CN117618602A - Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method - Google Patents

Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method Download PDF

Info

Publication number
CN117618602A
CN117618602A CN202311744495.4A CN202311744495A CN117618602A CN 117618602 A CN117618602 A CN 117618602A CN 202311744495 A CN202311744495 A CN 202311744495A CN 117618602 A CN117618602 A CN 117618602A
Authority
CN
China
Prior art keywords
antigen
component
cancer
vaccine
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311744495.4A
Other languages
Chinese (zh)
Inventor
刘金生
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Ersheng Biopharmaceutical Co Ltd
Original Assignee
Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Ersheng Biopharmaceutical Co Ltd filed Critical Suzhou Ersheng Biopharmaceutical Co Ltd
Priority to CN202311744495.4A priority Critical patent/CN117618602A/en
Publication of CN117618602A publication Critical patent/CN117618602A/en
Pending legal-status Critical Current

Links

Landscapes

  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention relates to a terminal sterilization method of a cancer particle vaccine loaded with a cancer cell lysate component and application thereof, belonging to the technical field of immunotherapy. The terminal sterilization method adopting specific irradiation sterilization can be used for preparing vaccine freeze-dried preparations, and after the vaccine freeze-dried preparations are sealed, irradiation sterilization such as rays and the like is used, so that the operation is more convenient, and the requirements on sites, instruments and the like are lower. Proved by verification, the preparation after irradiation treatment meets the requirements of Chinese pharmacopoeia aseptic inspection method, meets the aseptic requirements of injection preparation, and is stable and can be stored for a long time. Although the irradiation sterilization treatment can have a certain influence on the components contained in the irradiated substances, the efficacy of the nanometer/micrometer vaccine or particles for loading the lysate components is not reduced, and surprisingly, the efficacy of the nanometer/micrometer vaccine directly injected into the body or activated other immune cells in vitro after the irradiation sterilization is better than the efficacy of the nanometer/micrometer vaccine prepared in a whole-process sterile way, and the method has important application prospect in the field of tumor treatment.

