded with the cancer cell whole cell fraction degrade and present a broad spectrum of whole cell antigens in the phagocytosed nanoparticle loaded cancer cell whole cell fraction, and cancer cell epitopes presented by antigen-presenting cells to the cell membrane surface have been associated with Major Histocompatibility Complex (MHC) molecules. After the antigen presenting cells are mechanically disrupted, the cell membrane fraction of the antigen presenting cells contains an epitope bound to MHC. By centrifugation and/or filtration through a filter membrane with a certain aperture and/or by co-action with nano-particles or micro-particles, cell membrane components in the antigen presenting cells can form nano-particles or micro-particles, and are loaded with MHC molecules and cancer cell antigen epitopes to be presented by degradation, so that the cancer cell specific T cells can be directly activated for detecting the cells without the assistance of the antigen presenting cells.

Example 2 particles based on antigen presenting cells for 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 aid in the detection of cancer cell-specific T cells. In this embodiment, a B16F10 melanoma tumor tissue is lysed to prepare a water-soluble component and a water-insoluble component of the tumor tissue, then a nanoparticle system loaded with the water-soluble component and the water-insoluble component of the tumor tissue is prepared by a solvent evaporation method using PLGA as a nanoparticle backbone material and poly (I: C) and CpG1018 as immunoadjuvants, then antigen-presenting cells are activated using the nanoparticles, and the activated antigen-presenting cells are prepared into nanoparticles to detect cancer cell-specific T cells in peripheral blood immune cells.

(1) Lysis of tumor tissue and Collection of fractions

Subcutaneous inoculation of 1.5X 10 in the back of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissue was removed. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and freeze thawing is repeated for 5 times, and ultrasonic waves are accompanied to destroy the lysed cells. After the cells are lysed, centrifuging the lysate for 5 minutes at the rotating speed of 5000g, and taking supernatant fluid which is a water-soluble component soluble in pure water; the addition of 8M urea to the resulting precipitate portion dissolves the precipitate portion, thereby converting the water-insoluble components insoluble in pure water to soluble in an 8M aqueous urea solution. Mixing the water-soluble component and the water-insoluble component according to the mass ratio of 1.

(2) Preparation of nanoparticle systems

In this example, the nanoparticles were prepared by a solvent evaporation method. The molecular weight of PLGA material prepared from nanoparticle 1 (NP 1) loaded with whole cell components is 7Da-17KDa, the adopted immunologic adjuvant is poly (I: C) and CpG1018, and the adjuvant is loaded in the nanoparticle. As mentioned above, in the preparation process, the antigen and adjuvant are loaded inside the nanoparticles by using a multiple emulsion method, after the antigen (lysis component) is loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are freeze-dried for 48 hours. The average particle diameter of the nano particles 1 is about 280 nm; each 1mg PLGA nano particle is loaded with about 100 mug protein and polypeptide components, and each 1mg PLGA nano particle uses 0.02mg of poly (I: C) and CpG1018 immunologic adjuvant; nanoparticle 1 is also known as nano-vaccine 1. In the embodiment, the polypeptide nanoparticles 2 loaded with four polypeptide neoantigens B16-M20 (Tubb 3, FRRKAFLHWYTGEAMDEMEDEFESNOM), B16-M24 (Dag 1, TAVITPTTTKTKKARVSTPKPATPSTD), B16-M46 (Actn 4, NHSGLVTFQAFFIDVMSRETTDTADQ) and TRP2:180-188 (SVYDFFVWL) in equal mass are used as the contrast nanoparticles, the preparation material and the preparation method are the same as those of the nanoparticles 1, the particle diameter of the contrast nanoparticles 2 is about 280nm, 100 mu g of polypeptide components are loaded, and equal amount of adjuvant is loaded. The blank nanoparticle 3 has the same preparation material and preparation method as the nanoparticle 1, the particle size is about 280nm, and only the same amount of immunologic adjuvant is loaded but no antigen component is loaded.

(3) Preparation of antigen-presenting cells

Bone marrow-derived dendritic cells (BMDCs) and B cells were used as antigen-presenting cells. BMDC was prepared as in example 1. The B cell extraction procedure was as follows: taking spleen of mouse after killing mouse, preparing single cell suspension of mouse spleen cell, and separating CD19 from single cell suspension of spleen cell by magnetic bead sorting method ⁺ B cells. BMDCs and B cells were mixed at a quantitative ratio of 1.

(4) Activation of antigen presenting cells

Nanoparticle 1 (500. Mu.g) or polypeptide nanoparticle 2 (500. Mu.g) or blank nanoparticle 3 (500. Mu.g) + free lysate was co-incubated with 2000 ten thousand mixed antigen presenting cells (1000 ten thousand BMDC +1000 ten thousand B cells) in 15mL RPMI1640 complete medium for 48 hours (37 ℃,5% CO ₂ ) (ii) a The incubation system contains a combination of cytokines: IL-15 (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL), IL-12 (1000U/mL).

Or co-incubation of nanoparticle 1 with 2000 million BMDCs in 15mL RPMI1640 complete Medium for 48 hours (37 ℃,5% ₂ ) (ii) a The incubation system contains a combination of cytokines: IL-15 (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL), IL-12 (1000U/mL).

(5) Preparation of antigen-presenting cell-based nanoparticles

2000 ten thousand mixed antigen presenting cells (1000 ten thousand BMDCs +1000 ten thousand B cells) after incubation were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice using physiological saline, and after resuspending the cells in physiological saline, the cells were disrupted using low-power 7.5W ultrasound for 10 minutes at 4 ℃ to prepare a sample containing a cell membrane fraction. And then filtering the sample through a filter membrane with the aperture of 50 microns, 10 microns, 5 microns, 1 micron, 0.45 microns and 0.22 microns at a time, collecting the obtained filtrate, co-incubating with the corresponding nanoparticle 1 (50 mg) or polypeptide nanoparticle 2 (50 mg) or blank nanoparticle 3 (50 mg) loaded with the cancer cell whole cell component prepared in the step (2) for 10 minutes, repeatedly co-extruding by using the filter membrane with the aperture of 0.45 microns, centrifuging the extruded liquid at 15000g for 60 minutes, discarding the supernatant, and re-suspending the obtained precipitate by using physiological saline to obtain the nanoparticle. Wherein, the nano particle 4 is prepared by the coaction of the mixed antigen presenting cell membrane component activated by the nano particle 1 and the nano particle 1, and the particle size is 300nm; the nano particle 5 prepared by using the mixed antigen presenting cell membrane component activated by the polypeptide nano particle 2 and the polypeptide nano particle 2 to act together has the particle size of 300nm; the nano particle 6 prepared by the combined action of the mixed antigen presenting cell membrane component activated by the blank nano particle 3 and the blank nano particle 3 has the particle size of 300nm.

Alternatively, 2000 ten thousand BMDCs after incubation with the nanoparticle 1 were collected by centrifugation at 400g for 5 minutes, and then the BMDCs were washed twice with physiological saline, and after resuspending the cells in physiological saline, the cells were disrupted using low-power 7.5W ultrasound for 10 minutes at 4 ℃ to prepare a sample containing a cell membrane fraction. Then, the sample is filtered by a filter membrane with the aperture of 50 μm, 10 μm, 5 μm, 1 μm, 0.45 μm and 0.22 μm at a time, the obtained filtrate is collected and incubated with the nano particles 1 (50 mg) prepared in the step (2) for 10 minutes, then the co-extrusion is repeatedly carried out by using the filter membrane with the aperture of 0.45 μm, the extruded liquid is centrifuged at 15000g for 60 minutes, the supernatant is discarded, and the obtained precipitate is the nano particles 7 which are re-suspended by using physiological saline and have the particle size of 300nm.

(6) Detection of cancer cell-specific T cells

On day 0, each C57BL/6 mouse was inoculated subcutaneously into the back of 1.5X 10 ⁵ Injecting 100 mu L of 1mgPLGA nano vaccine loaded with cancer cell whole cell component or 100 mu L PBS into the mice subcutaneously on the 10 th day and the 14 th day by B16F10 cells; the growth rate of the tumors in the PBS and vaccine groups was monitored over the course of the procedure. Mice were sacrificed on day 18, peripheral blood of the mice was collected, peripheral Blood Mononuclear Cells (PBMCs) were separated from the peripheral blood of the mice using density gradient centrifugation, and then flow cytometry was usedIsolation of CD3 from PBMC by cytometry ⁺ T cells. CD3 in which group was treated with Nanovaccine 1 ⁺ T cells were vaccine-treated groups, and T cells of PBS-treated groups were PBS control groups.

The 100. Mu.g of the antigen-presenting cell-based nanoparticles prepared in step (5) (nanoparticle 4, or nanoparticle 5, or nanoparticle 6, or nanoparticle 7) were co-incubated with B cells (500 ten thousand) and 40 ten thousand T cells from tumor-infiltrating lymphocytes (vaccine group or PBS group) in 5mL of RPMI1640 complete medium for 24 hours (37 ℃,5% CO) ₂ ) Then sorting the incubated CD3 by flow cytometry ⁺ IFN-γ ⁺ T cells. At the same time, CD3 was analyzed ⁺ IFN-γ ⁺ IFN-gamma in T cells ⁺ Mean fluorescence intensity of the attached fluorescent probes (MFI). The stronger the fluorescence intensity is, the more the lethal substance is expressed by the cancer cell specific T cell, and the higher the sensitivity and the better the accuracy in detection are.

Or co-incubating 100. Mu.g of the nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3) prepared in step (2) with B cells (500 ten thousand) and 40 ten thousand T cells (vaccine group or PBS group) from tumor infiltrating lymphocytes in 5mL of RPMI1640 complete medium for 24 hours (37 ℃,5% ₂ ) Then sorting the incubated CD3 by flow cytometry ⁺ IFN-γ ⁺ T cells. At the same time, CD3 was analyzed ⁺ IFN-γ ⁺ IFN-gamma in T cells ⁺ Mean Fluorescence Intensity (MFI) of the attached fluorescent probes.

Or co-incubating 100. Mu.g of the antigen presenting cell based nanoparticles prepared in step (5) (nanoparticle 4, or nanoparticle 5, or nanoparticle 6, or nanoparticle 7) with 40 ten thousand T cells from tumor infiltrating lymphocytes (vaccine group or PBS group) in 5mL of RPMI1640 complete medium for 24 hours (37 ℃,5% ₂ ) Then sorting the incubated CD3 by flow cytometry ⁺ IFN-γ ⁺ T cells. At the same time, CD3 was analyzed ⁺ IFN-γ ⁺ IFN-gamma in T cells ⁺ Mean fluorescence intensity of the attached fluorescent probes (MFI).

Or co-incubating 100. Mu.g of the nanoparticles prepared in step (2) (nanoparticle 1) with 40 ten thousand T cells from tumor infiltrating lymphocytes (vaccine group) in 5mL of RPMI1640 complete medium for 48 hours (37 ℃,5% CO% ₂ ) Then sorting the incubated CD3 by flow cytometry ⁺ IFN-γ ⁺ T cells.

(7) Results of the experiment

As shown in a and b of fig. 3, the growth rate of the tumor of the PBS group mice was significantly faster than that of the vaccine group, which indicates that cancer cell-specific T cells in the vaccine group mice were significantly more than that in the PBS group, and thus the tumor volume growth of the mice could be delayed and controlled. The PBS group was not vaccine-induced and therefore contained fewer T cells specific for cancer cells, while the vaccine group was vaccine-induced and then contained more T cells specific for cancer cells, as shown in fig. 3, the PBS control group and the vaccine group had different results after different nanoparticle detection. Nanoparticle 4 was most effective and the most comprehensive cancer cell-specific T cells could be detected. Nanoparticle 1 failed to detect cancer cell-specific T cells without the aid of antigen presenting cells. The

nanoparticles

4, 5 and 7 can detect cancer cell-specific T cells without the assistance of antigen presenting cells, and the effect of the nanoparticle 4 is obviously better than that of the other two nanoparticles. Furthermore, the effect of cancer cell-specific T cells detected by nanoparticles 4 without antigen presenting cell help was better than that detected by nanoparticles 7 with antigen presenting cell help. Furthermore, the cancer cell-specific T cells detected by

nanoparticles

4, 5 and 7 with or without the assistance of antigen presenting cells were not very different. This demonstrates that nanoparticles prepared using nanoparticle-activated antigen-presenting cells loaded with cancer cell whole-cell antigens are useful for better detection of cancer cell-specific T cells; and the effect of the mixed antigen presenting cells of the DC and the B cells is better than that of a single DC. Furthermore, the methods of the invention for detecting broad spectrum cancer cell-specific T cells can be independent of antigen presenting cells. Moreover, in the process of detecting cancer cell-specific T cells, the same effect as that of detecting cancer cell-specific T cells by adding antigen-presenting cells can be obtained without adding antigen-presenting cells, which is an advantage of the present invention in detecting cancer cell-specific T cells by using nanoparticles or microparticles prepared from activated antigen-presenting cells.

