A kit for detecting cancer cell-specific t cells

Abstract

The present application relates to a kit for detecting cancer cell-specific T cells, which comprises the following steps: co-incubating immune cells containing T cells in peripheral blood, peripheral immune organs or tumor infiltrating lymphocytes with particles loaded with antigen components of multiple cancer cell lines and / or allogeneic tumor tissues to activate cancer cell-specific T cells which can recognize and kill cancer cells; detecting and analyzing the number and proportion of activated cancer cell-specific T cells; and evaluating the strength of specific immune ability of the patient to recognize and kill cancer cells. The present application overcomes the difficulty in clinic that how to prepare nano / micro particles and effectively screen and detect specific T cells in peripheral blood, peripheral immune organs or tumor infiltrating lymphocytes when the patient's own tumor tissue cannot be obtained. The content of detected cancer cell-specific T cells can be used as a biomarker for the therapeutic effect of cancer immunotherapy or radiotherapy, and has the characteristics of easy separation and high specificity.

Description

Technical Field

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

[0002] T cells, especially cancer cell-specific T cells, play a crucial role in the fight against cancer. T cells are the main cells in the body that specifically recognize and kill cancer cells, and each clone of a cancer cell-specific T cell can specifically recognize an antigenic epitope. Cancer patients, especially those who have undergone immunotherapy or radiotherapy, contain a certain number of cancer cell-specific T cells. Therefore, detecting the number of cancer cell-specific T cells in cancer patients is particularly important, especially how to accurately and comprehensively detect them. The inventors previously proposed using nanoparticles or microparticles loaded with whole-cell components of cancer cells to assist in the detection of cancer cell-specific T cells (application number 202011027741.0, Detection Method for Tumor-Specific T Cells). However, the above detection system uses cancer cell lines or tumor tissues of the same type and subtype of cancer. In practical applications, it may not be possible to obtain the same cancer cell or tumor tissue samples from the patient being tested. Therefore, how to accurately and comprehensively detect cancer cell-specific T cells in cancer patients when it is not possible to obtain their own cancer cells or tumor tissue becomes a challenge. To solve the above problems, the applicant proposes this invention. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a method for indirectly activating cytotoxic (effective) cancer cell-specific T cells (T cells) by using nanoparticles (NPs) or microparticles (MPs) loaded with whole-cell antigens from multiple cancer cells or multiple patient tumor tissues. eff After activation, specific markers are used to qualitatively and quantitatively detect these cancer cell-specific T cells. This effectively solves the problem of how to prepare nanoparticles or microparticles loaded with cancer cell antigens for detecting broad-spectrum and polyclonal cancer cell-specific T cells with the ability to recognize and kill cancer cells in peripheral blood, peripheral immune organs, or tumor-infiltrating lymphocytes when it is not possible to obtain cancer cells or tumor tissue from cancer patients themselves.

[0004] The first objective of this invention is to provide a detection kit for cancer cell-specific T cells, the kit comprising nanoparticles and / or microparticles loaded with antigen components, wherein the nanoparticles or microparticles contain particle preparation materials and antigen components, the antigen components being selected from one or more of the following (i), (ii), and (iii):

[0005] (i) Whole-cell components of various cancer cell lines and / or whole-cell components of allogeneic tumor tissues.

[0006] (ii) Protein and polypeptide components obtained from lysates of various cancer cell lines and / or allogeneic tumor tissues after separation, purification, and / or immunogenicity enhancement treatment.

[0007] (iii) Protein peptide components and RNA components (preferably mRNA components) obtained by separating, purifying and / or enhancing the immunogenicity of lysates from various cancer cell lines and / or allogeneic tumor tissues.

[0008] The multiple cancer cell lines are cancer cell lines of different subtypes of the same cancer; the allogeneic tumor tissues are tumor tissues of the same subtype and / or different subtypes of the same tumor from different patients.

[0009] Whole-cell components of various cancer cell lines and / or whole-cell components of allogeneic tumor tissues may contain water-soluble and insoluble components, with the insoluble components dissolved in a solvent containing a solvent.

[0010] Protein and polypeptide components obtained from lysates of various cancer cell lines and / or allogeneic tumor tissues after separation, purification, and / or immunogenicity enhancement treatment are dissolved in a solvent containing a solvent.

[0011] The detection kit of the present invention may contain, in addition to containing one or more of (i), (ii), and (iii), one or more of the following (iv) and (v):

[0012] (iv) Artificially synthesized polypeptides containing antigenic polypeptide epitopes, wherein the antigenic polypeptide epitopes are cancer-specific or cancer-related antigenic polypeptide epitopes;

[0013] (v) It can express nucleic acids containing antigenic polypeptide epitopes, which are cancer-specific or cancer-associated antigenic polypeptide epitopes, and the nucleic acids are mRNA or DNA.

[0014] Furthermore, the detection kit described in this disclosure is characterized in that the various cancer cell lines or allogeneic tumor tissues are loaded onto nanoparticles and / or microparticles after being treated by any of the following methods:

[0015] (1) After mixing multiple cancer cell lines or allogeneic tumor tissues, their structures are inactivated and destroyed to obtain the component to be loaded; or,

[0016] (2) After inactivating and destroying the structure of multiple cancer cell lines or allogeneic tumor tissues, they are mixed to obtain the component to be loaded; or,

[0017] (3) Collect water-soluble components from various cancer cell lines or allogeneic tumor tissues, mix the water-soluble components to obtain the component to be loaded; or,

[0018] (4) Dissolve the insoluble components from various cancer cell lines or allogeneic tumor tissues in a dissolving solution containing a solvent, collect the dissolved insoluble components, and mix the insoluble components to obtain the component to be loaded; or,

[0019] (5) Collect water-soluble components from various cancer cell lines or allogeneic tumor tissues, and dissolve insoluble components using a solvent containing a solvent. Collect the dissolved insoluble components, and mix the water-soluble and insoluble components to obtain the component to be loaded; or,

[0020] (6) Using a solvent-containing solution to directly treat various cancer cell lines or allogeneic tumor tissues, and mixing the treatment solutions to obtain the component to be loaded; or

[0021] (7) Use any one of (1)-(6) to mix with (iv) a synthetic polypeptide containing an antigenic polypeptide epitope and / or (v) a nucleic acid that can express an antigenic polypeptide epitope to obtain the antigen component to be loaded.

[0022] The detection kit described in this disclosure is characterized in that the various cancer cell lines or allogeneic tumor tissues are loaded onto nanoparticles and / or microparticles after being treated by any of the following methods:

[0023] (1) After mixing multiple cancer cell lines or allogeneic tumor tissues, their structures are inactivated and destroyed, and the protein and polypeptide components are separated and extracted to obtain the antigen component to be loaded; or,

[0024] (2) After inactivating and destroying the structure of various cancer cell lines or allogeneic tumor tissues, and separating and extracting protein and polypeptide components, the components are mixed to obtain the antigen component to be loaded; or,

[0025] (3) After mixing multiple cancer cell lines or allogeneic tumor tissues, their structures are inactivated and destroyed. Protein and polypeptide components, as well as RNA components (preferably mRNA components), are then separated and extracted to obtain the antigen component to be loaded; or,

[0026] (4) Inactivate and destroy the structure of various cancer cell lines or allogeneic tumor tissues, and separate and extract protein and polypeptide components and RNA components (preferably mRNA components), then mix them to obtain the antigen component to be loaded; or,

[0027] (5) Collect water-soluble components from various cancer cell lines or allogeneic tumor tissues, then separate and extract the protein polypeptide components and / or RNA components (preferably mRNA components), and then mix the protein polypeptide components and / or RNA components (preferably mRNA components) from the water-soluble components to obtain the antigen component to be loaded; or,

[0028] (6) Treat the insoluble components from various cancer cell lines or allogeneic tumor tissues with a dissolving solution containing a solubilizing agent, collect the dissolved insoluble components, and then separate and extract the protein polypeptide components and / or RNA components (preferably mRNA components). Mix the protein polypeptide components and / or RNA components (preferably mRNA components) from the insoluble components to obtain the antigen component to be loaded; or,

[0029] (7) Collect water-soluble components from various cancer cell lines or allogeneic tumor tissues, and dissolve insoluble components using a dissolving solution containing a solubilizing agent. Collect the dissolved insoluble components, and separate and extract the protein, polypeptide, and / or RNA components (preferably mRNA components) from the water-soluble and insoluble components respectively. Then, mix them to obtain the antigen component to be loaded; or,

[0030] (8) Various cancer cell lines or allogeneic tumor tissues are directly treated with a dissolving solution containing a solvent. After mixing the treatment solutions, the protein and / or RNA components (preferably mRNA components) are separated and extracted to obtain the antigen component to be loaded; or

[0031] (9) Use any one of (1)-(8) with (iv) a synthetic polypeptide containing an antigenic polypeptide epitope and / or

[0032] (v) The antigen component to be loaded can be obtained by mixing nucleic acids containing antigenic polypeptide epitopes.

[0033] Cancer cells and cancer cell antigens exhibit high heterogeneity; even within the same type of cancer, the mutations and antigen types of cancer cells in two different patients can differ significantly. Due to this high heterogeneity, using allogeneic cancer cells or cancer cell lines may not be sufficient to cover the antigens in a particular patient. In this invention, nanoparticles and / or microparticles loaded with whole-cell antigens of cancer cells or tumor tissue encompass the whole-cell antigens of cancer cells from multiple cancer cell lines or tumor tissues from multiple allogeneic patients, thus achieving the unexpected effect of covering the cancer cell antigens of the cancer patient themselves. These nanoparticles and / or microparticles are then used to activate cancer cell-specific T cells in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes. The activation of these T cells is then indirectly achieved by detecting specific substances highly expressed or secreted intracellularly or on the cell surface of these activated cancer cell-specific T cells. Flow cytometry and other techniques are used to analyze the number and proportion of diverse and broad-spectrum cancer-specific T cells capable of recognizing and killing cancer cells.

[0034] When the delivery particles are loaded with whole-cell components, the loaded whole-cell components contain water-soluble components and non-water-soluble components dissolved using a solution containing a specific solubilizer; when the delivery particles are loaded with whole-cell antigens, the whole antigen components contain protein and polypeptide components from cell lysates and / or RNA components (or mRNA components) from cell lysates.

[0035] The process for preparing whole-cell components loaded with delivery particles includes: first lysing cancer cells or tumor tissue, then collecting water-soluble and insoluble components from the lysate, dissolving the insoluble components in a solution containing a specific solvent, and then using them together with the water-soluble components.

[0036] Alternatively, the process for preparing the whole-cell component loaded with delivery particles may include: first lysing cancer cells or tumor tissue with a solution containing a specific solubilizer, and then dissolving the whole-cell lysate with a solution containing a specific solubilizer before use.

[0037] Alternatively, the preparation process of the whole antigen component in the whole-cell component loaded with the delivery particle includes: first lysing cancer cells or tumor tissue, then collecting the water-soluble and insoluble components from the lysate separately, and treating all water-soluble components by one or more of the following methods: salting out, heating, enzymatic treatment (such as enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, irradiation, and radiation; the precipitated portion is then redissolved using a solvent containing a solvent; the insoluble portion is directly treated using a solvent containing a solvent. The precipitate is dissolved in a solvent, or dissolved in a solvent containing a solvent and then treated by one or more of the following methods: salting out, heating, enzyme treatment (such as enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, irradiation, radiation, etc. The precipitate is then dissolved a second time in a solvent containing a solvent. The protein and polypeptide components in the above water-soluble components, together with the insoluble components, or the antigen components that have been purified and then dissolved a second time, constitute the whole antigen components in the whole cell components.

[0038] Alternatively, the preparation method of the whole antigen component loaded onto the delivery particle includes: lysing cancer cells and / or tumor tissues using a lysing solution containing a solvent, dissolving the lysate component using a lysing solution containing a solvent, and then treating the obtained dissolved lysate component by one or more of the following methods: salting out, heating, enzymatic treatment (such as enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, irradiation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, and radiation. The resulting precipitated component is then redissolved a second time using a lysing solution containing a solvent.

[0039] Furthermore, the cancer cell lines are of at least two types, and when used, the mass ratio is 1-20:1-20:1-20… (meaning that the ratio of each group is 1-20), preferably 1:1:1…; the allogeneic tumor tissues are of at least two sources, and when used, the mass ratio is 1-20:1-20:1-20… (meaning that the ratio of each group is 1-20), preferably 1:1:1….

[0040] Furthermore, the water-soluble components and the insoluble components are mixed in a mass ratio of 1-5:1-5.

[0041] Furthermore, the antigen components, insoluble components, whole-cell components, or whole-cell antigen components, proteins and peptides treated by appropriate methods such as precipitation and / or denaturation are dissolved in a solvent containing a solvent and then loaded onto delivery nano / micro particles.

[0042] Furthermore, the solvent is one or more of the following: a compound containing structural formula 1, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein-degrading enzyme, albumin, lecithin, Triton, Tween, amino acids, polypeptides, glycosides, and choline;

[0043] Structure 1 is shown below:

[0044]

[0045] Wherein, R1 is C, N, S or O, and R2 to R5 are independently selected from hydrogen, alkyl, carboxyl, substituted or unsubstituted amino, mercapto, substituted or unsubstituted guanidine.

[0046] Compounds containing structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, guanidine isothiocyanate, urea, guanidine hydrochloride, other compounds containing a guanidine group, guanidine carbonate, arginine, guanidineacetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, aminourea hydrochloride, carbamoylurea, acetylurea, sulfonylureas (glibenclamide, gliclazide, glimepiride, glimepiride, etc.), thiourea compounds (thiouracil derivatives, imidazole derivatives, etc.), and nitrosoureas.

[0047] Further, the mass ratio of protein to polypeptide components in the particle preparation backbone material and the loaded antigen component is 1:0.001-10; preferably, the mass ratio of protein to polypeptide components in the particle preparation backbone material is 1:0.01-2; most preferably, the mass ratio of protein to polypeptide components in the particle preparation backbone material is 1:0.05-1.

[0048] Furthermore, when the nanoparticles or microparticles are also loaded with mRNA, the mass ratio of the particle preparation framework material to the mRNA component is 1:0.001-10; preferably, the mass ratio of the particle preparation framework material to the mRNA component is 1:0.01-2; most preferably, the mass ratio of the particle preparation framework material to the mRNA component is 1:0.05-1.

[0049] Furthermore, the nanoparticles and / or microparticles are prepared from at least one of organic synthetic polymer materials, natural polymer materials, and inorganic materials.

[0050] Furthermore, organic synthetic polymer materials include, but are not limited to, PLGA, PLA, PEG-PLGA, PEG-PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, and synthetic peptides; natural polymer materials include, but are not limited to, lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, and peptides; and inorganic materials include, but are not limited to, ferric oxide, ferric oxide, carbonates, and phosphates.

[0051] In this invention, the particles are selected to be nano- or micro-sized to ensure that they are phagocytosed by antigen-presenting cells. To improve phagocytosis efficiency, the particle size must be within a suitable range. The particle size of nanoparticles (NP) is 1 nm-1000 nm, more preferably 50 nm-500 nm, and most preferably 100 nm-300 nm; the particle size of microparticles (MP) is 1 μm-1000 μm, more preferably 1 μm-100 μm, more preferably 1 μm-10 μm, and most preferably 1 μm-5 μm.

[0052] Nanoparticles / microparticles used to deliver antigens can also be bacteria and viruses. When the antigen delivery particles are bacteria and viruses, the bacteria and viruses can express tumor-specific antigens and / or tumor-associated antigens, or the bacteria and viruses contain DNA and / or mRNA that can express tumor-specific antigens and / or tumor-associated antigens.

[0053] Furthermore, the inactivation and destruction of the structure of various cancer cell lines or allogeneic tumor tissues are carried out by one or more of the following methods: irradiation, radiation, heating, oxidation, ultrasound, homogenization, repeated freeze-thaw cycles, homogenization, high-speed stirring, high-pressure destruction, high-shear destruction, swelling, chemical substances, and shrinkage.

[0054] Before lysis, the cancer cells or tumor tissue can be co-incubated with specific chemical substances to stimulate them, and then the cancer cells or tumor tissue is lysed. These specific substances include, but are not limited to, small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), plant extracts (such as important extracts of ginseng, plant root and stem extracts, etc.), growth factors, cytokines, chemokines, interferon, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate and stimulate cancer cells or tumor tissue is to induce the cancer cells to produce more antigenic components.

[0055] Furthermore, the specific method is as follows: various cancer cell lines or allogeneic tumor tissues are frozen at -20℃ to -273℃, and after adding water or a solution without solvent, they are repeatedly freeze-thawed and lysed. The supernatant obtained is the water-soluble component, and the precipitate is treated with a solvent containing solvent. The part that becomes soluble after dissolution is the non-water-soluble component. The water-soluble and non-water-soluble components are combined to obtain the whole cell component of cancer cells.

[0056] Furthermore, the kit also includes antigen-presenting cells.

[0057] Furthermore, the antigen-presenting cells include at least one of B cells, dendritic cells (DCs), and macrophages. Preferably, two or more types of cells, including dendritic cells, are included, and more preferably, a combination of three types of cells are used.

[0058] Furthermore, the nanoparticles and / or microparticles are also loaded with immune-enhancing adjuvants.

[0059] In this invention, immune-enhancing adjuvants include, but are not limited to, pattern recognition receptor agonists, BCG, BCG cell wall cytoskeleton, BCG methanol extract residue, BCG cell wall acyl dipeptide, Mycobacterium tumefaciens, polyantigen A, mineral oil, virus-like particles, immune-enhancing regenerated influenza virus bodies, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, newborn bovine liver active peptide, miquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant polyICLC, Corynebacterium breviculae vaccine, hemolytic streptococcal preparations, coenzyme Q10, levamisole, polycytosine, manganese adjuvant, aluminum adjuvant, calcium adjuvant, cytokines, interleukins, interferon, polyinosinic acid, polyadenylate, alum, aluminum phosphate, lanolin, squalene, vegetable oil, endotoxin, liposome adjuvant, MF59, double-stranded RNA, double-stranded DNA, CAF01, and the active ingredients of ginseng and astragalus.

[0060] Preferably, the immune-enhancing adjuvant includes (1) Poly(I:C) or Poly(ICLC); (2) CpG-ODN, wherein the CpG-ODN is at least one of type A CpG-ODN, type B CpG-ODN and type C CpG-ODN, and at least one of them is type B CpG-ODN or type C CpG-ODN. Among them, Class A CpG-ODN is selected from CpG-ODN 2216, CpG-ODN 1585 or CpG-ODN 2336; Class B CpG-ODN is selected from CpG-ODN 1018, CpG-ODN 2006, CpG-ODN 1826, CpG-ODN 1668, CpG-ODN 2007, CpG-ODN BW006 or CpG-ODN SL01; and Class C CpG-ODN is selected from CpG-ODN 2395, CpG-ODN SL03 or CpG-ODNM362.

[0061] Furthermore, the nanoparticles and / or microparticles are also loaded with substances that promote lysosomal escape.

[0062] Furthermore, substances that promote lysosomal escape include positively charged polypeptides (such as KALA polypeptide, RALA polypeptide, bee venom peptide, etc.), arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH4HCO3, protamine, or histone.

[0063] Furthermore, the nanoparticles and / or microparticles are also loaded with targets that actively target antigen-presenting cells.

[0064] Furthermore, the target includes, but is not limited to, mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, or CD44 antibody.

[0065] In this invention, cancer cell-specific T cells include CD4. + T cells and / or CD8 + T cells, preferably including CD4+ + T cells and CD8 + T cells.

[0066] When using the kit, nanoparticles and / or microparticles loaded with various cancer cell lines and / or allogeneic tumor tissues are co-incubated with antigen-presenting cells and test cells. Antigen-presenting cells indirectly activate antigen-specific T cells. Then, specific markers inside or on the surface of the test cells or secreted by them are detected using certain detection methods. The detection of cancer cell-specific T cells is indirectly achieved by analyzing the content of specific markers outside or inside the cells.

[0067] Furthermore, the process of activating cancer cell-specific T cells with nanoparticles / microparticles involves simultaneously co-incubating nanoparticles / microparticles with antigen-presenting cells and T cells to activate T cells; or the process of activating cancer cell-specific T cells with nanoparticles / microparticles involves first co-incubating nanoparticles / microparticles with antigen-presenting cells to activate antigen-presenting cells (the antigen-presenting cells that have already been activated by antigen can be used directly or after appropriate inactivation treatment), and then co-incubating the activated antigen-presenting cells with T cells to activate T cells.

[0068] After activating antigen-presenting cells by co-incubating them with nanoparticles / microparticles, the activated antigen-presenting cells can be used directly to activate T cells, or the activated antigen-presenting cells can be appropriately inactivated before being used to activate T cells. Inactivation methods include, but are not limited to, fixation (such as using chemical substances such as formaldehyde or paraformaldehyde) and radiation (such as using ultraviolet light, X-rays, alpha rays, beta rays, gamma rays, etc.) to inactivate cells.

[0069] Furthermore, the concentration of the nanoparticles / microparticles during co-incubation with antigen-presenting cells is from 0.001 mg / mL to 50 mg / mL, preferably 0.01 mg / mL to 2 mg / mL; the co-incubation time is from 1 h to 72 h, preferably 5 to 48 h; and the incubation temperature is from 4 to 40 °C, preferably 37 °C.

[0070] Furthermore, the concentration of the nanoparticles / microparticles during co-incubation with antigen-presenting cells and T cells is from 0.001 mg / mL to 50 mg / mL, preferably from 0.01 mg / mL to 2 mg / mL; the co-incubation time is from 1 h to 72 h, preferably from 5 to 48 h; and the incubation temperature is from 4 to 40 °C, preferably from 37 °C.

[0071] Furthermore, the specific markers include proteins, peptides, nucleic acids, carbohydrates, or lipids.

[0072] Furthermore, when the specific biomarker is a protein, it includes, but is not limited to, one or a combination of the following biomarkers: interferon-γ, interleukin, granzyme, perforin, CD69, FAS, FASL, CD107, IL-2R, FASL, HLA, CD137, CD40L, CD25, OX40 (CD134), TCF-1, CD25, IP-10, CD39, CD38, CD27, CD29, CXCL13, CXCL6, PTPRC, CD103, NCR1, CD56, CD279, CD278, CD244, CD27, CD154, TCF-1, CD102, CD107, CD137, CD44, IL-2R, F ASL, CD28, HLA-DR, CD127(IL-7R), CD150, CD107A, CD83, CD166, CD178, CD212, CD229, CD100, CD107b, CD1 08. CD109, CD113, CD122, CD126, CD253, CD197, PD-1, TIM3, LAG-3, TIGIT, CD62L, CD70, CTLA-4(CD152), CD27, CD26, CD30, TNFRSF9, CD74, PD-L1(CD274), CD258, CD261, 4-1BB, CD154, ICAM-1, LFA-1, LFA-2, VLA -4, CD160, CD71, CXCR3, TNFRSF14, TNFRSF13, DUSP10, EMB, CHD3, TIM1, TCF-7, CD40, SELL, ICAM4, TNFSF1 3. PODXL, LEF1, TNFRSF18, TNFSF4, TNFSF9, TNFSF14, CD11a, CD101, CD48, CD244, CD49a, CD95, CD44, CXCR 1. CXCR 6, CXCR 13, CD103, CD45RO, ICOS(CD278), VTCN1, HHLA2, LGAL59, CCR7, CCR5, CD357, BCL6, TCF-1, etc. Techniques for analyzing and detecting the number and proportion of cancer cell-specific T cells using specific biomarkers include, but are not limited to, flow cytometry, magnetic bead sorting, enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and multi-cytokine assay. When using specific biomarkers to analyze and detect the number and proportion of cancer cell-specific T cells, at least one biomarker can be used.