Description

Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method
Technical Field
The invention relates to the field of immunotherapy, in particular to a terminal sterilization method of a nano vaccine (delivery particles) or a micro vaccine (delivery particles) loaded with all components or partial components in cancer cells and/or tumor tissue lysates and application thereof.
Background
Cancer vaccines are one of the important approaches to cancer immunotherapy. Major factors affecting cancer vaccines include tumor antigens, adjuvants, and delivery dosage forms. Wherein, the antigen can induce and activate specific immune response, the adjuvant can amplify specific immune response, and the dosage form can influence the phagocytosis of antigen-presenting cells (APCs) on vaccine and the subsequent efficiency of activating antigen-specific T cells. Among the three, the antigen is the main component capable of eliciting a specific immune response and is thus the most critical component. Cancer cells and/or tumor tissue contain all of the cancer cell-specific antigen and the cancer cell-associated antigen, and thus the cancer cell or tumor tissue lysate is the best raw material for preparing a cancer vaccine. The inventor develops nano vaccine or micron vaccine loaded with cancer cell/tumor tissue whole cell lysate component or partial lysate component, and the prepared nano vaccine or micron vaccine has particle size of hundreds of nanometers to microns. Vaccines are generally administered by injection, which requires a sterile formulation, a relatively strict sterility requirement, and either terminal sterilization or the whole process is guaranteed to be sterile. The process sterility is more demanding on enterprise sites, equipment, instruments, etc., and thus costs and management requirements are also higher. For terminal sterilization, filtration is typically performed using a 0.22 μm (220 nm) filter. Filter sterilization using a 0.22 μm filter is feasible for nanovaccines with diameters less than 150nm and not feasible for nanovaccines with diameters greater than 200nm using a 0.22 μm filter. Therefore, research on a feasible terminal sterilization method is needed, which is important for industrialized application of the nano vaccine, cost reduction, efficiency improvement and the like.
Disclosure of Invention
Problems to be solved by the invention
In order to solve the problems that the whole process of aseptically preparing the nano vaccine has higher requirements on sites, equipment, management and the like and higher cost and the like, the invention provides a terminal sterilization method by using specific irradiation sterilization, which can be used for preparing vaccine freeze-dried preparations and sealing, and then using rays and the like for irradiation sterilization, so that the operation is more convenient and the requirements on sites, instruments and the like are lower. It was verified that the irradiation treatment had a certain effect on the components contained in the irradiated material, but did not reduce the efficacy of the nano/micro vaccine or particles carrying the lysate components. Moreover, the effect of directly injecting the nanometer/micrometer vaccine (particles) into the body or activating other immune cells in vitro after irradiation sterilization is better than that of the nanometer/micrometer vaccine (particles) prepared in a whole-process sterile way, and unexpected effects are obtained.
Solution for solving the problem
A terminal sterilization method of cancer nano particles (vaccine) or micro particles (vaccine) loaded with antigen components in lysate of cancer cells or tumor tissues, wherein the sterilization mode is radiation sterilization for 1-48 hours; the irradiation is one or more treatments of X-rays, gamma-rays, alpha-rays, beta-rays and 1-100MeV electron beams with the dosage of 12-60 kGy;
The nanoparticle or microparticle has: (i) A nanoparticle and/or microparticle scaffold formed from a particle preparation material, (ii) a whole cell lysate component from cancer cells and/or tumor tissue, and/or a partial cell lysate component comprising an antigen component; wherein the whole cell lysate component and/or the antigen component-containing partial cell lysate component from cancer cells and/or tumor tissue is supported within and/or on the surface of the scaffold structure;
wherein all of the whole cell fraction from the cancer cell and/or tumor tissue lysate fraction is lysed using a lysis solution comprising a lysing agent; or the whole cell fraction from the cancer cell and/or tumor tissue lysate fraction comprises a water-soluble fraction and a non-water-soluble fraction solubilized using a solubilization solution comprising a solubilization agent; the antigen component contained in the partial cell lysate component comprises protein and polypeptide components in cancer cells and/or tumor tissue lysates and/or RNA components or mRNA components in cell lysates, and the partial lysate component containing the antigen component is separated and purified from the lysate by using a proper method;
the cancer nano-particles (vaccine) or micro-particles (vaccine) carrying antigen components in the lysate of cancer cells or tumor tissues after irradiation terminal sterilization can be directly injected into an organism to be used as a vaccine or used as antigen delivery particles for activating other cells in vitro.
Further, the terminal sterilization method is radiation sterilization for 1 hour to 48 hours, 12 to 60kGy or 1 to 100MeV; preferably for 4-24 hours by irradiation sterilization; more preferably from 6 to 16 hours of irradiation.
Further, the irradiation is one or more treatments of different irradiation methods such as X-rays, gamma rays, alpha rays, electron beams, beta rays and the like; preferably, one or more of gamma rays, alpha rays, beta rays, electron beams, and X rays are used, and more preferably, gamma rays, X rays, or electron beams are used.
Further, the cancer nanovaccine or nanoparticle (antigen delivery nanoparticle) has a particle size of 1nm to 1000nm; preferably 50-500nm; more preferably 100-400nm; the particle size of the micrometer vaccine or micrometer particle (antigen delivery micrometer particle) is 1 μm-1000 μm; preferably 1-10 μm; more preferably 1-5. Mu.m.
Further, when a mixed vaccine (particle) of nano/micro vaccine (particle) is used, the particle size of the nano vaccine or nano particle (antigen delivery nano particle) is 100-600nm, and the particle size of the micro vaccine or micro particle (antigen delivery micro particle) is 1.5-5 μm; preferably the particle size of the nanovaccine or nanoparticle (antigen delivery nanoparticle) is 150-500nm and the particle size of the micrometer vaccine or micrometer (antigen delivery micrometer) is 2.0-3.5 μm.
Further, a proper amount of a substance for improving stability or a substance for promoting targeting is added to a nano vaccine or nano particle (antigen delivery nano particle) or a preparation material (main material such as PLGA or PLA) of a micro vaccine or micro particle (antigen delivery micro particle). Preferably, the PEG modified PLGA or PLA can better achieve the effects of long circulation and passive targeting; wherein the mass ratio of PEG modified PLGA or PLA or the like to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.
Further, the source of cancer cells according to the present invention is any method that can obtain cancer cells, including but not limited to cancer cell lines, cancer cells obtained by in vitro expansion of cancer cells isolated from tumor tissue, cancer cells obtained by expansion of circulating tumor cells isolated from blood, or cancer cells differentiated from stem cells, etc.
Preferably, the cancer cells are from one or more organisms, or from one or more cancer cell lines; the tumor tissue is from one or more organisms; the protein and polypeptide component/mRNA component (or RNA component) in the lysate of the cancer cells or the lysate of the tumor tissue contains an antigen component; the protein and polypeptide component/mRNA component (or RNA component) in the water-soluble component and the protein and polypeptide component/mRNA component (or RNA component) in the water-insoluble component/water-insoluble component in the cancer cell and/or tumor tissue contain an antigen component.
Further, the antigen component may be a protein polypeptide component, a mixture of a protein polypeptide component and a total RNA component, or a mixture of a protein polypeptide component and a total mRNA component.
Further, the nano/micro vaccine (particles) is further loaded with at least one component as shown below:
(iii) An mRNA component or an RNA component in the water-soluble component and/or the non-water-soluble component;
(iv) An immunoadjuvant;
(v) A substance having a positive charge is provided,
preferably, the immunoadjuvant comprises at least one of: pattern recognition receptor type agonists, toll-like receptor agonists, BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall dipeptide, mycobacterium phlei, polyoxin a, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virus minibodies, cholera enterotoxin, saponins and derivatives thereof, resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly (I: C), immune adjuvant poly ICLC, short rod bacterin, hemolytic streptococcus preparation, coenzyme QIO, levamisole, polycytidylic acid, interleukins, interferons, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokines, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin adjuvant, liposome, CAF01, yellow active ingredient;
More preferably, the immunoadjuvant comprises at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist;
most preferably, the immunoadjuvant comprises at least one of Poly (I: C), poly ICLC, A class CpG-OND, B class CpG-OND, and C class CpG-OND;
preferably, the positively charged substance is selected from positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and/or positively charged inorganics;
more preferably, the positively charged substance is selected from the group consisting of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH 4 HCO 3 Any one or more of the following.
Further, the mass ratio of the particle skeleton structure, the protein and the polypeptide components is 1:0.001-10;
more preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-2;
most preferably, the mass ratio of the particle framework, protein and polypeptide components is 1:0.05-1.
Further, the mass ratio of the particle skeleton structure to the RNA component or the mRNA component is 1:0.001-10;
More preferably, the mass ratio of the particle backbone structure to the RNA component or mRNA component is 1:0.01-2;
most preferably, the mass ratio of the particle backbone structure to the RNA component or mRNA component is 1:0.05-1.
Further, prior to lysing the cancer cells/tumor tissue, the cancer cells/tumor tissue may also be irradiated with radiation, including but not limited to gamma rays, X-rays, beta rays, alpha rays, and the like, to inactivate the cancer cells/tumor tissue.
The terminal sterilization method is used for preparing sterilized cancer nanometer vaccines (particles) or micrometer vaccines (particles) and comprises the following steps:
(1) Lysing cancer cells or tumor tissue using a lysing solution comprising a lysing agent, and then lysing the lysate component using a lysing solution comprising a lysing agent;
(2) Purifying or enhancing immunogenicity of lysate components dissolved in the dissolving solution, and then separating and purifying or enhancing immunogenicity components from the lysate by using the dissolving solution containing the dissolving agent for secondary dissolution;
(3) Directly loading the lysate component dissolved by the dissolution liquid obtained in the step (1) on nanometer/micrometer vaccine (particles); or the lysate component dissolved by the dissolution liquid obtained in the step (2) is subjected to purification or immunogenicity enhancing treatment and then is loaded on the nanometer/micrometer vaccine (particle).
Wherein the dissolving agent is independently selected from one or more of a compound shown in a structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline; wherein, structural formula 1 is as follows:
R 1 c, S, P, N or O, R 2 ～R 5 Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino;
preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea peroxide, guanidine hydrochloride, urea peroxide, guanidine sulfate, guanidine sulfonate, guanidine salts, urea, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, other guanidine group-containing compounds, semicarbazide hydrochloride, carbamyl urea, acetylurea, sulfonylurea-type compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea-type compounds (thiouracil, imidazoles, etc.), nitrosoureas, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzymes, polypeptides, amino acids, glycosides, and choline.
The terminal sterilization method of the invention is used for the preparation process of sterilized cancer nanometer vaccine or micrometer vaccine, and comprises the following steps:
(1) Firstly, using ultrapure water or aqueous solution to lyse cancer cells or tumor tissues, then collecting lysate and centrifuging, wherein the supernatant liquid part is water-soluble components, and using a dissolving solution containing a dissolving agent to solubilize and dissolve the precipitated non-water-soluble components;
(2) Purifying or enhancing immunogenicity of the water-soluble component and/or the non-water-soluble component, and then secondarily dissolving the water-soluble component and/or the non-water-soluble component in a dissolving solution containing a dissolving agent to separate and purify or enhance the immunogenicity of the components from the dissolving solution;
(3) And then directly loading the water-soluble component and/or the non-water-soluble component obtained in the step (1) on the nano/micron vaccine (particle) respectively or simultaneously, or loading the water-soluble component and/or the non-water-soluble component obtained in the step (2) on the nano/micron vaccine (particle) after purification or immunogenicity enhancement treatment.
Wherein the dissolving agent is independently selected from one or more of a compound shown in a structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline; wherein, structural formula 1 is as follows:
R 1 C, S, P, N or O, R 2 ～R 5 Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino;
preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea peroxide, guanidine hydrochloride, urea peroxide, guanidine sulfate, guanidine sulfonate, guanidine salts, urea, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, other guanidine group-containing compounds, semicarbazide hydrochloride, carbamyl urea, acetylurea, sulfonylurea-type compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea-type compounds (thiouracil, imidazoles, etc.), nitrosoureas, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzymes, polypeptides, amino acids, glycosides, and choline.
Further, the cancer cells or tumor tissue may also be suitably treated prior to lysis of the cancer cells or tumor tissue to enhance the immunogenicity of the antigenic components in the cancer cells or tumor tissue, treatment methods to enhance immunogenicity include, but are not limited to, one or more of heating (greater than 50 ℃), co-incubation with specific substances that stimulate cancer cells, irradiation, fixation treatment, co-action with hapten substances, enzymatic treatment, oxidation, reduction, irradiation, fixation, mineralization, and the like.
After lysing the cancer cells or tumor tissue, all or a portion of the lysate fraction of the cancer cells or tumor tissue may also be subjected to suitable treatments prior to loading the lysate fraction thereof into the nano/micro vaccine (particles) to purify the lysate fraction or enhance the immunogenicity of the antigen fraction in the cancer cells or tumor tissue, such treatments including, but not limited to, one or more of heating (greater than 50 ℃), oxidation, reduction, immobilization, mineralization, salting-out, dilution of a lytic agent, irradiation, co-action with hapten substances, enzymatic treatment, extraction of RNA fraction, extraction of whole protein fraction, extraction of mRNA fraction, ultrafiltration, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, etc.
The enzyme treatment methods include, but are not limited to, the use of one or more of nucleases, dnases, pepsin, chymotrypsin, trypsin, other proteolytic enzymes, protease inhibitors, and the like.
Wherein the oxidizing agent for oxidizing the antigen component includes but is not limited to hypochlorous acid, hydrogen peroxide (hydrogen peroxide), persulfate, KIO 3 、KBrO 3 Chlorine, dichromate, nitric acid, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, hydrogen peroxide, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium permanganate, nitric acid, clO 3 - 、ClO 4 - 、Na 2 O 2 、K 2 O 2 、MgO 2 、CaO 2 、BaO 2 、H 2 O 2 、NO 3 - 、MnO 4 - 、F 2 、Cl 2 、O 2 、Br 2 、I 2 、S、Si、HNO 3 、MnO 2 、FeCl 3 And the like, and one or more of various types of oxidizing agents. The oxidation may enhance the immunogenicity of a portion of the antigen component.
The specific substances for stimulating cancer cells include, but are not limited to, small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, chemokines, plant extracts (such as important extracts of ginseng, etc., plant rhizome extracts, etc.), interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of stimulating cancer cells or tumor tissue with a particular substance co-incubation with the cancer cells or tumor tissue is to allow the cancer cells to produce more antigenic components.
Among these, mineralization methods include, but are not limited to, methods using one or more mineralizations of siliconization, calcification, magnesian, biomineralization, and the like.
The reduction is to reduce the antigen component using a component that can reduce the antigen, and reducing agents for reducing the antigen component include, but are not limited to, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), and the like.
Wherein the irradiation is any commonly used irradiation method including, but not limited to, one or more of X-ray irradiation, alpha-ray irradiation, beta-ray irradiation, gamma-ray irradiation, etc.
The chromatography of the present invention includes, but is not limited to, column chromatography, gas chromatography, high pressure liquid chromatography, adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography, ion exchange chromatography, thin film chromatography, affinity chromatography, gel chromatography, etc.
The chromatography of the present invention includes, but is not limited to, column chromatography, thin layer chromatography, liquid chromatography, gas chromatography, supercritical fluid chromatography, and the like.
The electrophoresis method of the present invention includes, but is not limited to, SDS electrophoresis, isoelectric focusing electrophoresis, isotachophoresis, immunoelectrophoresis, serum protein electrophoresis, nucleic acid electrophoresis, DNA sequencing electrophoresis, gel electrophoresis, preparative electrophoresis, and the like.
The methods of the present invention for isolating and extracting mRNA or RNA components from cancer cells and/or tumor tissue include, but are not limited to, one or more of the methods of using mRNA isolation and extraction reagents, using RNA isolation and extraction kits, using DNA removal kits, using enzymes to degrade DNA, and the like.
The hapten substance is used for coacting with cells in cancer cells or tumor tissues for a certain time to modify antigen components in the cancer cells or the tumor tissues, and then the cancer cells and/or the tumor tissues are lysed to obtain lysate thereof; or first lysing cancer cells or tumor tissue to obtain a lysate thereof, and then modifying the antigen component in the cancer cells or tumor tissue lysate with a hapten substance.
The hapten substance is a substance which can increase the immunogenicity of a protein or polypeptide after coaction with the protein or polypeptide.
The hapten substances include, but are not limited to, 2, 4-Dinitrofluorobenzene (DNFB), 2, 4-Dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, ovalbumin (OVA), N-iodoacetyl-N' - (5-sulfo-1-naphthyl) ethylene diamide (AED), substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, and the like.
The materials that may be used to fix tissue/cells or for a sedimentation treatment include, but are not limited to, formaldehyde, paraformaldehyde, glutaraldehyde, other hapten materials containing aldehyde groups, ethanol, methanol, acetone, acetic acid, propionic acid, butyric acid, formic acid, formalin, dichromate, chromic acid, potassium permanganate, picric acid, zamboni, PLP, FPA, TAF, rosman, regaud, PLPD, PAPG, orth, muller, mcDoWell, AAF, hollande, gendre, aldehydes, mercury, alcohols, oxidants, neutral calcium formaldehyde, FAB, carney, clarke, B-5, bouin, finfix, a.g. (70% ethanol 80ml + 10ml of glacial acetic acid 10ml + methanol), helman, zelmer, or other bitterholder, kolmer, or other bitterholder of tissue or tissue.
The components obtained by the separation and purification treatment are protein and polypeptide components and/or RNA components (or mRNA components) in the lysate.
Preferably, the nano/micro vaccine (particles) may further comprise at least one component as shown below:
(a) Cancer cell membrane fraction derived from tumor tissue and/or tumor cells;
(b) An extracellular vesicle membrane fraction derived from extracellular vesicle lysates, the extracellular vesicles being secreted by bacteria or tumor cells;
(c) A bacterial membrane component derived from a bacterial lysate;
(d) A membrane fraction derived from antigen presenting cells;
preferably, the bacteria include at least one of: bacillus calmette-guerin, escherichia coli, bifidobacterium longum, bifidobacterium breve, bifidobacterium lactis, lactobacillus acidophilus, lactobacillus formans, lactobacillus reuteri, and lactobacillus rhamnosus.
Preferably, the particle material is selected from natural high molecular materials, synthetic high molecular materials and/or inorganic materials.
Preferably, the particulate material may be a component of bacterial or viral origin, including but not limited to one or more of bacterial cell walls, bacterial proteins, viral proteins, and the like.
Preferably, the shape of the nano/micro vaccine (particles) is any shape including, but not limited to, sphere, ellipsoid, barrel, polygon, rod, plate, line, worm, square, triangle, butterfly, disc, vesicle, etc.
The invention also provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises the cancer nano vaccine/micro vaccine or the cancer vaccine prepared according to the method;
optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.
The present invention also provides a method of preventing or treating a disease, wherein the method comprises administering to a subject a prophylactically or therapeutically effective amount of the cancer vaccine, or a cancer vaccine prepared according to the method, or the pharmaceutical composition.
The invention also provides a terminal sterilization method of the nano particles or the micro particles, or the nano particles or the micro particles which are prepared according to the method and load cancer cells/tumor tissue lysate components, and the application of the nano particles or the micro particles in at least one of the following steps:
(1) Preparing nano-particles or micro-particles which are directly injected into a body and used for preventing or treating diseases;
(2) Preparing antigen delivery particles for in vitro activation of antigen presenting cells and preparation of antigen presenting cell vaccines;
(3) Preparing antigen delivery particles for use in aiding activation of antigen-specific T cells (either indirectly or directly by antigen presenting cells) to detect antigen-specific T cell content;
(4) After preparing antigen-specific T cells for helper activation (either indirectly or directly through antigen presenting cells), isolating and expanding the activated antigen-specific T cells for use in preventing or treating a disease;
alternatively, the disease is cancer or tumor;
alternatively, the cancer or tumor is a solid tumor or hematological tumor.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention uses a specific ray radiation method to realize terminal sterilization of nano/micron vaccine (particles) loaded with lysate antigen components, overcomes the difficult problem that terminal sterilization cannot be carried out by using filtration sterilization, and the preparation after irradiation sterilization treatment accords with the regulations of Chinese pharmacopoeia aseptic inspection method, meets the aseptic requirements of injection preparation, keeps stable and can be stored for a long time, has good sterilization effect, has better effect of nano/micron vaccine (particles) after irradiation sterilization, and achieves unexpected effect.
Drawings
FIG. 1 is a schematic illustration of the terminal sterilization procedure of the present invention by irradiation of nanoparticles/microparticles loaded with cancer cell/tumor tissue lysate components.
FIG. 2 is a structure of formula 1, wherein R 1 C, S, P, N or O, R 2 ～R 5 Independently selected from any other group such as hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino, and the like.
FIGS. 3-9 are experimental results of tumor growth rate and survival time for the prevention or treatment of cancer using the nanovaccine and/or the minivaccine of examples 1-7; wherein a is the experimental result of the tumor growth rate (n is more than or equal to 8) when preventing or treating cancer; b is the result of a mice survival experiment (n.gtoreq.8) when cancer is prevented or treated, and each data point is the mean.+ -. Standard error (mean.+ -. SEM); wherein, the significant difference of the tumor growth inhibition experiment in the a graph is analyzed by adopting an ANOVA method, and the significant difference in the b graph is analyzed by adopting Kaplan-Meier and log-rank test; * P < 0.005 compared to PBS blank, with significant differences; * P < 0.01, showing significant differences compared to PBS blank; * Indicating a significant difference in p < 0.05 compared to the PBS blank; # # # indicates that the two groups have significant differences compared with p < 0.005; # represents that the two groups have significant differences compared with p < 0.01; # represents that the two groups have significant differences in comparison with p < 0.05.
Detailed Description
In order to make the technical scheme and the beneficial effects of the invention more obvious and understandable, the following detailed description is given by way of example. Wherein the drawings are not necessarily to scale, and wherein local features may be exaggerated or reduced to more clearly show details of the local features; unless defined otherwise, technical and scientific terms used herein have the same meaning as technical and scientific terms in the technical field to which this application belongs.
The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified, and for example, the experimental methods in the following examples are not specified under the specific conditions, and are generally performed under the conventional conditions or under the conditions recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.
In the claims and/or the specification of the present invention, the terms "a" or "an" or "the" may refer to "one" but may also refer to "one or more", "at least one" and "one or more".
As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.
The term "suffering from a disease" means: the body develops symptoms of the disease.
The term "treatment" refers to: after suffering from the disease, the subject is contacted (e.g., dosed) with a delivery particle, delivery system, vaccine, antigen-loaded drug, pharmaceutical composition, thereby alleviating the symptoms of the disease compared to when not contacted, and does not mean that the symptoms of the disease must be completely inhibited.
The term "preventing" refers to: by contacting (e.g., administering) a subject with the delivery particles, delivery systems, vaccines, antigen-loaded drugs, pharmaceutical compositions of the present invention prior to the onset of disease, the symptoms after the onset of disease are reduced compared to when not contacted, and do not mean that complete inhibition of the disease is necessary.
The vaccine of the present invention may be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes.
In the present invention, the route of administration can be varied or adjusted in any suitable manner to meet the nature of the drug, the convenience of the patient and medical personnel, and other related factors.