The effect of detecting cancer cell specific T cells by the nanoparticles prepared by the nanoparticle activated antigen presenting cells loaded with cancer cell whole cell antigens is better than that of detecting cancer cell specific T cells by the nanoparticles prepared by the nanoparticle activated antigen presenting cells loaded with four antigen polypeptides. This indicates that the types of cancer cell-specific T cell clones that can be detected by the nanoparticles prepared by loading four antigen-presenting cells activated by the nascent antigen polypeptide nanoparticles are limited. The nanoparticles prepared from the antigen presenting cells activated by the nanoparticles loaded with the cancer cell whole cell antigens can detect wider-spectrum cancer cell specific T cells, so that the number of T cell clones obtained after amplification is wider.

As shown in c in fig. 3, the stronger the fluorescence intensity is, the more the killer substance expressed by the cancer cell-specific T cell is, the more the sensitivity is and the better the accuracy is. When activated cancer cell-specific T cells are labeled with anti-mouse IFN- γ antibodies labeled with the same fluorescent probe, the fluorescent signal (mean fluorescence intensity, MFI) of the fluorescent probe to which IFN- γ antibodies are linked, which is detectable by cancer cell-specific T cells activated by nanoparticle 4 loaded with activated antigen-presenting cell membrane fraction and cancer cell whole cell fraction at the same time, is stronger than the fluorescent signal of the fluorescent probe to which IFN- γ antibodies are linked, which is detectable by cancer cell-specific T cells activated by nanoparticle 1 loaded with cancer cell whole cell fraction alone. And no matter whether antigen presenting cells exist in an incubation system in the detection and activation processes, the fluorescent signal detected by the cancer cell specific T cells activated by the nanoparticles 4 is stronger than that of the cancer cell specific T cells activated by the nanoparticles 1, which shows that the cancer cell specific T cells activated by the nanoparticles 4 express more specific markers and are easier to detect in the detection process.

Therefore, the nanoparticles prepared by the antigen presenting cells activated by the nanoparticles loaded with the whole cell components can better detect the cancer cell specific T cells with the capacity of identifying and killing cancer cells. The cancer cell whole cell antigen loaded by the nano particles can be degraded into antigen epitopes after being phagocytized by antigen presenting cells and then presented to the surface of the antigen presenting cells, specific T cells capable of identifying the cancer cell whole cell antigen can identify the cancer cell whole cell antigen epitopes and then be activated and express specific surface markers, and the proportion of the T cells highly expressing the specific surface markers is analyzed through flow cytometry, so that the quantity and proportion of the activated cancer cell specific T cells capable of identifying and having killing efficiency can be known.

Example 3 detection of cancer cell-specific T cells in peripheral splenocytes from mice

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

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

Tumor tissue was collected by first subcutaneously inoculating 1.5X 10 dorsal cells of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Killing mice and picking tumor tissues, cutting the tumor tissues into blocks, grinding, adding a proper amount of pure water through a cell filter screen, repeatedly freezing and thawing for 5 times, and destroying samples obtained by lysis with ultrasound; when the cultured B16F10 cancer cell lines were collected,centrifuging to remove the culture medium, washing twice by using PBS, centrifuging to collect cancer cells, resuspending the cancer cells in ultrapure water, repeatedly freezing and thawing for 3 times, and disrupting and cracking the cancer cells with ultrasound. After the tumor tissue or the cancer cells are cracked, centrifuging the lysate for 5 minutes at the rotating speed of 5000g, and taking supernatant fluid as a water-soluble component which can be dissolved in pure water; the addition of 8M urea to the resulting precipitate portion dissolves the precipitate portion, thereby converting the water-insoluble components insoluble in pure water to soluble in an 8M aqueous urea solution. Mixing water-soluble components of the tumor tissue and water-soluble components of the cancer cells according to a mass ratio of 1; the water-insoluble component of the tumor tissue and the water-insoluble component of the cancer cell are mixed in a mass ratio of 1. And (3) mixing the water-soluble component mixture and the water-insoluble component mixture according to the mass ratio of 1.

(2) Preparation of bacterial extracellular vesicles (OMVs)

Centrifuging Bifidobacterium longum at 5000g for 30min, discarding precipitate, collecting supernatant, filtering the supernatant with 1 μ M filter membrane, treating with 20W ultrasound at 4 deg.C for 5min, centrifuging at 16000g for 90 min, resuspending the precipitate in PBS to obtain collected bacterial outer vesicle membrane component, and lysing and dissolving the bacterial outer vesicle membrane component with 8M urea aqueous solution.

Or centrifuging Bifidobacterium longum at 5000g for 30min, discarding precipitate, collecting supernatant, filtering the supernatant with 1 μm filter membrane, treating with 20W ultrasound at 4 deg.C for 5min, centrifuging at 16000g for 90 min, resuspending the precipitate in PBS to obtain collected bacterial outer vesicle membrane component, and lysing and dissolving the bacterial membrane component with Tween 80 aqueous solution.

(3) Preparation of nanoparticles

In the embodiment, the nano-particle 1 is prepared by a multiple emulsion method, the molecular weight of the prepared material PLGA is 7KDa-17KDa, the adopted immunologic adjuvant is poly (I: C) and CpG2006, and the adjuvant is encapsulated in the nano-particle. The preparation method is as described above, in the preparation process, firstly, the lysate component and the adjuvant are loaded inside the nanoparticles by using a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used for resuspension, and then the resuspension is frozen and dried for 48 hours for later use. The average particle diameter of the nanoparticle 1 is about 250nm, each 1mg of PLGA nanoparticle 1 is loaded with about 130 mug of protein or polypeptide component, each 1mg of PLGA nanoparticle 1 is loaded with 0.02mg of poly (I: C) and CpG2006 immunoadjuvant, and the nanoparticle 1 is also called nano vaccine 1 during injection.

In this example, the material and method for preparing the nanoparticles 2 are the same as those of the nanoparticles 1. The antigen component prepared in the step (1) and the 8M urea-dissolved bacterial outer vesicle membrane component prepared in the step (2) are loaded in the nano particle 2 at the same time, and the mass ratio of the antigen component to the bacterial outer vesicle membrane component is 1. The immune adjuvants used are poly (I: C) and CpG2006 and the adjuvant is entrapped within the nanoparticle. In the preparation process, firstly, a compound emulsion method is adopted to carry a lysate component, a bacteria outer vesicle component and an adjuvant on tumor tissue in the nano particles, then 100mg of the nano particles are centrifuged for 20 minutes at 10000g, and are frozen and dried for 48 hours for later use after being resuspended by 10mL of ultrapure water containing 4% of trehalose. The average particle diameter of the nano-particle 2 is about 250nm, each 1mg of PLGA nano-particle 2 is loaded with about 130 mug protein or polypeptide component, and each 1mg of PLGA nano-particle 2 is loaded with 0.02mg of poly (I: C) and CpG2006 immunoadjuvant.

The preparation material and preparation method of the nanoparticles 3 in this example are the same as those of the nanoparticles 1. The antigen component prepared in the step (1) and the bacteria outer vesicle membrane component dissolved by the Tween 80 prepared in the step (2) are loaded in the nano-particle 3 at the same time, and the mass ratio of the antigen component to the bacteria outer vesicle membrane component is 1. The immune adjuvants used are poly (I: C) and CpG2006 and the adjuvant is entrapped within the nanoparticle. In the preparation process, firstly, a compound emulsion method is adopted to carry a lysate component, a bacteria outer vesicle component and an adjuvant in the tumor tissue inside the nano particles, then 100mg of the nano particles are centrifuged for 20 minutes at 10000g, and 10mL of ultrapure water containing 4% trehalose is used for resuspension, and then the mixture is frozen and dried for 48 hours for later use. The average particle diameter of the nano-particles 3 is about 250nm, each 1mg of PLGA nano-particles 3 is loaded with about 130 mug protein or polypeptide component, and each 1mg of PLGA nano-particles 3 are loaded with 0.02mg of poly (I: C) and CpG2006 immunoadjuvant respectively.

The blank nanoparticles 4 were prepared from the same materials and using the same method as the nanoparticles 1, but the blank nanoparticles 4 were loaded with the same amount of adjuvant and not with any tumor tissue lysate fraction. The particle diameter of the nanoparticles 4 is about 250 nm.

(4) Isolation of B cells

Killing C57BL/6 mouse, collecting mouse spleen, preparing mouse spleen cell single cell suspension, and separating CD19 from spleen cell by magnetic bead sorting method ⁺ B cells.

(5) Activation of antigen presenting cells

Incubating 500. Mu.g of

nanoparticle

1, or 500. Mu.g of

nanoparticle

2, or 500. Mu.g of

nanoparticle

3, or 500. Mu.g of nanoparticle 4, respectively, with B cells (1000 ten thousand) in 15mL of RPMI1640 complete medium for 48 hours (37 ℃,5% ₂ ) The incubation system contained GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), albumin (50 ng/mL), and CD80 antibody (10 ng/mL).

(6) Preparation of antigen-presenting cell-derived nanoparticles

Incubated B cells were harvested by centrifugation at 400g for 5 minutes, followed by three washes with PBS, resuspending the cells in PBS water followed by 15 minutes of sonication at low power (10W). And centrifuging the sample at 500g for 5 minutes, collecting supernatant, filtering the supernatant by sequentially passing through membranes with the pore diameters of 30 micrometers, 10 micrometers, 5 micrometers, 0.45 micrometers and 0.22 micrometers, centrifuging the obtained filtrate sample at 18000g for 60 minutes, removing supernatant, and re-suspending the precipitate by using PBS to obtain the nano particles. Wherein, the nano particle prepared by the antigen presenting cell activated by the nano particle 1 is a nano particle 5 with the particle diameter of 110 nanometers; the nano particle prepared by using the nano particle 2 activated antigen presenting cell is a nano particle 6, and the particle size is 110 nanometers; the nano particle prepared by using the nano particle 3 activated antigen presenting cell is a nano particle 7, and the particle size is 110 nanometers; the nanoparticles prepared using the nanoparticle 4 activated antigen presenting cells were nanoparticles 8, having a particle size of 110 nm.

(7) Detection of cancer cell-specific T cells

Each C57BL/6 mouse was inoculated subcutaneously into the back of the mouse at day 0 at 1.5X 10 ⁵ Injecting 0.7mg of PLGA nanoparticle 1 (Nanoprotein 1) prepared in step (3) into the mice subcutaneously on days 7, 12 and 17 of B16F10 cells respectively100 μ L PBS. Mice were sacrificed on day 21, spleens were harvested and single cell suspensions of the spleens were prepared, and CD3 was sorted from the splenocytes using magnetic bead sorting ⁺ T cells. Incubating the isolated T cells (400 million) with 100. Mu.g of the nanoparticles (nanoparticles 5, or nanoparticles 6, or nanoparticles 7, or nanoparticles 8) prepared in step (6) in 40mL of high-glucose DMEM complete medium for 48 hours (37 ℃,5% CO) ₂ ) Cells were then harvested by centrifugation at 400g for 5 minutes and then analyzed by flow cytometry for CD3 in incubated T cells after antibody labeling ⁺ IFNγ ⁺ The number and the proportion of T cells, namely cancer cell specific T cells which are specifically activated by the whole cell antigen of the cancer cells.

The cancer cell whole cell antigen loaded by the nano particle can be degraded into an antigen epitope after being phagocytized by an antigen presenting cell and is presented to the surface of an antigen presenting cell membrane, the nano particle prepared by the antigen presenting cell can be identified by a cancer cell specific T cell and can activate the cancer cell specific T cell, and a killer cell factor is secreted after the nano particle is activated. IFN-gamma is the most prominent cytokine secreted by antigen-specific T cells upon recognition of the antigen. CD3 analysis using flow cytometry ⁺ IFN-γ ⁺ The T cell is a cancer cell specific T cell which can recognize and kill cancer cells.