[0073] Furthermore, the cells to be tested can be T cells or a mixture of cells containing T cells, such as T cells or a mixture of cells containing T cells derived from peripheral blood, peripheral immune organs, or tumor-infiltrating lymphocytes.

[0074] Furthermore, before co-incubating the test cells with nanoparticles and / or microparticles, they can be sorted to separate T cells. Specifically, CD3+ cells can be sorted from peripheral blood, peripheral immune tissues, and tumor-infiltrating lymphocytes using flow cytometry or magnetic bead sorting. + Cells, sorted out CD45 + CD3 + Cells, sorted out CD3 + CD8 + Cells, sorted out CD45 + CD3 + CD8 + Cells, sorted out CD3 + CD4 + Cells or sorted CD45 + CD3 + CD4 + . cells.

[0075] Furthermore, before co-incubating the test cells with nanoparticles and / or microparticles, they can be sorted to separate T cells. Specifically, CD3+ cells can be sorted from peripheral blood, peripheral immune tissues, and tumor-infiltrating lymphocytes using flow cytometry or magnetic bead sorting. + Cells, sorted out CD45 + CD3 + Cells, sorted out CD3 + CD8 + Cells, sorted out CD45 + CD3 + CD8 + Cells, sorted out CD3 + CD4 + Cells or sorted CD45 + CD3 + CD4 + The cells. Then, before co-incubation, further purification can be performed to remove a very small number of T cells that are positive for specific target markers, such as removing CD25+ T cells or CD69-positive T cells before co-incubation.

[0076] Furthermore, the co-incubation system may contain cytokines or antibodies. Further, the cytokines include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF), 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, etc.; preferably, the co-incubation system contains granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), and interleukin-12 (IL-12). Furthermore, antibodies include, but are not limited to, αCD-3 antibodies, αCD-4 antibodies, αCD-8 antibodies, αCD-28 antibodies, αCD-40 antibodies, αOX-40 antibodies, and αOX-40L antibodies.

[0077] Furthermore, the cancer cells or tumor tissue used to prepare the antigen in the nanoparticles and / or microparticles are not derived from cancer patients of the sample to be tested.

[0078] This invention breaks through the limitations of existing detection methods, enabling particles to be loaded with all antigens and activated antigen-presenting cell membranes, thus enabling the detection of a broader and more diverse range of cancer cell-specific T cells with high specificity. It is particularly effective in immunoassays, thereby providing a potential biomarker detection method for immunotherapy.

[0079] The preparation steps of the whole-cell components of cancer cells and / or tumor tissue loaded with nanoparticles or microparticles are as follows: First, lyse the tumor tissue of each cancer cell line and / or each cancer patient to obtain its lysate. Then, use one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration to separate the water-soluble and insoluble components in the lysate, obtaining the water-soluble and insoluble components in the lysate separately. Then, dissolve the precipitate in a dissolving solution containing a solvent to obtain the dissolved insoluble component. Then, separate the water-soluble and insoluble components of each cancer cell line and / or each cancer patient. The components are mixed separately to obtain a mixture of water-soluble and insoluble components. Using both together constitutes the whole-cell component. Alternatively, multiple cancer cells and / or tumor tissues from multiple cancer patients are mixed and lysed. Then, one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration are used to separate the water-soluble and insoluble components in the lysate, obtaining a mixture of water-soluble and insoluble components in the lysate. The precipitate is then dissolved in a dissolving solution containing a solvent to obtain a dissolved mixture of insoluble components. Using both together constitutes the whole-cell component.

[0080] Alternatively, the preparation steps of the whole-cell components in cancer cells and / or tumor tissue loaded with nanoparticles or microparticles are as follows: first, each cancer cell line and / or each cancer patient's tumor tissue is lysed separately using a solvent containing a solvent to obtain its lysate; then, all lysate components are dissolved using a solvent containing a solvent to obtain the whole-cell components of each cancer cell line and / or each cancer patient's tumor tissue; then, the whole-cell components of one cancer cell line and / or each cancer patient's tumor tissue are mixed to obtain the whole-cell components; or, multiple cancer cells and / or multiple cancer patients' tumor tissues are mixed and lysed using a solvent containing a solvent, and then all lysates are dissolved using a solvent containing a solvent to obtain the whole-cell components.

[0081] Alternatively, the preparation steps for antigenic components in cancer cells and / or tumor tissues loaded with nanoparticles or microparticles are as follows: First, lyse each cancer cell line and / or each cancer patient's tumor tissue to obtain its lysate. Then, use one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration to separate the water-soluble and insoluble components in the lysate, obtaining the water-soluble and insoluble components separately. Then, dissolve the insoluble components directly in a solvent containing a solvent to obtain the dissolved insoluble components. Treat the water-soluble components in the obtained lysate through one or more methods such as salting out and / or heating, enzymatic treatment (e.g., enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, irradiation, and radiation to precipitate the protein and polypeptide components. Then, dissolve the precipitated protein and polypeptide components in a solvent containing a solvent to obtain the water-soluble protein and polypeptide components. Finally, mix the water-soluble protein and polypeptide components and insoluble components of each cancer cell line and / or each cancer patient separately to obtain the water-soluble components. The antigen component is a mixture of protein and polypeptide components and a mixture of insoluble components. Alternatively, multiple cancer cells and / or tumor tissues from multiple cancer patients are mixed and lysed. Then, one or more methods, such as centrifugation, filtration, dialysis, and ultrafiltration, are used to separate the water-soluble and insoluble components from the lysate, resulting in a mixture of water-soluble and insoluble components. The precipitate is then dissolved in a solvent containing a solvent to obtain the dissolved insoluble component mixture. The water-soluble component mixture is then treated with one or more methods, such as salting out and / or heating, enzymatic treatment (e.g., enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, irradiation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, or radiation, to precipitate or denature the protein and polypeptide components. The precipitated / denatured protein and polypeptide components are then dissolved in a solvent containing a solvent to obtain a mixture of water-soluble protein and polypeptide components. The antigen component is obtained by using both the insoluble and water-soluble component mixtures together.

[0082] The preparation steps of the whole-cell protein and polypeptide antigen components in cancer cells and / or tumor tissue loaded with nanoparticles or microparticles are as follows: First, multiple cancer cell lines and / or tumor tissues from multiple tumor patients are mixed. Then, the mixture of cancer cell lines and / or tumor tissues is lysed using a dissolving solution containing a solvent. Then, all lysates are dissolved using a dissolving solution containing a solvent. Then, the mixture is treated with one or more of the following methods: salting out and / or heating, enzyme treatment (such as enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, irradiation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, and radiation, so that the proteins and polypeptides are analyzed or denatured and precipitated / denatured and separated. Then, the antigen components are dissolved a second time using a dissolving solution containing a solvent. Alternatively, each cancer cell line and / or tumor tissue from each cancer patient can be mixed, and then the cancer cell line and / or tumor tissue can be lysed using a lysing solution containing a solvent. All lysates can then be dissolved using a lysing solution containing a solvent. The lysates can then be treated using one or more of the following methods: salting out and / or heating, enzymatic treatment (such as enzymatic digestion), oxidation, reduction, fixation, mineralization, irradiation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, or radiation, so that the proteins and peptides are analyzed or denatured and precipitated or separated. The lysates can then be dissolved a second time using a lysing solution containing a solvent and mixed to obtain the antigen component.

[0083] The preparation steps for the insoluble protein and polypeptide components in cancer cells and / or tumor tissue loaded with nanoparticles or microparticles are as follows: First, lyse the cancer cell line and / or tumor tissue to obtain its lysate; then, use one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration to separate the water-soluble and insoluble components in the lysate, respectively obtaining the water-soluble and insoluble components in the lysate; then, treat the insoluble components in the obtained lysate with one or more methods such as salting out and / or heating, enzymatic treatment (such as enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, irradiation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, and radiation to precipitate or denature the protein and polypeptide components; then, dissolve the precipitated protein and polypeptide components in a solvent containing a solvent to obtain the insoluble protein and polypeptide components. In use, the above-mentioned insoluble protein and polypeptide components are used together with the water-soluble protein and polypeptide components as whole-cell protein and polypeptide antigen components.

[0084] The above-mentioned whole-cell protein and polypeptide antigen components can also be used together with whole-cell mRNA antigen components. The preparation method is as follows: first, the protein and polypeptide components are separated and purified by one or more of the following methods: salting out and / or heating, enzyme treatment (such as enzymatic hydrolysis), oxidation, reduction, fixation, mineralization, irradiation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, and radiation. The mRNA component is located in the supernatant. The mRNA component in the supernatant is separated and purified using a specific method. The protein and polypeptide precipitate is dissolved in a dissolving solution containing a solvent. Then, the dissolved protein and polypeptide components are mixed with the mRNA component before use.

[0085] The methods for separating, purifying, and / or enhancing the immunogenicity of antigenic components described in this disclosure include, but are not limited to, one or more of the following methods: salting out, heating, enzyme treatment (e.g., enzymatic digestion, enzyme inhibition, etc.), using an RNA isolation kit, using an mRNA isolation kit, oxidation, reduction, fixation, mineralization, irradiation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, and radiation.

[0086] In the preparation process of the antigen component loaded with nanoparticles or microparticles, the cations contained in the reagents used for salting out include, but are not limited to, Al. 3+ Fe 3+ Fe 2+ Mg 2+ Sn 2+ Zn 2+ Ca 2+ Li + Na + NH4 + K + Cu 2+ Ag + Ba 2+ The reagents used for salting out contain anions including, but not limited to, Cl-. - SO4 2- NO3 - CO3 2- SiO3 2- S2O7 2- B4O7 2- PO4 3- RCOO - NO2 - S2O8 2- S 2- CrO4 2- MnO4 - P2O7 4- wait.

[0087] The heating process involves heating at a temperature above 40°C for at least 5 seconds.

[0088] The enzyme treatment method includes, but is not limited to, using one or more of the following: nuclease, DNase, pepsin, chymotrypsin, trypsin, other protein-degrading enzymes, and protease inhibitors.

[0089] The oxidation process involves treating the antigenic component with an oxidizing agent, which includes, but is not limited to, hypochlorous acid, persulfate, KIO3, KBrO3, chlorine, dichromate, nitric acid, hydrogen peroxide, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, hydrogen peroxide, sodium percarbonate, sodium perborate, potassium perborate, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium dichromate, potassium permanganate, nitric acid, and ClO3. - ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, H2O2, NO3 - MnO4 - One or more of various oxidizing agents such as F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3, etc.

[0090] The mineralization includes, but is not limited to, one or more of the following mineralization methods: silicification, calcification, magnesification, and biomineralization.

[0091] The reduction refers to reducing the antigen component using a component that can reduce the antigen.

[0092] The irradiation is a treatment using one or more different irradiation methods such as ultraviolet rays, X-rays, gamma rays, alpha rays, beta rays, and radioactive sources.

[0093] The chromatography described in this disclosure includes, but is not limited to, column chromatography, gas chromatography, high-performance liquid chromatography, adsorption chromatography, partition chromatography, thin-layer chromatography, high-performance liquid chromatography, ion exchange chromatography, thin film chromatography, affinity chromatography, gel chromatography, etc.

[0094] The chromatographic methods described in this disclosure include, but are not limited to, column chromatography, thin-layer chromatography, liquid chromatography, gas chromatography, and supercritical fluid chromatography.

[0095] The electrophoresis methods described in this disclosure include, but are not limited to, SDS electrophoresis, isoelectric point focusing electrophoresis, isovelocity electrophoresis, immunoelectrophoresis, serum protein electrophoresis, nucleic acid electrophoresis, DNA sequencing electrophoresis, gel electrophoresis, and preparative electrophoresis.

[0096] The methods for isolating and purifying RNA or mRNA components include, but are not limited to, using DNase to degrade DNA, using appropriate kits to remove DNA, using specific kits to isolate total RNA, and using specific kits to isolate mRNA.

[0097] The process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components during the preparation of antigen components loaded with nanoparticles or microparticles may include salting out; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include heating; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include enzymatic treatment (e.g., enzymatic hydrolysis); the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components. The process of separating and purifying antigenic components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include oxidation; the process of separating and purifying antigenic components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include reduction; the process of separating and purifying antigenic components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include fixation; the process of separating and purifying antigenic components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include mineralization, such as biomineralization, silicification, calcification, magnesification, etc.; the process of separating and purifying antigenic components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include irradiation.

[0098] The process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components during the preparation of antigen components loaded with nanoparticles or microparticles may include salting out; the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include heating; the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include enzyme treatment (such as enzymatic hydrolysis, enzyme inhibition, and other enzyme treatment methods); the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include enzyme treatment (such as enzyme hydrolysis, enzyme inhibition, and other enzyme treatment methods); The immunogenicity process may include oxidation; the process of enhancing the immunogenicity of antigenic components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include reduction; the process of enhancing the immunogenicity of antigenic components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include fixation; the process of enhancing the immunogenicity of antigenic components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include mineralization, such as biomineralization, calcification, magnesia, etc.; the process of enhancing the immunogenicity of antigenic components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include irradiation.

[0099] The above lists several commonly used methods for preparing cancer cell lines based on multiple cancer cell lines or tumor tissues from multiple patients. In practical applications, other relevant and feasible preparation methods can also be used in combination as needed.

[0100] The sources of cancer cells described in this disclosure are any methods by which cancer cells can be obtained, including but not limited to cancer cell lines, cancer cells obtained by in vitro expansion of cancer cells isolated and extracted from tumor tissue, cancer cells obtained by expansion of circulating tumor cells isolated and extracted from blood, or cancer cells differentiated and cultured from stem cells.

[0101] Alternatively, the whole-cell components or whole-cell antigen components prepared by the above methods can be mixed with in vitro synthesized peptides containing cancer-specific antigenic epitopes / cancer-associated antigenic epitopes and / or nucleic acids (DNA or mRNA) that can express cancer-specific antigenic epitopes / cancer-associated antigenic epitopes, and then used as mixed antigen components loaded onto nanoparticles or microparticles.

[0102] The particles described in this disclosure for detecting cancer cell-specific T cells are nanoparticles or microparticles, or a mixture of nanoparticles and microparticles.

[0103] Furthermore, the particle size of the nanoparticles is 1nm-1000nm, more preferably 30nm-1000nm, even more preferably 50nm-600nm; even more preferably 50-500nm; even more preferably 100-400nm. For example, the particle size of the nanoparticles is 10nm, 50nm, 100nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 400nm, 500nm, etc.

[0104] Furthermore, the particle size of the micron particles is 1μm-1000μm, more preferably 1μm-100μm, even more preferably 1μm-10μm, and even more preferably 1μm-5μm; for example, the particle size of the micron particles is 1μm, 1.1μm, 1.2μm, 1.3μm, 1.4μm, 1.5μm, 1.6μm, 1.7μm, 1.8μm, 1.9μm, 2μm, 2.5μm, 3μm, 5μm, 10μm, etc.

[0105] When using a mixture of nanoparticles and microparticles, the nanoparticles have a particle size of 100nm-500nm and the microparticles have a particle size of 1.0μm-5μm; more preferably, when using a mixture of nanoparticles and microparticles, the nanoparticles have a particle size of 150nm-400nm and the microparticles have a particle size of 1.5μm-4.0μm; even more preferably, when using a mixture of nanoparticles and microparticles, the nanoparticles have a particle size of 200nm-350nm and the microparticles have a particle size of 2.0μm-3.5μm.

[0106] The particles described in this disclosure load antigen components onto the surface of the antigen delivery particles in at least one of the following methods: adsorption, covalent bonding, charge interaction, hydrophobic interaction, one-step or multi-step solidification, mineralization, and encapsulation; the antigen components are loaded, individually or simultaneously, into the interior and / or surface of the delivered particles.

[0107] The exemplary embodiments of this disclosure use solvent evaporation to prepare antigen delivery particles. In practical applications, any other method that can prepare antigen delivery particles can also be used, including but not limited to precipitation, dialysis, dispersion, microfluidics, high-pressure homogenization, stirring, spray drying, phase separation, electrostatic spraying, emulsion polymerization, machine stirring and shearing, membrane emulsification, and other feasible methods.

[0108] By means of the above-described solution, the present invention has at least the following advantages:

[0109] This invention provides a technique for in vitro detection of cancer cell-specific T cells in immune cells using a nanoscale or microscale particle delivery system. The analyzed and detected cancer cell-specific T cells are broad-spectrum and highly specific, encompassing all clones of effector (cytotoxic) cancer cell-specific T cells (T cells). eff Furthermore, compared to nanoparticles and / or microparticles loaded with only one cancer cell line or tumor tissue from a single allogeneic cancer patient, nanoparticles and / or microparticles loaded with multiple cell lines or tumor tissue from multiple cancer patients exhibit higher levels of specific biomarkers secreted by activated T cells. This makes them easier to detect, avoiding situations where they cannot be detected due to weak or poor signals. Moreover, this invention can use tumor tissue from multiple other patients or multiple cancer cell lines to prepare nanoparticles / microparticles, overcoming the challenge of not being able to obtain tumor tissue from the patient being tested in most clinical settings. These advantages allow the particle detection method of this invention to avoid situations where some cancer cell-specific T cells cannot be detected due to weak expression of specific biomarkers after activation, thus resulting in higher detection accuracy. Furthermore, this invention optimizes the T cell incubation process and the loading material of the nanoparticles and / or microparticles, enabling the method to detect a more comprehensive range of cancer cell-specific T cells with stronger and more accurate signals.

[0110] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0111] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to certain specific embodiments and accompanying drawings.

[0112] Figure 1 The diagram illustrates certain preparation processes and applications of the detection system of the present invention; wherein, a is a schematic diagram of collecting and preparing nanoparticles or microparticles from water-soluble and insoluble components respectively; b is a schematic diagram of dissolving whole-cell antigens of cancer cells and preparing nanoparticles or microparticles using a solution containing a solvent; c is a schematic diagram of using the nanoparticles and / or microparticles prepared in a or b for detecting cancer cell-specific T cells after activation.

[0113] Figure 2-19The figures represent the results of experiments using nanoparticles or microparticles to detect cancer cell-specific T cells in Examples 1-18, respectively. * indicates a significant difference (p≤0.05) compared to before treatment; ** indicates a significant difference (p≤0.01) compared to before treatment; *** indicates a significant difference (p≤0.005) compared to before treatment; # indicates a significant difference (p≤0.05); ## indicates a significant difference (p≤0.01); ### indicates a significant difference (p≤0.005). Detailed Implementation

[0114] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0115] First, an antigen component is prepared, then nanoparticles and / or microparticles loaded with the antigen component are prepared, and then the nanoparticles or microparticles are used for the detection of cancer cell-specific T cells.

[0116] The present invention describes a method for detecting cancer cell-specific T cells in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes. During detection, these cancer cell-specific T cells are first co-incubated with nanoparticles and / or microparticles and antigen-presenting cells, or first co-incubated with activated antigen-presenting cells (contained by nanoparticles and / or microparticles). Then, flow cytometry, ELISPOT, ELISA, multi-cytokine detection, or magnetic bead sorting are used to analyze the specific molecules expressed or secreted by the cancer cell-specific T cells after antigen-specific activation, thus obtaining broad-spectrum information on cancer cell-specific T cells.

[0117] In this process, antigen-presenting cells are first activated by nanoparticles and / or microparticles loaded with whole-cell antigens or mixtures thereof from multiple patient tumor tissues and / or various cancer cells. The activated antigen-presenting cells then further activate cancer cell-specific T cells. The process and applications of detecting cancer cell-specific T cells include... Figure 1 As shown.

[0118] When preparing nanoparticles or microparticles to activate antigen-presenting cells, the water-soluble components and water-insoluble antigens can be collected separately after lysing the cells or tissues and then prepared into nanoparticle or microparticle systems; or the cells or tissues can be directly lysed and the whole-cell antigens of cancer cells can be dissolved using a dissolving solution containing a solvent to prepare nanoparticle or microparticle systems. The whole-cell antigen of cancer cells described in this invention can be prepared into nanoparticles or microparticles before or after cell lysis, either by inactivation or denaturation, co-incubation with compounds, solidification, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, mineralization, ionization, protein extraction, salting out, heating, chemical modification, nuclease treatment, protease endolysis or degradation, etc.; or by directly preparing nanoparticles or microparticles before or after cell lysis without any inactivation or denaturation, solidification, oxidation, reduction, co-incubation with compounds, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, biomineralization, heating, ionization, protein extraction, salting out, chemical modification, nuclease treatment, protease endolysis or degradation. In some embodiments of this invention, tumor tissue cells undergo inactivation and / or denaturation treatment before lysis. In actual use, inactivation and / or denaturation treatment can also be performed after cell lysis, or both before and after cell lysis. In some embodiments of this invention, the inactivation and / or denaturation treatment methods before and after cell lysis are ultraviolet irradiation and high-temperature heating. In actual use, methods including but not limited to radiation irradiation, high pressure, solidification, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, biomineralization, ionization, protein extraction, salting out, heating, chemical modification, nuclease treatment, collagenase treatment, protease endolysis or degradation, and freeze-drying can also be used. Those skilled in the art will understand that appropriate adjustments can be made according to specific circumstances during practical applications.

[0119] The solvent is one or more of the following: a compound containing structural formula 1, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein-degrading enzyme, albumin, lecithin, Triton, Tween, amino acids, polypeptides, glycosides, and choline.

[0120] Urea, guanidinosuccinic acid, aminourea hydrochloride, arginine, and guanidine hydrochloride contain structures in Formula 1. The inventors discovered that substances with the structure in Formula 1 can be used as solvents in solutions to dissolve insoluble components in cells or tumor tissues, or components precipitated after treatment such as salting out, heating, or enzymatic hydrolysis. Therefore, in addition to common compounds containing guanidino and urea groups, such as urea, guanidinosuccinic acid, aminourea hydrochloride, arginine, guanidine hydrochloride, and metformin, other compounds containing this structure also have the ability to dissolve insoluble components as solvents.