The term "individual", "patient" or "subject" as used in the context of the present invention includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors. The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.
In the preparation of the cancer vaccine, the antigen component can be prepared by using cancer cells/tumor tissues obtained by several methods such as tumor tissues, cancer cell lines, cancer cells obtained by culturing and amplifying cancer cells separated from the tumor tissues, cancer cells obtained by culturing and amplifying circulating tumor cells separated from peripheral blood, and the like, and in practical application, any other feasible scheme can be used for obtaining the cancer cells.
The cancer species in the embodiment of the invention is solid tumor, and the nano vaccine or the micro vaccine can be used for treating blood tumor, lymphoma and the like in practical application. Because the immune microenvironment of the hematological tumor and the lymphoma is not complicated by the solid tumor, the nano vaccine or the micro vaccine has better effect in the hematological tumor and the lymphoma.
A terminal sterilization method of cancer nano particles (vaccine) or micro particles (vaccine) loaded with antigen components in lysate of cancer cells or tumor tissues, wherein the sterilization mode is radiation sterilization for 1-48 hours; the irradiation is one or more treatments of different irradiation methods such as X-ray, gamma-ray, alpha-ray, beta-ray, 1-100MeV electron beam and the like with the dosage of 12-60 kGy;
The nanoparticle or microparticle has: (i) A nanoparticle and/or microparticle scaffold formed from a particle preparation material, (ii) a whole cell lysate component from cancer cells and/or tumor tissue, and/or a partial cell lysate component comprising an antigen component; wherein the whole cell lysate component and/or the antigen component-containing partial cell lysate component from cancer cells and/or tumor tissue is supported within and/or on the surface of the scaffold structure;
wherein all of the whole cell fraction from the cancer cell and/or tumor tissue lysate fraction is lysed using a lysis solution comprising a lysing agent; or the whole cell fraction from the cancer cell and/or tumor tissue lysate fraction comprises a water-soluble fraction and a non-water-soluble fraction solubilized using a solubilization solution comprising a solubilization agent; the antigen component contained in the partial cell lysate component comprises protein and polypeptide components in cancer cells and/or tumor tissue lysates and/or RNA components or mRNA components in cell lysates, and the partial lysate component containing the antigen component is separated and purified from the lysate by using a proper method;
the cancer nano-particles (vaccine) or micro-particles (vaccine) carrying antigen components in the lysate of cancer cells or tumor tissues after irradiation terminal sterilization can be directly injected into an organism to be used as a vaccine or used as antigen delivery particles for activating other cells in vitro.
Further, the lysate component in the cancer cells and/or tumor tissue loaded by the nano/micro vaccine (particles) may be one or more selected from the group consisting of: (1) The dissolution solution containing the dissolution agent solubilizes all lysate components dissolved; (2) The solubilization solution containing the solubilization agent solubilizes the protein polypeptide component of all lysate components solubilized; (3) Solubilization of the solubilized protein polypeptide component + RNA component (or mRNA component) in all lysate components with a solubilization agent; (4) a water-soluble component+a water-insoluble component; (5) Protein polypeptide component + water insoluble component in the water soluble component; (6) Protein polypeptide component in water-soluble component + protein polypeptide component in water-insoluble component; (7) Protein polypeptide component and RNA component (or mRNA component) in the water-soluble component) +protein polypeptide component and RNA component (or mRNA component) in the water-insoluble component. In some preferred embodiments, the mass ratio of the protein polypeptide in the water-soluble component to the protein polypeptide in the water-insoluble component/water-insoluble component is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Illustratively, the mass ratio of protein and polypeptide components in the water-soluble component to protein and polypeptide components in the water-insoluble component/water-insoluble component is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.
Further, the particle material may be PEG-modified or not PEG-modified, and preferably, when preparing the skeleton structure, the mass ratio of the PEG-modified particle material to the PEG-modified particle material is 25-200:1. The preparation material of the nanometer/micrometer vaccine (particle) can be prepared by adding a proper amount of PEG modified PLGA or PLA into main materials such as PLGA or PLA, and can better achieve the effects of long circulation and passive targeting; wherein the mass ratio of PEG modified PLGA or PLA or the like to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.
The nano/micron vaccine (particle) comprises the following components in percentage by mass: 0.001-10; preferably, the mass ratio of the components of the particle preparation framework material, protein and polypeptide is 1:0.01-2; most preferably, the particles produce a mass ratio of the framework material, protein and polypeptide components of 1:0.05-1.
The nano/micro vaccine (particle) surface of the invention can also be loaded with a membrane component, which can be one or more from antigen presenting cells, cancer cells, bacteria or extracellular vesicles.
The antigen presenting cells used to prepare the surface-loaded biofilm component of the nano/micro vaccine (particles) of the present invention may be derived from autologous or allogeneic sources, or from cell lines or stem cells. The antigen presenting cells may be DC cells, B cells, macrophages or any mixture of the three, or may be other cells having an antigen presenting function. Antigen presenting cells may be activated by antigen-loaded nanoparticles or microparticles.
When the biomembrane component loaded on the surface of the nano/micron vaccine (particle) is derived from extracellular vesicles, the biomembrane component can be one or more of extracellular vesicles of cancer cells, extracellular vesicles of bacteria or extracellular vesicles of antigen presenting cells.
Any nanoparticle, microparticle preparation method known to those skilled in the art may be used to prepare the nano/microparticle vaccine (particles) of the present invention described herein, including but not limited to solvent evaporation, dialysis, phase separation, spray drying, emulsion polymerization, machine stirring shear, membrane emulsification, microfluidic, ultrafiltration, homogenization emulsification, dispersion, precipitation, and the like.
In some specific embodiments, the present invention provides the following exemplary particle preparation and radiation sterilization methods thereof, taking solvent evaporation as an example:
step 1, lysing tumor tissues or cancer cells by using a lysis solution containing a lysis agent, and then, lysing a lysis solution component by using the lysis solution containing the lysis agent; alternatively, after lysing cancer cells/tumor tissue using ultrapure water or an aqueous solution, the lysate fraction is centrifuged, and the water-soluble fraction and the water-insoluble fraction solubilized by the lysate containing the lytic agent are collected, respectively. The lysate fraction obtained above may be directly loaded into the nano/micro vaccine (particles), or may be subjected to the following treatment (purification or enhancement of the immunogenicity of the antigen fraction) in step 2 and step 3.
Alternatively, prior to tissue or cell lysis, appropriate treatments may be performed to enhance the immunogenicity of the antigen component, treatment methods to enhance immunogenicity include, but are not limited to, irradiation with radiation, heating, oxidation, reduction, hapten modification, enzymatic treatment, mineralization, and the like.
The irradiation is for a period of time using one or more of the different irradiation methods of X-rays, gamma rays, alpha rays, beta rays, etc.
The cancer cells or tumor tissue may be co-incubated with specific chemicals prior to lysis to stimulate the cancer cells or tumor tissue, including, but not limited to, small molecule compounds (e.g., doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, plant extracts (e.g., important extracts of ginseng, plant rhizome extracts, etc.), chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of stimulating cancer cells or tumor tissue with a particular substance co-incubation with the cancer cells or tumor tissue is to allow the cancer cells to produce more antigenic components.
The hapten substance is a substance which can increase the immunogenicity of a protein or polypeptide after coaction with the protein or polypeptide.
The hapten substances include, but are not limited to, 2, 4-Dinitrofluorobenzene (DNFB), 2, 4-Dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, ovalbumin (OVA), N-iodoacetyl-N' - (5-sulfo-1-naphthyl) ethylene diamide (AED), substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, and the like.
Any oxidizing agent that oxidizes an antigenic component may be used in the present invention, including but not limited to hypochlorous acid, sulfate, hydrogen peroxide (hydrogen peroxide), KIO 3 、KBrO 3 Chlorine, dichromate, nitric acid, hydrogen peroxide, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxideOxygen, chlorine, sodium dichromate, potassium permanganate, nitric acid, clO 3 - 、ClO 4 - 、Na 2 O 2 、K 2 O 2 、MgO 2 、CaO 2 、BaO 2 、H 2 O 2 、NO 3 - 、MnO 4 - 、F 2 、Cl 2 、O 2 、Br 2 、I 2 、S、Si、HNO 3 、MnO 2 、FeCl 3 And the like.
The reducing agents described herein include, but are not limited to, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), and the like.
The enzyme treatment methods of the present invention include, but are not limited to, the use of one or more of nucleases, dnases, pepsin, chymotrypsin, trypsin, other proteolytic enzymes, protease inhibitors, and the like. Enzymatic hydrolysis in the present invention includes, but is not limited to, any viable enzymatic hydrolysis method using nucleases, pepsin, trypsin, protease inhibitors, chymotrypsin, dnase, and the like.
The methods of mineralization described herein include, but are not limited to, any mineralization or biomineralization method using siliconization, calcification, magnesian, and the like.
The cancer cells can be one or more cancer cells or a cancer cell line, can be obtained by culturing cancer cells in tumor tissues, and can be obtained by amplifying circulating tumor cells; the tumor tissue is tumor tissue derived from one body or a plurality of bodies. The lysis method is a common lysis method for cancer cells and/or tumor tissue, including but not limited to one or more of repeated freeze thawing, swelling, sonication, high pressure treatment, homogenization, extrusion, homogenization, high speed stirring, chemical treatment, high shear force treatment, ultrafiltration treatment, crimping, and the like.
The dissolving agent is selected from one or more of a compound containing a structure shown in a structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline; wherein, structural formula 1 is as follows:
R 1 c, S, P, N or O, R 2 ～R 5 Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino. The compounds containing the structure of formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, urea, other guanidino-containing compounds, guanidine carbonate, arginine, guanidinoacetic acid, guanidino-phosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazoles, etc.), nitrosoureas, etc.
And 2, heating, diluting a dissolving solution containing a dissolving agent to prepare a dissolved lysate component, salting out, oxidizing, reducing, coacting with a hapten substance, fixing, irradiating, enzyme treating, extracting a protein polypeptide component, extracting an RNA component, extracting an mRNA component, mineralizing and other methods are used for purifying the lysate component or enhancing the immunogenicity of the lysate component.
Step 3, secondarily dissolving the antigen component of step 2 (lysate component subjected to purification or immunogenicity enhancing treatment) using a dissolving solution containing a dissolving agent.
The dissolving agent in the dissolving solution used for secondarily dissolving the precipitate component is selected from one or more of a compound shown in a structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline; wherein, structural formula 1 is as follows:
R 1 c, S, P, N or O, R 2 ～R 5 Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino.The compounds containing the structure of formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, urea, other guanidino-containing compounds, guanidine carbonate, arginine, guanidinoacetic acid, guanidino-phosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazoles, etc.), nitrosoureas, etc.
And 4, using the solubilised antigen component solubilised by the solubilising solution containing the solubilising agent obtained in the step 3 as an initial water phase, and adding the initial water phase into an organic phase to prepare a initial emulsion sample.
In preparation, the initial aqueous phase is mixed with the organic phase, specifically by adding a first predetermined volume of an aqueous phase solution containing an antigen component at a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of the raw material for preparing particles.
In some embodiments, the aqueous phase solution may contain at least one of the following i) to ii): i) An antigen component in the lysate; ii) an antigenic component in the lysate and an immunopotentiating adjuvant. The antigen component in the lysate is separated and purified in the form of a solution containing urea or guanidine hydrochloride. The first predetermined concentration is the concentration of the protein and polypeptide contained in the aqueous solution or the concentration of the antigen component contained in the aqueous solution, and the first predetermined concentration requires a concentration of the protein and polypeptide of greater than 1ng/mL so as to be capable of supporting sufficient antigen component to activate the cells of interest. The concentration of immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