(5) Results of the experiment

As shown in fig. 4, the growth rate of the tumor in the PBS control group mice was fast, while the growth rate of the tumor in the nanoparticle treated group was slow, which indicates that the nanoparticles induce cancer cell-specific T cells to control the growth of the tumor. The nanoparticles 8 hardly activate cancer cell-specific T cells; whereas

nanoparticles

5, 6 and 7 were able to detect more cancer cell specific T cells. Furthermore, the effect of nanoparticle 6 is superior to that of nanoparticles 5 and 7, which suggests that antigen-presenting cells activated after loading of nanoparticles with bacterial outer vesicle components lysed and solubilized using appropriate methods are advantageous for the detection of cancer cell-specific T cells.

Example 4 detection of cancer cell-specific T cells by Nano-or microparticles

In this example, the whole cell antigen of B16F10 melanoma cancer cells was first lysed using 6M guanidinium hydrochloride. Then, PLGA is used as a microparticle scaffold material, cpG BW006 (class B), cpG2216 (class a) and Poly ICLC are used as immunoadjuvants to prepare a microparticle system loaded with cancer cell whole cell antigens. After the antigen presenting cells are activated by the microparticles, the antigen presenting cells are prepared into nano particles or microparticles for detecting the cancer cell specific T cells.

(1) Lysis of cancer cells

Collecting the cultured B16F10 melanoma cancer cell line, centrifuging for 5 minutes at 350g, then removing the supernatant, washing twice with PBS, then adopting 6M guanidine hydrochloride to resuspend and crack the cancer cells, and obtaining the antigen raw material source for preparing the micron particle system after the cancer cell whole cell antigen is cracked and dissolved in 6M guanidine hydrochloride.

(2) Preparation of microparticle systems

In this example, the microparticles were prepared by a multiple emulsion method. The molecular weight of PLGA material prepared from the adopted microparticle 1 is 38-54 KDa, and the adopted immunologic adjuvants are CpG BW006, CPG2216 and Poly ICLC. Poly ICLC is a Toll-like receptor 3 agonist, various CpG types are Toll-like receptor 9 agonists, and Toll-like receptor 3 and Toll-like receptor 9 are positioned in endocytotic membrane structures in cells. Firstly, loading a lysate component and an immunologic adjuvant in a micron particle, centrifuging for 15 minutes at 10000g, resuspending by using 10mL of ultrapure water containing 4% trehalose, and freeze-drying for 48 hours; particles were resuspended in 7mL PBS before use and 3mL of cancer cell lysate fraction (protein concentration 50 mg/mL) was added and allowed to act at room temperature for 10min to give lysate loaded microparticles 1 both inside and outside. The average particle diameter of the micron particles is about 2.50 mu m, and the surface potential is about-2 mV; about 140. Mu.g of protein or polypeptide component was loaded per 1mg of PLGA microparticles 1, and CpG BW006 (class B), CPG2216 (class A) and Poly ICLC were loaded at 0.02mg each.

The control microparticle 2 was prepared from the same materials and by the same method as the above, and the loaded immunoadjuvants were CpG2336 (class A), CPG2216 (class A) and Poly ICLC. The particle size of the control microparticle 2 is about 2.50 μm, the surface potential is about-2 mV, about 140 μ g of protein or polypeptide component is loaded on each 1mg of PLGA microparticle, and each 1mg of PLGA microparticle is loaded with 0.02mg of CpG2336 (class A), CPG2216 (class A) and Poly ICLC immunoadjuvant.

The control microparticle 3 was prepared using the same materials and methods as the control microparticle, and the loaded immunoadjuvants were CpG BW006 (class B) and CpG2216 (class a). The control microparticles 3 used 0.02mg adjuvant per 1mg PLGA microparticle, a particle size of about 2.50 μm, a surface potential of about-2 mV, about 140 μ g protein or polypeptide component loaded per 1mg PLGA microparticle, and 0.03mg each of CpG BW006 (class B) and CPG2216 (class A) loaded per 1mg PLGA microparticle.

(3) Preparation of antigen-presenting cells

Collecting lymph node and spleen of mouse after killing mouse, mincing and grinding lymph node or spleen of mouse, respectively filtering with cell screen to obtain single cell suspension, mixing lymph node single cell suspension and spleen single cell suspension, and separating CD19 from them by flow cytometry ⁺ B cell and CD11c ⁺ Of (3) is performed.

(4) Activation of antigen presenting cells

The cancer cell whole cell fraction-loaded microparticles (500. Mu.g) were incubated with the prepared DCs (1000 ten thousand) and B cells (1000 ten thousand) in 20mL of high-glucose DMEM complete medium for 72 hours (37 ℃,5% CO2) in an incubation system containing granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) and CD86 antibody (10 ng/mL).

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

The incubated DC and B cells were harvested by centrifugation at 400g for 5 minutes, then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water and sonicated at low power (22.5W) for 1 minute at 4 ℃. And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes and collecting supernatant, centrifuging at 16000g for 90 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano particles. Wherein, the nano particle prepared by the mixed antigen presenting cell activated by the micro particle 1 is the nano particle 1 with the particle diameter of 110 nanometers; wherein the nanoparticle prepared by the mixed antigen presenting cell activated by the microparticle 2 is the nanoparticle 2 with the particle size of 110 nanometers; wherein the nanoparticles prepared from the mixed antigen presenting cells activated by the microparticles 3 are nanoparticles 3 with the particle size of 110 nanometers.

Alternatively, the incubated DC and B cells were harvested by centrifugation at 400g for 5 minutes (activated with microparticle 1), and then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, and the cells were sonicated at 4 ℃ for 1 minute with low power (22.5W) after resuspension in PBS water. And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes and collecting supernatant, centrifuging at 16000g for 90 minutes, collecting the precipitate, discarding the supernatant, and re-suspending the precipitate in PBS to obtain the nano-particles. 20mg of nano particles and 100mg of micron particles 1 are mixed and incubated together for 15 minutes at room temperature, then ultrasonic treatment is carried out for 1 minute at 10W, then centrifugation is carried out for 15 minutes at 8000g, supernatant is discarded, precipitates are collected, and the precipitates are resuspended to obtain micron particles 4 with the particle size of about 2.55 microns.

(6) Detection of cancer cell-specific T cells

On day 0, each C57BL/6 mouse was inoculated subcutaneously into the back of 1.5X 10 ⁵ And B16F10 cells. Mice were treated with radiation (Radiotherapy) or injected with 100 μ L PBS on

days

10, 15, 20 using radiation to irradiate the tumor site. Mice were sacrificed on day 24, peripheral blood was collected from each group of mice, peripheral Blood Mononuclear Cells (PBMC) were prepared, and CD3 was sorted out using magnetic bead sorting ⁺ T cells. The sorted T cells (500 ten thousand), the nanoparticles 1-3 (100. Mu.g) prepared in step (5) or the microparticles 4 (100. Mu.g) were co-incubated in 2mL of RPMI1640 complete medium for 24 hours (37 ℃,5% CO) ₂ ) Thereafter, flow cytometry was used to detect CD3 in T cells ⁺ IFN-γ ⁺ T cells are cancer cell-specific T cells activated by cancer cell whole cell antigens. At the same time, CD3 was analyzed ⁺ IFN-γ ⁺ IFN-gamma in T cells ⁺ Mean Fluorescence Intensity (MFI) of the attached fluorescent probes.

(7) Results of the experiment

As shown in FIG. 5a, the effect of detecting cancer cell-specific T cells by the nanoparticles prepared from the microparticle-activated antigen-presenting cells loaded with the CpG adjuvant and the Poly ICLC mixed adjuvant is better than that of the nanoparticles prepared from the microparticle-activated antigen-presenting cells loaded with the two CpG mixed adjuvants. Moreover, the effect of the nanoparticle prepared by the microparticle activated antigen presenting cell loaded with one B class CpG, one A class CpG and PolyICLC mixed adjuvant is better than that of the nanoparticle prepared by the microparticle activated antigen presenting cell loaded with two A class CpG and PolyICLC mixed adjuvants; moreover, the particles prepared from the activated antigen-presenting cell membrane have a better effect of loading the cancer cell whole cell component inside. CD3 ⁺ IFN-γ ⁺ IFN-gamma in T cells ⁺ The analysis of the Mean Fluorescence Intensity (MFI) of the attached fluorescent probes was consistent with the above trend (FIG. 5 b). This shows that the nanoparticles prepared from the microparticles activated antigen-presenting cells loaded with the mixed adjuvant of two different Toll-like receptors have better effect, and the nanoparticles prepared from the microparticles activated antigen-presenting cells containing B-class CpG and Toll-like receptor 3 agonist as the mixed adjuvant have better effect.

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

In this example, B16F10 melanoma tumor tissue was first lysed using 8M urea and the tumor tissue lysate fraction was solubilized. Then, PLGA is used as a nanoparticle framework material, poly (I: C), cpG2006 (B type) and CpGSL01 (B type) are used as immunologic adjuvants to prepare nanoparticles loaded with cancer cell whole-cell antigens, the nanoparticles are used for activating antigen presenting cells to prepare the nanoparticles, and then cancer cell specific T cells in tumor tissues are detected.

(1) Collection and lysis of tumor tissue

C57BL/6 mice were each inoculated subcutaneously on the back with 1.5X 10 ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of 8M urea is added through a cell filter screen to lyse cells and lyse cell lysates. The above is the antibody for preparing the nanoparticle systemThe raw material source is as follows.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a solvent evaporation method. The molecular weight of PLGA used as a preparation material of the nano particle 1 is 7KDa-17KDa, the adopted immunologic adjuvants are Poly (I: C), cpG2006 and CpGSL01, and the lysate component and the adjuvant are loaded in the nano particle. The preparation method is as described above, after loading lysate components and adjuvants in the nanoparticles, centrifuging 100mg of nanoparticles at 12000g for 20 minutes, resuspending the nanoparticles with 10mL of ultrapure water containing 4% trehalose, and freeze-drying for 48 hours to obtain lyophilized powder for later use. The average particle size of the nano particles is about 270nm, and the surface potential of the nano particles is about-3 mV; about 80 μ g of protein or polypeptide component is loaded per 1mg of PLGA nanoparticle, and 0.02mg of Poly (I: C), cpG2006 and CpGSL01 are used per 1mg of PLGA nanoparticle.

The preparation material and the preparation method of the control nano particle 2 are the same as those of the control nano particle 2, the particle diameter is about 270nm, the same amount of lysate components are loaded, the loaded immune adjuvant is Poly (I: C), and 0.06mg of Poly (I: C) is loaded to every 1mg of PLGA.

The preparation material and the preparation method of the control nano particle 3 are the same as those of the control nano particle, the particle diameter is about 270nm, the equivalent lysate components are loaded, the loaded immunologic adjuvants are Poly (I: C), cpG1585 (A class) and CpG2216 (A class), and 0.02mg of Poly (I: C), cpG1585 (A class) and CpG2216 (A class) are loaded on each 1mg of PLGA.

(3) Preparation of DC and B cells

Mouse lymph nodes are removed after C57BL/6 is sacrificed, a mouse lymph node single cell suspension is prepared, and then CD11C is separated from the lymph node single cell suspension by using flow cytometry ⁺ DC and CD19 ⁺ B cells.

(4) Activation of antigen presenting cells

Nanoparticle 1 (500. Mu.g), nanoparticle 2 (500. Mu.g) or nanoparticle 3 (500. Mu.g) loaded with cancer cell whole cell components was incubated with DC (500 ten thousand) and B cells (500 ten thousand) in 20mL of high-glucose DMEM complete medium for 72 hours (37 ℃, 5-percent CO ₂ ) (ii) a 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) andCD86 antibody (10 ng/mL).