[0121] Structure 1 is shown below:

[0122]

[0123] Among them, R1, R2, R3, R4 and R5 are arbitrary chemical structures.

[0124] Compounds containing the structure in Formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, other compounds containing a guanidine group, guanidine carbonate, arginine, guanidineacetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, aminourea hydrochloride, carbamoylurea, acetylurea, sulfonylureas (glibenclamide, gliclazide, glimepiride, glimepiride, etc.), thiourea compounds (thiouracil derivatives, imidazole derivatives, etc.), and nitrosoureas.

[0125] Furthermore, inactivating and destroying the structure of cancer cells or tumor tissue includes, but is not limited to, one or more of the following: irradiation, radiation, heating, oxidation, ultrasound, homogenization, repeated freeze-thaw cycles, homogenization, high-speed stirring, high-pressure destruction, high-shear destruction, swelling, chemical substances, and shrinkage. In some embodiments of this invention, repeated freeze-thaw cycles, swelling with ultrapure water, and ultrasound treatment are used. In practical applications, any other method capable of inactivating and destroying the structure of cells or tumor tissue can also be used.

[0126] The preparation steps of the whole-cell components of cancer cells and / or tumor tissue loaded with nanoparticles or microparticles are as follows: First, lyse the tumor tissue of each cancer cell line and / or each cancer patient to obtain its lysate. Then, use one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration to separate the water-soluble and insoluble components in the lysate, obtaining the water-soluble and insoluble components in the lysate separately. Then, dissolve the precipitate in a dissolving solution containing a solvent to obtain the dissolved insoluble component. Then, separate the water-soluble and insoluble components of each cancer cell line and / or each cancer patient. The components are mixed separately to obtain a mixture of water-soluble and insoluble components. Using both together constitutes the whole-cell component. Alternatively, multiple cancer cells and / or tumor tissues from multiple cancer patients are mixed and lysed. Then, one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration are used to separate the water-soluble and insoluble components in the lysate, obtaining a mixture of water-soluble and insoluble components in the lysate. The precipitate is then dissolved in a dissolving solution containing a solvent to obtain a dissolved mixture of insoluble components. Using both together constitutes the whole-cell component.

[0127] Alternatively, the preparation steps of the whole-cell components in cancer cells and / or tumor tissue loaded with nanoparticles or microparticles are as follows: first, each cancer cell line and / or each cancer patient's tumor tissue is lysed separately using a solvent containing a solvent to obtain its lysate; then, all lysate components are dissolved using a solvent containing a solvent to obtain the whole-cell components of each cancer cell line and / or each cancer patient's tumor tissue; then, the whole-cell components of one cancer cell line and / or each cancer patient's tumor tissue are mixed to obtain the whole-cell components; or, multiple cancer cells and / or multiple cancer patients' tumor tissues are mixed and lysed using a solvent containing a solvent, and then all lysates are dissolved using a solvent containing a solvent to obtain the whole-cell components.

[0128] Alternatively, the preparation steps for antigenic components in cancer cells and / or tumor tissues loaded with nanoparticles or microparticles are as follows: First, lyse the tumor tissue of each cancer cell line and / or each cancer patient to obtain its lysate. Then, use one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration to separate the water-soluble and insoluble components in the lysate, obtaining the water-soluble and insoluble components separately. Then, dissolve the insoluble components directly in a solvent containing a solvent to obtain the dissolved insoluble components. Treat the water-soluble components in the obtained lysate using one or more methods such as salting out, heating, enzymatic treatment (e.g., enzyme degradation and enzyme inhibition), chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, radiation, oxidation, reduction, fixation, mineralization, and irradiation to precipitate the protein and polypeptide components. Then, dissolve the precipitated protein and polypeptide components in a solvent containing a solvent to obtain the water-soluble protein and polypeptide components. Finally, mix the water-soluble protein and polypeptide components and insoluble components of each cancer cell line and / or each cancer patient separately to obtain the water-soluble components. The antigen component is a mixture of protein and polypeptide components and a mixture of insoluble components. Using both together constitutes the antigen component. Alternatively, multiple cancer cells and / or tumor tissues from multiple cancer patients are mixed and lysed. Then, one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration are used to separate the water-soluble and insoluble components from the lysate, resulting in a mixture of water-soluble and insoluble components. The precipitate is then dissolved in a solvent containing a solvent to obtain the dissolved insoluble component mixture. The water-soluble component mixture is then treated with one or more methods such as salting out, heating, enzymatic treatment (e.g., enzymatic hydrolysis, enzyme inhibition), chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, radiation, oxidation, reduction, fixation, mineralization, and irradiation to precipitate or denature the protein and polypeptide components. The precipitated / denatured protein and polypeptide components are then dissolved in a solvent containing a solvent to obtain a mixture of water-soluble protein and polypeptide components. Using both the insoluble and water-soluble component mixtures together constitutes the antigen component.

[0129] The preparation steps of the whole-cell protein and polypeptide antigen components in cancer cells and / or tumor tissues loaded with nanoparticles or microparticles are as follows: First, multiple cancer cell lines and / or tumor tissues from multiple tumor patients are mixed. Then, the mixture of cancer cell lines and / or tumor tissues is lysed using a dissolving solution containing a solvent. Then, all lysates are dissolved using a dissolving solution containing a solvent. Then, one or more of the following methods are used to treat the protein and polypeptide components to extract or denature them to form precipitates / denatured components. Finally, the antigen components are dissolved a second time using a dissolving solution containing a solvent. Alternatively, the cancer cell lines and / or tumor tissues from each cancer patient can be mixed, and then the cancer cell lines and / or tumor tissues can be lysed using a lysing solution containing a solvent. All lysates can then be dissolved using a lysing solution containing a solvent. The lysates can then be treated using one or more of the following methods: salting out, heating, enzymatic treatment (such as enzymatic digestion and enzyme inhibition), chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, radiation, oxidation, reduction, fixation, mineralization, or irradiation, so that the proteins and peptides are analyzed or denatured and precipitated or separated. The lysates can then be dissolved a second time using a lysing solution containing a solvent and mixed to obtain the antigen component.

[0130] The preparation steps for the insoluble protein and polypeptide components in cancer cells and / or tumor tissue loaded with nanoparticles or microparticles are as follows: First, lyse the cancer cell line and / or tumor tissue to obtain its lysate; then, use one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration to separate the water-soluble and insoluble components in the lysate, respectively obtaining the water-soluble and insoluble components in the lysate; then, treat the insoluble components in the obtained lysate with one or more methods such as salting out, heating, enzymatic treatment (e.g., enzymatic hydrolysis), oxidation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, reduction, fixation, mineralization, and irradiation to precipitate or denature the protein and polypeptide components; then, dissolve the precipitated protein and polypeptide components in a solvent containing a solvent to obtain the insoluble protein and polypeptide components. In use, the insoluble protein and polypeptide components are used together with the water-soluble protein and polypeptide components as whole-cell protein and polypeptide antigen components.

[0131] The above-mentioned whole-cell protein and polypeptide antigen components can also be used together with whole-cell mRNA antigen components. The preparation method is as follows: first, the protein and polypeptide components are separated and purified by one or more of the following methods: salting out, heating, enzymatic treatment (such as enzymatic hydrolysis), oxidation, reduction, fixation, radiation, mineralization, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, irradiation, etc. After the RNA component or mRNA component is in the supernatant, the RNA component or mRNA component in the supernatant is separated and purified using a specific method. The protein and polypeptide precipitate is dissolved in a dissolving solution containing a solvent, and then the dissolved protein and polypeptide components are mixed with the RNA component or mRNA component before use.

[0132] The methods for separating, purifying, and / or enhancing the immunogenicity of antigenic components described in this disclosure include, but are not limited to, one or more of the following methods: salting out, heating, enzyme treatment (e.g., enzymatic digestion, enzyme inhibition, etc.), using an RNA isolation kit, using an mRNA isolation kit, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, fixation, mineralization, and irradiation.

[0133] In the preparation process of the antigen component loaded with nanoparticles or microparticles, the cations contained in the reagents used for salting out include, but are not limited to, Al. 3+ Fe 3+ Fe 2+ Mg 2+ Sn 2+ Zn 2+ Ca 2+ Li + Na + NH4 + K + Cu 2+ Ag + Ba 2+ The reagents used for salting out contain anions including, but not limited to, Cl-. - SO4 2- NO3 - CO3 2- SiO3 2- S2O7 2- B4O7 2- PO4 3- RCOO - NO2 - S2O8 2- S 2- CrO4 2- MnO4 - P2O7 4- wait.

[0134] The heating process involves heating at a temperature above 40°C for at least 5 seconds.

[0135] The enzyme treatment method includes, but is not limited to, using one or more of the following: nuclease, DNase, pepsin, chymotrypsin, trypsin, other protein-degrading enzymes, and protease inhibitors.

[0136] The oxidation process involves treating the antigenic component with an oxidizing agent, which includes, but is not limited to, hypochlorous acid, persulfate, KIO3, KBrO3, chlorine, dichromate, nitric acid, hydrogen peroxide, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, hydrogen peroxide, sodium percarbonate, sodium perborate, potassium perborate, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium dichromate, potassium permanganate, nitric acid, and ClO3. - ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, H2O2, NO3 - MnO4 - One or more of various oxidizing agents such as F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3, etc.

[0137] The mineralization includes, but is not limited to, one or more of the following mineralization methods: silicification, calcification, magnesification, and biomineralization.

[0138] The reduction refers to reducing the antigen component using a component that can reduce the antigen.

[0139] The irradiation is a treatment using one or more different irradiation methods such as ultraviolet light, X-rays, gamma rays, alpha rays, beta rays, and radioactive sources.

[0140] The methods for isolating and purifying mRNA include, but are not limited to, using DNase to degrade the DNA, using an appropriate kit to remove the DNA, using a specific kit to isolate total RNA, and using a specific kit to isolate mRNA.

[0141] The chromatography described in this disclosure includes, but is not limited to, column chromatography, gas chromatography, high-performance liquid chromatography, adsorption chromatography, partition chromatography, thin-layer chromatography, high-performance liquid chromatography, ion exchange chromatography, thin film chromatography, affinity chromatography, gel chromatography, etc.

[0142] The chromatographic methods described in this disclosure include, but are not limited to, column chromatography, thin-layer chromatography, liquid chromatography, gas chromatography, and supercritical fluid chromatography.

[0143] The electrophoresis methods described in this disclosure include, but are not limited to, SDS electrophoresis, isoelectric point focusing electrophoresis, isovelocity electrophoresis, immunoelectrophoresis, serum protein electrophoresis, nucleic acid electrophoresis, DNA sequencing electrophoresis, gel electrophoresis, and preparative electrophoresis.

[0144] The process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components during the preparation of antigen components loaded with nanoparticles or microparticles may include salting out; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include heating; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include enzymatic treatment (e.g., enzymatic hydrolysis); the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include oxidation; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or from whole-cell lysate components may include reduction; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components may include... The process of separating and purifying antigen components from water-soluble and / or water-insoluble components or whole-cell lysate components may include fixation; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or whole-cell lysate components may include mineralization, such as biomineralization, silicification, calcification, magnesification, etc.; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or whole-cell lysate components may include irradiation; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or whole-cell lysate components may include chromatography; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or whole-cell lysate components may include electrophoresis; the process of separating and purifying antigen components from water-soluble and / or water-insoluble components or whole-cell lysate components may include chromatography.

[0145] The process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components during the preparation of antigen components loaded with nanoparticles or microparticles may include salting out; the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include heating; the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include enzyme treatment (e.g., enzymatic hydrolysis, enzyme inhibition, etc.); the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include oxidation; the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components or in whole-cell lysate components may include reduction; the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components may include reduction; the process of enhancing the immunogenicity of antigen components in water-soluble and / or water-insoluble components may include reduction. The process of enhancing the immunogenicity of the original component or the antigenic component in the whole-cell lysate component may include fixation; the process of enhancing the immunogenicity of the antigenic component in the water-soluble and / or water-insoluble components or the antigenic component in the whole-cell lysate component may include mineralization, such as biomineralization, calcification, magnesification, etc.; the process of enhancing the immunogenicity of the antigenic component in the water-soluble and / or water-insoluble components or the antigenic component in the whole-cell lysate component may include irradiation; the process of enhancing the immunogenicity of the antigenic component in the water-soluble and / or water-insoluble components or the antigenic component in the whole-cell lysate component may include chromatography; the process of enhancing the immunogenicity of the antigenic component in the water-soluble and / or water-insoluble components or the antigenic component in the whole-cell lysate component may include electrophoresis; the process of enhancing the immunogenicity of the antigenic component in the water-soluble and / or water-insoluble components or the antigenic component in the whole-cell lysate component may include chromatography.

[0146] The cancer cell sources described in this disclosure are any methods by which cancer cells can be obtained, including but not limited to cancer cell lines, cancer cells obtained by in vitro expansion of cancer cells isolated and extracted from tumor tissue, cancer cells obtained by expansion of circulating tumor cells isolated and extracted from blood, or cancer cells differentiated and cultured from stem cells.

[0147] Before lysis, the cancer cells or tumor tissue can be co-incubated with specific chemical substances to stimulate them, and then the cancer cells or tumor tissue is lysed. These specific substances include, but are not limited to, small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), plant extracts (such as important extracts of ginseng, plant root and stem extracts, etc.), growth factors, cytokines, chemokines, interferon, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate and stimulate cancer cells or tumor tissue is to induce the cancer cells to produce more antigenic components.

[0148] The above lists several commonly used methods for preparing antigenic components from cancer cell lysates derived from multiple cancer cell lines or tumor tissues from multiple patients. In practical applications, these methods can be combined or other relevant and feasible preparation methods can be used as needed. Alternatively, cancer cells from tumor tissue can be isolated and amplified before preparing antigenic components; or circulating tumor cells from peripheral blood can be isolated and amplified before using the amplified circulating tumor cells to prepare antigenic components.

[0149] After the antigen component is prepared, it can be loaded onto nanoparticles or microparticles.

[0150] In practical applications, the nanoparticles / microparticles used to deliver antigens can also be bacteria and viruses. When the antigen delivery particles are bacteria and viruses, the bacteria and viruses can express tumor-specific antigens and / or tumor-associated antigens, or the bacteria and viruses contain DNA and / or mRNA that can express tumor-specific antigens and / or tumor-associated antigens.

[0151] The following example illustrates how to prepare nanoparticles and / or microparticles loaded with antigen components using the solvent evaporation method. In practical applications, any other feasible method can be used to prepare nanoparticles or microparticles, including but not limited to precipitation, dialysis, dispersion, microfluidics, high-pressure homogenization, stirring, spray drying, phase separation, electrostatic spraying, emulsion polymerization, machine stirring and shearing, and membrane emulsification.

[0152] The following example illustrates the specific preparation and detection methods using the double emulsion method in solvent evaporation to prepare nanoparticles / microparticles and their application in detecting cancer cell-specific T cells. The steps are as follows:

[0153] Step 1: Add a first predetermined volume of aqueous solution containing a first predetermined concentration to a second predetermined volume of organic phase containing a second predetermined concentration of raw material for preparing particles.

[0154] In some embodiments, the aqueous phase solution may contain antigenic components (or simultaneously contain an immunomodulatory adjuvant) derived from lysates of multiple cancer cell lines / tumor tissues from multiple patients; the antigenic components in the lysates are, during preparation, water-soluble components and / or originally water-insoluble components dissolved in a solution containing a solvent such as urea, guanidine hydrochloride, or glycosides, or water-soluble and water-insoluble components simultaneously dissolved in a solution containing a solvent. Alternatively, the components may be obtained after separation, purification, or immunogenicity enhancement of the above components. The concentration of the water-soluble components or the originally water-insoluble components in the aqueous phase solution, or the concentration of the water-soluble components plus the water-insoluble components, i.e., the first predetermined concentration, requires a protein / peptide concentration greater than 1 ng / mL, sufficient to load sufficient antigen to activate relevant cells. If an immunomodulatory adjuvant is contained, its concentration in the initial aqueous phase is greater than 0.01 ng / mL.

[0155] In some embodiments, dichloromethane is selected as the organic solvent. Other solvents, such as ethyl acetate or acetonitrile, may also be used in practical applications. Furthermore, in some embodiments, the second predetermined concentration for preparing the particulate raw material ranges from 0.5 mg / mL to 5000 mg / mL, preferably from 10 mg / mL to 100 mg / mL.

[0156] In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase. In this invention, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably from 1:1.5 to 10. During implementation, the first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0157] Preferably, when the aqueous phase solution is a lysate component solution, the concentration of protein and peptide is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL; when the aqueous phase solution is a lysate component / immunoadjuvant solution, the concentration of protein and peptide is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of immunoadjuvant is greater than 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. In the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0158] Step 2: The mixture obtained in Step 1 is subjected to ultrasonic treatment for more than 2 seconds, or stirring, homogenization, or microfluidic treatment for more than 1 minute. Preferably, when stirring is mechanical or magnetic, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm and the stirring time is 0.1 hours to 24 hours; when ultrasonic treatment is performed, the ultrasonic power is greater than 5W and the time is greater than 0.1 seconds, for example, 2 to 200 seconds; when homogenizing, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used, with the pressure being greater than 5 psi when using a high-pressure / ultra-high-pressure homogenizer, for example, 20 psi to 100 psi, and the rotation speed being greater than 100 rpm when using a high-shear homogenizer, for example, 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, for example, 0.1 mL / min to 100 mL / min. Nanoparticles and / or microparticles can be nano-sized or micronized by ultrasonication, stirring, homogenization, or microfluidic processing. The duration of ultrasonication, stirring speed, or homogenization pressure and time can control the size of the prepared nanoparticles or microparticles. If the particles are too large or too small, they will cause changes in particle size.

[0159] Step 3 involves adding the mixture obtained in Step 2 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of emulsifier and subjecting it to ultrasonic treatment for more than 2 seconds, or stirring for more than 1 minute, or homogenization or microfluidic treatment. In this step, the mixture obtained in Step 2 is added to the emulsifier aqueous solution and further ultrasonicated or stirred to achieve nano- or micron-sized structures. In this invention, the ultrasonic time is greater than 0.1 seconds, for example, 2 to 200 seconds; the stirring speed is greater than 50 rpm, for example, 50 to 500 rpm; and the stirring time is greater than 1 minute, for example, 60 to 6000 seconds. Preferably, when stirring is mechanical or magnetic, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, for example, a stirring speed of 50 rpm to 1500 rpm and a stirring time of 0.5 hours to 5 hours; when ultrasonic treatment is performed, the ultrasonic power is 50 W to 500 W and the time is greater than 0.1 seconds, for example, 2 to 200 seconds; when homogenizing, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used, with a pressure greater than 20 psi when using a high-pressure / ultra-high-pressure homogenizer, for example, 20 psi to 100 psi, and a rotation speed greater than 1000 rpm when using a high-shear homogenizer, for example, 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, for example, 0.1 mL / min to 100 mL / min. Nanoparticles or microparticles are produced through ultrasonication, stirring, homogenization, or microfluidic treatment. The duration of ultrasonication, stirring speed, or homogenization pressure and time can control the size of the prepared nanoparticles or microparticles; excessively large or small particles will lead to changes in particle size.

[0160] In some embodiments, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg / mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In this invention, the ratio of the second predetermined volume to the third predetermined volume is set in the range of 1:1.1 to 1:1000, preferably 1:1.5 to 5. In specific implementations, the ratio of the second predetermined volume to the third predetermined volume can be adjusted to control the size of nanoparticles or microparticles. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution, and the concentration in this step are all determined to obtain nanoparticles or microparticles of suitable size.

[0161] Step 4: Add the liquid obtained after step 3 to a fourth predetermined volume of an emulsifier aqueous solution of a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.

[0162] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0163] The fourth predetermined concentration is 5 mg / mL. The selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size. The selection of the fourth predetermined volume is determined by the ratio of the third predetermined volume to the fourth predetermined volume. In this invention, the ratio of the third predetermined volume to the fourth predetermined volume ranges from 1:1.5 to 1:2000, preferably from 1:4 to 10. In specific implementations, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the size of the nanoparticles or microparticles.

[0164] In this invention, the predetermined stirring condition for this step is until the organic solvent has completely evaporated, that is, the dichloromethane in step 1 has completely evaporated.

[0165] Step 5: After centrifuging the mixture from Step 4, which meets the predetermined stirring conditions, at a speed greater than 100 RPM for more than 1 minute, remove the supernatant and resuspend the remaining precipitate in a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of lyophilization protectant, or in a sixth predetermined volume of PBS (or physiological saline). Alternatively, after ultrafiltration, replace the liquid in the system with an aqueous solution containing lyophilization protectant to obtain the desired suspension.

[0166] Step 6: After freeze-drying the suspension containing the freeze-drying protectant obtained in Step 5, the freeze-dried material is ready for use.

[0167] Step 7: Use the nanoparticle-containing suspension obtained in step 5, resuspended in PBS (or physiological saline) in the sixth predetermined volume, or use the freeze-dried material containing nanoparticles or microparticles and a freeze-drying protectant obtained in step 6, resuspended in the sixth predetermined volume of PBS (or physiological saline) directly; or use the above sample after mixing with the water-soluble component or the dissolved original non-water-soluble component in the seventh predetermined volume.

[0168] In this invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.

[0169] Step 8: Obtain peripheral blood, peripheral immune tissue, or tumor tissue, and collect immune cells containing T cells from these tissues. Alternatively, sort the T cells and antigen-presenting cells from the above tissues, and then mix them in a certain ratio, with the ratio of antigen-presenting cells to T cells being 1:0.1-20.

[0170] Step 9: Mix the nano and / or micro particles prepared in Step 5, 6, or 7 with the immune cells containing T cells and antigen-presenting cells obtained in Step 8 and co-incubate for a certain period of time to activate cancer cell-specific T cells; or first co-incubate the nano and / or micro particles prepared in Step 5, 6, or 7 with antigen-presenting cells (isolated and purified antigen-presenting cells or mixed cells containing antigen-presenting cells) for a certain period of time, and then co-incubate the incubated antigen-presenting cells (inactivated or non-inactivated) with T cells for a certain period of time to activate cancer cell-specific T cells.

[0171] When nanoparticles and / or microparticles are co-incubated with the cells to be tested, the concentration of nanoparticles and / or microparticles is 0.001 mg / mL to 50 mg / mL, preferably 0.005 mg / mL to 5 mg / mL, more preferably 0.01 to 2 mg / mL; the co-incubation time is 3 h to 72 h, preferably 5 to 48 h; and the incubation temperature is a temperature that can allow the cells to survive and be activated, preferably 4 to 40 °C, and most preferably 37 °C.

[0172] Antigen-presenting cells co-incubated with nanoparticles or microparticles and T cells can be derived from the same autologous or allogeneic source as the T cells, or from cell lines or stem cells. Antigen-presenting cells can be dendritic cells (DCs), B cells, macrophages, or any mixture of the above, or other cells with antigen-presenting function.

[0173] When using whole-cell antigen components loaded with tumor tissue from multiple patients and / or multiple cancer cells to activate cancer cell-specific T cells via antigen-presenting cells, the system may contain cytokines and / or antibodies to enhance activation efficiency.

[0174] In some implementations, nanoparticles or microparticles loaded with whole-cell antigen components from multiple cancer cells or tumor tissues from multiple patients are first phagocytosed and presented by antigen-presenting cells, and then cancer cell-specific T cells in peripheral blood, peripheral immune tissues, or tumor-infiltrating lymphocytes are activated and detected by antigen-presenting cells.

[0175] During co-incubation, only nanoparticles, only microparticles, or a mixture of nanoparticles and microparticles can be used.

[0176] In some embodiments of this disclosure, 250 nm nanoparticles and 2.5 μm microparticles are used as mixed particles. In practical applications, nanoparticles and microparticles of other particle sizes can also be mixed to prepare mixed particles. When preparing mixed particles, the preferred particle size range of nanoparticles is 150 nm-500 nm, and the preferred particle size range of microparticles is 2.0 μm-3.5 μm.

[0177] When antigen-presenting cells are first activated by co-incubating with antigen delivery particles, and then co-incubated with a sample containing T cells, the antigen-presenting cells can be pre-isolated and purified, or they can be used directly as a mixed cell line containing antigen-presenting cells for co-incubation with antigen delivery particles without prior isolation and purification. The antigen-presenting cells activated after co-incubation with antigen delivery particles can be inactivated before co-incubation with a sample containing T cells, or the activated antigen-presenting cells can be directly co-incubated with a sample containing T cells for a certain period without inactivation to activate cancer cell-specific T cells for detection. Methods for inactivating antigen-presenting cells activated by antigen delivery particles include, but are not limited to, one or more methods of cell inactivation such as fixation and irradiation.

[0178] Step 10: Analyze the content and proportion of antigen-activated cancer cell-specific T cells using methods such as flow cytometry, enzyme-linked immunospot assay, enzyme-linked immunosorbent assay, multi-cytokine detection, or magnetic bead sorting.

[0179] This disclosure demonstrates that after cancer cell-specific T cells are activated, they express activation markers such as CD69 and CD25 on their surface and simultaneously secrete cytotoxic substances. Previously, no researchers had demonstrated the degree of overlap between the structural expression of activation markers and the functional secretion of cytotoxic substances by activated T cells. This disclosure confirms that after cancer cell-specific T cells are activated by cancer antigens, they simultaneously begin to express both structural activation markers and functional cytotoxic molecules. Furthermore, this study confirms the overlap between structural activation markers such as CD25 and CD69 and secreted cytotoxic substances such as IFN-γ and FASL, revealing structural and functional consistency. This disclosure demonstrates that either structural activation markers alone or functional cytotoxic substances alone can serve as activation markers for both structurally and functionally activated cancer cell-specific T cells.The examples used structural and functional activation markers such as IFN-γ, IL-2R, CD25, CD69, CD137, FASL, CD40L, HLA, and CD107. In practical applications, markers that can also be used include, but are not limited to, one or more combinations of the following: granzymes (e.g., granzyme B), perforin, CD69, FAS, FASL, CD107, CD40L, CD25, OX40 (CD134), TCF-1, CD25, IP-10, and CD40L. 39. CD38, CD29, CD27, CXCL13, CXCL6, CXCL7, PTPRC, CD103, NCR1, CD56, CD279, CD278, CD244, CD27, CD154, TCF- 1. TCF-7, CD102, CD44, IL-2R, FASL, CD28, HLA-DR, CD127(IL-7R), CD150, CD107A, CD83, CD166, CD178, CD212, C D229, CD100, CD107b, CD108, CD109, CD113, CD122, CD126, CD253, CD197, PD-1, TIM3, LAG-3, TIGIT, CD62L, CD70 , CTLA-4(CD152), CD27, CD26, CD30, TNFRSF9, CD74, PD-L1(CD274), CD258, CD261, 4-1BB, CD154, ICAM-1, LFA-1 , LFA-2, VLA-4, CD160, CD71, CXCR3, TNFRSF14, TNFRSF13, DUSP10, EMB, CHD3, TIM1, TCF-7, CD40, SELL, ICAM4, T NFSF13, PODXL, LEF1, TNFRSF18, TNFSF4, TNFSF9, TNFSF14, CD11a, CD101, CD48, CD244, CD49a, CD95, CD44, CXCR 1. CXCR13, CXCR6, CD103, CD45RO, ICOS(CD278), VTCN1, HHLA2, LGAL59, CCR5, CCR7, CD357, BCL6, TCF-1, etc.

[0180] In practical applications, activation markers can be a single marker or a combination of multiple markers.

[0181] Techniques for analyzing and detecting the number and proportion of cancer cell-specific T cells using specific biomarkers include, but are not limited to, flow cytometry, magnetic bead sorting, enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and multi-cytokine assay. When using specific biomarkers to analyze and detect the number and proportion of cancer cell-specific T cells, at least one biomarker can be used.

[0182] Other markers that can structurally and functionally demonstrate the specific activation of cancer cell-specific T cells by cancer antigens. Furthermore, these markers can be located intracellularly, on the cell surface, or secreted extracellularly.

[0183] In some embodiments of this invention, methods such as salting out, heating, enzymatic hydrolysis, and oxidation are used to separate and purify antigenic components in cancer cell / tumor tissue lysates or enhance the immunogenicity of antigenic components. In practical applications, methods such as phase separation, chromatographic separation, removal of DNA components from lysates, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, mineralization, and irradiation can also be used to separate and purify antigenic components in cancer cell / tumor tissue lysates or enhance the immunogenicity of antigenic components therein.

[0184] Example 1: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients

[0185] This embodiment uses nanoparticles (NPs) to detect cancer cell-specific T cells in cancer patients. In this embodiment, water-soluble and insoluble components from various human lung cancer cell lines are loaded onto nanoparticles, which are then used to assist in the detection of cancer cell-specific T cells in peripheral immune organs of non-small cell lung cancer patients.

[0186] (1) Preparation of antigen components based on lysates of various lung cancer cell lines

[0187] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. Among them, A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma cell line of the lung cancer subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma cell line of the lung cancer subtype.

[0188] After collecting the eight cell lines, the eight lung cancer cell lines were mixed in a cell number ratio (equivalent to cell mass ratio) of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to further lyse the cancer cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. Adding 8M urea to the resulting precipitate dissolved the insoluble component, converting it into a soluble component in an 8M urea aqueous solution. These water-soluble and insoluble components served as the antigenic raw materials for preparing nanoparticle 1 (NP1). The water-soluble component complex of the eight cancer cell lines was used as the antigenic raw material for preparing nanoparticle 2 (NP2).

[0189] Alternatively, only A549 cells can be collected, centrifuged, and the culture medium removed. The cell pellet is then resuspended in ultrapure water and subjected to five freeze-thaw cycles, with sonication used during the freeze-thaw process to more thoroughly lyse the cancer cells. After cell lysis, the lysate is centrifuged at 5000g for 5 minutes, and the supernatant is collected as the water-soluble component that is soluble in pure water. Adding 8M urea to the resulting precipitate dissolves the insoluble component, converting it into a soluble component in an 8M urea aqueous solution. These water-soluble and insoluble components constitute the antigenic raw material for preparing nanoparticles 3 (NP3).

[0190] Alternatively, tumor tissue samples can be collected from surgically removed tumor tissue of patient A, a non-small cell lung cancer patient. This patient had excellent response to immunotherapy, with complete tumor disappearance after treatment. The tumor tissue from this patient was minced and filtered through a cell sieve. Then, an appropriate amount of ultrapure water was added, and the mixture was repeatedly frozen and thawed five times. During the freeze-thaw process, ultrasonic disruption was used to more thoroughly lyse the cells in the tumor tissue. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. Adding 8M urea to the resulting precipitate dissolved the insoluble component in pure water, converting it into a soluble component in an 8M urea aqueous solution. These water-soluble and insoluble components serve as the antigenic raw materials for preparing nanoparticles 4 (NP4).

[0191] (2) Preparation of nanoparticles

[0192] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method in the solvent evaporation process. During preparation, nanoparticles loaded with the water-soluble components of whole-cell antigen from cancer cells and nanoparticles loaded with the insoluble components of whole-cell antigen from cancer cells were prepared separately and then used together. The PLGA nanoparticle material used had a molecular weight of 10 kDa-30 kDa, and the preparation method was as described above. In the preparation process, the antigen components were first loaded inside the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, and each 1 mg of PLGA nanoparticles loaded approximately 100 μg of protein or polypeptide components, the components of which are described in step (1) of the preparation method.

[0193] The preparation process of nanoparticle 2 (NP2) is the same as that of nanoparticle 1. The average particle size of nanoparticle 2 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 100 μg of protein or peptide components.

[0194] The preparation process of nanoparticle 3 (NP3) is the same as that of nanoparticle 1. The average particle size of nanoparticle 3 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 100 μg of protein or peptide components.

[0195] The preparation process of nanoparticle 4 (NP4) is the same as that of nanoparticle 1. The average particle size of nanoparticle 4 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 100 μg of protein or peptide components.

[0196] Control nanoparticle 5 (NP5) is a control nanoparticle loaded with multiple peptides, specifically three lung cancer neoantigen peptides: MAGE-A3 (sequence: FLWGPRALV), CEA (sequence: YLSGANLNL), and MUC1 (sequence: TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP). The mass ratio of the three neoantigen peptides is 1:1:1. The preparation process of nanoparticle 5 is the same as that of nanoparticle 1, but it uses neoantigen peptides instead of any cancer cell lysate components. Nanoparticle 5 has an average particle size of approximately 280 nm and loads 100 μg of lung cancer neoantigen peptides, but does not load any cancer cell lysate components.

[0197] Control nanoparticle 6 (NP6) is a blank control nanoparticle. Its preparation process is the same as that of nanoparticle 1, but no cancer cell lysate components are used in its preparation. The average particle size of nanoparticle 6 is about 280 nm and it does not carry any cancer cell lysate components.

[0198] (3) Detection of cancer cell-specific T cells

[0199] Patient A, a non-small cell lung cancer patient, recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation. The separation process of PBMCs is as follows: Divide 10 mL of blood into two 5 mL portions. Add PBS to the 5 mL of fresh blood to dilute the blood at a 1:1 ratio. Take 5 mL of lymphocyte separation solution into a 15 mL sterile centrifuge tube for later use. Aspirate the diluted blood and slowly add it along the tube wall 1 cm above the separation solution, so that the diluted blood overlaps the separation solution and forms a clear interface with the separation solution. Centrifuge the obtained sample at 2000 rpm for 20 min at room temperature. At this time, 5 layers are formed in the centrifuge tube: the top layer is plasma, and the layer between the plasma layer and the lymphocyte separation solution is a lymphocyte layer in the form of a white membrane. Carefully aspirate the middle layer of mononuclear cells in the white membrane, and try to aspirate all the mononuclear cells. Add more than 5 times the volume of PBS to the sample and wash twice, centrifuging at 1500 rpm for 10 min each time. Then discard the supernatant and add 1 mL of 1640 culture medium and mix well. Take a small amount of PBMCs for cell counting and use later.

[0200] Nanoparticle 1 (50 μg nanoparticles loaded with water-soluble components + 50 μg nanoparticles loaded with insoluble components), or nanoparticle 2 (100 μg nanoparticles loaded with water-soluble components), or nanoparticle 3 (50 μg nanoparticles loaded with water-soluble components + 50 μg nanoparticles loaded with insoluble components), or nanoparticle 4 (50 μg nanoparticles loaded with water-soluble components + 50 μg nanoparticles loaded with insoluble components), or nanoparticle 5 (100 μg), or blank nanoparticle 6 (without any lysate components) + an equal volume of free mixed cancer cell lysate (the same lysate used to prepare nanoparticle 1) and PBMCs (500,000 units) were co-incubated with PBMCs in 10 mL of serum-free AIM V medium for 72 hours (37°C, 5% CO2); or PBMCs (500,000 units) were incubated alone in 10 mL of serum-free AIM V medium for 72 hours (37°C, 5% CO2). Cells were collected and centrifuged at 400g for 5 minutes. After resuspending in PBS, T cells were first treated with Fc blocks to avoid nonspecific cell load. Extracellular staining with CD3, CD4, and CD8 antibodies was then performed. Cells were subsequently fixed and permeabilized, and intracellular staining with FN-γ antibody was performed. Flow cytometry was then used to analyze the T cell samples. CD4+ was analyzed separately. + T cells that are activated and can secrete IFN-γ are present in all CD4 cells. + The proportion of T cells and CD8 +T cells that are activated and can secrete IFN-γ are present in all CD8 cells. + The proportion of T cells. Above CD4 + IFN-γ + T cells and CD8 + IFN-γ + T cells are cancer cell-specific T cells.

[0201] Alternatively, T cells can be sorted from the PBMCs obtained above using magnetic bead sorting. Then, 500,000 T cells and nanoparticles 1 (50 μg nanoparticles loaded with water-soluble components + 50 μg nanoparticles loaded with insoluble components) are co-incubated in 10 mL of AIM V serum-free medium for 72 hours (37°C, 5% CO2). After cell collection, the cells are centrifuged at 400g for 5 minutes, resuspended in PBS, and treated with Fc blocks to avoid non-specific loading. Extracellular staining with CD3, CD4, and CD8 antibodies is then performed. Cells are then fixed and permeabilized, and intracellular staining with FN-γ antibody is performed. Flow cytometry is then used to analyze the sample T cells. CD4... + T cells that are activated and can secrete IFN-γ are present in all CD4 cells. + The proportion of T cells and CD8 + T cells that are activated and can secrete IFN-γ are present in all CD8 cells. + The proportion of T cells. Above CD4 + IFN-γ + T cells and CD8 + IFN-γ + T cells are cancer cell-specific T cells.

[0202] After nanoparticles / microparticles loaded with whole-cell components of tumor tissue and / or cancer cells are phagocytosed by antigen-presenting cells, the antigens are degraded into polypeptide epitopes and bound to major histocompatibility complex (MHC) molecules, which are then presented to the surface of the antigen-presenting cell membrane. Since whole-cell antigens loaded on nanoparticles and microparticles can be cross-presented, cancer cell antigen epitopes can be presented to the surface of the antigen-presenting cell membrane via both MHC I and MHC II pathways. After the cell membrane of antigen-presenting cells is prepared into nanoparticles or microparticles, the loaded MHC molecules and antigen polypeptide complexes can directly bind to T cell surface receptors that specifically recognize cancer cell antigens. Furthermore, if the T cell is a specific T cell capable of killing cancer cells, the cancer cell will begin to secrete cytotoxic substances such as IFN-γ, granzymes, and perforin. By analyzing the number and proportion of T cells that can secrete cytotoxic substances, the content of effector cancer cell-specific T cells with cancer cell recognition and killing capabilities can be obtained.

[0203] (4) Experimental Results

[0204] like Figure 2 As shown, no cancer cell-specific T cells were detected when PBMCs were incubated alone. Few cancer cell-specific T cells were detected after co-incubation with T cells and nanoparticle 1 alone, and very few were detected using nanoparticle 5 loaded with multiple peptides. However, nanoparticles 1, 2, 3, 4, and 6+ free lysate fractions all helped detect a certain amount of cancer cell-specific T cells. Nanoparticles 1 and 4 showed the best results, and their effects were superior to those of nanoparticles 2, 3, and 6+ free lysate fractions.

[0205] The detection results of co-incubating PBMCs with nanoparticle 1 were significantly better than those of co-incubating T cells and nanoparticle 1 alone, indicating that antigen-presenting cells are required for activation during the detection process. Nanoparticle 1 and nanoparticle 4 showed similar results, indicating that the mixed cancer cell lines contain and cover antigens from the cancer cells of non-small cell lung cancer patients' own tumor tissue. Nanoparticle 1 and nanoparticle 4 were superior to nanoparticle 3, indicating that a single cancer cell line cannot contain and cover antigens from most cancer patients' cancer cells, while using multiple cancer cell lines can diversify the types of cancer cell antigens, which is superior to using a single cancer cell line. Nanoparticle 1 was superior to nanoparticle 2, indicating that loading whole-cell antigen components onto nanoparticles is better than loading only water-soluble antigen components onto nanoparticles. Nanoparticle 1 was superior to nanoparticle 6 + free lysate components, indicating that lysate antigen components loaded onto nanoparticles are more easily phagocytosed and presented by antigen-presenting cells than in their free state, thus better activating and detecting cancer cell-specific T cells.

[0206] In summary, the particle system described in this invention can more accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0207] Example 2: Detection of cancer cell-specific T cells using nanoparticles from multiple cancer patient tumor tissues.

[0208] In this embodiment, nanoparticles (NPs) were prepared using tumor tissues from multiple cancer patients, and these nanoparticles were used to assist in the detection of cancer cell-specific T cells. Using PLGA as the framework material and poly(I:C) and CpG7909 as immunoadjuvants, nanoparticles loaded with water-soluble and insoluble components of tumor tissue were prepared by solvent evaporation. These nanoparticles were then used to activate and detect cancer cell-specific T cells in peripheral blood immune cells.

[0209] (1) Preparation of antigenic components based on tumor tissue lysates

[0210] Tumor tissues were collected from surgically removed lung tissues of 20 patients with non-small cell lung cancer and 10 patients with small cell lung cancer. Tumor tissue from each patient was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to a freeze-thaw cycle five times, possibly accompanied by sonication, to destroy lysed cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved by adding 6M guanidine hydrochloride aqueous solution, thus converting the water-insoluble component into a soluble component. Water-soluble components from tumor tissues of 30 cancer patients were mixed separately at a 1:1 mass ratio to obtain a mixture of water-soluble components from the 30 cancer patients. Similarly, water-insoluble components soluble in 6M guanidine hydrochloride from tumor tissues of the 30 cancer patients were mixed separately at a 1:1 mass ratio to obtain a mixture of water-insoluble components from the 30 cancer patients. The antigenic raw material for preparing nanoparticle 1 (NP1) is obtained by mixing the water-soluble component mixture and the insoluble component mixture at a mass ratio of 1:3.

[0211] The tumor tissue obtained from the surgical resection of the lung of one of the 20 non-small cell lung cancer patients, A, was processed according to the above method to obtain the water-soluble component and the non-water-soluble component dissolved in 6M guanidine hydrochloride aqueous solution of the tumor tissue of patient A. The water-soluble component and the non-water-soluble component were mixed at a mass ratio of 1:3, which is the source of antigen raw materials for preparing nanoparticle 2 (NP2).

[0212] Tumor tissue obtained from the surgical resection of the lung of a non-small cell lung cancer patient (Patient B) was collected. Patient B was not included in the aforementioned 30 cancer patients. The tumor tissue from Patient B was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to five freeze-thaw cycles, possibly accompanied by sonication, to destroy and lyse the cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved by adding 6M guanidine hydrochloride aqueous solution, thus converting the water-insoluble component into a soluble component. The water-soluble and water-insoluble components were mixed at a mass ratio of 1:3 to obtain the antigenic raw material for preparing nanoparticles 3 (NP3).

[0213] Tumor tissue obtained from the surgical resection of the lung of a non-small cell lung cancer patient (C) was collected. Patient C was not included in the aforementioned 30 cancer patients. The tumor tissue from patient C was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to five freeze-thaw cycles, possibly accompanied by sonication, to destroy and lyse the cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved by adding 6M guanidine hydrochloride aqueous solution, thus converting the water-insoluble component into a soluble component. The water-soluble and water-insoluble components were mixed at a mass ratio of 1:3 to obtain the antigenic raw material for preparing nanoparticles 4 (NP4).

[0214] Nanoparticles 5 (NP5) were prepared using multiple neoantigen peptides for lung cancer. The loaded peptides were three neoantigen peptides for lung cancer: MAGE-A3 (sequence: FLWGPRALV), CEA (sequence: YLSGANLNL), and MUC1 (sequence: TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP). The mass ratio of the three neoantigen peptides was 1:1:1.

[0215] (2) Preparation of nanoparticles

[0216] In this embodiment, the nanoparticles were prepared using the double emulsion method in the solvent evaporation process.

[0217] Nanoparticle 1 (NP1), loaded with whole-cell components from tumor tissues of 30 cancer patients, was prepared using PLGA nanoparticles with a molecular weight of 7 Da-17 kDa. The immunoadjuvants used were poly(I:C) and CpG7909, with the adjuvants encapsulated within the nanoparticles. The preparation method was as described previously. First, the antigen and adjuvant were loaded internally into the nanoparticles using a double emulsion method. After loading the antigen (lysed components), 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. The average particle size of this nanoparticle 1 was approximately 100 nm; each mg of PLGA nanoparticles loaded approximately 10 μg of protein and polypeptide components, and each mg of PLGA nanoparticles used 0.01 mg of poly(I:C) and CpG7909 immunoadjuvants.

[0218] Nanoparticle 2 (NP2), loaded with whole-cell components of tumor tissue from patient A, was prepared using the same materials and methods as nanoparticle 1. The average particle size of nanoparticle 2 was approximately 100 nm; each 1 mg of PLGA nanoparticles was loaded with approximately 10 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles used 0.01 mg each of poly(I:C) and CpG7909 immunoadjuvant.

[0219] Nanoparticles 3 (NP3) loaded with whole-cell components from tumor tissue of non-small cell lung cancer patient B were prepared using the same materials and methods as nanoparticles 1. The average particle size of nanoparticles 3 (NP3) was approximately 100 nm; each 1 mg of PLGA nanoparticles was loaded with approximately 10 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles used 0.01 mg each of poly(I:C) and CpG7909 immunoadjuvant.

[0220] Nanoparticles 4 (NP4) loaded with whole-cell components of tumor tissue from patient C were prepared using the same materials and methods as nanoparticles 1. The average particle size of nanoparticles 4 was approximately 100 nm; each 1 mg of PLGA nanoparticles was loaded with approximately 10 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles used 0.01 mg each of poly(I:C) and CpG7909 immunoadjuvant.

[0221] The materials and preparation method for the whole-cell component nanoparticles 5 (NP5) loaded with peptides are the same as those for nanoparticles 1. The average particle size of nanoparticles 5 is about 100 nm; each 1 mg of PLGA nanoparticles is loaded with about 10 μg of lung cancer neoantigen peptide component, and each 1 mg of PLGA nanoparticles uses 0.01 mg of poly(I:C) and CpG7909 immunoadjuvant.

[0222] (3) Detection of cancer cell-specific T cells

[0223] Patient B with non-small cell lung cancer (NSCLC) recovered after immunotherapy. Patient C with NSCLC showed no significant improvement after immunotherapy. Peripheral blood samples of 10 mL were collected from NSCLC patient B before and two weeks after immunotherapy in both patients. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation. The PBMC isolation process was as described previously.