In some embodiments, the organic solvent is selected from dichloromethane. Additionally, in some embodiments, the second predetermined concentration of the starting material for the preparation of particles ranges from 0.5mg/mL to 5000mg/mL, preferably 100mg/mL.
In practice, the second predetermined volume of the organic phase is set according to the ratio of the same to the first predetermined volume of the aqueous phase, in the present invention the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1:10. In a specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as required to adjust the size of the prepared nanoparticles or microparticles.
In some embodiments, the aqueous solution is a solution comprising the antigenic component in the cell and/or tissue lysate, wherein the concentration of the protein and polypeptide is greater than 1ng/mL, preferably between 1mg/mL and 100mg/mL. In some embodiments, the aqueous solution is a solution comprising the antigen component of the lysate and the immunoadjuvant, wherein the concentration of the protein and the polypeptide is greater than 1ng/mL, preferably between 1mg/mL and 100mg/mL, and the concentration of the immunoadjuvant is greater than 0.01ng/mL, preferably between 0.001mg/mL and 20mg/mL. In some embodiments, the solvent in the organic phase solution is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, and the like, preferably dichloromethane; the concentration of the organic phase is 0.5 mg/mL-5000 mg/mL, preferably 100mg/mL.
In some embodiments, when the aqueous solution is a solution comprising the antigen component in the lysate, the concentration of the protein and polypeptide components therein is greater than 0.01ng/mL, preferably between 1 μg/mL and 1mg/mL. In some embodiments, when the aqueous solution is a solution comprising the antigen component and the immunoadjuvant, wherein the concentration of the protein and polypeptide component is greater than 1ng/mL, preferably between 1 μg/mL and 1mg/mL, the concentration of the immunoadjuvant is greater than 0.01ng/mL, preferably between 0.001mg/mL and 20mg/mL. In some embodiments, the solvent in the organic phase solution is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, and the like, preferably dichloromethane; the concentration of the organic phase is 0.5 mg/mL-5000 mg/mL, preferably 100mg/mL.
And 5, performing any one of the following treatments on the mixed solution obtained in the step 4: i) Sonication for greater than 2 seconds; ii) stirring for more than 1 minute; iii) Homogenizing; iv) microfluidic processing. Preferably, the stirring speed is greater than 50rpm and the stirring time is greater than 1 minute, such as 50rpm to 1500rpm and 0.1 to 24 hours, when mechanically or magnetically stirring; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 seconds, such as 2-200 seconds; the high pressure/ultra-high pressure homogenizer or high shear homogenizer is used for the homogenization treatment, the pressure is more than 5psi, such as 20 psi-100 psi, and the rotating speed is more than 100rpm, such as 1000 rpm-5000 rpm; the microfluidic processing flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. The nano-scale and/or micro-scale is carried out by ultrasonic or stirring or homogenizing treatment or microfluidic treatment, the ultrasonic time or stirring speed or homogenizing treatment pressure and time can control the size of the prepared nano-scale particles or micro-scale particles, and the particle size change can be caused by the excessive or excessive small size.
Step 6, adding the mixture obtained after the treatment in step 5 into a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and performing any one of the following treatments: i) Sonication for greater than 2 seconds; ii) stirring for more than 1 minute; iii) Homogenizing; iv) microfluidic processing. The mixture obtained in the step 2 is added into the aqueous solution of the emulsifier to continue ultrasonic treatment or stirring or homogenization or mixing so as to carry out nanocrystallization or microminiaturization. In the present invention, the ultrasound time is greater than 0.1 seconds, such as 2 to 200 seconds; the stirring speed is greater than 50rpm, such as 50 rpm-500 rpm; the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm-1500 rpm, and the stirring time is 0.5-5 hours; during ultrasonic treatment, the ultrasonic power is 50-500W, and the time is more than 0.1 seconds, such as 2-200 seconds; the high pressure/ultra-high pressure homogenizer or high shear homogenizer is used for the homogenization treatment, the pressure is more than 20psi, such as 20 psi-100 psi, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm; the microfluidic processing flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. The ultrasonic treatment, stirring or homogenizing treatment or micro-fluidic treatment is performed to carry out nanocrystallization or microminiaturization, the ultrasonic time, stirring speed, homogenizing treatment pressure, homogenizing treatment time and the like can control the size of the prepared nano particles or micro particles, and the particle size change can be brought by too large or too small.
In some embodiments, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20mg/mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present invention, the ratio of the second predetermined volume to the third predetermined volume ranges from 1:1.1 to 1:1000, preferably may be 2:5. In order to control the size of the nano-or micro-particles during the implementation process, the ratio of the second predetermined volume to the third predetermined volume may be adjusted. Similarly, the ultrasonic time or stirring time or homogenizing time, the volume of the aqueous solution of the emulsifier and the concentration of the aqueous solution of the emulsifier are taken as values according to the step, so that the nano-particles or the micro-particles with proper size can be obtained.
And 7, adding the liquid obtained after the treatment in the step 6 into a fourth preset volume of a fourth preset concentration emulsifier water solution, and stirring until a preset stirring condition is met.
In this step, the aqueous emulsifier solution is a PVA solution or other solution.
The fourth preset concentration is 5mg/mL, and the nano particles or the micro particles with proper size are obtained through the selection of the fourth preset concentration. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the fourth predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles or microparticles in the implementation process.
In the present invention, the predetermined stirring conditions of this step are until the evaporation of the organic solvent is completed, that is, the evaporation of methylene chloride in step 1 is completed.
Step 8, centrifuging the mixed solution which meets the preset stirring condition and is obtained in the step 7 at a rotating speed of more than 100RPM for more than 1 minute, removing supernatant, and re-suspending the rest precipitate in a fifth preset volume of fifth preset concentration aqueous solution containing a lyoprotectant or a sixth preset volume of PBS (or physiological saline); the solution in the system is replaced with a fifth predetermined volume of aqueous solution or a sixth predetermined volume of PBS (or physiological saline) containing a fifth predetermined concentration of lyoprotectant while removing substances such as free PVA by ultrafiltration centrifugation or dialysis capable of removing substances of a specific molecular weight.
And 9, sub-packaging the suspension containing the freeze-drying protective agent obtained in the step 8 into SKJYLEAN (Su Ke) 20m1 transparent sample bottles, and performing freeze-drying treatment to obtain a freeze-dried substance for later use.
And 10, irradiating the freeze-dried nano particles or micro particles which are sub-packaged in freeze-dried sample bottles with gamma rays, alpha rays, beta rays and other radioactive rays for a certain time to sterilize the prepared nano particles or micro particles loaded with the antigen component.
The nanometer/micrometer particles after irradiation sterilization treatment are used as vaccines for preventing or treating diseases; or using the nanoparticle microparticles as antigen delivery particles to activate antigen presenting cells (such as dendritic cells or mixed antigen presenting cells) in vitro to prepare antigen presenting cell vaccines; or using nanoparticles or microparticles to assist in activating antigen-specific T cells to detect the content of antigen-specific T cells; or after the antigen-specific T cells are activated with the aid of nano-particles or micro-particles, sorting or amplifying the antigen-specific T cells, and using the sorted or amplified antigen-specific T cells for preventing or treating diseases.
EXAMPLE 1 nanovaccine for the treatment of pancreatic cancer
In the embodiment, the antigen component is derived from Pan02 mouse pancreatic cancer cell line, organic polymer material PLGA is used as nanoparticle skeleton material, polyinosinic-polycytidylic acid (poly (I: C)), cpG7909 and CpG2395 are used as immunoadjuvants, and a solvent volatilization method is used for preparing the nanometer vaccine.
(1) Preparation of antigenic components
Collect 1.5X10 7 Pan02 mice pancreatic cancer cells were resuspended in 1mL of PBS and then subjected to ultraviolet light for 30 minutes, then an appropriate amount of ultrapure water was added and freeze thawing was repeated 5 times (accompanied by ultrasound) to lyse the cancer cells . Centrifuging the lysate at 5000g for 5 min and collecting supernatant to obtain water soluble component soluble in pure water; adding 8M urea aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert the insoluble component insoluble in pure water into soluble component in the 8M urea aqueous solution. The water-soluble component and the water-insoluble component are mixed according to the mass ratio of 1:2 and then used as an antigen component 1 for preparing the nano vaccine 1.
(2) Preparation of nanovaccine
In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted vaccine preparation material PLGA is 10kDa-20kDa, and the adopted immunoadjuvants are poly (I: C), cpG7909 and CpG2395. All the substances are filtered by a 0.22 mu m filter membrane before being used, and the whole process is operated in a B-level sterile clean-level space in the preparation process. Preparation method As previously described, antigen component 1 and adjuvant were co-supported in the nanoparticle by the multiple emulsion method in the preparation process, and 100mg of the nanoparticle was centrifuged at 12000g for 30 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48 hours. The average particle size of the nanoparticle 1 is about 250nm, and each 1mg of PLGA nanoparticle is loaded with about 1.5mg of protein and polypeptide components, and 0.05mg of poly (I: C), cpG7909 and CpG2395.
In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted vaccine preparation material PLGA is 10kDa-20kDa, and the adopted immunoadjuvants are poly (I: C), cpG7909 and CpG2395. All the substances are filtered through a 0.22 mu m filter membrane before being used, and the preparation process is operated in a C-class clean grade space. Preparation method As previously described, antigen component 1 and adjuvant were co-supported in the nanoparticle by the multiple emulsion method in the preparation process, and 100mg of the nanoparticle was centrifuged at 12000g for 30 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48 hours. The average particle size of the nanoparticle 1 is about 250nm, and each 1mg of PLGA nanoparticle is loaded with about 1.5mg of protein and polypeptide components, and 0.05mg of poly (I: C), cpG7909 and CpG2395. After freeze-drying, the nanoparticles were subjected to irradiation sterilization using gamma rays for 24 hours (24 kGy). The preparation after irradiation sterilization meets the requirements of Chinese pharmacopoeia aseptic inspection method, meets the aseptic requirements of injection preparation, and the sterilized preparation is kept stable.
In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted vaccine preparation material PLGA is 10kDa-20kDa, and the adopted immunoadjuvants are poly (I: C), cpG7909 and CpG2395. All the substances are filtered through a 0.22 mu m filter membrane before being used, and the preparation process is operated in a C-class clean grade space. Preparation method As previously described, antigen component 1 and adjuvant were co-supported in the nanoparticle by the multiple emulsion method in the preparation process, and 100mg of the nanoparticle was centrifuged at 12000g for 30 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48 hours. The average particle size of the nanoparticle 1 is about 250nm, and each 1mg of PLGA nanoparticle is loaded with about 1.5mg of protein and polypeptide components, and 0.05mg of poly (I: C), cpG7909 and CpG2395. After freeze-drying, the nanoparticles were subjected to irradiation sterilization using gamma rays for 48 hours (48 kGy). The preparation after irradiation sterilization meets the requirements of Chinese pharmacopoeia aseptic inspection method, meets the aseptic requirements of injection preparation, and the sterilized preparation is kept stable.
(3) Nanometer vaccine for treating cancer
Preparation of pancreatic cancer tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 6 Pan 02 cells. On day 3, 6, 9, 14, 20 and 27 after tumor inoculation, 2mg of nanovaccine 1 (radiation sterilized and non-radiation sterilized) or 100 μl of PBS was injected subcutaneously in mice, respectively. The mice were monitored for tumor growth rate and mice survival. In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3. Tumor volume was calculated using the formula v=0.52×a×b 2 Calculation, where v is tumor volume, a is tumor length, and b is tumor width. For ethical reasons in animal experiments, the tumor volume of the mice exceeds 2000mm in the life cycle test of the mice 3 I.e. the mice were regarded as dead and euthanized.
(4) Experimental results