(5) Preparation of antigen-presenting cell-based nanoparticles

The incubated DC and B cells were harvested by centrifugation at 400g for 5 minutes, then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water and disrupted at 4 ℃ using a homogenizer at 2000rpm for 25 minutes. Then centrifuging the sample at 3000g for 15 minutes and collecting the supernatant, centrifuging the supernatant at 8000g for 15 minutes and collecting the supernatant, then centrifuging at 15000g for 30 minutes and discarding the supernatant and collecting the precipitate, and re-suspending the precipitate in PBS to obtain the nano-particles. Wherein, the antigen presenting cell activated by the nano particle 1 is used for preparing nano particles 4 with the particle diameter of 150 nanometers; the antigen presenting cells activated by the nano particles 2 are used for preparing nano particles 5 with the particle size of 150 nanometers; prepared using the nanoparticle 3-activated antigen-presenting cells was nanoparticle 6, with a particle size of 150 nm.

(6) Detection of cancer cell-specific T cells

On day 0, each C57BL/6 mouse was inoculated subcutaneously into the back of 1.5X 10 ⁵ Mice were injected subcutaneously with 100. Mu.L of α PD-1 antibody (10 mg/kg) or 100. Mu.L PBS on

days

8, 10, 12, 14, and 16, respectively, for B16F10 cells. Killing mice on day 20, collecting tumor tissues of the mice respectively, cutting the tumor tissues into small pieces, filtering the small pieces through a cell screen to prepare single cell suspension of the tumor tissues, and sorting the single cell suspension of the tumor tissues by using a magnetic bead sorting method to obtain CD3 ⁺ T cells. Then, the sorted T cells (50 ten thousand) were co-incubated with allogeneic-derived B cells (250 ten thousand), the nanoparticles prepared in step (5) (100. Mu.g) or in 20mL RPMI1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After centrifugation at 400g for 5 minutes, the supernatant was collected and analyzed for IFN-. Gamma.concentration by ELISA.

In the ELISA detection method, the cancer cell-specific T cells are activated to secrete a killer substance such as IFN-gamma. The concentration of the killing substances represents the content of activated cancer specific T cells and the killing capacity of the cancer cells.

(7) Results of the experiment

As shown in FIG. 6, more IFN-. Gamma.was detected after co-incubation of nanoparticles prepared from cancer cell whole-cell antigen loaded nanoparticles-activated antigen-presenting cells compared to PBS control. Moreover, the effect of the nanoparticles prepared by the nanoparticle activated antigen presenting cells loaded with two B-class CpG and Poly (I: C) as mixed adjuvants is better than that of the nanoparticles loaded with two A-class CpG and Poly (I: C) as mixed adjuvants or the nanoparticles activated antigen presenting cells loaded with only Poly (I: C) as adjuvants.

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 nanoparticles prepared using nanoparticle-activated antigen-presenting cells detect a broad spectrum of cancer cell-specific T cells. The colon cancer tumor tissue and the lung cancer cells are first lysed to prepare water-soluble components and water-insoluble components, and the antigen is first degraded into polypeptides in vitro using proteases. Other enzymes or other methods may be used in practice to degrade the proteins in the whole cell fraction into polypeptides. Then, a water-soluble component mixture (mass ratio of 1. Then, PLA is used as a nanoparticle framework material, cpGM362, CPG1018 and Poly ICLC are used as immune adjuvants to prepare nanoparticles, and the nanoparticles are used for detecting cancer cell specific T cells in vitro.

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

Each C57BL/6 mouse was inoculated subcutaneously at the back 2X 10 ⁶ The MC38 cells in the tumor grow to a volume of about 1000mm ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and freeze thawing is repeated for 5 times, and ultrasonic waves are accompanied to destroy the lysed cells. After the cells are lysed, centrifuging the lysate for 5 minutes at a rotating speed of more than 5000g and taking supernatant fluid as a water-soluble component which can be dissolved in pure water; in the obtained precipitation partThe precipitation fraction is dissolved by adding 8M urea to convert the water-insoluble fraction insoluble in pure water to soluble fraction in 8M urea aqueous solution. Trypsin (Trypsin, 0.5 mg/mL) and Chymotrypsin (Chymotrypsin, 0.5 mg/mL) were added to the water soluble fraction (80 mg/mL) and incubated for 1 hour, followed by heating at 95 ℃ for 10 minutes to inactivate the protease for use.

The cultured LLC lung cancer cell lines were harvested and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, then the cells were resuspended with ultrapure water and freeze-thawed repeatedly 5 times, with ultrasound optionally to disrupt the lysed cells. After cell lysis, centrifuging the lysate for 6 minutes at a rotating speed of 3000g and taking supernatant fluid as a water-soluble component soluble in pure water; the addition of 8M urea to the resulting precipitate portion dissolves the precipitate portion, thereby converting the water-insoluble components insoluble in pure water to soluble in an 8M aqueous urea solution. Trypsin (Trypsin, 0.5 mg/mL) and Chymotrypsin (Chymotrypsin, 0.5 mg/mL) were added to the water soluble fraction (80 mg/mL) and incubated for 1 hour, followed by heating at 95 ℃ for 10 minutes to inactivate the protease for use.

Mixing water-soluble components from colon cancer tumor tissue and lung cancer cells according to a mass ratio of 1; the water-insoluble components dissolved in 8M urea were also mixed in a mass ratio of 1. And then mixing the water-soluble component mixture and the water-insoluble component mixture according to the mass ratio of 1.

(2) Lysis and solubilization of BCG

Collecting BCG, using 8M urea aqueous solution to crack BCG, and dissolving cracking components for later use.

(3) Preparation of nanoparticles

In this example, the nanoparticles 1 were prepared by a solvent evaporation method. The molecular weight of PLA of the material prepared from the nano particles 1 is 20KDa, the interior of the nano particles is loaded with tumor tissues, cancer cell lysate, bacterial lysate and immunologic adjuvant, and the surface is loaded with components of the tumor tissues and the cancer cell lysate. The adopted immunoadjuvants are CpGM362, CPG1018 and poly ICLC, the adjuvants are loaded in the nanoparticles, and the mass ratio of tumor tissue and cancer cell lysate to bacterial lysate used for preparing the nanoparticles is 1. Preparation method As mentioned above, during the preparation process, firstly, the lysate mixture, the bacterial lysate component and the adjuvant are loaded inside the nanoparticles by using the multiple emulsion method, after the lysate and the adjuvant are loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are frozen and dried for 48 hours. Before use, 20mg of nanoparticles were resuspended in 0.9mL of PBS and incubated for 5 minutes at room temperature with 0.1mL of a sample containing equal amounts of the cancer cell and tumor tissue lysate mixture and bacterial lysate fraction (80 mg/mL). The average particle diameter of the nanoparticle 1 is about 290nm, about 140 mug of protein or polypeptide component is loaded on each 1mg of PLGA nanoparticle 1, and each 1mg of PLGA nanoparticle contains 0.04mg of CpGM362, CPG1018 and Poly ICLC immunoadjuvant.

(4) Preparation of antigen-presenting cells

Peripheral blood was collected from mice after C57BL/6 sacrifice, peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood, and CD11C was then sorted from PBMC using flow cytometry ⁺ DC and CD19 ⁺ B cells. In this example, BMDC and BMDM were used together as antigen-presenting cells. BMDC was prepared as in example 1. The BMDM was prepared as follows:

anaesthetizing C57 mice, dislocating, killing, sterilizing the mice with 75% ethanol, cutting a small opening on the back of the mice with scissors, directly tearing the skin to the position of the lower leg joint of the mice with hands, and removing the foot joints and the skin of the mice. The hind limb was removed with scissors along the greater trochanter of the mouse thigh, the muscle tissue was removed and placed in a 75% ethanol containing petri dish and soaked for 5min, and the 75% ethanol containing petri dish was replaced and transferred to a clean bench. The leg bone soaked in ethanol is transplanted into cold PBS for soaking, and the ethanol on the surfaces of the tibia and the femur is washed off, and the process can be repeated for 3 times. The cleaned femur and tibia are separated, the two ends of the femur and tibia are cut off by scissors, the cold induction medium is sucked by a 1mL syringe to blow out bone marrow from the femur and tibia, and purging is carried out for 3 times until the leg bone is not obviously red. The medium containing bone marrow cells was repeatedly blown up with a 5mL pipette to disperse cell clumps, the cells were then sieved using a 70 μm cell filter, transferred into a 15mL centrifuge tube, centrifuged at 1500rpm/min for 5min, the supernatant was discarded, the red blood cell lysate was added, resuspended and allowed to stand for 5min, then centrifuged at 1500rpm/min for 5min, the supernatant discarded and resuspended in cold prepared bone marrow macrophage induction medium (DMEM high-glucose medium containing 15% L929 medium), and plated. Cells are cultured overnight to remove other contaminating cells that adhere faster such as fibroblasts and the like. Collecting non-adherent cells, and seeding into a dish or cell culture plate according to the experimental design. Macrophage colony-stimulating factor (M-CSF) was used at a concentration of 40ng/mL to stimulate differentiation of bone marrow cells to mononuclear macrophages. Culturing for 8 days, and observing the macrophage morphological change under a light microscope. Digesting and collecting cells after 8 days, incubating for 30min at 4 ℃ in a dark place by using an anti-mouse F4/80 antibody and an anti-mouse CD11b antibody, and identifying the proportion of successfully induced macrophages by using flow cytometry.

(5) Activation of antigen presenting cells

Co-incubation of nanoparticle 1 (1000. Mu.g) with peripheral blood-derived DCs (2000 ten thousand) and BMDCs (2000 ten thousand) in RPMI1640 complete medium for 72 hours (37 ℃,5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL), IL-12 (500U/mL), and CD80 antibody (10 ng/mL).

Alternatively, nanoparticle 1 (1000. Mu.g) was incubated with peripheral blood-derived DCs (1000 ten thousand), BMDCs (1000 ten thousand), BMDM (1000 ten thousand) and B cells (1000 ten thousand) in 20mL of RPMI1640 complete medium for 48 hours (37 ℃,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 (10 ng/mL).

(6) Preparation of antigen presenting cell based nanoparticles

The incubated peripheral blood-derived DCs (2000 ten thousand) and BMDCs (2000 ten thousand) were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, and treated in a high pressure homogenizer (5000 bar) for 5 minutes after resuspending the cells in PBS water. And (2) centrifuging the sample at 2000g for 15 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, incubating the supernatant and the nanoparticle 1 (50 mg) prepared in the step (3) at 4 ℃ for 16 hours, repeatedly co-extruding the supernatant by using a 0.45-micrometer filter membrane, centrifuging the extruded liquid at 13000g for 20 minutes, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nanoparticle 2 with the particle size of 310 nanometers.

Alternatively, the incubated peripheral blood-derived DCs (1000 ten thousand), BMDCs (1000 ten thousand), B cells (1000 ten thousand) and BMDMs (1000 ten thousand) were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with a Phosphate Buffered Saline (PBS) containing a protease inhibitor at 4 ℃, resuspended in PBS water, and then treated in a high-pressure homogenizer (5000 bar) for 5 minutes. And (2) centrifuging the sample at 2000g for 15 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, incubating the supernatant and the nanoparticle 1 (50 mg) prepared in the step (3) at 4 ℃ for 16 hours, repeatedly co-extruding the supernatant by using a 0.45-micrometer filter membrane, centrifuging the extruded liquid at 13000g for 20 minutes, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nanoparticle 3 with the particle size of 310 nanometers.

(7) Detection of cancer cell-specific T cells

Day 0, each C57BL/6 mouse was inoculated subcutaneously into the back at 1.5X 10 ⁵ MC38 cells, mice were subcutaneously injected with 100 μ L of 1mg PLGA nanoparticle 1 at day 10, day 15 and day 21, respectively, to activate cancer cell-specific T cells in mice. Mice were sacrificed on day 24, draining lymph nodes and spleens of the mice were collected, single cell suspensions of draining lymph nodes and splenocytes were prepared and T cells were sorted therefrom using the magnetic bead method.

The resulting T cells (400 million), 200. Mu.g of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3), IL-2 (500U/mL), IL-7 (500U/mL), and IL-15 (5000U/mL) were co-incubated in 5mL DMMEM complete medium for 24 hours, and then the incubated CD3 was sorted by flow cytometry ⁺ IFN-γ ⁺ T cells are cancer cell-specific T cells activated by cancer cell whole cell antigens. At the same time, CD3 was analyzed ⁺ IFN-γ ⁺ IFN-gamma in T cells ⁺ Mean Fluorescence Intensity (MFI) of the attached fluorescent probes. The stronger the fluorescence intensity is, the more the lethal substance is expressed by the cancer cell specific T cell, the higher the sensitivity is in the detectionThe better the accuracy will be.