[0224] Nanoparticles 1 (2 mg), 2, 3, 4, or 5 (2 mg) were incubated with 1 million PBMCs from patient B or patient C in 1 mL of serum-free AIM V medium for 4 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were first treated with Fc blocks to avoid nonspecific cell loading. Extracellular staining with CD3, CD4, and CD8 antibodies was then performed. Cells were then fixed and permeabilized, and intracellular staining with FN-γ antibody was performed. Flow cytometry was then used to analyze the T cell samples. CD4 levels were analyzed separately. + T cells that are activated and can secrete IFN-γ are present in all CD4 cells. + The proportion of T cells and CD8 + T cells that are activated and can secrete IFN-γ are present in all CD8 cells. + The proportion of T cells. Above CD4 + IFN-γ + T cells and CD8 + IFN-γ + T cells are cancer cell-specific T cells.

[0225] (4) Experimental Results

[0226] like Figure 3As shown, stronger fluorescence intensity indicates a greater amount of cytotoxic substances expressed by the cancer cell-specific T cells, leading to higher sensitivity and accuracy in detection. Nanoparticles 1 and 3 showed the best detection results when detecting cancer cell-specific T cells in the peripheral blood of patient B; nanoparticles 1 and 4 showed the best results when detecting cancer cell-specific T cells in the peripheral blood of patient C. This indicates that detection nanoparticles prepared using mixed tumor tissues from multiple patients have similar effects to those prepared using tumor tissues from individual patients. This is because a mixture of tumor tissues from multiple cancers contains more diverse cancer cell antigens, which can cover the cancer cell antigens contained in the patient's own tumor tissue. The more comprehensive the coverage of cancer cell antigens, the more comprehensive and accurate the detection of cancer cell-specific T cells.

[0227] When detecting cancer cell-specific T cells in the peripheral blood of patient B, nanoparticles 2, 4, and 5 did not perform as well as nanoparticles 1 and 3. This indicates that detection nanoparticles prepared from a single allogeneic patient's tumor tissue and those loaded with multiple neoantigen peptides are not as effective as those prepared from autologous tumor tissue. This is because the cancer antigens contained in tumor tissue from a single allogeneic cancer patient differ somewhat from those contained in autologous tumor tissue from a cancer patient.

[0228] When detecting cancer cell-specific T cells in the peripheral blood of patient C, nanoparticles 2, 3, and 5 were less effective than nanoparticles 1 and 4. This indicates that detection nanoparticles prepared from a single allogeneic patient's tumor tissue and those loaded with multiple neoantigen peptides are not as effective as those prepared from autologous tumor tissue.

[0229] Furthermore, before immunotherapy, the levels of cancer cell-specific T cells in the peripheral blood of cancer patients B and C were similar and low. However, after immunotherapy, the number of cancer cell-specific T cells in the peripheral blood of cancer patient B (who responded well to immunotherapy) increased significantly, while the number of cancer cell-specific T cells in the peripheral blood of cancer patient C (who responded poorly to immunotherapy) did not increase significantly. This indicates that the increase in the level of cancer cell-specific T cells in the peripheral blood of cancer patients is positively correlated with the efficacy of cancer immunotherapy. The nanoparticle detection of changes in the level of cancer cell-specific T cells described in this invention can be used to predict the efficacy and prognosis of immunotherapy in cancer patients.

[0230] Therefore, the nanoparticles described in this invention can better detect cancer cell-specific T cells capable of recognizing and killing cancer cells. After being phagocytosed by antigen-presenting cells, the nanoparticles can be degraded into antigenic epitopes and presented on the surface of antigen-presenting cells. These epitopes can recognize and activate cancer cell-specific T cells and express specific surface markers. By analyzing the proportion of T cells highly expressing specific surface markers using techniques such as flow cytometry, the number and proportion of activated cancer cell-specific T cells capable of recognizing and killing cancer cells can be determined.

[0231] Example 3: Detection of cancer cell-specific T cells using micron-sized particles from multiple lung cancer patient tumor tissues.

[0232] In this embodiment, micron particles (MPs) were prepared using tumor tissues from multiple cancer patients, and these MPs were used to assist in the detection of cancer cell-specific T cells. Micron particles loaded with water-soluble and insoluble components of tumor tissue were prepared using PLGA as the backbone material via a solvent evaporation method. These micron particles were then used to activate and detect cancer cell-specific T cells in peripheral blood immune cells.

[0233] (1) Preparation of antigenic components based on tumor tissue lysates

[0234] Tumor tissues were collected from surgically removed lung tissues of 12 patients with non-small cell lung cancer and 3 patients with small cell lung cancer. Tumor tissue from each patient was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to a freeze-thaw cycle five times, possibly accompanied by sonication, to destroy lysed cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved by adding 6M guanidine hydrochloride aqueous solution, thus converting the water-insoluble component into a soluble component. Water-soluble components from tumor tissues of 15 cancer patients were mixed separately at a 1:1 mass ratio to obtain a mixture of water-soluble components from the 15 cancer patients. Similarly, water-insoluble components soluble in 6M guanidine hydrochloride from tumor tissues of 15 cancer patients were mixed separately at a 1:1 mass ratio to obtain a mixture of water-insoluble components from the 15 cancer patients. The antigen raw material for preparing microparticle 1 (MP1) is obtained by mixing the water-soluble component mixture and the insoluble component mixture at a mass ratio of 5:1.

[0235] The tumor tissue obtained from the surgical resection of the lung of one of the 12 non-small cell lung cancer patients, A, was processed according to the above method to obtain the water-soluble component and the non-water-soluble component dissolved in 6M guanidine hydrochloride aqueous solution of the tumor tissue of patient A. The water-soluble component and the non-water-soluble component were mixed at a mass ratio of 5:1, which is the source of antigen raw materials for preparing micron particles 2 (MP2).

[0236] Tumor tissue obtained from the surgical resection of the lung of another non-small cell lung cancer patient B (not included in the aforementioned 15 cancer patients) was collected. The tumor tissue from patient B was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to five freeze-thaw cycles, possibly accompanied by sonication, to destroy lysed cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved by adding 6M guanidine hydrochloride aqueous solution, thus converting the water-insoluble component into a soluble component. The water-soluble and water-insoluble components were mixed at a mass ratio of 5:1 to obtain the antigenic raw material for preparing micron-sized particles 3 (MP3).

[0237] Tumor tissue obtained from the surgical resection of the lung of another non-small cell lung cancer patient, C, was collected. Patient C was not included in the aforementioned 15 cancer patients. The tumor tissue from patient C was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to five freeze-thaw cycles, possibly accompanied by sonication, to destroy lysed cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved by adding 6M guanidine hydrochloride aqueous solution, thus converting the water-insoluble component into a soluble component. The water-soluble and water-insoluble components were mixed at a mass ratio of 5:1 to obtain the antigenic raw material for preparing micron-sized particles 4 (MP4).

[0238] Microparticles 5 (MP5) were prepared using a variety of lung cancer neoantigen peptides. The loaded peptides were three lung cancer neoantigen peptides: MAGE-A3 (sequence: FLWGPRALV), CEA (sequence: YLSGANLNL), and MUC1 (sequence: TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP). The mass ratio of the three neoantigen peptides was 1:1:1.

[0239] (2) Preparation of micron-sized particles

[0240] In this embodiment, the micron-sized particles were all prepared using a solvent evaporation method.

[0241] Microparticle 1 (MP1), loaded with whole-cell components from tumor tissues of 15 cancer patients, was prepared using PLGA with a molecular weight of 38 Da-54 kDa. The preparation method was as described previously. First, antigen was loaded into the microparticles using a double emulsion method. Then, 100 mg of microparticles were centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 16 h. The average particle size of these microparticles was approximately 1.5 μm; each mg of PLGA microparticles loaded approximately 200 μg of protein and peptide components.

[0242] The materials and preparation method for microparticles 2 (MP2) loaded with whole-cell components of tumor tissue from patient A are the same as those for microparticles 1. The average particle size of microparticles 2 is about 1.5 μm; each 1 mg of PLGA microparticles is loaded with approximately 200 μg of protein and polypeptide components.

[0243] The materials and preparation method for microparticles 3 (MP3) loaded with whole-cell components from tumor tissue of non-small cell lung cancer patient B are the same as those for microparticles 1. The average particle size of microparticles 3 is about 1.5 μm; each 1 mg of PLGA microparticles is loaded with approximately 200 μg of protein and peptide components.

[0244] The materials and preparation method for microparticles 4 (MP4) loaded with whole-cell components of tumor tissue from patient C are the same as those for microparticles 1. The average particle size of microparticles 4 is about 1.5 μm; each 1 mg of PLGA microparticles is loaded with approximately 200 μg of protein and peptide components.

[0245] The materials and preparation method for the whole-cell component loaded with peptides, microparticles 5 (NP5), are the same as those for microparticles 1. The average particle size of microparticles 5 is about 1.5 μm; each 1 mg of PLGA microparticles is loaded with approximately 200 μg of lung cancer neoantigen peptide component.

[0246] (3) Detection of cancer cell-specific T cells

[0247] Patient B with non-small cell lung cancer (NSCLC) recovered after immunotherapy. Patient C with NSCLC showed poor response to immunotherapy, with no significant therapeutic effect. Peripheral blood samples of 10 mL were collected from NSCLC patient B before immunotherapy and again two weeks after immunotherapy in both patients. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation. The PBMC isolation process was as described previously.

[0248] Microparticles 1 (0.1 mg), 2 (0.1 mg), 3 (0.1 mg), 4 (0.1 mg), or 5 (0.1 mg) were incubated with 1 million PBMCs in 1 mL of serum-free AIM V medium for 4 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were treated with an Fc block to prevent non-specific adsorption. Cells were then stained with CD3, CD4, CD8, and IL-2 receptor (IL-2R) antibodies. Flow cytometry was then used to analyze the T cell samples. CD4... +T cells that are activated and highly express the IL-2 receptor are present in all CD4+ cells. + The proportion of T cells and CD8 + T cells that are activated and highly express the IL-2 receptor are present in all CD8 cells. + The proportion of T cells. Above CD4 + IL-2R + T cells and CD8 + IL-2R + T cells are cancer cell-specific T cells.

[0249] (4) Experimental Results

[0250] like Figure 4 As shown, microparticles 1 and 3 exhibited the best detection results when detecting cancer cell-specific T cells in the peripheral blood of patient B; while microparticles 1 and 4 showed the best detection results when detecting cancer cell-specific T cells in the peripheral blood of patient C. This indicates that the detection microparticles prepared using mixed tumor tissues from multiple patients have similar detection results to those prepared using individual tumor tissues from a single patient.

[0251] When detecting cancer cell-specific T cells in the peripheral blood of patient B, microparticles 2, 4, and 5 were less effective than microparticles 1 and 3. This indicates that microparticles prepared from a single allogeneic patient's tumor tissue and microparticles loaded with multiple neoantigen peptides are less effective than microparticles prepared from autologous tumor tissue. Similarly, when detecting cancer cell-specific T cells in the peripheral blood of patient C, microparticles 2, 3, and 5 were less effective than microparticles 1 and 4.

[0252] Furthermore, before immunotherapy, the levels of cancer cell-specific T cells in the peripheral blood of cancer patients B and C were similar and low. However, after immunotherapy, the number of cancer cell-specific T cells in the peripheral blood of cancer patient B (who responded well to immunotherapy) increased significantly, while the number of cancer cell-specific T cells in the peripheral blood of cancer patient C (who responded poorly to immunotherapy) did not increase significantly. This indicates that the increase in the level of cancer cell-specific T cells in the peripheral blood of cancer patients is positively correlated with the efficacy of cancer immunotherapy. The micron-particle detection of changes in the level of cancer cell-specific T cells described in this invention can be used to predict the efficacy and prognosis of immunotherapy in cancer patients.

[0253] Example 4: Detection of cancer cell-specific T cells in colon cancer

[0254] In this embodiment, microparticles were prepared using tumor tissue from multiple cancer patients, and these microparticles were used to assist in the detection of cancer cell-specific T cells. Microparticles loaded with tumor tissue components were prepared using PLA as the backbone material via a solvent evaporation method. Then, the microparticles were used to activate and detect cancer cell-specific T cells in peripheral blood immune cells.

[0255] (1) Preparation of antigenic components based on tumor tissue and cancer cell lysates

[0256] Tumor tissues were collected from six patients with colon cancer after surgical resection. Each patient's tumor tissue was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to five freeze-thaw cycles, possibly accompanied by sonication, to disrupt the lysed cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. 8M urea was added to the resulting precipitate to dissolve the insoluble component, converting it into a soluble component in a 10% octyl glucoside aqueous solution. The water-soluble component (80 mg / mL) was incubated with trypsin (0.5 mg / mL) and chymotrypsin (0.5 mg / mL) for 1 hour, followed by heating at 95°C for 10 minutes to inactivate the proteases. Water-soluble components from the tumor tissues of 6 cancer patients were mixed separately at a 1:1 mass ratio to obtain a mixture of water-soluble components from 15 cancer patients. Insoluble components dissolved in 10% octyl glucoside from the tumor tissues of 6 cancer patients were mixed separately at a 1:1 mass ratio to obtain a mixture of insoluble components from 6 cancer patients. The mixture of water-soluble and insoluble components was then mixed at a 5:1 mass ratio to obtain the antigenic raw material for preparing micron-sized particles 1 (MP1).

[0257] Tumor tissue obtained from the surgical resection of another colon cancer patient, A (not included in the aforementioned six cancer patients), was collected. The tumor tissue from patient A was cut into blocks, ground, and filtered through a cell filter with an appropriate amount of pure water. The mixture was then subjected to five freeze-thaw cycles, possibly accompanied by sonication, to destroy lysed cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. 8M urea was added to the resulting precipitate to dissolve the insoluble component, converting it into a soluble component in a 10% octyl glucoside aqueous solution. Trypsin (0.5mg / mL) and chymotrypsin (0.5mg / mL) were added to the water-soluble component (80mg / mL), and the mixture was incubated for 1 hour, followed by heating at 95°C for 10 minutes to inactivate the proteases. The water-soluble and insoluble components were mixed at a mass ratio of 5:1 to obtain the antigenic raw material for preparing microparticles 2 (MP2).

[0258] (2) Preparation of micron-sized particles

[0259] In this embodiment, microparticles 1 were prepared using a solvent evaporation method. The materials used to prepare microparticles 1 were PEG2000-PLA (molecular weight 40 kDa) and PLA (molecular weight 40 kDa), with a mass ratio of PEG2000-PLA (molecular weight 40 kDa) to PLA (molecular weight 40 kDa) of 19:1. The preparation method was as described above. First, a double emulsion method was used to load a mixture of lysates inside the microparticles. Then, 100 mg of microparticles was centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 24 h. The average particle size of microparticles 1 was approximately 5.0 μm. Each mg of PLA microparticles 1 loaded approximately 240 μg of protein or polypeptide components, and each mg of PLA microparticles contained 0.04 mg each of CpGM362, CPG1018, and Poly ICLC immunoadjuvant.

[0260] In this embodiment, microparticles 2 were prepared by solvent evaporation. The materials used to prepare microparticles 2 were PEG2000-PLA (molecular weight 40 kDa) and PLA (molecular weight 40 kDa), with a mass ratio of PEG2000-PLA (molecular weight 40 kDa) to PLA (molecular weight 40 kDa) of 19:1. The preparation method was as described above. In the preparation process, the lysate was first loaded into the microparticles using a double emulsion method. Then, 100 mg of microparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 24 h. The average particle size of microparticles 2 was approximately 5.0 μm. Each mg of PLA microparticles 2 loaded approximately 240 μg of protein or peptide components, and each mg of PLA microparticles contained 0.04 mg each of CpGM362, CPG1018, and Poly ICLC immunoadjuvant.

[0261] (3) Detection of cancer cell-specific T cells

[0262] Patient A underwent immunotherapy after surgical removal of the tumor tissue, and the treatment showed excellent efficacy, with the tumor continuously shrinking. Peripheral blood samples (5 mL) were collected from the cancer patient before immunotherapy and again three weeks after immunotherapy. Peripheral blood cells (PBMCs) were first isolated from the PBMCs, and then T cells and B cells were sorted from the PBMCs using a magnetic bead method.

[0263] First, the obtained B cells (1 million cells) and 500 μg of microparticles (microparticle 1 or microparticle 2), IL-2 (100 U / mL), IL-7 (500 U / mL), and IL-15 (50 U / mL) were incubated in 5 mL of serum-free AIM V medium for 3 hours (37°C, 5% CO2). After incubation, the B cells were collected and centrifuged at 400g for 5 minutes. Then, the incubated B cells were resuspended and incubated with the corresponding T cells (500,000 cells) in 1 mL of serum-free AIM V medium for 3 hours (37°C, 5% CO2). The supernatant was then collected, and the concentration of IFN-γ in the supernatant was detected using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The concentration of IFN-γ indicates the amount of cancer cell-specific T cells: a higher concentration of IFN-γ indicates a higher number of cancer cell-specific T cells; a lower concentration of IFN-γ indicates a lower number of cancer cell-specific T cells.

[0264] (4) Experimental Results

[0265] like Figure 5 As shown, there was no significant difference in the concentration of IFN-γ detected in the patient's peripheral blood between micron-particle 1 and micron-particle 2, indicating that the detection capabilities of the two types of micron-particles are similar. Furthermore, the number of cancer cell-specific T cells in the peripheral blood of patients significantly increased after immunotherapy. This demonstrates that the nanoparticles or micron-particles described in this invention can predict the efficacy of cancer immunotherapy by detecting cancer cell-specific T cells.

[0266] Example 5: Detection of cancer cell-specific T cells in colon cancer

[0267] In this embodiment, tumor tissues from multiple cancer patients were used to prepare micron-sized particles, which were then used to assist in the detection of cancer cell-specific T cells. Micron-sized particles loaded with tumor tissue components were prepared using PLGA as the backbone material via a solvent evaporation method. These micron-sized particles were then used to activate and detect cancer cell-specific T cells in peripheral blood immune cells.

[0268] (1) Preparation of antigenic components based on tumor tissue and cancer cells

[0269] Tumor tissues were collected from six colon cancer patients after surgical resection. Each patient's tumor tissue was cut into blocks, ground, and passed through a cell filter before being lysed with an appropriate amount of 2M aminourea hydrochloride. After cell lysis, the whole-cell lysate components were dissolved using a 2M aminourea hydrochloride aqueous solution. The tumor tissue lysate components from the six cancer patients were mixed at a mass ratio of 1:1:1:1:1:1 to obtain a mixture of tumor tissue lysates from the six cancer patients, which served as the antigenic raw material source for preparing microparticle 1 (MP1).

[0270] Tumor tissue obtained from the surgical resection of another colon cancer patient, A (not included in the aforementioned six cancer patients), was collected. The tumor tissue from patient A was cut into pieces, ground, and passed through a cell filter. An appropriate amount of 2M aminourea hydrochloride was added to lyse the cells. After cell lysis, the whole-cell lysate components were dissolved using a 2M aminourea hydrochloride aqueous solution, which became the antigenic raw material for preparing microparticles 2 (MP2).

[0271] (2) Preparation of micron-sized particles

[0272] In this embodiment, microparticles 1 (MP1) were prepared by solvent evaporation. The PLGA material used to prepare microparticles 1 has a molecular weight of 24-38 kDa. The preparation method is as described above. First, a double emulsion method is used to load a mixture of lysed products inside the microparticles. Then, 100 mg of microparticles is centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 12 h. The average particle size of microparticles 1 is approximately 2.5 μm, and each mg of PLGA microparticles 1 loads approximately 20 μg of protein or peptide components.

[0273] In this embodiment, microparticles 2 (MP2) were prepared by solvent evaporation. The molecular weight of PLGA used to prepare microparticles 2 was 24-38 kDa. The preparation method was as described above. In the preparation process, the lysate was first loaded inside the microparticles using a double emulsion method. Then, 100 mg of microparticles were centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 12 h. The average particle size of microparticles 2 was about 2.5 μm, and each 1 mg of PLGA microparticles 2 was loaded with approximately 20 μg of protein or peptide components.

[0274] (3) Detection of cancer cell-specific T cells

[0275] Patient A underwent radiotherapy after surgical removal of the tumor tissue, and the treatment was very effective, with the tumor mass disappearing. Peripheral blood samples of 15 mL were collected from the cancer patient before immunotherapy and again 4 weeks after radiotherapy, and PBMCs were isolated from the peripheral blood.

[0276] This embodiment uses enzyme-linked immunospot assay (ELISPOT) to detect IFN-γ secreted by activated cancer cell-specific T cells. First, adsorbed antibody (IFN-γ) is pre-coated into 96-well plates. Then, 20,000 PBMC cells and 10 μg of microparticles (microparticle 1 or microparticle 2) are added to 200 μL of serum-free AIM V medium and incubated for 12 hours (37°C, 5% CO2). After incubation, the cells are discarded, and the detection antibody and subsequent reagents are added. Finally, spots are formed in the wells, and the number of spots in each well is analyzed using a detector. More spots indicate a higher number of cancer cell-specific T cells, and vice versa.

[0277] (4) Experimental Results

[0278] like Figure 6 As shown, there was no significant difference in the number of cancer cell-specific T cells detected in the patient's peripheral blood by micron-particles 1 and 2, indicating that the detection capabilities of the two types of micron-particles are similar. Furthermore, the number of cancer cell-specific T cells in the peripheral blood of patients significantly increased after radiotherapy. This demonstrates that the nanoparticles or micron-particles described in this invention can predict the effectiveness of cancer radiotherapy by detecting cancer cell-specific T cells.

[0279] Example 6: Detection of cancer cell-specific T cells in breast cancer

[0280] In this embodiment, nanoparticles were prepared using tumor tissues from multiple breast cancer patients, and these nanoparticles were used to assist in the detection of cancer cell-specific T cells in peripheral blood or peripheral immune organs.

[0281] (1) Preparation of antigen components

[0282] Tumor tissues were collected from surgically removed tumors of 12 patients with triple-negative breast cancer. Each patient's tumor tissue was cut into blocks, ground, filtered through a cell filter, and added to ultrapure water. The mixture was then subjected to five freeze-thaw cycles accompanied by sonication to lyse the cancer cells. 1 mg / mL of nuclease was added to the lysed cancer cells to degrade the nucleic acids in the lysate. The nuclease was then inactivated by heating at 95°C for 10 minutes. The supernatant was collected after centrifugation at 5000g for 5 minutes; this was the water-soluble component. The precipitate was dissolved in 10% sodium deoxycholate to obtain the water-insoluble component. The water-soluble component lysates from the 12 breast cancer patients were mixed at a 1:1 mass ratio to obtain a water-soluble component mixture. The water-insoluble component lysates from the 12 breast cancer patients were also mixed at a 1:1 mass ratio to obtain a water-insoluble component mixture. The water-soluble component mixture and the water-insoluble component mixture were then mixed at a 5:1 mass ratio to obtain antigen component 1 for preparing nanoparticle 1 (NP1).

[0283] Tumor tissue was collected from surgically removed tumor tissue from another triple-negative breast cancer patient, A. Patient A was not included in the aforementioned 12 cancer patients. The tumor tissue was cut into blocks, ground, filtered through a cell filter, and added to ultrapure water. The mixture was then subjected to five freeze-thaw cycles accompanied by sonication to lyse the cancer cells. 1 mg / mL of nuclease was added to the lysed cancer cells to degrade the nucleic acids in the lysate. The nuclease was then inactivated by heating at 95°C for 10 minutes. The supernatant was collected after centrifugation at 5000g for 5 minutes; this was the water-soluble component. The precipitate was dissolved in 10% sodium deoxycholate to obtain the water-insoluble component. The water-soluble and water-insoluble components were mixed at a mass ratio of 5:1 to obtain antigen component 2 for preparing nanoparticle 2 (NP2).

[0284] (2) Preparation of nanoparticles

[0285] In this embodiment, the nanoparticles were prepared using a double emulsion method. The PLGA backbone material of nanoparticle 1 has a molecular weight of 10 kDa-20 kDa, and the immunoadjuvants used are CpG2395 (class C), CpG1018 (class B), and Poly I:C. During preparation, nanoparticles internally loaded with antigen component 1 and adjuvants were prepared using a double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 12000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 h before use. The nanoparticles have a particle size of approximately 280 nm, with each 1 mg of PLGA nanoparticles loading approximately 150 μg of protein or peptide components, and 0.02 mg each of CpG2395, CpG1018, and Poly (I:C).

[0286] The PLGA nanoparticle backbone material has a molecular weight of 10 kDa-20 kDa, and the immunoadjuvants used are CpG2395 (class C), CpG1018 (class B), and Poly I:C. Nanoparticles loaded with antigen component 2 and adjuvants were prepared using a double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 12000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 h before use. The nanoparticles have a particle size of approximately 280 nm, with each mg of PLGA nanoparticle loading approximately 150 μg of protein or peptide component, and 0.02 mg each of CpG2395, CpG1018, and Poly (I:C).

[0287] (3) Detection of cancer cell-specific T cells

[0288] Patient A underwent cancer immunotherapy after surgical removal of the tumor tissue, and the treatment was very effective, with the tumor gradually shrinking. 10 mL of peripheral blood was drawn from the patient before or after immunotherapy. PBMCs were then isolated from the peripheral blood.

[0289] 6 million PBMCs, 10 ng IL-7, 10 ng IL-15, and 20 μg nanoparticles (nanoparticle 1 or nanoparticle 2, respectively) were incubated together in 2 mL of serum-free AIM V medium for 72 hours (37°C, 5% CO2). After incubation, cells were collected and sequentially labeled with flow cytometry antibodies, followed by flow cytometry analysis of CD3. + CD25 + T cells in total CD3 + The number of T cells is the number of cancer cell-specific T cells.

[0290] Alternatively, 6 million PBMCs, 10 ng IL-7, 10 ng IL-15, and 5 ng nanoparticles (nanoparticle 1 or nanoparticle 2, respectively) were co-incubated in 2 mL of serum-free AIM V medium for 72 hours (37°C, 5% CO2). After incubation, cells were collected and sequentially labeled with flow cytometry antibodies, followed by flow cytometry analysis of CD3. + CD25 + T cells in total CD3 + The number of T cells is the number of cancer cell-specific T cells.

[0291] Alternatively, PBMCs (6 million units), IL-7 (10 ng), IL-15 (10 ng), and 100 mg of nanoparticles (nanoparticle 1 or nanoparticle 2, respectively) were co-incubated in 2 mL of serum-free AIM V medium for 72 hours (37°C, 5% CO2). After incubation, cells were collected and sequentially labeled with flow cytometry antibodies, followed by flow cytometry analysis of CD3. + CD25 + T cells in total CD3 + The number of T cells is the number of cancer cell-specific T cells.

[0292] (4) Experimental Results

[0293] like Figure 7 As shown, compared with the control group, both types of nanoparticles can assist in the detection of cancer cell-specific T cells, with nanoparticle 1 and nanoparticle 2 showing similar effects. Moreover, the detection effect using an appropriate concentration (10 μg / mL) of nanoparticles is significantly better than using too low a concentration (2.5 ng / mL) or too high a concentration (50 mg / mL).

[0294] Example 7: Detection of cancer cell-specific T cells in pancreatic cancer

[0295] This example uses a CD19 antibody as a target to illustrate how to use active targeting nanoparticles to assist in the detection of cancer cell-specific T cells in peripheral blood.

[0296] (1) Preparation of antigen components

[0297] Tumor tissues were collected from surgically removed pancreatic cancer patients. Each patient's tumor tissue was cut into blocks, ground, filtered through a cell filter, and added to ultrapure water. The mixture underwent five freeze-thaw cycles accompanied by sonication to lyse cancer cells. After heating at 95°C for 10 minutes, the mixture was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved in 10% sodium dodecyl sulfate (SDS) to obtain the water-insoluble component. The water-soluble component lysates from the 10 pancreatic cancer patients were mixed at a 1:1 mass ratio to obtain a water-soluble component mixture. The water-insoluble component lysates from the 10 pancreatic cancer patients were also mixed at a 1:1 mass ratio to obtain a water-insoluble component mixture. The water-soluble and water-insoluble component mixtures were then mixed at a 1:1 mass ratio to obtain the antigen component for preparing nanoparticle 1.

[0298] Tumor tissue was collected from the surgical resection of another pancreatic cancer patient, A. Patient A is not included in the aforementioned 10 cancer patients. The tumor tissue was cut into blocks, ground, filtered through a cell filter, and added to ultrapure water. The mixture was then subjected to five freeze-thaw cycles with sonication to lyse the cancer cells. After heating at 95°C for 10 minutes, the mixture was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. The precipitate was dissolved in 10% sodium dodecyl sulfate (SDS) to obtain the water-insoluble component. The water-soluble and water-insoluble components were mixed at a mass ratio of 1:1 to obtain the antigen component for preparing nanoparticle 2.

[0299] (2) Preparation of nanoparticles

[0300] In this embodiment, the nanoparticles were prepared using a double emulsion method. The backbone material of nanoparticle 1 was a mixture of PLGA (molecular weight 20kDa-40kDa) and CD19 antibody-PEG5000-PLGA (20-40kDa) in a 9:1 ratio. During preparation, nanoparticles internally loaded with lysate components were prepared using a double emulsion method. Then, 100mg of nanoparticles were centrifuged at 12000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use. The nanoparticles had a particle size of approximately 350nm, and each 1mg of PLGA nanoparticles loaded approximately 50μg of protein or peptide components.

[0301] The nanoparticles were prepared using a mixture of PLGA (molecular weight 20-40 kDa) as the backbone material and CD19 antibody-PEG5000-PLGA (20-40 kDa) in a 9:1 ratio. The nanoparticles were prepared using a double emulsion method to internally load the lysate components. Then, 100 mg of nanoparticles were centrifuged at 12000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 h before use. The nanoparticles had a particle size of approximately 350 nm, and each mg of PLGA nanoparticles loaded approximately 50 μg of protein or peptide components.

[0302] (3) Detection of cancer cell-specific T cells

[0303] Patient A underwent cancer immunotherapy after surgical removal of tumor tissue, and the treatment was very effective, with the tumor gradually shrinking. 12 mL of peripheral blood was drawn from the patient before or after immunotherapy. PBMCs were then isolated from the peripheral blood. 10 million PBMCs and 500 μg of nanoparticles (nanoparticle 1, nanoparticle 2, or nanoparticle 3, respectively) were incubated together in 2 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2). After incubation, cells were collected and sequentially labeled with flow cytometry antibodies, followed by CD3 analysis using flow cytometry. + CD69 + T cells in total CD3 + The number of T cells is the number of cancer cell-specific T cells.

[0304] (4) Experimental Results

[0305] like Figure 8 As shown, nanoparticles 1 and 2 exhibit similar effects. This demonstrates that nanoparticles prepared from tumor tissues of multiple allogeneic cancer patients demonstrate comparable effectiveness in detecting cancer cell-specific T cells to nanoparticles prepared from tumor tissues of individual cancer patients. In this implementation, CD19 antibody was used as the active targeting head. However, in practical applications, any target head capable of targeting specific cells, such as mannose, CD32 antibody, CD20 antibody, mannan, or CD205 antibody, can also be used.

[0306] Example 8: Detection of cancer cell-specific T cells in melanoma using nanoparticles

[0307] (1) Preparation of antigenic components based on tumor tissue and cancer cells

[0308] Tumor tissues were collected from five melanoma patients. The tumor tissues from the five patients were mixed in a mass ratio of 1:1:1:1:1, cut into pieces, and ground. After passing through a cell filter, an appropriate amount of 8M urea aqueous solution was added to lyse the cells. The tumor tissue lysate was completely dissolved using 8M urea aqueous solution, which is the antigen component 1 for preparing nanoparticles 1 and 2.

[0309] Tumor tissue was collected from another melanoma patient, A. Patient A is not included in the above 5 cancer patients. The tumor tissue from patient A was cut into pieces and ground, then passed through a cell filter and an appropriate amount of 8M urea aqueous solution was added to lyse the cells. The tumor tissue lysate components were completely dissolved using 8M urea aqueous solution, which is the antigen component 2 for preparing nanoparticles 3 and 4.

[0310] (2) Particle preparation

[0311] In this embodiment, nanoparticle 1 (NP1) was prepared using a double emulsion method. The nanoparticles used were PLGA nanoparticles with molecular weights ranging from 10 kDa to 30 kDa, and included KALA peptide (WEAKLAKALAKALAKHLAKALAKALKACEA), a substance that increases lysosomal immune escape. The lysate components and KALA peptide were encapsulated within the nanoparticles. The preparation method was as described above. First, a double emulsion method was used to load the lysate components and KALA peptide inside the nanoparticles. Then, 100 mg of nanoparticles were centrifuged at 12000 g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of these nanoparticles was approximately 250 nm. Each mg of PLGA nanoparticles loaded approximately 100 μg of protein or peptide components, and each mg of PLGA nanoparticles loaded 0.15 mg of KALA peptide.

[0312] Nanoparticle 2 (NP2) is prepared using the same materials and methods. Its particle size is about 250 nm. Each 1 mg of PLGA nanoparticles is loaded with about 0.01 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.15 mg of KALA peptide.

[0313] The materials and preparation methods for nanoparticles 3 (NP3) are the same, with a size of about 250 nm. Each 1 mg of PLGA nanoparticles is loaded with about 100 μg of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.15 mg of KALA peptide.

[0314] The materials and preparation methods for nanoparticles 4 (NP4) are the same. They are about 250 nm in size. Each 1 mg of PLGA nanoparticles is loaded with about 0.01 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.15 mg of KALA peptide.

[0315] (3) Detection of cancer cell-specific T cells

[0316] Patient A underwent cancer immunotherapy after surgical removal of tumor tissue, and the treatment was very effective, with the tumor gradually shrinking. 12 mL of peripheral blood was drawn from the patient before or after immunotherapy. PBMCs were then isolated from the peripheral blood. 10 million PBMCs and 100 μg of nanoparticles (nanoparticle 1, nanoparticle 2, or nanoparticle 3, respectively) were incubated together in 10 mL of serum-free AIM V medium for 96 hours (37°C, 5% CO2). After incubation, cells were collected and sequentially labeled with flow cytometry antibodies, followed by CD3 analysis using flow cytometry. + HLA-DR + T cells in total CD3 + The number of T cells is the number of cancer cell-specific T cells.

[0317] (4) Experimental Results

[0318] like Figure 9 As shown, there was no significant difference in detection performance between nanoparticles prepared from a mixture of tumor tissues from multiple allogeneic cancer patients and nanoparticles prepared from autologous tumor tissues from cancer patients. Furthermore, nanoparticles loaded with appropriate antigenic components performed better than nanoparticles loaded with only low amounts of antigenic components.

[0319] Example 9: Detection of cancer cell-specific T cells in lung cancer

[0320] In this embodiment, lung cancer tumor tissue was first inactivated and denatured, then cells were lysed, and the whole-cell components of the lysed tumor tissue were dissolved in a 6M guanidine hydrochloride aqueous solution. Then, using PLGA as the nanoparticle framework material, CpG2007, CpG1018, and Poly ICLC as immunoadjuvants, and polyarginine and polylysine as substances to enhance lysosomal escape, nanoparticles loaded with cancer cell whole-cell antigens were prepared for the detection of cancer cell-specific T cells.

[0321] (1) Preparation of antigen components

[0322] Tumor tissues were collected from 10 patients with non-small cell lung cancer. The tumor tissues from the 10 patients were mixed in a mass ratio of 1:1:1:1:1:1:1:1:1:1, cut into pieces, and ground. After passing through a cell filter, an appropriate amount of 6M guanidine hydrochloride aqueous solution was added to lyse the cells. The lysate components of the tumor tissue were completely dissolved using 6M guanidine hydrochloride aqueous solution, which is the source of antigen raw materials for the preparation of nanoparticle 1 (NP1).

[0323] Tumor tissue was collected from non-small cell lung cancer patient A. Patient A was not included in the above 10 cancer patients. The tumor tissue from patient A was cut into pieces and ground, then passed through a cell filter and an appropriate amount of 8M urea aqueous solution was added to lyse the cells. The tumor tissue lysate components were completely dissolved using 8M urea aqueous solution, which is the source of antigen raw materials for preparing nanoparticles 2.

[0324] (2) Particle preparation

[0325] In this embodiment, nanoparticle 1 (NP1) was prepared using a double emulsion method. The PLGA backbone material of nanoparticle 1 has a molecular weight of 10 kDa-20 kDa. The immunoadjuvants used were CpG7909, CpG1018, and Poly ICLC. The lysosomal escape-enhancing substances used were polyarginine and polylysine. The preparation process involved first using a double emulsion method to prepare nanoparticles internally loaded with antigen components, adjuvants, and lysosomal escape-enhancing agents. Then, 100 mg of nanoparticles were centrifuged at 13000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use. The nanoparticles had a particle size of approximately 500 nm. Each 1 mg of PLGA nanoparticles loaded approximately 50 μg of protein or polypeptide components, containing 0.01 mg each of CpG7909, CpG1018, and Poly ICLC, and 0.02 mg each of polyarginine and polylysine.

[0326] In this embodiment, nanoparticle 2 (NP2) was prepared in the same manner as nanoparticle 1. Nanoparticle 2 has a particle size of about 500 nm, and each 1 mg of PLGA nanoparticles is loaded with about 50 μg of protein or polypeptide components, containing 0.01 mg each of CpG7909, CpG1018 and Poly ICLC, and 0.02 mg each of polyarginine and polylysine.

[0327] (3) Detection of cancer cell-specific T cells

[0328] Patient A underwent cancer immunotherapy after surgical removal of tumor tissue, and the treatment was very effective, with the tumor gradually shrinking. 12 mL of peripheral blood was drawn from the patient before or after immunotherapy. PBMCs were then isolated from the peripheral blood. 10 million PBMCs and 100 μg of nanoparticles (nanoparticle 1, nanoparticle 2, or nanoparticle 3, respectively) were incubated together in 20 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2). After incubation, cells were collected and stained with CD3, CD4, CD8, and CD69 antibodies modified with different fluorescent probes. Flow cytometry was then used to detect CD8+ in the cells. + CD69 + CD34 + T cells account for CD8 +The proportion of T cells and CD4 + CD69 + CD34 + T cells account for CD4+ + The proportion of T cells is the number of cancer cell-specific T cells that can recognize the full-cell antigen of cancer cells.

[0329] (4) Experimental Results

[0330] like Figure 10 As shown, there was no significant difference in the detection performance of nanoparticles prepared by loading a mixture of tumor tissues from multiple allogeneic cancer patients with that of nanoparticles prepared using autologous tumor tissues from cancer patients.

[0331] Example 10: Nanoparticles used to detect cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0332] This embodiment uses nanoparticles (NPs) to detect cancer cell-specific T cells in cancer patients. In this embodiment, water-soluble and insoluble components from various human lung cancer cell lines are loaded onto nanoparticles, which are then used to assist in the detection of cancer cell-specific T cells in peripheral immune organs of non-small cell lung cancer patients.

[0333] (1) Preparation of antigen components based on multiple lung cancer cell lines

[0334] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. Among them, A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma cell line of the lung cancer subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma cell line of the lung cancer subtype.

[0335] After collecting the above eight cell types separately, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed in a cell ratio of 5:5:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to further lyse the cancer cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. Adding 0.3M sodium guanidinosuccinate to the resulting precipitate dissolved the insoluble component, converting it into a soluble component in 0.3M sodium guanidinosuccinate aqueous solution. The water-soluble and insoluble components were mixed in a 1:1 mass ratio to obtain antigen component 1 for preparing nanoparticle 1 (NP1).

[0336] After collecting the above eight cell types separately, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed in a cell ratio of 5:5:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to further lyse the cancer cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. Adding 0.3M sodium guanidinosuccinate to the resulting precipitate dissolved the insoluble component, converting it into a soluble component in 0.3M sodium guanidinosuccinate aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysis buffer. After complete precipitation, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitate was dissolved in 0.3M sodium guanidinosuccinate aqueous solution for later use. The supernatant was heated at 95℃ for 10 minutes, and the resulting sample was centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the precipitate was dissolved in 0.3M sodium guanidinosuccinate aqueous solution. The precipitate dissolved with 0.3M sodium guanidinosuccinate and the heated precipitate were then combined to obtain the protein and polypeptide antigen component in the water-soluble component. The above insoluble and water-soluble protein and polypeptide components were mixed at a mass ratio of 1:1 to obtain antigen component 2 for preparing nanoparticle 2 (NP2).

[0337] After collecting the above eight cell types separately, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed in a cell ratio of 5:5:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to further lyse the cancer cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component. Adding 0.3M sodium guanidinosuccinate to the resulting precipitate dissolved the insoluble component, converting it into a soluble component in 0.3M sodium guanidinosuccinate aqueous solution. Saturated ammonium carbonate aqueous solution was added dropwise to the water-soluble component in the lysis buffer. After precipitation was complete, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitate was dissolved in 0.3M sodium guanidinosuccinate aqueous solution for later use. The supernatant was heated at 95℃ for 10 minutes, and the resulting sample was centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the precipitate was dissolved in 0.3M sodium guanidinosuccinate aqueous solution. The precipitate dissolved with 0.3M sodium guanidinosuccinate and the heated precipitate were then combined to obtain the protein and polypeptide antigen component in the water-soluble component. The above insoluble and water-soluble protein and polypeptide components were mixed at a mass ratio of 1:1 to obtain antigen component 3 for the preparation of nanoparticle 3 (NP3).

[0338] Alternatively, a tumor tissue sample can be collected from the surgically removed tumor of patient A, a non-small cell lung cancer patient. This patient had excellent response to immunotherapy, with the tumor completely disappearing after treatment. The tumor tissue from this patient was minced and filtered through a cell sieve. Then, an appropriate amount of ultrapure water was added, and the mixture was repeatedly frozen and thawed five times. During the freeze-thaw process, ultrasonic disruption was used to more thoroughly lyse the cells in the tumor tissue. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. Adding 0.3M sodium guanidinosuccinate to the resulting precipitate dissolved the precipitate, thus converting the water-insoluble component into a soluble component in a 0.3M sodium guanidinosuccinate aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysis buffer. After complete precipitation, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitate was dissolved in 0.3M sodium guanidinosuccinate aqueous solution for later use. The supernatant was heated at 95℃ for 10 minutes, and the resulting sample was centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the precipitate was dissolved in 0.3M sodium guanidinosuccinate aqueous solution. The precipitate dissolved with 0.3M sodium guanidinosuccinate and the heated precipitate were then combined to obtain the protein and polypeptide antigen component in the water-soluble component. The above insoluble and water-soluble protein and polypeptide components were mixed at a mass ratio of 1:1 to obtain antigen component 4 for the preparation of nanoparticle 4 (NP4).

[0339] Alternatively, a tumor tissue sample surgically removed from patient A, a non-small cell lung cancer patient, can be collected. This patient had excellent response to immunotherapy, with the tumor completely disappearing after treatment. The tumor tissue from this patient was minced and filtered through a cell sieve. Then, an appropriate amount of ultrapure water was added, and the mixture was repeatedly frozen and thawed five times. During the freeze-thaw process, ultrasonic disruption was used to more thoroughly lyse the cells in the tumor tissue. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. Adding 0.3M sodium guanidinosuccinate to the resulting precipitate dissolved the precipitate, converting the water-insoluble component into a soluble component in a 0.3M sodium guanidinosuccinate aqueous solution. This is the preparation of antigen component 5 for nanoparticle 5 (NP5).

[0340] (2) Preparation of nanoparticles loaded with antigen components

[0341] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 10 kDa-30 kDa. The preparation method was as described previously. First, the antigen component and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, and each 1 mg of PLGA nanoparticles loaded approximately 5 μg of protein or peptide component.

[0342] The preparation process of nanoparticle 2 (NP2) is the same as that of nanoparticle 1. The average particle size of nanoparticle 2 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 5 μg of protein or peptide components.

[0343] The preparation process of nanoparticle 3 (NP3) is the same as that of nanoparticle 1. The average particle size of nanoparticle 3 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 5 μg of protein or peptide components.

[0344] The preparation process of nanoparticle 4 (NP4) is the same as that of nanoparticle 1. The average particle size of nanoparticle 4 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 5 μg of protein or peptide components.

[0345] The preparation process of nanoparticle 5 (NP5) is the same as that of nanoparticle 1. The average particle size of nanoparticle 5 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 5 μg of protein or peptide components.

[0346] Control nanoparticle 6 (NP6) is a control nanoparticle loaded with multiple peptides, specifically three lung cancer neoantigen peptides: MAGE-A3 (sequence: FLWGPRALV), CEA (sequence: YLSGANLNL), and MUC1 (sequence: TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP). The mass ratio of the three neoantigen peptides is 1:1:1. Nanoparticle 5 was prepared using the same process as nanoparticle 1, but instead of any cancer cell lysate components, it used neoantigen peptides. This nanoparticle 6 has an average particle size of approximately 280 nm and loads 5 μg of lung cancer neoantigen peptides, but without any cancer cell lysate components.

[0347] (3) Detection of cancer cell-specific T cells

[0348] Patient A with non-small cell lung cancer recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0349] Nanoparticles 1 (10 mg), 2 (10 mg), 3 (10 mg), 4 (10 mg), 5 (10 mg), or 6 (10 mg) were incubated with PBMCs (1 million units) in 1 mL of AIM V serum-free medium for 12 hours (37°C, 5% CO2); or, as a control group, PBMCs (1 million units) were incubated alone in 1 mL of AIM V serum-free medium for 12 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were first treated with Fc blocks to avoid nonspecific loading, and then stained with CD3 antibody and granzyme B antibody. Flow cytometry was then used to analyze the sample T cells. CD3 analysis was performed. + GranzymeB + T cells in all CD3 + The proportion of T cells is cancer cell-specific T cells.

[0350] (4) Experimental Results

[0351] like Figure 11As shown, PBMCs incubated alone did not produce any activated cancer cell-specific T cells. Using nanoparticles 6 loaded with multiple peptides, a small number of cancer cell-specific T cells could be detected. Nanoparticles 1, 2, 3, 4, and 5 all helped detect more cancer cell-specific T cells. Furthermore, nanoparticles 1 and 5 showed similar effects, as did nanoparticles 2 and 4, with nanoparticles 2 and 4 showing better effects than nanoparticles 1 and 5. This indicates that using a specific method to separate and purify the protein and peptide components before loading them onto nanoparticles yields better results. Moreover, nanoparticle 2 performed better than nanoparticle 3, indicating that using a specific salting-out reagent was more effective. In summary, the particle system described in this invention can more accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0352] Example 11: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients

[0353] This embodiment uses nanoparticles (NPs) to detect cancer cell-specific T cells in cancer patients. In this embodiment, water-soluble and insoluble components from various human lung cancer cell lines are loaded onto nanoparticles, which are then used to assist in the detection of cancer cell-specific T cells in peripheral immune organs of non-small cell lung cancer patients.

[0354] (1) Preparation of antigen components based on multiple lung cancer cell lines

[0355] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. Among them, A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma cell line of the lung cancer subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma cell line of the lung cancer subtype.

[0356] After collecting the above eight cell types separately, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed at a cell ratio of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cells were resuspended in ultrapure water containing 0.1% protease inhibitor. The cells were then subjected to five freeze-thaw cycles, and 6M guanidine hydrochloride was added to lyse the cancer cells. The lysate was then dissolved in a 6M guanidine hydrochloride aqueous solution. The lysate was then mixed with a solution containing the cancer-associated antigen WT1. 235-243 -IO102( GASAYGSLCYTWNQMNLDTLLKALLEIASCLEKALQVF CYTWNQ The peptides and containing the cancer-associated antigen WT1 294-312 IO103(FRGIQDVRRVSGVAPTLVR- EQCLSAFTL - FMTYWHLLNAFTVTVPKDL The polypeptides were mixed at a mass ratio of 10:1:1 to obtain antigen component 1 for preparing nanoparticle 1 (NP1).

[0357] After collecting the above eight cell types, A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 cells were mixed at a cell ratio of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cells were resuspended in ultrapure water containing 0.1% protease inhibitor. The cells were then subjected to five freeze-thaw cycles, and 6M guanidine hydrochloride was added to lyse the cancer cells. The lysate was then dissolved in a 6M guanidine hydrochloride aqueous solution. Saturated sodium chloride aqueous solution was then added dropwise to the sample. The sample was centrifuged at 10000g for 25 minutes, the supernatant was discarded, and the precipitate was dissolved a second time in a 6M guanidine hydrochloride aqueous solution. The resulting fraction was then mixed with a solution containing the cancer-associated antigen WT1. 235-243 -IO102( GASAYG SL CYTWNQMNLDTLLKALLEIASCLEKALQVF CYTWNQ The peptides and containing the cancer-associated antigen WT1 294-312 IO103(FRGIQDVRRVSGVAPTLVR- EQCLSAFTL - FMTYWHLLNAFTVTVPKDL The polypeptides were mixed at a mass ratio of 10:1:1 to obtain antigen component 2 for preparing nanoparticles 2 (NP2).

[0358] After collecting the above eight cell types, A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 cells were mixed at a cell ratio of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cells were resuspended in ultrapure water containing 0.1% protease inhibitor. The cells were then subjected to five freeze-thaw cycles, and 6M guanidine hydrochloride was added to lyse the cancer cells. The lysate was then dissolved in 6M guanidine hydrochloride aqueous solution. Saturated magnesium carbonate aqueous solution was then added dropwise to the sample. The sample was centrifuged at 10000g for 25 minutes, the supernatant was discarded, and the precipitate was dissolved again in 6M guanidine hydrochloride aqueous solution. The resulting fraction was then mixed with a solution containing the cancer-associated antigen WT1. 235-243 -IO102( GASAYG SLCYTWNQMNLDTLLKALLEIASCLEKALQVF CYTWNQ The peptides and containing the cancer-associated antigen WT1 294-312 IO103(FRGIQDVRRVSGVAPTLVR- EQCLSAFTL - FMTYWHLLNAFTVTVPKDL The polypeptides were mixed at a mass ratio of 10:1:1 to obtain antigen component 3 for preparing nanoparticles 3 (NP3).

[0359] Alternatively, a tumor tissue sample surgically removed from patient A, a non-small cell lung cancer patient, could be collected. This patient had excellent response to immunotherapy, with complete tumor disappearance after treatment. The tumor tissue from this patient was minced and filtered through a cell sieve. The cells were then resuspended in ultrapure water containing 0.1% protease inhibitor, and subjected to five freeze-thaw cycles. 6M guanidine hydrochloride was added to lyse the cancer cells, and the lysate was dissolved in a 6M guanidine hydrochloride aqueous solution. Saturated sodium chloride aqueous solution was then added dropwise to the sample. The sample was then centrifuged at 10000g for 25 minutes, the supernatant was discarded, and the precipitate was dissolved a second time in a 6M guanidine hydrochloride aqueous solution. The resulting fraction was then mixed with a sample containing the cancer-associated antigen WT1. 235-243 -IO102( GASAYGSL CYTWNQMNLDTLLKALLEIASCLEKALQVF CYTWNQ The peptides and containing the cancer-associated antigen WT1 294-312 IO103(FRGIQDVRRVSGVAPTLVR- EQCLSAFTL - FMTYWHLLNAFTVTVPKDL The polypeptides were mixed at a mass ratio of 10:1:1 to obtain antigen component 4 for preparing nanoparticles 4 (NP4).

[0360] Alternatively, tumor tissue samples can be collected from surgically removed tumor tissue of patient A, a non-small cell lung cancer patient. This patient had excellent response to immunotherapy, with complete tumor disappearance after treatment. The tumor tissue from this non-small cell lung cancer patient was minced and filtered through a cell sieve. The cells were then resuspended in ultrapure water containing 0.1% protease inhibitor, and subjected to five freeze-thaw cycles. 6M guanidine hydrochloride was added to lyse the cancer cells. The lysate was then dissolved in a 6M guanidine hydrochloride aqueous solution. The lysate was then mixed with a sample containing the cancer-associated antigen WT1. 235-243 -IO102( GASAYGSL CYTWNQMNLDTLLKALLEIASCLEKALQVF CYTWNQ The peptides and containing the cancer-associated antigen WT1 294-312 IO103(FRGIQDVRRVSGVAPTLVR- EQCLSAFTL - FMTYWHLLNAFTVTVPKDLThe polypeptides were mixed at a mass ratio of 10:1:1 to obtain antigen component 5 for preparing nanoparticles 5 (NP5).

[0361] (2) Preparation of particles loaded with antigen components

[0362] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method in the solvent evaporation process. During preparation, nanoparticles loaded with the water-soluble components of whole-cell antigen from cancer cells and nanoparticles loaded with the insoluble components of whole-cell antigen from cancer cells were prepared separately and then used together. The PLGA nanoparticle material used had a molecular weight of 10 kDa-20 kDa. The preparation method was as described above. In the preparation process, the antigen components and adjuvants were first loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 180 nm, and each 1 mg of PLGA nanoparticles loaded approximately 500 μg of protein or polypeptide components, the components of which are described in step (1) of the preparation method.

[0363] The preparation process of nanoparticle 2 (NP2) is the same as that of nanoparticle 1. The average particle size of nanoparticle 2 is about 180 nm, and each 1 mg of PLGA nanoparticles is loaded with about 500 μg of protein or peptide components.

[0364] The preparation process of nanoparticle 3 (NP3) is the same as that of nanoparticle 1. The average particle size of nanoparticle 3 is about 180 nm, and each 1 mg of PLGA nanoparticles is loaded with about 500 μg of protein or peptide components.

[0365] The preparation process of nanoparticle 4 (NP4) is the same as that of nanoparticle 1. The average particle size of nanoparticle 4 is about 180 nm, and each 1 mg of PLGA nanoparticles is loaded with about 500 μg of protein or peptide components.

[0366] The preparation process of nanoparticle 5 (NP5) is the same as that of nanoparticle 1. The average particle size of nanoparticle 5 is about 180 nm, and each 1 mg of PLGA nanoparticles is loaded with about 500 μg of protein or peptide components.

[0367] (3) Detection of cancer cell-specific T cells

[0368] Patient A, a non-small cell lung cancer patient, recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0369] First, flow cytometry was used to analyze CD3 in PBMCs.+ CD25 + T cells were removed, and the remaining cells from 500,000 PBMCs were then incubated with nanoparticles 1 (25 μg), 2 (25 μg), 3 (25 μg), 4 (25 μg), or 5 (25 μg) in 10 mL of AIM V serum-free medium for 16 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were first treated with Fc blocks to avoid nonspecific loading. Extracellular staining with CD3 and CD25 antibodies was then performed. CD3 analysis was performed. + CD3+ is activated in T cells. + CD25 + The T cells are cancer cell-specific T cells.

[0370] (4) Experimental Results

[0371] like Figure 12 As shown, nanoparticles 1, 2, 3, 4, and 5 can all assist in the detection of cancer cell-specific T cells. Furthermore, nanoparticles 1 and 5 have similar effects, as do nanoparticles 2 and 4, but nanoparticles 2 and 4 are more effective than nanoparticles 1 and 5. This indicates that using a specific method to separate and purify the protein and peptide components before loading them onto the nanoparticles yields better results. Moreover, nanoparticle 2 is better than nanoparticle 3, indicating that using a specific salting-out reagent is more effective. In summary, the particle system described in this invention can more accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0372] In summary, the particle system described in this invention can more accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0373] Example 12: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0374] This embodiment uses nanoparticles (NPs) to detect cancer cell-specific T cells in cancer patients. In this embodiment, water-soluble and insoluble components from various human lung cancer cell lines are loaded onto nanoparticles, which are then used to assist in the detection of cancer cell-specific T cells in peripheral immune organs of non-small cell lung cancer patients.

[0375] (1) Preparation of antigen components based on multiple lung cancer cell lines

[0376] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170 and H520 were cultured separately.

[0377] After collecting the above eight cell types separately, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed at a cell ratio of 20:20:2:2:2:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to further lyse the cancer cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component in pure water. Adding 6M guanidine sulfate to the resulting precipitate dissolved the insoluble component in pure water, thus converting it into a soluble component in a 6M guanidine sulfate aqueous solution. After heating the water-soluble components of the lysis buffer at 95°C for 10 minutes, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitated protein and peptide components were dissolved in a 6M guanidine sulfate aqueous solution. The mRNA in the supernatant was extracted using an mRNA extraction kit. The mRNA component was then mixed with the protein and peptide components dissolved in 6M guanidine sulfate to obtain the antigen component in the water-soluble component. Next, saturated ammonium sulfate solution was added to the insoluble component dissolved in 6M guanidine sulfate to salt out the protein and peptide components. The precipitated precipitate was then dissolved a second time in 6M guanidine sulfate to obtain the antigen component in the insoluble component. The antigen components in the insoluble and water-soluble components were mixed at a mass ratio of 1:1 to obtain antigen component 1 for preparing nanoparticle 1 (NP1).

[0378] Alternatively, a tumor tissue sample can be collected from the surgically removed tumor of patient A, a non-small cell lung cancer patient. This patient had excellent response to immunotherapy, with the tumor completely disappearing after treatment. The tumor tissue from this patient was minced, filtered through a cell sieve, resuspended in ultrapure water, and subjected to five freeze-thaw cycles. During the freeze-thaw process, ultrasonic disruption was used to further lyse the cancer cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component in pure water. Adding 6M guanidine sulfate to the resulting precipitate dissolved the precipitate, thus converting the water-insoluble component into a soluble component in a 6M guanidine sulfate aqueous solution. After heating the water-soluble components of the lysis buffer at 95°C for 10 minutes, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitated protein and peptide components were dissolved in a 6M guanidine sulfate aqueous solution. The mRNA in the supernatant was extracted using an mRNA extraction kit. The mRNA component was then mixed with the protein and peptide components dissolved in 6M guanidine sulfate to obtain the antigen component in the water-soluble component. Next, saturated ammonium sulfate solution was added to the insoluble component dissolved in 6M guanidine sulfate to salt out the protein and peptide components. The precipitated precipitate was then dissolved a second time in 6M guanidine sulfate to obtain the antigen component in the insoluble component. The antigen components in the insoluble and water-soluble components were mixed at a mass ratio of 1:1 to obtain antigen component 2 for preparing nanoparticle 2 (NP2).

[0379] Alternatively, tumor tissue samples can be collected from surgically removed tumor tissue of patient A, a non-small cell lung cancer patient. This patient had excellent response to immunotherapy, with complete tumor disappearance after treatment. The tumor tissue was minced, filtered through a cell sieve, resuspended in ultrapure water, and subjected to repeated freeze-thaw cycles five times. During the freeze-thaw process, ultrasonic disruption was used to further lyse the cancer cells. After cell lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. Adding 6M guanidine sulfate to the resulting precipitate dissolved the insoluble component, converting it into a soluble component in 6M guanidine sulfate aqueous solution. Mixing the insoluble and water-soluble components at a 1:1 mass ratio yields antigen component 3 for preparing nanoparticle 3 (NP3).

[0380] (2) Preparation of nanoparticles loaded with antigen components

[0381] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 20 kDa-40 kDa. The preparation method was as described previously. First, the antigen component and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 380 nm, and each 1 mg of PLGA nanoparticles loaded approximately 800 μg of protein or peptide components.

[0382] The preparation process of nanoparticle 2 (NP2) is the same as that of nanoparticle 1. The average particle size of nanoparticle 2 is about 380 nm, and each 1 mg of PLGA nanoparticles is loaded with about 800 μg of protein or peptide components.

[0383] The preparation process of nanoparticle 3 (NP3) is the same as that of nanoparticle 1. The average particle size of nanoparticle 3 is about 380 nm, and each 1 mg of PLGA nanoparticles is loaded with about 800 μg of protein or peptide components.

[0384] (3) Detection of cancer cell-specific T cells

[0385] Patient A with non-small cell lung cancer recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0386] Nanoparticle 1 (0.5 mg), nanoparticle 2 (0.5 mg), or nanoparticle 3 (0.5 mg) were co-incubated with PBMCs (1 million units) in 1 mL of AIM V serum-free medium for 12 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were first treated with Fc blocks to avoid nonspecific cell loading. Extracellular staining with CD3 and FASL antibodies was then performed. Cells were subsequently fixed and permeabilized, and intracellular staining with FN-γ antibody was performed. Flow cytometry was then used to analyze the T cell samples. CD3 analysis was performed. + T cells that are activated and can secrete IFN-γ, as well as T cells that express FASL, are found in all CD3 cells. + The proportion of T cells.

[0387] (4) Experimental Results

[0388] like Figure 13As shown, both nanoparticles 1 and 2 can assist in the detection of cancer cell-specific T cells. Furthermore, nanoparticles 1 and 2 have similar effects, and the activated T cells that secrete IFN-IFN-γ overlap with T cells that express FASL. This indicates that the particle system described in this invention can more accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0389] Example 13: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0390] This embodiment uses nanoparticles (NPs) to detect cancer cell-specific T cells in cancer patients. In this embodiment, water-soluble and insoluble components from various human lung cancer cell lines are loaded onto nanoparticles, which are then used to assist in the detection of cancer cell-specific T cells in peripheral immune organs of non-small cell lung cancer patients.

[0391] (1) Preparation of antigen components

[0392] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170 and H520 were cultured separately.

[0393] After collecting the above eight cell types, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed at a cell ratio of 2:2:2:2:2:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended and lysed with 4M guanidine isothiocyanate. Then, an appropriate amount of 4M guanidine isothiocyanate was added to dissolve the lysate. Saturated sodium chloride aqueous solution was added to salt out the protein and peptide components. The precipitated precipitate was dissolved a second time with 4M guanidine isothiocyanate. The obtained antigen component was mixed with lung cancer neoantigen peptide MAGE-A3 (sequence: FLWGPRALV) and lung cancer neoantigen peptide MUC1 (sequence: TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP) at a mass ratio of 5:1:1. This is the antigen component 1 for preparing nanoparticle 1 (NP1).

[0394] Alternatively, tumor tissue samples surgically removed from patient A of the non-small cell lung cancer (NSCLC) can be collected. This NSCLC patient showed excellent response to immunotherapy, with the tumor completely disappearing after treatment. The tumor tissue from this NSCLC patient was minced and filtered through a cell sieve, then resuspended and lysed using 4M guanidine isothiocyanate. An appropriate amount of 4M guanidine isothiocyanate was added to dissolve the lysate. Saturated sodium chloride solution was then added to salt out protein and peptide components. The precipitated precipitate was dissolved a second time using 4M guanidine isothiocyanate. The resulting antigen component was then mixed with lung cancer neoantigen peptide MAGE-A3 (sequence: FLWGPRALV) and lung cancer neoantigen peptide MUC1 (sequence: TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP) at a mass ratio of 5:1:1. This mixture constitutes antigen component 2 for preparing nanoparticle 2 (NP2).

[0395] Alternatively, a tumor tissue sample surgically removed from patient A of the non-small cell lung cancer (NSCLC) can be collected. This NSCLC patient responded well to immunotherapy, and the tumor completely disappeared after treatment. The tumor tissue from this NSCLC patient was minced, filtered through a cell sieve, then resuspended and lysed using 4M guanidine isothiocyanate. An appropriate amount of 4M guanidine isothiocyanate was then added to dissolve the lysate, which is the antigen component 3 for preparing nanoparticles 3 (NP2).

[0396] (2) Preparation of nanoparticles loaded with antigen components

[0397] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 10 kDa-30 kDa. The preparation method was as described previously. First, the antigen component and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, and each 1 mg of PLGA nanoparticles loaded approximately 200 μg of protein or peptide components.

[0398] The preparation process of nanoparticle 2 (NP2) is the same as that of nanoparticle 1. The average particle size of nanoparticle 2 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 200 μg of protein or peptide components.

[0399] The preparation process of nanoparticle 3 (NP3) is the same as that of nanoparticle 1. The average particle size of nanoparticle 3 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 200 μg of protein or peptide components.

[0400] (3) Detection of cancer cell-specific T cells

[0401] Patient A with non-small cell lung cancer recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0402] Nanoparticle 1 (5 mg), nanoparticle 2 (5 mg), or nanoparticle 3 (5 mg) were co-incubated with 10 million PBMCs in 10 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were first treated with Fc blocks to avoid nonspecific cell loading. Cells were then stained with CD3 and CD40L antibodies, and the samples were analyzed by flow cytometry. CD3 analysis was performed. + CD40L + T cells in all CD3 + The proportion of T cells is the content of cancer cell-specific T cells.

[0403] (4) Experimental Results

[0404] like Figure 14 As shown, nanoparticles 1, 2, and 3 can all effectively assist in the detection of cancer cell-specific T cells. Furthermore, nanoparticles 1 and 2 show similar effects, with nanoparticles 1 and 2 performing slightly better than nanoparticle 3. This indicates that the particle system described in this invention can more accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0405] Example 14: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0406] This embodiment uses nanoparticles (NPs) to detect cancer cell-specific T cells in cancer patients. In this embodiment, water-soluble and insoluble components from various human lung cancer cell lines are loaded onto nanoparticles, which are then used to assist in the detection of cancer cell-specific T cells in peripheral immune organs of non-small cell lung cancer patients.

[0407] (1) Collection of various components of multiple lung cancer cell lines

[0408] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170 and H520 were cultured separately.

[0409] After collecting the above eight cell types, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed in a cell ratio of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in 1M aminourea hydrochloride aqueous solution and lysed. The lysate was then dissolved in 1M aminourea hydrochloride and 0.5M arginine water-soluble solution, which is the antigen component 1 for preparing nanoparticle 1 (NP1).

[0410] Alternatively, a tumor tissue sample surgically removed from patient A of the non-small cell lung cancer (NSCLC) could be collected. This NSCLC patient responded well to immunotherapy, with the tumor completely disappearing after treatment. The tumor tissue from this NSCLC patient was minced, filtered through a cell sieve, resuspended in a 1M aminourea hydrochloride aqueous solution, and then lysed. The lysate components were then dissolved using 1M aminourea hydrochloride and 0.5M arginine water-soluble solvents, which became antigen component 2 for preparing nanoparticle 2 (NP2).

[0411] (2) Preparation of nanoparticles loaded with antigen components

[0412] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 10 kDa-30 kDa. The preparation method was as described previously. First, the antigen component and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, and each 1 mg of PLGA nanoparticles loaded approximately 50 μg of protein or peptide components.

[0413] The preparation process of nanoparticle 2 (NP2) is the same as that of nanoparticle 1. The average particle size of nanoparticle 2 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 50 μg of protein or peptide components.

[0414] (3) Preparation of nanoparticles loaded with antigen-presenting cell membrane components

[0415] Peripheral blood was collected from patient A, and PBMCs were isolated. CD11c was then sorted from the PBMCs using flow cytometry. + Dendritic cells (DCs) and CD19 +For B cells, 1 million DCs and 5 million B cells were mixed at a ratio of 1:10. The cells were then washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4°C. The cells were resuspended in PBS and sonicated at low power (22.5W) for 1 minute at 4°C. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was then centrifuged at 8000g for 15 minutes and the supernatant was collected. After centrifugation at 16000g for 90 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS and mixed with 5 mg of nanoparticles (nanoparticle 1 or nanoparticle 2) prepared in step (3). The mixture was incubated for 10 minutes and then repeatedly co-extruded through a 0.3 μm filter membrane. The extruded material was then centrifuged at 13000g for 30 minutes and resuspended in 10 mL of ultrapure water containing 4% trehalose. The mixture was then freeze-dried for 48 h for later use. Nanoparticle 3 is obtained by co-interacting the membrane component of antigen-presenting cells with nanoparticle 1. The average particle size is about 300 nm, and each 1 mg of PLGA nanoparticles is loaded with about 60 μg of protein or peptide components. Nanoparticle 4 is obtained by co-interacting the membrane component of antigen-presenting cells with nanoparticle 2.

[0416] (3) Detection of cancer cell-specific T cells

[0417] Patient A with non-small cell lung cancer recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0418] Nanoparticles 3 (5 mg) or 4 (5 mg) were incubated with PBMCs (1 million units) in 5 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were first treated with an Fc block to avoid nonspecific loading. Cells were then stained with CD3 and CD107A antibodies, followed by flow cytometry analysis of the sample T cells. CD3 analysis was performed. + CD107A + T cells in all CD3 + The proportion of T cells is the content of cancer cell-specific T cells.

[0419] (4) Experimental Results

[0420] like Figure 15As shown, both nanoparticles 3 and 4 can assist in the detection of cancer cell-specific T cells, indicating that the particle system described in this invention can more accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0421] This embodiment uses a co-extrusion method to load the antigen-presenting cell membrane onto the nanoparticle surface. In practical applications, any other method such as sonication, co-incubation, centrifugation, ultrafiltration, stirring, or homogenization can also be used to load the antigen-presenting cell membrane onto the nanoparticle surface. This embodiment uses autologous antigen-presenting cells; in practical applications, allogeneic antigen-presenting cells, antigen-presenting cell lines, or antigen-presenting cells prepared through other methods can also be used. This embodiment uses both DC and B cell membranes; in practical applications, only one type or multiple antigen-presenting cell membranes can be used. This embodiment uses membrane components of antigen-presenting cells; in practical applications, other membrane components such as extracellular vesicle membrane components secreted by antigen-presenting cells and bacterial membrane components can also be used. The antigen-presenting cells used in this embodiment were not activated with antigen; in practical applications, whole-cell component antigen or delivery particles loaded with whole-cell component antigen can be used to first activate the antigen-presenting cells before loading the cell membrane onto the delivery particle surface.

[0422] Example 15: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0423] (1) Collection of various components of multiple lung cancer cell lines

[0424] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. Among them, A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma cell line of the lung cancer subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma cell line of the lung cancer subtype.

[0425] After collecting the above eight cell types, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed in a cell ratio of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to further lyse the cancer cells. Then, 1% hypochlorous acid was added to oxidize the lysate components, followed by dropwise addition of saturated sodium chloride aqueous solution to salt out the protein and polypeptide components. The sample was then heated at 95°C for 1 minute and centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the precipitate was dissolved in 6M guanidine hydrochloride aqueous solution to obtain antigen component 1 for the preparation of nanoparticle 1 (NP).

[0426] Alternatively, tumor tissue samples can be collected from surgically removed tumor tissue of patient A, a non-small cell lung cancer patient. This patient had excellent response to immunotherapy, and the tumor completely disappeared after treatment. The tumor tissue from this non-small cell lung cancer patient was minced and filtered through a cell sieve. Then, an appropriate amount of ultrapure water was added, and the mixture was repeatedly frozen and thawed 5 times. During the freeze-thaw process, ultrasonic disruption was used to more thoroughly lyse the cells in the tumor tissue. Then, 1% hypochlorous acid was added to oxidize the lysate components, followed by dropwise addition of saturated sodium chloride aqueous solution to salt out protein and polypeptide components. The sample was then heated at 95°C for 1 minute, and the resulting sample was centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the precipitate was dissolved in 6M guanidine hydrochloride aqueous solution, which is the antigen component 2 for preparing nanoparticle 2 (NP2).

[0427] (2) Preparation of nanoparticles loaded with antigen components

[0428] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 10 kDa-30 kDa. The preparation method was as described previously. First, the antigen component and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, and each 1 mg of PLGA nanoparticles loaded approximately 5 μg of protein or peptide component.

[0429] The preparation process of nanoparticle 2 (NP2) is the same as that of nanoparticle 1. The average particle size of nanoparticle 2 is about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 5 μg of protein or peptide components.

[0430] (3) Detection of cancer cell-specific T cells

[0431] Patient A with non-small cell lung cancer recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0432] Nanoparticle 1 (30 mg) or nanoparticle 2 (30 mg) was co-incubated with PBMCs (1 million units) in 1 mL of serum-free AIM V medium for 24 hours (37°C, 5% CO2), 1 hour (37°C, 5% CO2), or 144 hours (37°C, 5% CO2); or, as a control group, PBMCs (1 million units) were incubated alone in 1 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2). Cells were then collected, centrifuged at 400 g for 5 minutes, resuspended in PBS, and treated with Fcblock to avoid nonspecific loading. Staining was then performed using CD3 and IFN-γ antibodies. Flow cytometry was then used to analyze the sample T cells. CD3 analysis was performed. + IFN-γ + T cells in all CD3 + The proportion of T cells is cancer cell-specific T cells.

[0433] (4) Experimental Results

[0434] like Figure 16 As shown, PBMCs incubated alone did not produce any activated cancer cell-specific T cells; however, both nanoparticles 1 and 2 detected a certain number of cancer cell-specific T cells. Furthermore, co-incubation for 24 hours resulted in the detection of even more cancer cell-specific T cells, indicating that a certain co-incubation duration is necessary; both excessively long and short co-incubation times are undesirable. In conclusion, the particle system described in this invention can accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0435] In this embodiment, hypochlorous acid was used to oxidize the antigen component to enhance its immunogenicity. In practical applications, hydrogen peroxide, potassium permanganate, or other oxidizing agents can also be used to oxidize the antigen component and enhance its immunogenicity. Alternatively, in practical applications, reducing agents such as dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) can be used to reduce the antigen component.

[0436] Example 16: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0437] (1) Preparation of antigen components

[0438] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. Among them, A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma cell line of the lung cancer subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma cell line of the lung cancer subtype.

[0439] After collecting the above eight cell types, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed in a cell ratio of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to further lyse the cancer cells. Then, saturated ammonium sulfate aqueous solution was added dropwise to salt out the protein and polypeptide components. The sample was then heated at 40°C for 10 minutes and centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the pellet was dissolved in 6M guanidine hydrochloride aqueous solution to obtain antigen component 1 for the preparation of nanoparticle 1 (NP).

[0440] (2) Preparation of nanoparticles or microparticles loaded with antigen components

[0441] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 10 kDa-30 kDa. The preparation method was as described previously. First, the nanoparticles were loaded with cellular components and adjuvants using the double emulsion method. After loading the cellular lysis components, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 250 nm, and each mg of PLGA nanoparticles loaded approximately 500 μg of protein or peptide components.

[0442] In this embodiment, micron-sized particles 1 (MP1) were prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 30 kDa-50 kDa. The preparation method was as described previously. First, the antigen component and adjuvant were loaded inside the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of these nanoparticles 1 was approximately 2.5 μm, and each mg of PLGA nanoparticles loaded approximately 500 μg of protein or peptide components.

[0443] Nanoparticles 1 and microparticles 1 are mixed at a mass ratio of 1:1 and used as mixed particles (MixP).

[0444] (3) Detection of cancer cell-specific T cells

[0445] Patient A with non-small cell lung cancer recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0446] The mixed particles (0.2 mg) were incubated with 200,000 PBMCs in 0.1 mL of serum-free AIM V medium for 24 hours (37°C, 5% CO2), or mixed particles 1 (0.2 mg) were incubated with 1000 PBMCs in 0.1 mL of serum-free AIM V medium for 24 hours (37°C, 5% CO2), or mixed particles 1 (0.2 mg) were incubated with 50 million PBMCs in 0.1 mL of serum-free AIM V medium for 24 hours (37°C, 5% CO2); or, as a control group, 200,000 PBMCs were incubated alone in 0.1 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2). Cells were then collected and centrifuged at 400 g for 5 minutes. After resuspending the cells in PBS, T cells were first treated with Fc blocks to avoid non-specific loading, and then stained with CD3 and IFN-γ antibodies. Flow cytometry was then used to analyze the sample T cells. CD3 analysis was performed. + IFN-γ + T cells in all CD3 + The proportion of T cells is cancer cell-specific T cells.

[0447] (4) Experimental Results

[0448] like Figure 17 As shown, PBMCs incubated alone did not produce any activated cancer cell-specific T cells; however, a certain amount of cancer cell-specific T cells could be detected using nanoparticles 1. Furthermore, during co-incubation, both excessively high and excessively low cell concentrations in the system were undesirable. In summary, the particle system described in this invention can accurately detect cancer cell-specific T cells in the peripheral blood of cancer patients.

[0449] Example 17: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0450] (1) Preparation of antigen components

[0451] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. Among them, A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma cell line of the lung cancer subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma cell line of the lung cancer subtype.

[0452] After collecting the above eight cell types, A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed in a cell ratio of 1:1:1:1:1:1:1:1. After centrifugation, the culture medium was removed, and the cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles. During the freeze-thaw process, sonication was used to lyse the cancer cells. Nuclease was then added and reacted for 5 minutes to degrade the nucleic acid. The lysate components were then dissolved in 8M urea aqueous solution. The sample was then heated at 65°C for 1 minute, and saturated sodium chloride aqueous solution was added dropwise to salt out the protein and polypeptide components. The resulting sample was centrifuged at 3000g for 5 minutes, the supernatant was discarded, and the pellet was dissolved a second time in 8M urea aqueous solution to obtain antigen component 1 for the preparation of nanoparticle 1 (NP).

[0453] (2) Preparation of nanoparticles loaded with antigen components

[0454] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 10 kDa-30 kDa. The preparation method was as described previously. First, antigen component 1 was loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, and each mg of PLGA nanoparticles loaded approximately 5 μg of protein or peptide component.

[0455] (3) Detection of cancer cell-specific T cells

[0456] Patient A with non-small cell lung cancer (NSCLC) recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation. CD19 cells were isolated from the PBMCs using flow cytometry. + B cells and CD11c +Dendritic cells (DCs) were then used as mixed antigen-presenting cells after B cells and DCs were mixed at a ratio of 19:1.

[0457] Nanoparticle 1 (2 mg) was co-incubated with 2 million mixed antigen-presenting cells (1.9 million sorted B cells and 100,000 sorted DCs) in 2 mL of serum-free AIM V medium for 6 hours (37°C, 5% CO2). Cells were then collected and fixed with 4% paraformaldehyde. The nanoparticles were removed by centrifugation at 400 g, and the fixed mixed antigen-presenting cells were washed with PBS. 1 million fixed mixed antigen-presenting cells (co-incubated with nanoparticles) were mixed with 1 million PBMCs and co-incubated with 2 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2); alternatively, in the control group, 2 million PBMCs were incubated alone in 2 mL of serum-free AIM V medium for 12 hours (37°C, 5% CO2). Cells were then collected by centrifugation, resuspended in PBS, and treated with Fc blocks to avoid non-specific cell loading before staining with CD3, CD8, and CD137 antibodies. Subsequently, flow cytometry was used to analyze T cells in the samples. CD8+ was analyzed. + CD137 + T cells in CD8 + The proportion of T cells is cancer cell-specific T cells.

[0458] (4) Experimental Results

[0459] like Figure 18 As shown, no activated cancer cell-specific T cells were observed when PBMCs were incubated alone, but a certain number of cancer cell-specific T cells could be detected using nanoparticles 1.

[0460] Example 18: Nanoparticles for detecting cancer cell-specific T cells in peripheral immune organs of lung cancer patients.

[0461] (1) Preparation of antigen components

[0462] Each human lung cancer cell line—A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520—was co-incubated with doxorubicin hydrochloride (0.05 μM) for 16 hours during culture. After co-incubation, each cell line was centrifuged at 400g for 5 minutes to remove the doxorubicin. The eight cell lines were then collected separately. A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 cells were mixed at a cell ratio of 2:2:1:1:1:1:1:1, centrifuged, and the culture medium was removed. The cell pellet was resuspended in ultrapure water and subjected to five freeze-thaw cycles, with sonication used during these cycles to lyse the cancer cells. The lysate was then heated at 65°C for 1 minute. The lysate was then dissolved in an 8M urea (containing 0.1M arginine) aqueous solution. The protein and polypeptide components were then salted out dropwise by adding a saturated sodium sulfate aqueous solution. The protein and polypeptide components were then salted out dropwise by adding a saturated sodium chloride aqueous solution. The supernatant was then discarded, and the precipitate was dissolved in an 8M urea (containing 0.1M arginine) aqueous solution. This was the antigen component 1 for preparing nanoparticle 1 (NP).

[0463] (2) Preparation of nanoparticles loaded with antigen components

[0464] In this embodiment, nanoparticle 1 (NP1) was prepared using the double emulsion method within the solvent evaporation process. The PLGA nanoparticle material used had a molecular weight of 20 kDa-40 kDa. The preparation method was as described previously. First, antigen component 1 was loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h. The average particle size of nanoparticle 1 was approximately 280 nm, and each mg of PLGA nanoparticles loaded approximately 500 μg of protein or peptide component.

[0465] (3) Detection of cancer cell-specific T cells

[0466] Patient A with non-small cell lung cancer recovered after immunotherapy. Peripheral blood samples of 10 mL were collected from patient A before and two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 mL of peripheral blood using gradient centrifugation.

[0467] Nanoparticle 1 (20 mg) and 10 million PBMCs were co-incubated in 2 mL of AIM V serum-free medium for 24 hours (37°C, 5% CO2); or, in the control group, 10 million PBMCs were incubated alone in 2 mL of AIM V serum-free medium for 24 hours (37°C, 5% CO2). Cells were then collected by centrifugation, resuspended in PBS, and treated with an Fc block to avoid nonspecific loading. Staining was then performed using CD3 and CD134 antibodies. Flow cytometry was then used to analyze the sample T cells. CD3 analysis was performed. + CD134 + T cells in CD3 + The proportion of T cells is cancer cell-specific T cells.

[0468] (4) Experimental Results

[0469] like Figure 19 As shown, no activated cancer cell-specific T cells were observed when PBMCs were incubated alone, but a certain number of cancer cell-specific T cells could be detected using nanoparticles 1.

[0470] Before lysis, the cancer cells or tumor tissue can be co-incubated with specific chemical substances to stimulate the cancer cells or tumor tissue, and then the cancer cells or tumor tissue can be lysed. The specific substances include, but are not limited to, small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), plant extracts (such as important extracts of ginseng, plant root and stem extracts, etc.), growth factors, cytokines, chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc.

[0471] In this embodiment, the concentration of doxorubicin hydrochloride used is 0.05 μM. In practical applications, other concentrations, such as 0.01 nM to 2 μM, are used.

[0472] In some embodiments of this disclosure, antigen-presenting cells activated after co-incubation with particles are treated with paraformaldehyde. In practical applications, irradiation or other methods can also be used to inactivate antigen-presenting cells activated after co-incubation with particles, and then the antigen-presenting cells are co-incubated with samples containing T cells. In some embodiments of this disclosure, PBMCs containing antigen-presenting cells are first co-incubated with particles to activate the antigen-presenting cells, then the incubated cells are inactivated, and then the inactivated antigen-presenting cells are co-incubated with samples containing T cells. In practical applications, activated antigen-presenting cells can also be directly co-incubated with samples containing T cells without inactivation. In some embodiments of this disclosure, PBMCs containing antigen-presenting cells are first co-incubated with particles to activate the antigen-presenting cells, then the incubated cells are inactivated, and then the inactivated antigen-presenting cells are co-incubated with a sample containing T cells. In practical applications, antigen-presenting cells can also be purified first, then the purified antigen-presenting cells are co-incubated with antigen delivery particles to activate the antigen-presenting cells, and then the activated antigen-presenting cells (activated or unactivated) are co-incubated with a sample containing T cells.

[0473] In some embodiments of this disclosure, 250 nm nanoparticles and 2.5 μm microparticles are used as mixed particles. In practical applications, nanoparticles and microparticles of other particle sizes can also be mixed to prepare mixed particles. When preparing mixed particles, the preferred particle size range of nanoparticles is 150 nm-500 nm, and the preferred particle size range of microparticles is 2.0 μm-3.5 μm.

[0474] In some embodiments of this disclosure, experiments were conducted in solid tumors such as lung cancer. In practical applications, it can also be used to detect cancer cell-specific T cells in patients with other types of solid tumors, and it can also be used to detect cancer cell-specific T cells in patients with non-solid tumor cancers such as hematologic malignancies.

[0475] The embodiments disclosed herein detect cancer cell-specific T cells in peripheral blood. In practical applications, it can also be used to detect cancer cell-specific T cells in other peripheral immune organs (such as lymph nodes) or tumor tissues.

[0476] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A detection kit for cancer cell-specific T cells, characterized in that, The detection kit includes nanoparticles and / or microparticles loaded with antigen components. The nanoparticles or microparticles contain particle preparation materials and antigen components, and the antigen components are selected from one or more of the following (i), (ii), and (iii): (i) Whole-cell components of various cancer cell lines, (ii) Protein and polypeptide components obtained from lysates of various cancer cell lines after separation, purification, and / or immunogenicity enhancement treatment. (iii) Protein and polypeptide components and RNA components obtained from lysates of various cancer cell lines after separation, purification and / or immunogenicity enhancement treatment; Among them, the various cancer cell lines are cancer cell lines of different subtypes of the same cancer; Nanoparticles and / or microparticles are prepared from at least one of organic synthetic polymers, natural polymers, and inorganic materials; The various cancer cell lines mentioned are human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520; the cell mass ratio of the human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 is 1:1:1:1:1:1:1:

1.

2. The detection kit according to claim 1, wherein the antigen component further comprises one or more of the following (iv) and (v): (iv) Artificially synthesized polypeptides containing antigenic polypeptide epitopes, wherein the antigenic polypeptide epitopes are cancer-specific or cancer-related antigenic polypeptide epitopes; (v) can express nucleic acids containing antigenic polypeptide epitopes, which are cancer-specific or cancer-associated antigenic polypeptide epitopes, and the nucleic acids are mRNA or DNA.

3. The detection kit according to claim 1, characterized in that, The various cancer cell lines were processed using any of the following methods and then loaded onto nanoparticles and / or microparticles: (1) After mixing multiple cancer cell lines, their structures are inactivated and destroyed to obtain the component to be loaded; or, (2) After inactivating and destroying the structure of multiple cancer cell lines, they are mixed to obtain the component to be loaded; or, (3) Collect water-soluble components from various cancer cell lines, mix the water-soluble components to obtain the component to be loaded; or, (4) Dissolve the non-water-soluble components in various cancer cell lines using a solvent containing a solvent, thereby collecting the dissolved non-water-soluble components, and mixing the non-water-soluble components to obtain the component to be loaded; or, (5) After collecting water-soluble components from various cancer cell lines, the insoluble components are dissolved in a dissolving solution containing a solvent to collect the dissolved insoluble components. The water-soluble and insoluble components are then mixed to obtain the component to be loaded; or, (6) Multiple cancer cell lines were directly treated with a solution containing a solvent, and the treatment solutions were mixed to obtain the component to be loaded; or, (7) Use any one of (1)-(6) to mix with (iv) a synthetic polypeptide containing an antigenic polypeptide epitope and / or (v) a nucleic acid that can express an antigenic polypeptide epitope to obtain the antigen component to be loaded.

4. The detection kit according to claim 1, characterized in that, The various cancer cell lines were processed using any of the following methods and then loaded onto nanoparticles and / or microparticles: (1) After mixing multiple cancer cell lines, their structures are inactivated and destroyed, and the protein and polypeptide components are separated and extracted to obtain the antigen component to be loaded; or, (2) After inactivating and destroying the structure of various cancer cell lines and separating and extracting protein and polypeptide components, the components are mixed to obtain the antigen component to be loaded; or, (3) After mixing multiple cancer cell lines, their structures are inactivated and destroyed, and the protein, polypeptide, and RNA components are separated and extracted to obtain the antigen component to be loaded; or, (4) Inactivate and destroy the structure of various cancer cell lines, separate and extract protein, polypeptide, and RNA components, and then mix them to obtain the antigen component to be loaded; or, (5) Collect water-soluble components from various cancer cell lines, then separate and extract the protein, polypeptide, and / or RNA components, and then mix the protein, polypeptide, and / or RNA components from the water-soluble components to obtain the antigen component to be loaded; or, (6) Dissolve the insoluble components from various cancer cell lines using a dissolving solution containing a solvent, collect the dissolved insoluble components, then separate and extract the protein, polypeptide, and / or RNA components, and mix the protein, polypeptide, and / or RNA components from the insoluble components to obtain the antigen component to be loaded; or, (7) Collect water-soluble components from various cancer cell lines, and dissolve insoluble components in a solvent containing a solvent. Collect the dissolved insoluble components, and separate and extract the protein, polypeptide, and / or RNA components from the water-soluble and insoluble components respectively. Then mix them to obtain the antigen-loading component; or, (8) Various cancer cell lines are directly treated with a solution containing a solvent. After mixing the treatment solutions, the protein, polypeptide, and / or RNA components are separated and extracted to obtain the antigen component to be loaded; or (9) Use any one of (1)-(8) with (iv) a synthetic polypeptide containing an antigenic polypeptide epitope and / or (v) a nucleic acid that can express an antigenic polypeptide epitope to obtain the antigen component to be loaded.

5. The detection kit according to claim 1, characterized in that, Methods for isolating, purifying, or enhancing the immunogenicity of antigenic components from cancer cells include one or more of the following: salting out, heating, oxidation, enzymatic treatment, reduction, chromatography, electrophoresis, chromatography, recrystallization, extraction, precipitation, fixation, irradiation, and mineralization.

6. The detection kit according to claim 3 or 4, characterized in that, The water-soluble and insoluble components are mixed in a mass ratio of 1:5 to 5:

1.

7. The detection kit according to claim 3 or 4, characterized in that, The solvent is one or more of the following: a substance containing structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein-degrading enzyme, albumin, lecithin, Triton, Tween, amino acids, polypeptides, glycosides, and choline; Structure 1 is shown below: Wherein, R1 is C, N, S or O, and R2 to R5 are independently selected from hydrogen, alkyl, carboxyl, substituted or unsubstituted amino, mercapto, substituted or unsubstituted guanidine.

8. The detection kit according to claim 1, characterized in that, Cancer cells are stimulated by co-incubating with specific chemicals before being lysed.

9. The detection kit according to claim 1 or 2, characterized in that, The test kit also includes antigen-presenting cells.

10. The detection kit according to claim 9, characterized in that, The process of activating cancer cell-specific T cells by nanoparticles / microparticles involves co-incubating nanoparticles / microparticles with antigen-presenting cells and T cells simultaneously to activate T cells; or first co-incubating nanoparticles / microparticles with antigen-presenting cells to activate antigen-presenting cells, and then co-incubating the activated antigen-presenting cells with T cells to activate T cells.

11. The detection kit according to claim 9, characterized in that, The concentration of the nanoparticles / microparticles in the co-incubation system is 0.001 mg / mL to 50 mg / mL; the concentration of the antigen-presenting cells or T cells in the co-incubation system is 10,000 cells / mL to 500 million cells / mL.

12. The detection kit according to claim 1, characterized in that, Nanoparticles and / or microparticles are also loaded with targets that actively target antigen-presenting cells.

13. The detection kit according to claim 1, characterized in that, Nanoparticles and / or microparticles are also loaded with immune-enhancing adjuvants and / or substances that promote lysosomal escape.

Images