As shown in fig. 3, the tumor volume of PBS group mice rapidly increased and the mice died rapidly. The mice using the nano vaccine 1 (nanovaccinee 1) have obviously slowed tumor growth speed and obviously prolonged survival period, and most of the mice recover without tumor. Furthermore, the effect of the irradiation sterilized Nanovaccine 1 (Nanovaccine irradiate) was slightly better than that of the Nanovaccine 1 (nanovaccinee 1) which was not subjected to irradiation sterilization.
EXAMPLE 2 micron vaccine for preventing brain cancer
(1) Preparation of antigenic components
Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 6 GL261 cells, growing to a volume of about 1000mm in each tumor 3 When the antigen component 1 is obtained, mice are killed, tumor tissues are taken, a proper amount of ultrapure water is added for resuspension, repeated freezing and thawing are carried out for 5 times, then tumor tissue lysate is heated at 90 ℃ for 5 minutes, then a heated sample is centrifuged at 5000g for 5 minutes, all precipitate parts are collected after supernatant liquid is removed, and the precipitate parts are dissolved by using 8M urea aqueous solution.
(2) Preparation of micrometer vaccine
In this example, micrometer vaccine 1 (Micronvaccine 1) was prepared by the multiple emulsion method of the solvent evaporation method. The molecular weight of the adopted micron particle preparation material PLA is 30kDa-50kDa, and the adopted immunological adjuvants are poly (I: C), cpG 1018 and CpG2395. All the substances are filtered by a 0.22 mu m filter membrane before being used, and the whole process is operated in a B-level sterile clean-level space in the preparation process. Preparation method As previously described, antigen component 1 and adjuvant were loaded inside microparticles during the preparation, and 100mg microparticles were centrifuged at 8000g for 20 minutes, resuspended in 10mL of 4% trehalose-containing ultrapure water and lyophilized for 48h. The average particle size of the micrometer vaccine 1 is about 2.0 mu m, about 20 mu g of protein or polypeptide component is loaded per 1mg of PLA micrometer particle, and 0.01mg of poly (I: C), cpG 1018 and CpG2395 are loaded per 1mg of PLA micrometer particle. After freeze drying, taking part of the freeze-dried micrometer vaccine, and performing irradiation sterilization by using gamma rays for irradiation for 16 hours (16 kGy); after freeze drying, taking part of the freeze-dried micrometer vaccine, and irradiating with ultraviolet rays of 280-315nm for 16 hours for irradiation sterilization;
(3) Micron vaccine for preventing cancer
Tumor bearing mice were prepared by selecting 6-8 week female C57BL/6 as model mice, and subcutaneously injecting 4mg of micrometer vaccine 1 (with or without irradiation) or 100 μl of PBS in the mice on day-35, day-28, day-21, day-14 and day-7, respectively, prior to tumor inoculation of the mice. Day 0, 1.5X10 s were subcutaneously inoculated on the lower right back of each mouse 6 And GL261. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.
(9) Experimental results
As shown in fig. 4, the PBS group mice rapidly grew in tumor volume and the mice died quickly. Mice using micrometer vaccine 1 (Micronvaccine 1) had significantly slower tumor growth rates, significantly longer survival, and most mice were tumor-free. The effect of the micrometer vaccine 1 irradiated by gamma rays (gamma-ray) is better than that of the micrometer vaccine 1 which is not irradiated and the micrometer vaccine 1 irradiated by ultraviolet rays (Ultraviolet irradiate), so that the irradiation sterilization can not reduce or influence the curative effect of the micrometer vaccine loaded with tumor tissue lysate components, and the proper irradiation method can improve the vaccine effect.
EXAMPLE 3 nanometer vaccine for treatment of liver cancer
(1) Preparation of antigenic components
The back of each C57BL/6 mouse was inoculated subcutaneously with 1.5X10 6 The Hepa 1-6 liver cancer cells grow to a volume of about 1000mm in the tumor 3 The mice are killed and tumor tissues are taken out, the tumor tissues are irradiated under X rays for 20 minutes, then tumor tissue single cell suspension is prepared, a proper amount of 6M guanidine hydrochloride aqueous solution is used for cracking the tumor tissue single cell suspension, then 6M guanidine hydrochloride aqueous solution is used for dissolving lysate components, 40% ammonium sulfate aqueous solution is added dropwise for salting out protein polypeptide components in the lysate components dissolved by the 6M guanidine hydrochloride aqueous solution, the protein polypeptide components are separated out after precipitation and are centrifuged at 14000RPM for 30 minutes, and after the separated protein polypeptide components are collected, the separated protein polypeptide components are secondarily dissolved out by using 6M guanidine hydrochloride, thus the antigen component 1 for preparing the nano vaccine 1 is obtained.
(2) Preparation of nanovaccine
In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted nanometer vaccine preparation material PLGA is 20kDa-30kDa, the molecular weight of PEG5000-PLGA is 25kDa-35kDa, and the mass ratio of PLGA to PEG5000-PLGA is 99:1. The immunoadjuvants used were poly (I: C), cpG 2395 and CpG SL03. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, and then centrifuging 100mg of the nanoparticle at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension using 10mL of ultra-pure water containing 4% trehalose. The average particle size of the nanoparticle 1 is about 300nm, and about 500 mug of protein or polypeptide components are loaded per 1mg of PLA nanoparticle, and 0.1mg of poly (I: C), cpG 2395 and CpG SL03 are loaded. After freeze drying, irradiating half of the nano vaccine 1 with electron beam (25 MeV) for 1 hr for irradiation sterilization; the other half is directly used without any irradiation treatment.
(3) Nanometer vaccine for treating cancer
Preparation of tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 6 And (3) liver cancer cells of the Hepa 1-6. 100. Mu.L of 0.4mg nanovaccine 1 (irradiated or non-irradiated) or 100. Mu.L of PBS was injected subcutaneously in mice on day 3, 6, 9, 14, 19 and 25, respectively, after tumor inoculation. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.
(4) Experimental results
As shown in fig. 5, the PBS group mice rapidly grew in tumor volume and the mice died quickly. The survival time of the mice using the nano vaccine 1 (nanovaccinee 1) is obviously prolonged, and most of the mice are healed without tumor. And the effect of the irradiated nano vaccine is better.
EXAMPLE 4 nanovaccine for treatment of esophageal cancer
(1) Preparation of antigenic components
The back of each C57BL/6 mouse was inoculated subcutaneously with 1.5X10 6 AKR esophageal cancer cells, respectively, grow to a volume of about 1000mm in tumor 3 Mice were sacrificed at that time and the tumors were removedTumor tissue was then prepared as a single cell suspension of tumor tissue, then 60 μm hypochlorous acid was added and incubated for 1 hour to oxidize the tumor tissue but the cell suspension, then 8M urea in PBS water was added to lyse the oxidized single cell suspension of tumor tissue, and 8M urea in PBS water was used to lyse the lysate components. Then adding 10 times of volume of ultrapure water into the lysate component dissolved in the 8M urea PBS aqueous solution to dilute the urea concentration, standing for 1 hour, centrifuging at 3000RPM for 10 minutes after the protein polypeptide component is separated out, and collecting supernatant and a precipitation part respectively. Collecting the precipitate, and secondarily dissolving the precipitate component by using 8M urea PBS aqueous solution; to the supernatant after centrifugation, 30% ammonium sulfate was added dropwise to salt out the protein polypeptide component, and then centrifugation was carried out at 15000RPM for 25 minutes, and after discarding the supernatant, the precipitate precipitated after salting out was dissolved in 8M urea in PBS aqueous solution for the second time. And then mixing the precipitation parts which are secondarily dissolved by using the 8M urea PBS aqueous solution twice, thus obtaining the antigen component 1.
(2) Preparation of nanovaccine
In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted nanometer vaccine preparation material PLGA is 20kDa-40kDa. The immunoadjuvants used were poly (I: C), cpG 1018, and CpG 7909. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, and then centrifuging 100mg of the nanoparticle at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension using 10mL of ultra-pure water containing 6% trehalose. The average particle size of the nanoparticle 1 is about 260nm, and about 100 mug of protein or polypeptide components are loaded per 1mg of PLGA nanoparticle, and 0.005mg of poly (I: C), cpG 1018 and CpG 7909 are loaded. After freeze drying, one third of the nano vaccine 1 is directly used without irradiation sterilization; the other two thirds of the vaccines were used after irradiation sterilization by beta-radiation for 12 hours (12 KGy) or 36 hours (36 KGy), respectively.
(3) Nanometer vaccine for treating cancer
Preparation of esophageal cancer tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 6 AKR esophageal cancer cells. Inoculation in mice On day 3, 6, 9, 14, 19 and 25 post-tumor, 100 μl of 2mg nanovaccine 1 or 100 μl of PBS was injected subcutaneously in mice, respectively. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.
(4) Experimental results
As shown in fig. 6, the PBS group mice rapidly grew in tumor volume and the mice died quickly. The survival time of the mice using the nano vaccine 1 (nanovaccinee 1) is obviously prolonged, and most of the mice are healed without tumor.
EXAMPLE 5 nanoparticle-activated antigen presenting cell vaccine for treatment of colon cancer
(1) Preparation of antigenic components
The back of each C57BL/6 mouse was inoculated subcutaneously with 1.5X10 6 MC38 colon cancer cells, which grow to a volume of about 1000mm each 3 Mice were sacrificed and tumor tissues were harvested, the tumor tissues were fixed in 75% ethanol aqueous solution (containing 2% hydrogen peroxide) for 2 hours, then centrifuged at 1500RPM5 for 5 minutes, the supernatant was discarded, an appropriate amount of 8M aqueous urea peroxide solution was added to the pellet to lyse tumor tissue cells, and 8M aqueous urea peroxide solution was used to lyse tumor tissue lysate components, then 40 volumes of ultrapure water was added to dilute the 8M aqueous urea peroxide solution containing lysate components, and the pellet was left for 6 hours to stand for precipitation. And then centrifuging at 3500RPM for 10 minutes, and secondarily dissolving the precipitation part by using an aqueous solution of 8M urea peroxide, namely, preparing the antigen component 1 of the antigen delivery particle 1.
(2) Preparation of antigen-loaded component antigen delivery nanoparticles
In this example, nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the adopted nanometer vaccine preparation material PLGA is 10kDa-20kDa. The immunoadjuvants used were poly (I: C), cpG SL01 and CpG SL03. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, and then centrifuging 100mg of the nanoparticle at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension using 10mL of ultra-pure water containing 6% trehalose. The average particle size of the nanoparticle 1 is about 280nm, and each 1mg of PLGA nanoparticle is loaded with about 2.0mg of protein or polypeptide component, and 0.02mg of poly (I: C), cpG 1018 and CpG 7909. Half of the nanoparticles 1 were then irradiated with gamma rays for 18 hours (18 kGy); the other half was used directly without irradiation.
(3) In vitro activation of antigen presenting cells (dendritic cells+B cells) using antigen delivery particles 1 (irradiated or non-irradiated)
This example illustrates how DCs can be prepared by taking the preparation of dendritic cells from mouse bone marrow cells as an example. Firstly, taking C57BL/6 mice with 6-8 weeks of age, killing cervical dislocation, taking out tibia and femur of the rear leg after operation, putting the tibia and femur into PBS, and removing muscle tissues around the bone by scissors and forceps. Cutting off two ends of bone with scissors, extracting PBS solution with syringe, inserting needle into bone marrow cavity from two ends of bone, and repeatedly flushing bone marrow into culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and 1mL of erythrocyte lysate was added for lysis. Lysis was terminated by adding 3mL of RPMI 1640 (10% FBS) medium, centrifuging 400g for 3min, and discarding the supernatant. Cells were placed in 10mm dishes and cultured using RPMI 1640 (10% FBS) complete medium with the addition of recombinant mouse GM-CSF (20 ng/mL) at 37℃with 5% CO 2 Culturing for 7 days. The flask was gently shaken on day 3 and the same volume of RPMI1640 (10% FBS) medium containing GM-CSF (20 ng/mL) was supplemented. On day 6, the medium was subjected to half-volume liquid exchange. On day 7, small amounts of suspended and semi-adherent cells were collected and assayed by flow as CD86 + CD80 + Cell in CD11c + The ratio of cells is 15-20%, and the induced BMDC can be used for the next experiment.
B cells derived from mouse spleen cells were used. The preparation method comprises killing mice, preparing single cell suspension of spleen cells, and separating by magnetic bead separation method to obtain CD19 + B cells of (a).
BMDC (1000 ten thousand) and B cells (1000 ten thousand) were mixed in a quantitative ratio of 1:1 and co-incubated with 0.5mg of nanoparticle 1 (irradiated or not) in 10mL of RPM I1640 complete medium containing 20ng/mL of interleukin 15 (IL-15) for 24 hours (37 ℃,5% CO) 2 ). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove any excess cells in the systemThe nanoparticles were isolated and then the mixed cells activated by antigen delivery particles were used as a mixed Cell vaccine (Cell vaccinee). Wherein, as the mixed Cell vaccine 1 (Cell vaccinee 1), the mixed cells activated by the antigen delivery particles after irradiation are used; mixed cells activated with antigen delivery particles 1 not subjected to irradiation treatment were used as mixed Cell vaccine 2 (Cell vaccinee 2).
(4) DC vaccine for treating cancer
Preparation of tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 6 Individual MC38 colon cancer cells. 100 ten thousand cell vaccine1 or 100 μl of PBS was injected subcutaneously in mice on day 3, 6, 9, 14, 19 and 25 after tumor inoculation. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.
(5) Experimental results