Or the obtained T cells (400 ten thousand), peripheral blood-derived DCs (100 ten thousand), BMDCs (100 ten thousand), B cells (100 ten thousand), BMDM (100 ten thousand), and 200. Mu.g of nanoparticles (nanoparticle 1 or nanoparticle 3), IL-2 (500U/mL), IL-7 (500U/mL), and IL-15 (5000U/mL) were co-incubated in 5mL DMMEM complete medium for 24 hours, and then the incubated CD3 was sorted by flow cytometry ⁺ IFN-γ ⁺ T cells are cancer cell-specific T cells activated by cancer cell whole cell antigens. At the same time, CD3 was analyzed ⁺ IFN-γ ⁺ IFN-gamma in T cells ⁺ Mean Fluorescence Intensity (MFI) of the attached fluorescent probes.

(8) Results of the experiment

As shown in fig. 7, nanoparticle 3 outperforms both nanoparticle 1 and nanoparticle 2. In the detection of T cells without antigen presenting cells, nanoparticle 1 was not detectable, whereas both nanoparticle 2 and nanoparticle 3 were detectable. When the antigen presenting cells are contained to detect T cells, the

nano particles

1, 2 and 3 can detect the T cells, and the

nano particles

2 and 3 are better than the nano particles 1 and the nano particles 3 are better than the nano particles 2. Furthermore, even when T cells are detected without antigen-presenting cells, the effect of the nanoparticle 2 is better than the effect of the nanoparticle 1 and the nanoparticle 2 when T cells are detected with antigen-presenting cells. The analysis result of the average fluorescence intensity was consistent with the above-described tendency. This demonstrates that both loading of activated antigen-presenting cell membrane components on the particle surface and the use of mixed antigen-presenting cell membrane components can enhance the detection of cancer cell-specific T cells by nanoparticles or microparticles.

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 microparticles loaded with cancer cell whole cell antigens activate antigen-presenting cells and then prepared into microparticles 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, followed by repeated freeze-thawing 5 times with sonication to lyse cancer cells. Adding 1mg/mL nuclease into the lysed cells to degrade nucleic acid in the lysate, heating at 95 ℃ for 10 minutes to inactivate the nuclease, centrifuging at 5000g for 5 minutes to collect supernatant as a water-soluble component, dissolving the precipitate with 10% sodium deoxycholate (containing 10M arginine) to obtain a water-insoluble component, and mixing the water-soluble component with the water-insoluble component according to a mass ratio of 3:1 mixing to obtain the raw material source for preparing the particle system.

(2) Preparation of microparticle systems

In this example, the method of multiple emulsion was used to prepare microparticles. The molecular weight of PLGA, the microparticle 1 matrix material, is 38-54 KDa, and the adopted immunologic adjuvants are CpG2395 (C class), cpGM362 (C class) and Poly (I: C). The preparation method comprises the steps of preparing the microparticles internally loaded with the lysate component and the adjuvant by a multiple emulsion method, centrifuging 100mg of the microparticles at 9000g for 20 minutes, using 10mL of ultrapure water containing 4% trehalose to resuspend, and drying for 48 hours for later use. The average particle diameter of the micron particle system is about 2.5 mu m, and the surface potential is about-6 mV; each 1mg PLGA microparticle was loaded with about 110. Mu.g of protein or polypeptide components, 0.02mg each of CpG2395, cpGM362 and Poly (I: C). The preparation material and the preparation method of the reference micrometer particle 2 are the same, the particle size is about 2.5 micrometers, and the surface potential is about-6 mV; each 1mg PLGA microparticle was loaded with about 110. Mu.g of protein or polypeptide components, and each 1mg PLGA was loaded with 0.02mg of each of CpG1585 (class A), cpG2336 (class A) and Poly (I: C).

(3) Preparation of B cells

B cells from peripheral splenocytes were used. Collecting spleen after killing mice, preparing mouse spleen cell single cell suspension, and sorting CD19 in the single cell suspension by magnetic bead sorting method ⁺ B cells.

(4) Activation of antigen presenting cells

Incubating cancer cell whole cell antigen fraction-loaded microparticles (800. Mu.g) with the B cells (1000 ten thousand) prepared in step (3) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃,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 (10 ng/mL).

(5) Preparation of antigen presenting cell based microparticles

Incubated B cells (1000 ten thousand) were collected by centrifugation at 400g for 5min, then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water, sonicated at 4 ℃ for 1 min with low power (20W) and then treated with a homogenizer at 1000rpm for 3 min. And (3) centrifuging the sample at 3000g for 15 minutes and collecting a supernatant, centrifuging the supernatant at 8000g for 15 minutes and collecting the supernatant, carrying out ultrasonic treatment on the supernatant and the micrometer particles (60 mg) prepared in the step (2) and DSPE-PEG-mannose (1 mg) at 50W for 2 minutes, centrifuging at 8000g for 20 minutes, collecting and discarding the supernatant, collecting a precipitate, and carrying out resuspension on the precipitate in PBS to obtain the micrometer particles. Wherein the microparticles prepared by the coaction of the antigen presenting cell membrane component activated by the microparticles 1 and the microparticles 1 are microparticles 3 with the particle size of 2.6 μm; the microparticles prepared using the microparticle 2-activated antigen-presenting cell membrane fraction to co-act with microparticle 2 were microparticle 4, having a particle size of 2.6 μm.

(6) Detection of cancer cell-specific T cells

Day 0 each BALB/c mouse was inoculated subcutaneously on the back at 1X 10 ⁶ 4T1 cells, mice were injected subcutaneously with 100. Mu.L of 1mg PLGA microparticle 1 (micrometer vaccine 1) or 100. Mu.L PBS on

days

10, 14 and 18, respectively. Mice were sacrificed on day 22, spleens of the mice were collected, and single cell suspensions of splenocytes were prepared. Incubating single cell suspensions (600 ten thousand) of splenocytes, DC2.4 (200 ten thousand), and microparticles (50 μ g) in 2mL DMEM complete medium for 72 hours (37 ℃,5% ₂ ) Then separating out CD3 from the mixture by flow cytometry ⁺ IFN-γ ⁺ T cells are cancer cell-specific T cells activated by cancer cell whole cell antigens.

(7) Results of the experiment

As shown in fig. 8, microparticles prepared from microparticle-activated antigen-presenting cells were effective in detecting more cancer cell-specific T cells in the treated mice than in the control group. Furthermore, microparticles 3 prepared using microparticle 1-activated antigen-presenting cells with two C class CpGs and Poly (I: C) as mixed adjuvants were more effective than microparticles 4 prepared using microparticle 2-activated antigen-presenting cells with two A class CpGs and Poly (I: C) as mixed adjuvants. In the micro vaccine of the embodiment, mannose is used as a target head for active targeting, and any target head having the ability to target cells, such as a CD32 antibody, mannan, a CD205 antibody, a CD19 antibody, and the like, can be used in practical applications.

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

This example targets mannose to demonstrate how to detect cancer cell-specific T cells in peripheral blood using nanoparticles prepared by actively targeting nanoparticle-activated antigen-presenting cells.

(1) Lysis of cancer cells

After the cultured Pan02 pancreatic cancer cells were collected, the cancer cells were lysed using 10% octyl glucoside and the cancer cell whole cell antigen derived from the cancer cells was lysed.

(2) Preparation of nanoparticles

The nanoparticle system in this example was prepared using a multiple emulsion process. The nano particle preparation materials are PLGA and PLGA modified by mannose, and the molecular weights of the PLGA and the PLGA are both 7KDa-17KDa. When the target head-carrying nano particles are prepared, the mass ratio of the target head-carrying nano particles to the target head-carrying nano particles is 4. The immunological adjuvants used were Poly (I: C) and CpG SL03. The preparation method is as described above, the lysate component and the adjuvant are loaded inside the nanoparticles by a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used for resuspension, and then the mixture is frozen and dried for 48 hours for later use. The average particle diameter of the nanoparticle 1 with the target head is about 270nm, and each 1mg of PLGA nanoparticle is loaded with about 80 mug protein and polypeptide components, and contains 0.04mg of Poly (I: C) and CpGSL03 respectively. The particle diameter of the nanoparticle 2 without adjuvant but with mannose target is about 270nm, the same amount of cell lysis component but without any immunologic adjuvant is adopted during preparation, and about 80 mug of protein and polypeptide component are loaded per 1mg of PLGA nanoparticle.

(3) Preparation of antigen-presenting cells

This example uses BMDCs and BMDMs as antigen presenting cells. BMDC and BMDM were prepared as above.

(4) Activation of antigen presenting cells

Incubation of nanoparticle 1 (1000. Mu.g) or nanoparticle 2 (1000. Mu.g) with BMDC (1000 ten thousand), BMDM (1000 ten thousand) and IL-7 (500U/mL), respectively, in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃,5% CO ₂ )。

Alternatively, BMDCs (1000 ten thousand), BMDM (1000 ten thousand) and IL-7 (500U/mL) were co-incubated in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃, 5%. Both of the above incubation systems contained IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN-. Gamma. (500U/mL), and CD80 antibody (10 ng/mL).

(5) Preparation of antigen-presenting cell-derived nanoparticles

The incubated DCs and macrophages were harvested by centrifugation at 400g for 5 minutes, followed by two washes of the cells using 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspension of the cells in PBS water followed by low power (10W) sonication at 4 ℃ for 20 minutes. Centrifuging the sample at 3000g for 15 minutes, collecting supernatant, filtering the supernatant by membranes with the pore diameters of 30 microns, 10 microns, 5 microns, 2 microns, 1 micron, 0.45 microns and 0.22 microns in sequence, collecting filtrate, centrifuging the filtrate at 18000g for 50 minutes, collecting the supernatant, discarding precipitate, resuspending the precipitate in 4% trehalose aqueous solution, and then freeze-drying for 48 hours to obtain the nano particles. Wherein, the mixed antigen presenting cells which are not activated by the nano particles are used for preparing nano particles 3, and the particle size is 110 nanometers; the mixed antigen presenting cells activated by the nano particles 2 are used for preparing nano particles 4 with the particle size of 110 nanometers; the mixed antigen presenting cells activated by the nanoparticles 1 are used to prepare nanoparticles 5 with a particle size of 110 nm.

(6) Detection of cancer cell-specific T cells

Each C57BL/6 mouse was inoculated subcutaneously into the back of the patient at day 0 at 1X 10 ⁶ On each of Pan02 pancreatic cancer cells, mice were subcutaneously injected with 100. Mu.L of 1mg PLGA nanoparticles on

days

10, 15, 20 and 27, respectively. Mice were sacrificed and collected on day 24Peripheral blood, peripheral Blood Mononuclear Cells (PBMC) prepared from peripheral blood, and CD3 isolated from PBMC using flow cytometry ⁺ T cells. Incubating T cells (500 ten thousand) with 100. Mu.g of nanoparticles (nanoparticle 1, or nanoparticle 3, or nanoparticle 4, or nanoparticle 5) in a DMEM high-glucose medium for 72 hours (37 ℃,5% ₂ ) The incubation system contained IL-2 (500U/mL) and IL-7 (500U/mL). CD3 was then separated from the incubated cells by flow cytometry ⁺ IFN-γ ⁺ T cells are cancer cell specific T cells.

(7) Results of the experiment

As shown in fig. 9,

nanoparticles

4 and 5 are more effective than

nanoparticles

1 and 3. And the nanoparticles 5 are better than the nanoparticles 4. In conclusion, the prepared nanoparticles can effectively detect cancer cell-specific T cells regardless of whether the antigen-presenting cells are activated by the nanoparticles with the adjuvant, but the nanoparticles prepared by the antigen-presenting cells activated by the nanoparticles with the adjuvant have better effect.

Example 9 nanoparticle detection of lung cancer cell-specific T cells

This example illustrates that calcified nanoparticles detect cancer cell-specific T cells in mouse splenocytes, and that other biomineralization techniques, cross-linking, gelation, and other like modifications of the particles can be used in practice. In this example, a mouse lung cancer tumor tissue is lysed with 8M urea (containing 200mM sodium chloride), dissolved and loaded on a nanoparticle system, and after the particles are used to activate antigen-presenting cells, the antigen-presenting cells are prepared into nanoparticle-specific T cells for detecting cancer cells.

(1) Lysis of tumor tissue and cancer cells

Female C57BL/6 mice at 6-8 weeks were inoculated at 1X 10 in back ⁶ Lung cancer cells of LLC mouse with tumor size of 1000mm ³ Killing mouse, collecting tumor tissue, cutting, grinding, filtering with cell screen to obtain single cell suspension, irradiating with ultraviolet for 5min, heating at 80 deg.C for 10min, and lysing and dissolving single cells of tumor tissue with 8M urea (containing 200mM sodium chloride)Suspending to obtain cancer cell whole cell antigen.

(2) Preparation of nanoparticles

This example of the bio-calcification nanoparticles after loading cancer cell whole-cell antigens inside and on the surface of the nanoparticles. In the embodiment, the nano-particles are prepared by a solvent volatilization method, the molecular weight of PLGA (polylactic-co-glycolic acid) used as a nano-particle preparation material is 7KDa-17KDa, and immune adjuvants CpG2006 and Poly (I: C) are loaded in the nano-particles. The preparation method is as follows, firstly loading antigen in the nano particles by a multiple emulsion method, after loading lysis component in the nano particles, centrifuging 100mg PLGA nano particles for 20min at 13000g, then using 18mL PBS for heavy suspension, then adding 2mL tumor tissue and cancer cell lysate (60 mg/mL) dissolved in 8M urea, after acting for 10min at room temperature, centrifuging at 12000g for 20min, and then collecting the precipitate. The 100mg PLGA nanoparticles were then resuspended in 20mL DMEM medium, followed by 200. Mu.L CaCl ₂ (1 mM) and reacted at 37 ℃ for two hours. And centrifuging at 10000g for 20 minutes, collecting the precipitate, resuspending with ultrapure water, and centrifuging and washing twice to obtain the nano particles 1 with the average particle size of about 290 nm. Each 1mg PLGA nanoparticle 1 was loaded with approximately 140. Mu.g protein or polypeptide components, cpG2006 and Poly (I: C) 0.03mg each.

(3) Preparation of antigen-presenting cells

This example uses BMDC and B as antigen presenting cells. The BMDC was prepared as in example 1.B cells were derived from mouse peripheral blood PBMCs, prepared as described above. And mixing the BMDC and the B cells according to the quantitative ratio of 1.

(4) Activation of antigen presenting cells

Incubating the nanoparticles loaded with cancer cell whole cell fractions (1000. Mu.g) with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃,5% ₂ ) (ii) a The incubation system contains a cytokine component 1: IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN-. Gamma. (500U/mL).

Or as a control, nanoparticles (1000. Mu.g) loaded with cancer cell whole cell components were mixed with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL of high-glucose DMEM complete mediumMiddle incubation for 48 hours (37 ℃,5% CO) ₂ ) (ii) a The incubation system does not contain any cytokines and antibodies.

Incubation of cancer cell whole cell fraction-loaded nanoparticles (1000. Mu.g) with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃,5% CO) ₂ ) (ii) a The incubation system contains cytokine combination 2: IL-4 (500U/mL), IL-10 (200U/mL), IL-37 (200U/mL), TGF-. Beta.s (500U/mL).

(5) Preparation of antigen-presenting cell-based nanoparticles

Incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by low power (20W) sonication for 2 minutes at 4 ℃. And (2) centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 5000g for 10 minutes and collecting supernatant, filtering the supernatant through a 0.45-micron membrane, then performing ultrafiltration centrifugal filtration and concentration by using an ultrafiltration membrane (with the molecular weight cut-off of 50 KDa), mixing the filtered and concentrated sample with the nanoparticles 1 prepared in the step (2), treating the mixture for 3 minutes by using a high-pressure homogenizer (10000 bar), centrifuging at 13000g for 30 minutes, removing the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nanoparticles. When the particles are used for activating the antigen presenting cells, the system contains the antigen presenting cells of the cytokine combination 1, and the prepared nanoparticles 2 have the particle size of 300 nanometers; when the particles are used for activating the antigen presenting cells, the antigen presenting cells which do not contain cytokines in the system are prepared into nano particles 3, and the particle size is 300 nanometers; when the particles are used for activating the antigen presenting cells, the system contains the antigen presenting cells of the cytokine combination 2, the prepared nano particles 4 are nano particles, and the particle size is 300 nanometers.

(6) Detection of cancer cell-specific T cells

Each C57BL/6 mouse was inoculated subcutaneously into the back of the mouse at day 0 at 0.5X 10 ⁶ On

days

10, 15 and 20, mice were subcutaneously injected with 100. Mu.L of 1mg PLGA nanoparticles. Mice were sacrificed on day 24 and splenocytes were harvested and prepared as single cell suspensions, which were then run using flowCytometric CD45 isolation ⁺ CD3 ⁺ T cells.

Co-incubation of T cells (500 million) with nanoparticles 2 (100. Mu.g), nanoparticles 3 (100. Mu.g) or nanoparticles 4 (100. Mu.g) prepared from antigen-presenting cells in DMEM high-glucose medium for 12 hours (37 ℃,5% ₂ ) IL-2 (500U/mL) and IL-7 (500U/mL) were present in the system during incubation;

or co-incubating T cells (500 ten thousand) with nanoparticles 2 (100. Mu.g) prepared from antigen-presenting cells in DMEM high-glucose medium for 12 hours (37 ℃, 5%. The CO2) without any cytokines or antibodies in the incubation system;

or incubating T cells (500 ten thousand), 1000 ten thousand of the antigen-presenting cells mixed prepared in step (3), and nanoparticle 1 (100. Mu.g) in a DMEM high-glucose medium for 12 hours (37 ℃,5% ₂ ) IL-2 (500U/mL) and IL-7 (500U/mL) were included in the system during incubation.

The incubated cells were then collected, centrifuged at 400g for 5 minutes, labeled with the corresponding flow antibody, and then examined for CD3 by flow cytometry ⁺ IFN-γ ⁺ T cells are cancer cell specific T cells.

(7) Results of the experiment

As shown in fig. 10, the nanoparticles prepared from the calcified nanoparticle-activated antigen-presenting cells can detect more cancer cell-specific T cells than the control group. Moreover, when the cancer cell whole cell antigen-loaded nanoparticle activates an antigen presenting cell, the system contains the cytokine combination 1 which is superior to the system without the cytokine and/or antibody; when the nano particles activate the antigen presenting cells, the effect of the system containing the cell factor combination 1 or the system without the cell factors is better than that of the system containing the cell factor combination 2; moreover, when the nano particles prepared by the antigen presenting cells and T cells are incubated together, the system containing the cytokine combination 2 is better than the system without the cytokine.

Example 10 nanoparticle detection of cancer cell-specific T cells in melanoma

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

Tumor tissue was collected by first subcutaneously inoculating 1.5X 10 dorsal cells of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Killing mice and picking tumor tissues, cutting the tumor tissues into blocks, grinding, passing through a cell filter screen to prepare single cell suspension, adding ultrapure water, repeatedly freezing and thawing and carrying out ultrasonic lysis on the cells, adding nuclease for 5 minutes, and then inactivating the nuclease at 95 ℃ for 10 minutes. Centrifuging at 8000g for 3min to obtain supernatant as water soluble component; the precipitate was dissolved with 10% aqueous sodium deoxycholate to dissolve the water insoluble fraction. Mixing and dissolving the water-soluble component and the water-insoluble component dissolved by the sodium deoxycholate according to the mass ratio of 1.

(2) Preparation of nanoparticle systems

In the embodiment, the nanoparticles are prepared by a multiple emulsion method, and have the capability of targeting dendritic cells. The adopted nano particle preparation materials are PLGA and mannan-modified PLGA, the molecular weights of the PLGA and the mannan-modified PLGA are both 24KDa-38KDa, and the mass ratio of the unmodified PLGA to the mannan-modified PLGA in use is 9. The immunological adjuvant is poly (I: C), cpG1018 and CpG2216, the substance for increasing lysosome immune escape is KALA polypeptide (WEAKLAKALAKAKAKHLAKALKACEA), and the adjuvant and KALA polypeptide are coated in the nanoparticle. The preparation method is as described above, in the preparation process, firstly, the lysate component, the adjuvant and the KALA polypeptide are loaded inside the nanoparticles by a multiple emulsion method, after the components are loaded inside, 100mg of the nanoparticles are centrifuged at 12000g for 25 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are frozen and dried for 48 hours. The average particle diameter of the nano particles is about 250nm, and the surface potential is about-5 mV; each 1mg PLGA nano particle is loaded with about 100 mug protein or polypeptide component, each 1mg PLGA nano particle is loaded with 0.02mg of poly (I: C), cpG1018 and CpG2216 immunologic adjuvant, and each 1mg PLGA nano particle is loaded with 0.05mg of KALA polypeptide. The preparation material and the preparation method of the nanoparticle 2 are the same, the particle size is about 250nm, the surface potential is about-5 mV, KALA polypeptide is not loaded, and an adjuvant and a cell lysis component with the same quantity are loaded. The preparation material and the preparation method of the nano particles 3 are the same, the particle size is about 250nm, and the surface potential is about-5 mV; each 1mg PLGA nanoparticle is loaded with about 100 mug protein and polypeptide components, each 1mg PLGA nanoparticle is loaded with 0.02mg poly (I: C), 0.04mg CpG1018 and 0.05mg KALA polypeptide.

(3) Preparation of antigen-presenting cells

This example uses BMDC and BMDM as antigen presenting cells. BMDC and BMDM were prepared as above.

(4) Activation of antigen presenting cells

Incubating the nanoparticles loaded with cancer cell whole cell components (1000. Mu.g) with BMDCs (1000 ten thousand) and BMDMs (1000 ten thousand) in 15mL of high glucose DMEM complete medium for 48 hours (37 ℃,5% ₂ ) (ii) a The incubation system contained 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 (10 ng/mL).

(5) Preparation of antigen-presenting cell-based nanoparticles

The incubated BMDCs and BMDM were collected by centrifugation at 400g for 5 minutes, then washed three times by centrifugation at 1200rpm for 3min in 30mM pH 7.0Tris-HCl buffer containing 0.0759M sucrose and 0.225M mannitol, followed by ultrasonic mechanical disruption of antigen-presenting cells in the presence of phosphatase and protease inhibitors. After centrifugation, the resulting cell membrane was washed with a solution of 10mM Tris-HCl pH 7.5 and 1mM EDTA. And (3) filtering the sample by sequentially passing through membranes with the pore diameters of 30 microns, 10 microns, 5 microns, 2 microns and 0.45 microns, centrifuging the filtrate for 35 minutes at 16000g, discarding supernatant, collecting precipitates, re-suspending the precipitates by using PBS, co-incubating the re-suspended precipitates with the nanoparticles prepared in the step (2) for 10 minutes, repeatedly co-extruding the re-suspended solution by using a filter membrane with the diameter of 0.45 micron, centrifuging the extruded solution for 25 minutes at 12000g, discarding supernatant, collecting precipitates, re-suspending the precipitates in 4% trehalose aqueous solution, and freeze-drying the re-suspended precipitates to obtain the nanoparticles. Wherein the antigen presenting cell membrane component activated by the nano particle 1 and the nano particle 1 are used for coaction to prepare a nano particle 4 with the particle size of 260 nanometers; wherein the antigen presenting cell membrane component activated by the nano particle 2 and the nano particle 2 are used for coaction to prepare the nano particle 5, and the particle diameter is 260 nanometers; wherein the antigen presenting cell membrane component activated by the nano particle 3 and the nano particle 3 are used for coaction to prepare the nano particle 6, and the particle diameter is 260 nanometers.

(6) Detection of cancer cell-specific T cells

Female C57BL/6 mice from 6-8 weeks were selected and subcutaneously injected with 0.5mg PLGA nanoparticles (loaded lysate fraction, poly (I: C), and two CpG adjuvants and KALA polypeptide) on days 0, 7, 14, 21 and 28, respectively. Mice were sacrificed on day 32 and peripheral blood and lymph nodes of the mice were collected. PBMC in peripheral blood of mice was isolated by gradient centrifugation, lymph nodes were cut into small pieces and ground through a cell mesh to prepare single cell suspensions, and PBMC and lymph node cell single cell suspensions were then mixed. CD45 was then sorted out using magnetic bead sorting ⁺ CD3 ⁺ The T cell of (1). Sorting the obtained CD3 ⁺ T cells (500 million) and nanoparticles (40. Mu.g) were co-incubated with IL-7 (10 ng/mL) in 2mL RPMI1640 complete medium for 96 hours. Sorting of CD3 in incubated T cells by flow cytometry ⁺ IFN-γ ⁺ T cells are cancer cell specific T cells which can recognize cancer cell whole cell antigens.

(7) Results of the experiment

As shown in fig. 11, nanoparticles prepared from nanoparticle-activated antigen-presenting cells loaded with a whole cell fraction can detect more cancer cell-specific T cells than the control group. Moreover, the effect of the nano-particle 4 prepared by adding the nano-particle activated antigen presenting cell for increasing the lysosome escaping substance is better than that of the nano-particle 5 prepared by the nano-particle activated antigen presenting cell which is not added with the lysosome escaping substance; the effect of the nanoparticle 4 prepared from the nanoparticle-activated antigen-presenting cells using two CpG and Poly (I: C) adjuvants as mixed adjuvants is better than that of the nanoparticle 6 prepared from the nanoparticle-activated antigen-presenting cells using only one CpG and Poly (I: C) adjuvant.

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 cancer cell-specific T cells can be detected using microparticles prepared from microparticle-activated antigen-presenting cell membrane fractions and cancer cell membrane fractions loaded with cancer cell whole cell antigens. In this example, the breast cancer cells were first inactivated and denatured, and then lysed, and the water-insoluble components in the cancer cells were lysed with octyl glucoside lysis. Then, PLGA is used as a microparticle framework material, cpG2007, cpG1018 and Poly ICLC are used as immunologic adjuvants, poly-arginine and Poly-lysine are used as substances for enhancing escape of lysosomes, the microparticles loaded with cancer cell whole cell antigens are prepared, the microparticles are used for activating antigen presenting cells, then mixed microparticles based on antigen presenting cell membrane components and cancer cell membrane components are prepared, and then the microparticles are used for detecting 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 are respectively subjected to inactivation and denaturation treatment by adopting ultraviolet rays and high temperature heating at 60 ℃ for 5 minutes, then ultrapure water is added and freeze thawing is carried out repeatedly for 5 times, ultrasonic cracking is assisted, the cell lysate is centrifuged for 10 minutes at 5000g, the supernatant is a water-soluble component, the precipitate is dissolved by 10% of octyl glucoside to obtain a dissolved original water-insoluble component, and the water-soluble component and the water-insoluble component are mixed according to the mass ratio of 2 to 1 to obtain a lysate component required for preparing the micron particles.

(2) Preparation of microparticles

In the embodiment, a multiple emulsion method is adopted for preparing the micrometer particles 1, PLGA serving as a framework material of the micrometer particles has the molecular weight of 38KDa-54KDa, cpG2007, cpG1018 and Poly ICLC are adopted as immune adjuvants, and polyarginine and polylysine are adopted as lysosome escape increasing substances. The preparation method comprises preparing micrometer particles loaded with lysate component, adjuvant and KALA polypeptide by multiple emulsion method, centrifuging 100mg micrometer particles at 9000g for 20min, resuspending with 10mL ultrapure water containing 4% trehalose, and drying for 48h. The average particle size of the micrometer particle 1 system is about 3.1 micrometers, and the surface potential of the micrometer particle system is about-7 mV; each 1mg PLGA microparticle loaded about 110. Mu.g of protein or polypeptide component, 0.01mg each of CpG2007, cpG1018 and Poly ICLC, and 0.02mg each of polyarginine and polylysine.

(3) Preparation of antigen-presenting cells

This example used BMDC and DC2.4 as antigen presenting cells. The BMDC was prepared as in example 1.

(4) Activation of antigen presenting cells

The cancer cell whole cell fraction-loaded microparticles (1000. Mu.g) were incubated with BMDCs (500 ten thousand) and DC2.4 cells (500 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃,5% ₂ ) (ii) a The incubation system contained 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 (10 ng/mL).

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

1000 ten thousand post-incubation DCs were collected by centrifugation at 400g for 5 minutes, mixed with 1000 ten thousand 4T1 cells and washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water and sonicated at 4 ℃ for 2 minutes with low power (20W). Centrifuging 3000g of the sample for 15 minutes and collecting supernatant, centrifuging 5000g of the supernatant for 10 minutes and collecting supernatant, repeatedly co-extruding the supernatant through a 0.22-micron membrane, adding 30mg of the micron particles 1 prepared in the step (2), performing ultrasonic treatment at 10W for 10 seconds, incubating for 10 minutes, repeatedly co-extruding the supernatant through a 5-micron membrane, centrifuging 9000g of the extruded liquid for 120 minutes, collecting the discarded supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the micron particles 2 with the particle size of 3.15 microns.

Alternatively 2000 million post-incubation DCs were collected by centrifugation at 400g for 5 minutes, the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing protease inhibitors, resuspended in PBS water and sonicated at low power (20W) for 2 minutes at 4 ℃. Centrifuging 3000g of the sample for 15 minutes and collecting supernatant, centrifuging 5000g of the supernatant for 10 minutes and collecting supernatant, repeatedly co-extruding the supernatant through a 0.22-micron membrane, adding 30mg of the micron particles 1 prepared in the step (2), performing ultrasonic treatment at 10W for 10 seconds, incubating for 10 minutes, repeatedly co-extruding the supernatant through a 5-micron membrane, centrifuging 9000g of the extruded liquid for 120 minutes, collecting the discarded supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the micron particles 3 with the particle size of 3.15 microns.

(6) Detection of cancer cell-specific T cells

Female BALB/c mice of 6-8 weeks were selected and subcutaneously inoculated with 2X 10 mice in the back on day 0 ⁶ 4T1 breast cancer cells; microparticles 1 of 0.3mg PLGA were injected subcutaneously on days 7, 14 and 21, respectively. Mice were sacrificed on day 25, peripheral blood was collected from the mice, PBMCs were isolated from the peripheral blood, and CD3 was sorted from the PBMCs ⁺ T cells. Mixing CD3 ⁺ Co-incubation of T cells (100 million), 100. Mu.g of microparticles 2 or microparticles 3 in 10mL of RPMI1640 complete medium for 24 hours (37 ℃,5% ₂ ). Then, the cytomembrane staining of anti-mouse CD4 antibody and anti-mouse CD8 antibody modified by different fluorescent probes is firstly carried out, the intracellular staining of anti-mouse Granzyme B (Granzyme B) antibody modified by the fluorescent probes is carried out after the cells are fixed, and then the flow cytometry is adopted to detect the CD8 in the incubated cells ⁺ Granzyme B ⁺ T cell and CD4 ⁺ Granzyme B ⁺ T cells are cancer cell specific T cells which can recognize cancer cell whole cell antigens. After being activated by antigen, the cancer cell specific T cell can begin to synthesize and express killing substances, and granzyme B is one of the killing substances and has the strongest activity for inducing apoptosis. At the same time, analysis of CD8 ⁺ Granzyme B ⁺ Mean Fluorescence Intensity (MFI) of fluorescent probes attached to Granzyme B in T cells.

(7) Results of the experiment

As shown in fig. 12, the microparticles 2 prepared by mixing the microparticle-activated antigen-presenting cell membrane fraction with the cancer cell membrane fraction can better detect cancer cell-specific T cells than the microparticles 3, and the activated cancer cell-specific T cells can synthesize more lethal substances. Moreover, when detecting, the cancer cell specific T cells activated by the microparticles 2 can synthesize more killing substances, and the detection is more accurate because the killing substances are easier to detect.

Example 12 detection of cancer cell-specific T cells in Breast cancer

(1) Lysis of cancer cells and bacterial outer 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 are respectively inactivated and denatured by ultraviolet rays and high-temperature heating, and then 8M urea aqueous solution (containing 500mM sodium chloride) is used for cracking the cancer cells and dissolving a lysate component, namely the antigen component for preparing the micron particle system.

Lactobacillus acidophilus was centrifuged at 5000g for 30min, the precipitate was discarded and the supernatant was collected, the supernatant was filtered using a 1 μ M filter and then centrifuged at 16000g for 90 min, and the precipitate was lysed using 8M aqueous urea (containing 500mM sodium chloride) and the bacterial outer vesicle fraction was dissolved.

(2) Preparation of microparticles

In this example, the method of multiple emulsion was used to prepare microparticles. The framework material of the microparticle 1 is unmodified PLA and mannose-modified PLA, the molecular weight of the microparticle is 40KDa, and the ratio of the unmodified PLA to the mannose-modified PLA is 4. The adopted immune adjuvants are CpG2006, cpG2216 and Poly ICLC, and the adopted lysosome escape increasing substances are arginine and histidine. The mass ratio of the cancer cell lysate component to the bacterial outer vesicle component used in the preparation of the microparticles is 1. The preparation method comprises the steps of firstly preparing the micro-particles internally loaded with cancer cell lysate components, bacterial outer vesicle components, adjuvants, arginine and histidine by a multiple emulsion method, then centrifuging 100mg of the micro-particles at 9000g for 20 minutes, carrying out heavy suspension by using 10mL of ultrapure water containing 4% trehalose, and drying for 48 hours to obtain the micro-particles 1, wherein the average particle size is about 1.5 mu m, each 1mg of PLGA micro-particles 1 is loaded with about 100 mu g of protein or polypeptide components, each 0.02mg of each of CpG2006, cpG2216 and Poly ICLC, and each 0.05mg of arginine and histidine. The reference microparticle 2 was prepared from the same material and in the same manner as the microparticle 1, and had a particle size of about 1.5 μm, and loaded with the same amounts of arginine, histidine, and cancer cell lysate fraction and bacterial outer vesicle fraction, but without any adjuvant.

(3) Preparation of antigen-presenting cells

This example uses BMDCs, B cells, and BMDMs as antigen presenting cells. The preparation method of BMDC and BMDM is the same as above. B cells were derived from mouse peripheral blood PBMCs, prepared as described above. Mixing BMDC, B cells and BMDM according to the following quantity ratio of 2.

(4) Activation of antigen presenting cells

Mu.g of microparticles 1 or microparticles 2 were incubated with 4000 ten thousand mixed antigen presenting cells (containing 2000 ten thousand BMDCs, 1000 ten thousand B cells and 1000 ten thousand BMDM), respectively, in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃,5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN- γ (500U/mL), CD80 antibody (10 ng/mL), and CD40 antibody (20 mg/mL).

(5) Preparation of antigen-presenting cell-derived particles

4000 million mixed antigen presenting cells after incubation were collected by centrifugation at 400g for 5 minutes, then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water, and sonicated at 4 ℃ for 2 minutes at low power (20W). Centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 5000g for 10 minutes and collecting supernatant, filtering the supernatant through a 0.45 mu m membrane, then performing ultrafiltration centrifugal filtration and concentration by using an ultrafiltration membrane (with the molecular weight cutoff of 50 KDa), treating the filtered and concentrated sample for 3 minutes by using a high-pressure homogenizer (10000 bar), then centrifuging at 13000g for 30 minutes, removing the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano particles. Wherein, the mixed antigen presenting cells activated by the microparticles 1 are used for preparing nanoparticles 1 with the particle size of 250 nanometers; nanoparticles 2 were prepared using microparticle 2 activated mixed antigen presenting cells, with a particle size of 250 nm.

Alternatively, 4000 ten thousand mixed antigen presenting cells after incubation with microparticles 1 or microparticles 2 were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice with Phosphate Buffered Saline (PBS) containing protease inhibitors at 4 ℃, and the cells were resuspended in PBS water and then sonicated at low power (20W) for 2 minutes at 4 ℃. Centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 5000g for 10 minutes and collecting supernatant, filtering the supernatant through a 0.45 mu m membrane, then performing ultrafiltration centrifugal filtration and concentration by using an ultrafiltration membrane (with the molecular weight cut-off of 50 KDa), treating the filtered and concentrated sample for 3 minutes by using a high-pressure homogenizer (10000 bar), then performing coaction with 60mg of the

corresponding micron particles

1 or 2 prepared in the step (2) for 10 minutes, then repeatedly co-extruding by using a 2 mu m filter membrane, then centrifuging the extruded liquid at 10000g for 20 minutes, removing the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the micron particles. Wherein, the microparticles 3 with the particle diameter of 1.6 μm are prepared by the combined action of the mixed antigen presenting cell membrane components activated by the microparticles 1 and the microparticles 1; microparticles 4, having a particle size of 1.6 μm, were prepared using microparticle 2-activated mixed antigen-presenting cell membrane fraction to co-act with microparticle 2.

(6) Analysis of cancer cell-specific T cells

Female BALB/c mice of 6-8 weeks were selected and injected subcutaneously with 100. Mu.L of microparticles 1 containing 0.4mg of PLGA prepared in step (2) on days 0, 7, 14, 21 and 28, respectively. Mice were sacrificed on day 32, peripheral blood and spleen were collected, PBMC and splenocyte single cell suspensions were prepared and mixed, and CD3 was sorted therefrom using magnetic bead sorting ⁺ T cells. Sorting the obtained CD3 ⁺ T cells (200 ten thousand), 100. Mu.g nanoparticles or microparticles (nanoparticle 1, or nanoparticle 2, or microparticle 3 or microparticle 4), DC2.4 cells (100 ten thousand) were co-incubated in 10mL RPMI1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ) The incubation system contained IL-2 (200U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and CD80 antibody (10 ng/mL). The incubated CD3 was then analyzed by flow cytometry ⁺ CD3 in T cells ⁺ IFN-γ ⁺ T cells are cancer cell specific T cells which can recognize cancer cell whole cell antigens. At the same time, CD3 was analyzed ⁺ IFN-γ ⁺ Mean Fluorescence Intensity (MFI) of the fluorescent probes to which IFN-. Gamma.was attached in T cells.

Cancer cell whole cell antigen loaded by nano particles can be degraded into antigen epitope after being phagocytized by antigen presenting cells, and the antigen epitope is presented on the surface of antigen presenting cell membrane, and nano prepared by the antigen presenting cellsThe rice grains are loaded with the degraded and presented epitope, can be recognized by cancer cell specific T cells, activate the cancer cell specific T cells, and secrete killer cytokines after being activated. IFN-gamma is the most prominent cytokine secreted by antigen-specific T cells upon recognition of the antigen. CD3 analysis using flow cytometry ⁺ IFN-γ ⁺ The T cell is a cancer cell specific T cell which can recognize and kill cancer cells. At the same time, the Mean Fluorescence Intensity (MFI) of the fluorescent probes attached to IFN-. Gamma.in CD3+ IFN-. Gamma. + T cells was analyzed.

(7) Results of the experiment

As shown in FIG. 13, the high and low ratio of cancer cell-specific T cells correlated with the therapeutic effect of the particles of the present invention, indicating that T cells detected using the particles of the present invention are cancer cell-specific T cells that specifically recognize and kill cancer cells. Moreover, nanoparticles 1 are better than nanoparticles 2; microparticles 3 are better than microparticles 4. This demonstrates that particles prepared from micron-sized particle-activated antigen-presenting cells containing a substance that increases the lysosomal escape function and a mixed adjuvant detect cancer cell-specific T cells more effectively than particles prepared from micron-sized particle-activated antigen-presenting cells that contain only a substance that increases the lysosomal escape function and no mixed adjuvant. Furthermore, microparticles 3 are better than nanoparticles 1 and microparticles 4 are better than nanoparticles 2, indicating that solid particles loaded with cancer cell lysis components inside and activated antigen-presenting cell components on the surface are more effective in detecting cancer cell-specific T cells than just vesicular particles loaded with activated antigen-presenting cell components. Furthermore, the results of analysis of Mean Fluorescence Intensity (MFI) of IFN-. Gamma.linked fluorescent probes were consistent with the above results. Thus, the use of the mixed adjuvant and the internal loading of the cancer cell whole cell component both contribute to the detection of 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 nanoparticles prepared using nanoparticle-activated antigen-presenting cells loaded with colon cancer whole-cell antigen detect cancer cell-specific T cells. In this example, 8M urine was first usedThe aqueous solution of the hormone is used for cracking the colon cancer tumor tissue and dissolving the cracked components, then PLGA is used as a skeleton material, poly (I: C), cpG2336 and CpG2006 are used as adjuvants, and NH is used as an adjuvant ₄ HCO ₃ In order to increase lysosome escaping substances, nanoparticles are prepared, antigen presenting cells are activated by using the nanoparticles, then the antigen presenting cells are prepared into the nanoparticles, and then cancer cell specific T cells are detected by using the nanoparticles.

(1) Lysis of tumor tissue and Collection of fractions

Tumor tissue was collected by subcutaneous inoculation of 2X 10 cells into the back of each C57BL/6 mouse ⁶ The MC38 colon cancer cells grow to the tumor volume of about 1000mm ³ The mice were sacrificed and tumor tissue was harvested, the tumor tissue was cut into pieces and ground, the tumor tissue was understood by adding 8M urea aqueous solution through a cell strainer and the lysed components were dissolved. The above is the source of antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle systems

In this example, the nanoparticles were prepared by multiple emulsion method. The preparation material PLGA of the nano particles has the molecular weight of 7-17 KDa, takes Poly (I: C) and CpG as adjuvant and NH ₄ HCO ₃ To increase lysosome escaping substances, adjuvant and NH ₄ HCO ₃ Loaded within the nanoparticle; the preparation method is as described above, in the preparation process, firstly, a lysate component and an adjuvant are loaded inside the nano particles, then 100mg of the nano particles are centrifuged for 20 minutes at 10000g, and 10mL of ultrapure water containing 4% trehalose is used for resuspension and then is frozen and dried for 48 hours for later use; the average particle diameter of the nano particles is about 260nm, and the surface potential is about-7 mV; about 90 mug of protein and polypeptide components are loaded on each 1mg of PLGA nano particles, and 0.02mg of each of poly (I: C), cpG2336 and CpG2006 immunoadjuvants loaded on each 1mg of PLGA nano particles are loaded with NH ₄ HCO ₃ 0.01mg。

The preparation material and the preparation method of the nano-particle 2 are the same as those of the nano-particle 1, the particle diameter is about 260nm, the surface potential is about minus 7mV, about 90 mug of protein and polypeptide components are loaded on each 1mg of PLGA nano-particle, and NH is loaded on each 1mg of PLGA nano-particle ₄ HCO ₃ 0.01mg, cpG2336 and CpG2006 0.0 each3mg。

(3) Preparation of antigen-presenting cells

This example uses BMDCs and B as antigen presenting cells. BMDC was prepared as in example 1.B cells were derived from mouse peripheral blood PBMCs, prepared as described above.

(4) Activation of antigen presenting cells

Incubating the nanoparticles loaded with cancer cell whole cell fractions (1000. Mu.g) with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃,5% ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN- γ (500U/mL), and CD80 antibody (10 ng/mL) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen-presenting cell-based nanoparticles

Incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by low power (20W) sonication for 2 minutes at 4 ℃. Centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 5000g for 10 minutes and collecting supernatant, filtering the supernatant through a 0.45-micron membrane, then performing ultrafiltration centrifugal filtration and concentration by using an ultrafiltration membrane (with the molecular weight cutoff of 50 KDa), mixing the filtered and concentrated sample with the nano particles prepared in the step (2), then treating the mixture for 3 minutes by using a high-pressure homogenizer (10000 bar), then centrifuging the mixture for 30 minutes at 13000g, removing the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano particles. Wherein, the antigen presenting cell activated by the nano particle 1 and the nano particle 1 are used for coaction to prepare a nano particle 3, the particle diameter is 300 nanometers; the antigen presenting cells activated by the nano-particles 2 and the nano-particles 2 act together to prepare the nano-particles 4 with the particle size of 300 nanometers.

(6) Detection of cancer cell-specific T cells

Selecting 6-8 week female C57BL/6 mice, and injecting 100 μ L nanoparticles containing 0.5mg PLGA (loaded with lysate component, mixed adjuvant, and lysosome escape-increasing substance) or 100 μ LP subcutaneously on days 0, 7, 14, and 28 respectivelyAnd (5) the BS. Mice were sacrificed on day 32, peripheral blood was collected and peripheral blood mononuclear cells (PBM C) were isolated, and CD3 was sorted from PBMCs using flow cytometry ⁺ T cells.

This example uses Enzyme Linked Immunospot (ELISPOT) to detect cancer cell-specific T cells. Anti-mouse IFN-. Gamma.antibody a (coated antibody) was first coated in a 96-well plate for 12 hours, then subjected to a blocking treatment with addition of a medium for 1 hour, washed with PBS, and then subjected to the above-mentioned sorting in 100. Mu.L of RPMI1640 complete medium to give

nanoparticles

3 or 4 prepared by adding 50. Mu.g of antigen-presenting cells to each well and adding thereto 50. Mu.g of antigen-presenting cells, and then incubated at 37 ℃ (5% CO% ₂ ) Incubate under conditions for 24 hours. Thereafter, the mixture of cells and particles was discarded, the 96-well plate was washed and after addition of anti-mouse IFN-. Gamma.antibody b (detection antibody), CO was determined at 37 deg.C (5% ₂ ) Culturing in an incubator for more than 2 hours. The solution containing the anti-mouse IFN-. Gamma.antibody b was discarded, and after washing the 96-well plate, it was developed by a corresponding method to form spots on the surface of the 96-well plate. And reading the data by using an ELISPOT analyzer and analyzing the number of spots formed in each hole, namely the cancer cell specific T cells capable of recognizing the whole cell antigen of the cancer cells.

(7) Results of the experiment

As shown in fig. 14, compared to the PBS control group, the mice injected with the particle group loaded with the whole cell fraction contained more cancer cell-specific T cells, and the nanoparticles prepared from the antigen-presenting cells activated by the nanoparticles of the present invention could effectively detect the cancer cell-specific T cells. In addition, the effect of the cancer cell specific T cell assisted by the nanoparticles prepared from the nanoparticle activated antigen presenting cells loaded with the mixed adjuvant, the lysate component and the lysosome escape substance is better than that of the nanoparticles loaded with the lysate component, the two CpG adjuvants and the lysosome escape substance.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims

1. A method for detecting cancer cell-specific T cells from particles prepared from activated antigen presenting cells, comprising the steps of:

s1, co-incubating an antigen presenting cell and a first particle to obtain an activated antigen presenting cell; wherein the first particles are nanoparticles or microparticles carrying tissue and/or cellular whole cell components;

s2, preparing the activated cell membrane component of the antigen presenting cell into a nano vesicle; or enabling the activated cell membrane component of the antigen presenting cell to act with the second particle to obtain a second particle loaded with the cell membrane component; wherein the second particles are nanoparticles or microparticles carrying tissue and/or cellular whole cell components;

and S3, co-incubating the second particle of the nano vesicle and/or the loaded cell membrane component in the step S2 with a cell to be detected, detecting a marker in or on the surface of the cell to be detected, and detecting the cancer cell specific T cell by analyzing the T cell containing the marker.

2. The method of claim 1, wherein: the marker comprises a protein or a nucleic acid.

3. The method of claim 1, wherein: before the co-incubation of the nanovesicle and/or the second particle loaded with the cell membrane component with the cell to be tested, the method further comprises the step of sorting the T cell in the cell to be tested.

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

5. The method of claim 1, wherein: the first particle or the second particle is loaded with a bacterial component or a bacterial outer vesicle component.

6. The method of claim 5, wherein: the bacterial component or the bacterial outer vesicle component is obtained by cracking bacteria or bacterial outer vesicles through a cracking solution containing a cracking agent; the cracking agent is one or more of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, triton, tween, amino acid, glucoside and choline.

7. The method of claim 1, wherein: in step S2, the method further includes a step of mixing the cancer cell membrane fraction or extracellular vesicle membrane fraction with the activated cell membrane fraction of the antigen-presenting cell to prepare nanovesicles or to load the nanovesicles on second particles.

8. The method of claim 1, wherein: the first particle or the second particle is loaded with an immunopotentiating adjuvant.

9. The method of claim 8, wherein: the immune enhancing adjuvant comprises two or more Toll-like receptor agonists.

10. A kit for detecting a cancer cell-specific T cell, comprising at least one of the following (1) to (2):

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

wherein the content of the first and second substances,

the particles loaded with the activated antigen-presenting cell membrane components are obtained by enabling the cell membrane components of the activated antigen-presenting cells to act together with second particles so that the cell membrane components are loaded on the second particles;