As shown in fig. 7, the PBS group mice rapidly grew in tumor volume and the mice died quickly. The survival of mice treated with Cell vaccine1 (Cell vaccinee 1) and Cell vaccine 2 (Cell vaccinee 1) was significantly prolonged and the mice were mostly cured without tumor. And the effect of the cell vaccine1 is better than that of the cell vaccine 2.
Example 6 nanoparticle-assisted sorting of expanded T cells loaded with antigen component for treatment of breast cancer
(1) Preparation of antigenic components
The back of each C57BL/6 mouse was inoculated subcutaneously with 1.0X10 6 Breast cancer cells of E0771 mice grew to a volume of about 1000mm each in the tumor 3 The mice are killed and tumor tissues are picked, single cell suspension of the tumor tissues is prepared, then 4mL of 3% hydrogen peroxide (hydrogen peroxide) is added for oxidation for 1 hour, then 16 mL of ultrapure water is added for cracking the single cell suspension, then the protein polypeptide component is precipitated by heating at 95 ℃ for 5 minutes, then centrifugation is carried out at 5000RPM for 5 minutes, the supernatant is removed, the precipitate is collected, and a proper amount of 8M urea water solution precipitation component is added, thus obtaining the antigen component 1 for preparing the nano particles 1.
(2) Preparation of antigen delivery nanoparticles
In this example, nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the adopted nanometer vaccine preparation material PLGA is 20kDa-40kDa. The immunoadjuvants used were poly (I: C), cpG 1018, and CpG SL03. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, and then centrifuging 100mg of the nanoparticle at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension using 10mL of ultra-pure water containing 6% trehalose. The average particle size of the nanoparticle 1 is about 280nm, and about 600 mug of protein or polypeptide components are loaded per 1mg of PLGA nanoparticle, and 0.02mg of poly (I: C), cpG 1018 and CpG 7909 are loaded. Then half of the nanoparticles 1 were subjected to terminal sterilization by X-ray irradiation for 30 hours (30 kGy); the other half of the nanoparticles 1 were used without irradiation.
(3) Sorting expansion of antigen-specific T cells
Female C57BL/6 mice were selected for 6-8 weeks, and each mouse was inoculated subcutaneously with 1.0X10 on day 0 6 Each mouse was intraperitoneally injected with 150 μg of mouse PD-1 antibody on day 6, day 8, day 10, day 12, day 14, day 16, day 18, day 20, respectively, by E0771 cells. Mice were sacrificed on day 21, peripheral blood was collected from the mice, peripheral Blood Mononuclear Cells (PBMCs) were then isolated from the peripheral blood, then 500 ten thousand PBMC cells, 2mg nanoparticle 1 (irradiated or not) were co-incubated in 5ml of rpm 1640 complete medium for 36 hours, and then the incubated CD3 was sorted using flow cytometry + CD134 + T cells are antigen-specific T cells (tumor-specific) that recognize cancer cell antigens. 3 ten thousand CD3 s obtained by the separation + CD95 + T cells with IL-2 (20 ng/mL), IL-7 (10 ng/mL), IL-15 (10 ng/mL), and the alpha CD3 antibody (10 ng/mL) and the alpha CD28 antibody (10 ng/mL) in 10mL DMEM complete medium (37 ℃,5% CO) 2 ) Co-incubation was performed for 21 days to expand cancer cell-specific T cells (cell viability about 75%). Wherein, the cancer cell specific T cells which are amplified by using the irradiated nano particles 1 (nano vaccine 1) in an assisted sorting way are T cells 1 (T cells 1); the non-irradiated nanoparticle 1 (nanovaccine 1) was used to aid in sorting the expanded cancer cell-specific T cells to T cells 2 (T cells 2).
(4) Sorting expanded cancer cell antigen-specific T cells for treatment of cancer
Preparation of breast cancer tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.0X10 lower right back of each mouse on day 0 6 E0771 mouse breast cancer cells. On day 7, 10, 15, 20 and 25 after tumor inoculation, mice were intravenously injected with 100 ten thousand sorted expanded antigen-specific T cells 1 or T cells 2 (10000U of IL-2 were simultaneously intravenously injected every two days from day 7 to day 27) or 10000U of IL-2 or 100 μL of PBS were intravenously injected every two days from day 7 to day 27, respectively. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.
(5) Experimental results
As shown in fig. 8, the PBS group mice rapidly grew in tumor volume and the mice died quickly. Mice treated with either T cell 1 (T cells 1) or T cell 2 (T cells 2) had significantly prolonged survival, with the vast majority of mice recovering without tumors. And T cell 1 is better than T cell 2.
EXAMPLE 7 nanometer vaccine for treatment of Lung cancer
(1) Preparation of antigenic components
The back of each C57BL/6 mouse was inoculated subcutaneously with 2.0X10 6 LLC lung cancer cells, in which tumors grow to a volume of about 1000mm, respectively 3 Mice were sacrificed and tumor tissues were harvested, fixed with 75% ethanol for 24 hours, then centrifuged at 1000g for 5 minutes, the supernatant discarded, the pellet lysed with 8M urea in PBS and the lysate fraction lysed with 8M urea in PBS. Then adding 2 times of 75% ethanol for precipitating antigen components and the like into the lysate component dissolved by the 8M urea PBS aqueous solution, standing for 6 hours, centrifuging at 3000RPM for 10 minutes after the antigen components and the like are separated out, collecting an upper precipitation part, and secondarily dissolving the precipitation component by using the 8M urea PBS aqueous solution to obtain the antigen component 1.
(2) Preparation of nanovaccine
In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted nanometer vaccine preparation material PLGA is 20kDa-40kDa. The immunoadjuvants used were poly (I: C), cpG SL01 and CpG SL03. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, and then centrifuging 100mg of the nanoparticle at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension using 10mL of ultra-pure water containing 6% trehalose. The average particle diameter of the nanoparticle 1 is about 260nm, and each 1mg of PLGA nanoparticle is loaded with about 100 mug protein or polypeptide components, and 0.05mg of poly (I: C), cpG SL01 and CpG SL03 respectively. After freeze-drying, half of the nano vaccine 1 is not subjected to irradiation sterilization; the other half of the vaccine was terminally sterilized using alpha radiation for 48 hours (48 kGy).
(3) Nanometer vaccine for treating cancer
Preparation of lung cancer tumor-bearing mice by selecting 6-8 week female C57BL/6 as model mice, and subcutaneously inoculating 2.0X10 s on day 0 to the lower right back of each mouse 6 And LLC lung cancer cells. 100. Mu.L of 0.5mg nanovaccine 1 (irradiated or non-irradiated) or 100. Mu.L of PBS was injected subcutaneously in mice on day 3, 6, 9, 14, 19 and 25, respectively, after tumor inoculation. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.
(4) Experimental results
As shown in fig. 9, the PBS group mice rapidly grew in tumor volume and the mice died quickly. The survival time of the mice using the nano vaccine 1 (nano vaccine 1) is obviously prolonged, and the treatment effect of the irradiated nano vaccine 1 is further improved compared with that of the non-irradiated nano vaccine 1, and most of the mice are healed without tumor.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. A method of terminal sterilization of an antigen component-loaded cancer particle vaccine or antigen delivery particle, the terminal sterilization comprising: irradiating the packaged cancer particle vaccine or antigen delivery particles, wherein the irradiation is that X-rays, gamma rays, alpha rays, beta rays or 1-100MeV electron beams with the dosage of 12-60 kGy are used for treating for 1-48 hours; the cancer particle vaccine contains antigen delivery particles, wherein the antigen delivery particles are nano particles or micro particles;
the nanoparticle or microparticle has: (i) Nanoparticle and/or microparticle scaffold structures formed from the particle preparation material, (ii) whole cell fraction from cancer cells and/or tumor tissue lysate fraction and/or partial cell lysate fraction containing antigen fraction; wherein the whole cell fraction and/or the partial cell lysate fraction comprising the antigen fraction from the cancer cells and/or the tumor tissue is supported inside and/or on the surface of the scaffold structure.
2. Terminal sterilization method according to claim 1, characterized in that the specific steps of terminal sterilization are as follows:
s1, preparing antigen delivery particles, and re-suspending the antigen delivery particles in a buffer solution containing a freeze-drying protective agent to obtain suspension;
S2, packaging the suspension, freeze-drying the packaged product, and then carrying out irradiation treatment.
3. The terminal sterilization method according to claim 1, wherein the particle preparation material is selected from one or more of natural polymer materials, synthetic polymer materials, inorganic materials, biological materials of bacterial or viral origin.
4. A terminal sterilization process according to claim 3, characterized in that: the particle preparation material is polylactic acid-glycolic acid copolymer modified by polyethylene glycol and/or polylactic acid modified by polyethylene glycol.
5. The terminal sterilization method according to claim 1, wherein: the whole cell fraction from the cancer cell and/or tumor tissue lysate fraction is obtained by lysing the cancer cell and/or tumor tissue using a lysing solution containing a lysing agent in its entirety, or comprises a water-soluble fraction and a non-water-soluble fraction solubilized using a lysing solution containing a lysing agent; the antigen component contained in the partial cell lysate component comprises protein and polypeptide components of cancer cells and/or tumor tissue lysates and/or RNA components or mRNA components of cell lysates.
6. The terminal sterilization method according to claim 5, wherein the antigen delivery particles are prepared by the following method:
(1) Lysing cancer cells or tumor tissue using a lysing solution comprising a lysing agent, and then lysing the lysate component using a lysing solution comprising a lysing agent;
(2) Purifying or enhancing immunogenicity of lysate components dissolved in the dissolving solution, and then separating and purifying or enhancing immunogenicity components from the lysate by using the dissolving solution containing the dissolving agent for secondary dissolution;
(3) Directly loading the lysate component dissolved by the dissolution liquid obtained in the step (1) on antigen delivery particles; or the components of the lysate component dissolved by the dissolution liquid obtained in the step (2) are subjected to purification or immunogenicity enhancing treatment and then are loaded on antigen delivery particles;
wherein the dissolving agent is independently selected from one or more of a compound shown in a structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline; wherein, structural formula 1 is as follows:
R 1 c, S, P, N or O, R 2 ～R 5 Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino.
7. The terminal sterilization method according to claim 5, wherein the antigen delivery particles are prepared by the following method:
(1) Firstly, using ultrapure water or aqueous solution to lyse cancer cells or tumor tissues, then collecting lysate and centrifuging, wherein the supernatant liquid part is water-soluble components, and using a dissolving solution containing a dissolving agent to solubilize and dissolve the precipitated non-water-soluble components;
(2) Purifying or enhancing immunogenicity of the water-soluble component and/or the non-water-soluble component, and then secondarily dissolving the water-soluble component and/or the non-water-soluble component in a dissolving solution containing a dissolving agent to separate and purify or enhance the immunogenicity of the components from the dissolving solution;
(3) Then directly loading the water-soluble component and/or the non-water-soluble component obtained in the step (1) on antigen delivery particles respectively or simultaneously, or loading the components of which the water-soluble component and/or the non-water-soluble component are subjected to purification or immunogenicity enhancement treatment in the step (2) on the antigen delivery particles;
wherein the dissolving agent is independently selected from one or more of a compound shown in a structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline; wherein, structural formula 1 is as follows:
R 1 c, S, P, N or O, R 2 ～R 5 Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino.
8. Use of the terminal sterilization method according to any one of claims 1 to 7 or of the cancer particle vaccine or antigen delivery particles prepared by the terminal sterilization method for the preparation of a medicament for preventing or treating a tumor.
9. The use according to claim 8, characterized in that: the application is that cancer particle vaccine containing antigen delivery particles is injected into a body, or immune cells are activated in vitro by using the antigen delivery particles, and then the immune cells are infused back into the body.
10. The application according to claim 9, characterized in that the application comprises at least one of the following:
(1) Preparing antigen delivery particles for direct injection into the body for the prevention or treatment of disease;
(2) Preparing antigen delivery particles for in vitro activation of antigen presenting cells or preparation of antigen presenting cell vaccines;
(3) Preparing antigen delivery particles for assisting in activating antigen-specific T cells to detect the content of the antigen-specific T cells;
(4) After preparing antigen delivery particles for helper activation of antigen-specific T cells, isolating and expanding activated antigen-specific T cells for use in preventing or treating a disease;
wherein the disease is cancer or tumor;
wherein the cancer or tumor is a solid tumor or hematological tumor.
CN202311744495.4A 2023-12-18 2023-12-18 Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method Pending CN117618602A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311744495.4A CN117618602A (en) 2023-12-18 2023-12-18 Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311744495.4A CN117618602A (en) 2023-12-18 2023-12-18 Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method

Publications (1)

Publication Number Publication Date
CN117618602A true CN117618602A (en) 2024-03-01

Family

ID=90027007

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311744495.4A Pending CN117618602A (en) 2023-12-18 2023-12-18 Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method

Country Status (1)

Country Link
CN (1) CN117618602A (en)

Similar Documents

Publication Publication Date Title
US20230330199A1 (en) Targeting delivery system loaded with whole-cell components and use thereof
WO2021189678A1 (en) Whole-cell constituent transport system and application thereof
WO2023087441A1 (en) Vaccine system for preventing or treating cancer and use thereof
WO2023115676A1 (en) Dendritic cell cancer vaccine and use thereof
CN114288397B (en) Vaccine system for preventing or treating cancer based on whole cell components of various cancer cells and/or tumor tissues, preparation and application thereof
CN114225021A (en) Vaccine system for preventing or treating cancer based on one or more cancer cell and/or tumor tissue whole cell components or mixture thereof
CN114288398B (en) Application of cancer vaccine system based on whole cell component in preparation of medicine for cross prevention or treatment of heterogeneous cancer
CN113181354B (en) Foot-and-mouth disease bionic nanometer vaccine based on dendritic cells, and preparation method and application thereof
WO2023236330A1 (en) Cancer vaccine based on antigen-presenting cell membrane components, method for preparing same, and use thereof
WO2024000724A1 (en) Preparation method for vaccine loaded with cancer cell whole-cell component and mixed membrane component and use thereof
WO2023236331A1 (en) Method for preparing vaccine of autoimmune disease derived from pre-activated antigen-presenting cell and use thereof
CN116731964A (en) Specific T cell for preventing or treating cancer and preparation method thereof
CN117618602A (en) Terminal sterilization method of cancer particle vaccine loaded with cancer cell lysate component and application of terminal sterilization method
WO2023082454A1 (en) Vaccine system for preventing or treating diseases on basis of whole-cell components of one or more bacteria, and preparation method therefor and use thereof
CN117122678A (en) Method for preparing antigen delivery particles
CN117653720A (en) Extraction method of antigen component, delivery particle containing antigen component and application of delivery particle
CN117618382A (en) Preparation method and application of antigen delivery particles
WO2023201786A1 (en) Immunological adjuvant composition and cancer vaccine based on composition and application thereof
CN116942802A (en) Cancer vaccine based on cancer cell and/or tumor tissue lysate component and synthetic cancer antigen and preparation method thereof
CN116549620A (en) Cancer vaccine based on part of components in cancer cells or tumor tissues and preparation method thereof
WO2024016688A1 (en) Nucleic acid delivery particle based on activated antigen-presenting cells, nucleic acid delivery system and preparation method
CN116850275A (en) Mixed cell cancer vaccine containing dendritic cells and B cells activated in vitro and application thereof
WO2023201787A1 (en) Cancer-specific t cell-based cell system, lymphocyte drug and use thereof
WO2023206684A1 (en) Cell system and use thereof, and method for activating broad-spectrum cancer cell-specific t cells
WO2024000725A1 (en) Cancer cell-specific t cell vaccine and method for activating cancer cell-specific t cells

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination