CN117442710A - Tumor vaccine based on enucleated melanoma cells and preparation method and application thereof - Google Patents
Tumor vaccine based on enucleated melanoma cells and preparation method and application thereof Download PDFInfo
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- CN117442710A CN117442710A CN202311535752.3A CN202311535752A CN117442710A CN 117442710 A CN117442710 A CN 117442710A CN 202311535752 A CN202311535752 A CN 202311535752A CN 117442710 A CN117442710 A CN 117442710A
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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Abstract
The invention relates to the field of biological medicine, and discloses a tumor vaccine based on enucleated melanoma cells, and a preparation method and application thereof, wherein the tumor vaccine comprises the following components: removing a cell nucleus from a melanoma cell and a bacterial outer membrane vesicle coupled to the cell nucleus-removed melanoma cell; wherein the bacterial outer membrane vesicles are loaded with STING agonists. The tumor vaccine has the advantages of enhancing the immunogenicity of the whole cell tumor vaccine, improving the bioavailability of STING agonists, recruiting and activating antigen presenting cells, stimulating macrophages to convert into M1 type macrophages, reducing the weight of the tumor and the like, has remarkable effect of promoting the activation and proliferation of specific T cells in the tumor, and can be used for preventing and treating melanoma.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a tumor vaccine based on enucleated melanoma cells, and a preparation method and application thereof.
Background
The tumor vaccines such as cell vaccines, nucleic acid vaccines, protein polypeptide vaccines, genetic engineering vaccines and the like are widely studied clinically, and the tumor vaccines achieve great achievement in cancer treatment, in particular to tumor whole cell vaccines which bring great attention in preclinical researches of solid tumors. The tumor whole cell vaccine refers to a vaccine which changes or eliminates the tumorigenicity of the tumor whole cell or the tumor whole cell by treating the tumor whole cell or the tumor whole cell of a foreign body through physical factors, chemical factors and biological factors (virus infection, gene transfer and the like), and is often used in combination with an adjuvant and the like. The cell can express various tumor antigens, has no need of antigen identification, does not need to consider MHC individual difference, and can activate CD8 at the same time + T cells and CD4 + T helper cells produce a stronger overall anti-tumor response effect while reducing the chance of tumor escape.
In the related art, although the tumor whole cell vaccine has a plurality of advantages compared with other vaccines, the tumor whole cell belongs to tumor cells, has strong tumorigenicity, can secrete some soluble factors to inhibit the functions of dendritic cells and T cells, and has the defects of weak immunogenicity of expressed antigen, incapacity of inducing strong specific immune response and the like. There are certain limitations.
Based on this, the present invention proposes a tumor vaccine based on enucleated melanoma cells to solve the above-mentioned problems.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a tumor vaccine based on enucleated melanoma cells, which can effectively enhance the immunogenicity of the whole-cell tumor vaccine, improve the bioavailability of STING agonists, recruit and activate antigen presenting cells, stimulate macrophages to convert into M1 type macrophages, promote the activation and proliferation of specific T cells in tumors, and finally achieve the aim of inhibiting the growth of melanoma.
The invention also provides a preparation method of the tumor vaccine based on the enucleated melanoma cells.
The invention also provides application of the tumor vaccine based on the enucleated melanoma cells in preparation of a melanoma treatment and/or prevention product.
In a first aspect of the invention, there is provided a tumor vaccine based on enucleated melanoma cells, the tumor vaccine comprising:
a1 Removing the melanoma cells of the nucleus; the method comprises the steps of,
a2 A bacterial outer membrane vesicle coupled to the enucleated melanoma cells;
wherein the bacterial outer membrane vesicles are loaded with STING agonists.
The tumor vaccine according to the embodiment of the invention has at least the following beneficial effects:
(1) According to the invention, the bacterial outer membrane vesicle loaded with the STING agonist 2'3' -cGAMP is connected to the enucleated melanoma cells, and the good immunoadjuvant and biocompatibility characteristics of the bacterial outer membrane vesicle are utilized, so that the immunogenicity of the whole cell tumor vaccine is enhanced, the bioavailability of the STING agonist is improved, antigen presenting cells are recruited and activated, macrophages are stimulated to be converted into M1 type macrophages, activation and proliferation of specific T cells in tumors are promoted, and finally the purpose of inhibiting the growth of the melanoma is achieved.
(2) Compared with the single STING agonist or the single Enucleated cell, the preimmunization with the Enucleated tumor cell vaccine Envolbed B16F10-BDVs-cGAMP designed by the invention can protect healthy mice from being attacked by melanoma cells to a great extent and effectively increase CD44 + CD62L - Infiltration of effector memory T cells into tumors produces effective anticancer efficacy and long-term memory immunity.
(3) The invention adopts the enucleated melanoma cells, has the basic function of maintaining important cell organelles of cells relative to the conventional inactivation treatment of the melanoma cells, and has the advantages of retaining tumor antigens to the greatest extent, maintaining the immunogenicity of the tumor cells, and the like.
In some embodiments of the invention, the melanoma cells are selected from human melanoma cells or mouse melanoma cells.
In some embodiments of the invention, the human melanoma cells comprise at least one of RPMI-7951, meWo, hs 688 (a). T, COLO829, C32, a-375, hs 294T, hs 695T, hs 852T, A2058 cells.
In some embodiments of the invention, the mouse melanoma cells comprise B16F10-luc cells.
In some embodiments of the invention, the bacterial outer membrane vesicles are prepared from prokaryotic cells.
In some preferred embodiments of the invention, the bacterial outer membrane vesicles are prepared from E.coli.
In some preferred embodiments of the invention, the E.coli BL21 (DE 3) plysS.
In some embodiments of the invention, the escherichia coli is recombinant e.coli BL21 (DE 3) plysS.
In some embodiments of the invention, the bacterial outer membrane vesicles are obtained by expression induction and purification of escherichia coli recombinant expression vectors.
Bacterial outer membrane vesicles (Bacteria derived vesicles, hereinafter BDVs) are membranous vesicles comprising a variety of phospholipid, lipopolysaccharide, protein, etc., which when contacted with a pathogen-associated pattern molecule (Pathogen associated molecular pattern, PAMP) contained in BDVs, with a dendritic cell membrane surface pattern recognition receptor (Pattern recognition receptor, PRR) in a host, will initiate signal transduction pathways involved in immunomodulation, which may alter cytokine release, enhance expression of co-stimulatory molecules (e.g., CD40 and CD 80) while increasing DC adhesion upregulation to integrins (e.g., CD11 c) upon antigen presentation, activating the innate and adaptive immune systems. In addition, virulence factors in BDVs can be transmitted to host cells, stimulate bacterial-host cell interactions, and have inherent antitumor activity.
BDVs have significant advantages as drug carriers in tumor treatment: on the one hand, BDVs, as nano-scale vesicles, are able to maintain their ability to accumulate in tumors by enhancing the osmotic and retention effects; on the other hand, BDVs can be modified by genetic engineering and other means to have tumor-specific targeting. In addition, BDVs are immunogenic, and tumor antigens carried by BDVs further stimulate the organism to generate immune response, so that the problem of insufficient immunogenicity of the enucleated melanoma cells can be overcome, and the BDVs and the tumor antigens have synergistic effect.
In some embodiments of the invention, the recombinant expression vector expresses an Lpp protein and an OmpA protein.
Lpp is one of the major components of the outer membrane structure of bacteria, which can enhance the evasion ability of bacteria to the host immune system; ompA is an outer membrane protein that is involved in the adhesion and invasion process of bacteria.
In some embodiments of the invention, the recombinant expression vector expresses an Lpp-OmpA-GFP protein.
In some embodiments of the invention, the encoding nucleotide sequence of the Lpp-OmpA-GFP protein is shown in SEQ ID NO. 1.
In some embodiments of the invention, the bacterial outer membrane vesicle further comprises an Lpp protein and an OmpA protein.
In some embodiments of the invention, the STING agonist comprises cGAMP or a cGAMP derivative.
The cyclic dinucleotide (Cyclic guanosine monophosphate-adenosine monophosphate, hereinafter referred to as cGAMP) is used as a common STING agonist, can be used as a vaccine adjuvant to strengthen the effect of tumor vaccines and inhibit the growth of various tumor cells, but most STING agonists can only be injected in tumors due to poor cell membrane permeability and high toxicity, and cannot be administered systemically, so that the treatment effect of STING is limited to a great extent. The invention uses the bacterial outer membrane vesicle which can maintain the accumulation capacity of the bacterial outer membrane vesicle in the tumor by enhancing the permeation and retention effects as a carrier, thereby effectively improving the utilization effect of the STING agonist.
In some embodiments of the invention, the tumor vaccine further comprises a PD-1 antibody.
In some embodiments of the invention, the function of the tumor vaccine comprises any of B1) to B6):
b1 Activating STING pathway of antigen presenting cells;
b2 Promoting maturation and activation of dendritic cells;
b3 Inducing macrophage polarization;
b4 Increasing IFN-gamma in tumor tissue + CD8 + T cells, gzmB + CD8 + T cells, CD69 + CD8 + A number of at least one T cell in the T cells;
b5 Increasing CD69 in tumor tissue + CD8 + T cells, CD44 + CD69 + CD8 + T cells, CD44 + CD62L-CD8 + The expression proportion of at least one T cell in the T cells;
b6 Reducing melanoma volume and/or weight.
In a second aspect of the present invention, there is provided a method for preparing the tumor vaccine according to the first aspect, comprising the steps of:
s1, preparing cell nucleus-removed melanoma cells, constructing recombinant bacteria with STING agonist synthesis capacity, and extracting bacterial outer membrane vesicles;
s2, mixing the bacterial outer membrane vesicle with a dibenzocyclooctyne coupling agent for reaction to obtain a dibenzocyclooctyne coupling modified bacterial outer membrane vesicle;
s3, mixing azido sugar analogues with the cell nucleus-removed melanoma cells to obtain azide-modified enucleated melanoma cells, and then mixing the dibenzocyclooctyne-coupled modified bacterial outer membrane vesicles with the azide-modified enucleated melanoma cells to react to obtain the product.
The preparation method of the tumor vaccine provided by the embodiment of the invention has at least the following beneficial effects:
the tumor vaccine preparation is based on Click chemistry reaction, and the Enucleated melanoma cells are connected with the bacterial adventitia vesicles by using azido-alkyne cycloaddition reaction, so that the prepared tumor vaccine (Enucleated B16F 10-BDVs-cGAMP) for Enucleated melanoma cells has the advantage of good stability, and is beneficial to improving the treatment effect of the tumor vaccine.
In some embodiments of the invention, in S1, the method of preparing the enucleated melanoma cells comprises density gradient centrifugation.
In some embodiments of the invention, in S2, the dibenzocyclooctyne coupling agent comprises dibenzocyclooctyne-PEG 4 -N-hydroxysuccinimide ester (DBCO-PEG) 4- NHS Ester)。
In some embodiments of the invention, in S3, the azido sugar analog comprises tetrA-Acylated N-azidoacetate galactosamine.
In some embodiments of the invention, in S3, the temperature of the reaction is 15 to 37 ℃.
In some embodiments of the invention, in S3, the reaction time is 1 to 40 hours.
In a third aspect, the invention provides the use of the tumor vaccine based on enucleated melanoma cells according to the first aspect for the preparation of a product for the treatment and/or prevention of melanoma.
In some embodiments of the invention, the function of the product comprises any of B1) to B6):
b1 Activating STING pathway of antigen presenting cells;
b2 Promoting maturation and activation of dendritic cells;
b3 Inducing macrophage polarization;
b4 Increasing IFN-gamma in tumor tissue + CD8 + T cells, gzmB + CD8 + T cells, CD69 + CD8 + A number of at least one T cell in the T cells;
b5 Increasing CD69 in tumor tissue + CD8 + T cells, CD44 + CD69 + CD8 + T cells, CD44 + CD62L - CD8 + The expression proportion of at least one T cell in the T cells;
b6 Reducing melanoma volume and/or weight.
In some embodiments of the invention, the promoting dendritic cell maturation activation comprises promoting bone marrow derived dendritic cell maturation activation.
In some preferred embodiments of the invention, the promoting mature activation of bone marrow-derived dendritic cells comprises increasing CD40 + CD11c + Expression level of the cells.
In some embodiments of the invention, activating the STING pathway of the antigen presenting cell comprises increasing the expression levels of p-IRF3 and p-TBK1 proteins.
In some embodiments of the invention, the inducing macrophage polarization comprises inducing macrophage M1 polarization; preferably, the macrophages comprise bone marrow derived macrophages.
In some embodiments of the invention, the product comprises a pharmaceutical product.
In some embodiments of the invention, the pharmaceutical product further comprises a pharmaceutically acceptable excipient.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 shows the results of validation of enucleated B16F10 cells of the present invention, wherein a is the distribution of cellular components after density gradient centrifugation. B is the staining of cell membranes (WGA AF647, red) and nuclei (HoechsT 33342, blue) of B16F10 cells before and after enucleation, scale: 10 μm; c is the transmission electron microscope detection of B16F10 cells before and after enucleation, and the scale bar: 1 μm.
FIG. 2 shows the characterization results of GFP-BDVs and Enuceated B16F10-BDVs of the invention, where a is the fluorescent observation of E.coli GFP, scale bar: 2 μm; b is immunoblotting detection of GFP protein; c is a representative transmission electron microscope image of GFP-BDVs, scale bar: 200nm; confocal fluorescence detection of GFP-BDVs, scale bar: 5 μm; e is BDVszeta potential detection (n=3); f is the particle size distribution of GFP-BDVs; g is the confocal observations of different concentrations of GFP-BDVs bound to the Enucleated B16F10 cells, scale bar: 5 μm; h is Ac 4 GalNAz modified Envolved B16F10 cells and DBCO pretreated GFP-BDVs were assembled by click reaction to create schematic representation of Enucleated B16F10-BDVs. i is the scanning electron microscope image of the Enucleated B16F10-BDVs, the BDVs coupled to the Enucleated B16F10 cells are represented in the right magnified image by a green pseudo-color, scale bar: 200nm (magnified) and 1 μm; j is the validation of binding of the Enucleated B16F10 cells to GFP-BDVs using flow cytometry.
FIG. 3 is a graph showing the statistics of the efficiency of transfer and release of BDVs-cGAMP of the present invention, wherein a is the efficiency of BDVs loading with STING agonist and b is the efficiency of BDVs-cGAMP release.
FIG. 4 shows the result of the WB detection of the STING signal pathway of bone marrow-derived cells according to the present invention, wherein a is the result of the WB detection of the STING signal pathway of bone marrow-derived dendritic cells BMDC, and b is the result of the WB detection of the STING signal pathway of bone marrow-derived macrophages BMDM.
FIG. 5 shows the results of flow assays of bone marrow derived dendritic cell BMDC and bone marrow derived macrophage BMDM according to the present invention, wherein a is the activation of flow assay BMDC (n=4); b and c are the M1 (b) and M2 (c) polarizations (n=4) of the flow detection BMDM; d is PCR to detect the expression of BMDM polarized gene (n=3), E-B16-BDVs is the engeled B16F10-BDVs, E-B16-BDVs-cGAMP is the engeled B16F10-BDVs-cGAMP, ＊ P<0.05， ＊＊ P<0.01， ＊＊＊ P<0.001， ＊＊＊＊ P<0.0001。
fig. 6 shows the results of tumor growth curves, tumor weights and survival curves of melanoma mice from different treatment groups according to the invention, wherein a is the tumor growth curve of melanoma mice from different treatment groups (n=7); b is the average tumor growth curve (n=7) for each group of tumor-bearing mice; c is the tumor weight (n=4) of melanoma mice from different treatment groups; d is the survival curve (n=10) for each group of tumor-bearing mice; PBS (G1), cGAMP (G2), envolved B16F10 (G3), envolved B16F10-BDVs (G4), aPD-1 (G5), envolved B16F10-BDVs-cGAMP (G6), envolved B16F10-BDVs-cGAMP+aPD-1(G7)， ＊ P<0.05， ＊＊ P<0.01， ＊＊＊ P<0.001， ＊＊＊＊ P<0.0001。
FIG. 7 shows the results of T cell flow analysis of melanoma in mice of different treatment groups according to the invention, wherein a is CD8 of melanoma in mice of different treatment groups + T cell detection results, b is CD69 of melanoma of mice in different treatment groups + CD8 + T cell detection result, c is GzmB of melanoma of mice in different treatment groups + CD8 + T cell detection result, d is IFN-gamma of melanoma of mice in different treatment groups + CD8 + The results of the T cell assay were obtained, ＊ P<0.05， ＊＊ P<0.01， ＊＊＊ P<0.001， ＊＊＊＊ P<0.0001。
fig. 8 shows the results of T cell flow analysis of the growth curves, weight changes, and mouse melanoma tissues of different treatment groups according to the invention, where a is the tumor-free growth curve of the mice of the different treatment groups (n=7); b is the average tumor growth curve (n=7) for each group of tumor-bearing mice; c is the tumor weight (n=4) of melanoma mice in the different treatment groups, d is CD8 of melanoma tissue in mice in the different treatment groups + T cell assay (n=4), e is CD44 of melanoma tissue from mice of different treatment groups + CD69 + CD8 + T cell assay (n=4), f is CD44 of melanoma tissue from mice of different treatment groups + CD62L - CD8 + T cell detection results (n=4), ＊ P<0.05， ＊＊ P<0.01， ＊＊＊ P<0.001， ＊＊＊＊ P<0.0001。
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The words "preferably," "more preferably," and the like in the present invention refer to embodiments of the invention that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
In the embodiments of the present invention, the Enucleated B16F10 cells, enucleated melanoma cells, and Enucleated B16F10 refer to the melanoma cells after enucleation.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: preparation of tumor vaccine based on enucleated melanoma cells
1. Preparation and characterization of enucleated melanoma cells
1. Preparation of enucleated melanoma cells
Ficoll powder (BioFrox, 1345GR 100) was first prepared with PBS (pH 7.2-7.4) to a 25% Ficoll isolate, and then the 25% Ficoll isolate was diluted to 17%, 16%, 15%, 12.5% solutions, respectively, and 10. Mu.g/mL relaxin B was added.
Ficoll solutions of different concentrations (3 mL 25%, 3mL 17%, 0.5mL 16%, 0.5mL 15% and 2mL 12.5%) were carefully added sequentially to the ultracentrifuge tube. The collected B16F10-luc cells (2X 10) 7 individual/mL) was resuspended in 12.5% Ficoll separation with relaxin B, after incubation at 37 ℃ for 1h, carefully added to the prepared density gradient, the cells were centrifuged at 26000rpm for 70min in an ultracentrifuge preheated at 31 ℃, the centrifuge set to minimum acceleration, and the centrifuge turned off.
After centrifugation, cells at 17% -25% of the separation layer were collected (as shown in fig. 1 a), resuspended in PBS and washed 3 times, and finally Enucleated melanoma cells (Enucleated B16F 10) were obtained.
2. Characterization of enucleated melanoma cells
And respectively plating the melanoma cells (B16F 10) and the Enucleated melanoma cells (Enucleated B16F 10) on a laser confocal culture dish, then respectively adding WGA-Alexa-flow 647 and Hoechst33342 dye solution, respectively dyeing cell membranes and cell nuclei according to the proportion of 1:1000, incubating for 10min at room temperature and in a dark place, washing with PBS for three times, each time for 5min, and finally performing photographing detection under a laser confocal microscope. The results are shown in fig. 1 b, where red is the cell membrane and blue is the nucleus.
Further, the morphology of the melanoma cells B16F10 before and after enucleation was examined by a transmission electron microscope, and the results are shown as c in FIG. 1, which shows that the enucleated melanoma cells were successfully prepared according to the present invention.
2. Preparation and characterization of bacterial outer membrane vesicles
1. Plasmid construction
The expression vector pET21a (+) containing the gene coding for Lpp-OmpA-GFP protein is synthesized by Beijing England flourishing industry biosciences, wherein the gene sequence information of the gene coding for Lpp-OmpA-GFP protein is shown as SEQ ID NO. 1:
ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCCAGCAACGCTAAAATCGATCAGGGTATCAACCCGTATGTTGGCTTTGAAATGGGTTACGACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAAGCTCAGGGCGTTCAACTGACCGCTAAACTGGGTTACCCAATCACTGACGACCTGGACATCTACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAACGTTTATGGTAAAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGATCACTCCTGAAATCGCTACCCGTCTGGAATACCAGTGGACCAACAACATCGGTGACGCACACACCATCGGCACTCGTCCGGACAACGGTATCCCTGGTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG(SEQ ID NO.1)。
2. plasmid amplification
The expression vector pET21a (+) constructed as described above and containing the protein encoding Lpp-OmpA-GFP was transferred into E.coli BL21 (DE 3) plysS competent cells (TransGen Biotech, CD 701-02) as follows:
(1) E.coli BL21 (DE 3) plysS competent cells were thawed on ice, 500ng of pET21a (+) containing the expression vector encoding Lpp-OmpA-GFP protein was added under the condition of a super clean bench, mixed gently and allowed to stand on ice for 30min.
(2) Heat shock is carried out in a water bath at 42 ℃ for 50s, and ice bath is carried out for 3min.
(3) 200. Mu.L of LB medium was added to the super clean bench, and incubated for 1.5h at 37℃and 220rpm with a shaker.
(4) 200. Mu.L of the culture was pipetted onto an Amp plate medium and incubated in a bacterial incubator for 12h.
(5) Single colonies on the plate medium were picked up to a LB medium shaking tube containing 60. Mu.g/mL ampicillin, and shake cultured at 37℃and 220rpm for 12 hours.
(6) 200. Mu.L of bacterial liquid was aspirated per tube, and base sequencing was performed by the Optimus of Prinsepia. The residual bacterial liquid is temporarily stored in a refrigerator at 4 ℃. And (3) screening and storing the monoclonal strain with the sequencing result consistent with the base sequence of the designed sequence, namely, a recombinant strain E.coli BL21 (DE 3) plysS. Meanwhile, the bacterial liquid with the correct sequencing result is extracted by using a smallly extracted plasmid kit with high purity of smallness root, and the steps are carried out according to the specification. The extracted plasmid was measured for concentration and purity, requiring OD260/OD280>1.8, OD260/OD230>2. After the detection, the plasmid is stored at-20 ℃ for standby.
3. Induction expression of GFP
Recombinant strain e.coli BL21 (DE 3) plysS was shake cultured at 220rpm at 37 ℃ to an od=about 0.6. Then 1mM isopropyl beta-d-1-thiogalactopyranoside (IPTG) was added thereto, and the mixture was shake-cultured at 16℃and 220rpm for 6 hours to induce GFP expression. After induction, recombinant strains stably expressing GFP were collected by centrifugation at 5000 Xg for 10 minutes.
The GFP expression condition of the recombinant strain E.coli BL21 (DE 3) plysS is detected by a laser confocal microscope, and the specific method is as follows: 10. Mu.L of diluted bacteria were dropped onto a slide glass, then fixed with 4% paraformaldehyde at room temperature for 15min, then washed once with PBS, 1mL of DAPI dye solution of 1. Mu.g/mL was added, incubated at room temperature for 10min in the absence of light, and washed three times with PBS for 5min each. Finally, 20 mu L of anti-fluorescence quenching sealing tablet is added, and the mixture is taken into a machine for shooting.
The results are shown as a in FIG. 2, which shows that the present invention successfully constructs recombinant cells expressing Lpp-OmpA-GFP protein.
4. Preparation of bacterial outer membrane vesicles (BDVs)
(1) The above-collected recombinant cells stably expressing GFP were resuspended in pre-chilled PBS and washed 3 times, and then the cells were resuspended in cold HM lysis buffer (0.25M sucrose, 1mM EDTA, 20mM HEPES-NaOH, and 1.0mM protease inhibitor cocktail).
(2) The cell resuspension was transferred to a glass homogenizer, manually homogenized 500 times, after which the homogenate was collected and centrifuged at 1000g for 5 minutes, the supernatant was removed and the pellet was removed.
(3) The supernatant was centrifuged at 3000g for 5 minutes, the supernatant was taken, and then the supernatant was centrifuged at 15000g for 30 minutes to collect the pellet, resulting in bacterial outer membrane vesicles (BDVs) pellet.
(4) The BDVs pellet was resuspended in cold HM buffer and passed through 0.8 μm and 0.22 μm filters sequentially at least 10 times to obtain bacterial outer membrane vesicles (BDVs), -stored at 80 ℃.
5. Characterization of bacterial outer membrane vesicles
(1) Western Blot verification of GFP protein expression
SDS-PAGE gels were first prepared at a concentration of 10% and run at a voltage of 70V at 20. Mu.L per well. Then, the PVDF membrane is activated by methanol in advance, and a transfer membrane sandwich is manufactured according to the blackboard-sponge-filter paper-glue-PVDF membrane-filter paper-sponge-whiteboard. The film transfer tank was placed in ice, film transfer was performed at constant current, 250mA, and 2h. Then, 5% skimmed milk powder was prepared, and the PVDF membrane was sealed at room temperature for 1h. Cutting membranes, incubating EGFP antibody and betA-Actin antibody respectively, and shaking overnight at 4 ℃ at a slow speed. The membranes were washed three times with TBST on a shaker for 10min each. The secondary antibody was incubated at room temperature for 1h. The membranes were then washed three times with TBST for 10min each on a shaker. Finally, according to the following steps of 1:1 proportion of ECL luminous solution, and machine exposure.
The results of the Western Blot verification of GFP protein expression are shown in b in fig. 2, which shows that GFP protein expression was detected in both recombinant strain e.coli BL21 (DE 3) plysS and bacterial outer membrane vesicles (BDVs).
(2) Observation by transmission electron microscope
The bacterial outer membrane vesicles (BDVs) prepared above were observed by transmission electron microscopy as follows:
after the copper net is clamped by tweezers, the dovetail clamp is added for fixation, so that the copper net is prevented from falling off. The forceps were fixed on ice with an adhesive tape, 10. Mu.L of a cell membrane vesicle solution was dropped, left stand for 5min, the liquid was sucked from the edge of the copper mesh with filter paper, the dropping was repeated six times, then 10. Mu.L of 3% uranium acetate was dropped for 5min, and the liquid was sucked from the edge with filter paper. Finally, airing the copper net at room temperature, and observing the appearance of the vesicle and photographing by using a 120kV transmission electron microscope. In the experimental process, gun heads, filter papers and the like related to uranium acetate are treated according to safety standards.
The results are shown in FIG. 2, c, which shows that the produced bacterial outer membrane vesicles have a bilayer membrane structure.
(3) Fluorescent observation of BDVs
The bacterial outer membrane vesicles (BDVs) obtained were added dropwise to 10. Mu.L on a slide, covered with a cover slip, sealed with nail polish at four sides, and dried in the dark at room temperature. The vesicles were then observed for fluorescence under a confocal laser microscope.
The results are shown in fig. 2 d, which shows that the obtained bacterial outer membrane vesicles contain green fluorescent signals, indicating that the present invention successfully produced bacterial outer membrane vesicles.
(4) Zeta potential and particle size distribution detection of BDVs
The Zeta potential and particle size of the outer membrane vesicles of the bacteria were measured by a nanoparticle size and Zeta analyzer, and the results are shown as e and f in FIG. 2, wherein the potential of the outer membrane vesicles of the bacteria is-10.7 mV, and the particle size is about 120 nm.
3. Ligation and characterization of bacterial outer membrane vesicles and enucleated cells
(1) Ligation and characterization of BDVs with enucleated cells
First at a temperature of 1X 10 6 40. Mu.M tetrA-Acylated N-azidoacetylgalactosamine (Ac) was added to the medium of individual/mL enucleated B16F10 cells 4 GalNAz, purchased at Thermo Fisher Scientific, cat No. 88905), for 72h. Next 20. Mu.M dibenzocyclooctyne-PEG was used 4 -N-hydroxysuccinimide ester (DBCO-PEG) 4 -NHS Ester) treated at room temperature for 50. Mu.g BDVs 2h. DBCO-PEG is then added 4 The NHS ester treated BDVs were added to the azide-modified Enucleated B16F10 cells and incubated at room temperature for 2h to produce click chemistry, resulting in the encumbered B16F10-BDVs. Enuceated B16F10-BDVs were washed with PBS and centrifuged at 500gFor 5 minutes, the connection was observed with a confocal microscope and verified by scanning electron microscopy and flow technique.
The results are shown in FIG. 2 at g-j, where g is the confocal observation of different concentrations of GFP-BDVs bound to the Enuceated B16F10 cells, scale bar 5 μm; h is Ac 4 GalNAz modified Envolved B16F10 cells and DBCO pretreated GFP-BDVs were assembled into schematic representations of Enucleated B16F10-BDVs by click reactions; i is a scanning electron microscope image of an encumbered B16F10-BDVs, the BDVs coupled with encumbered B16F10 cells are represented by a green pseudo color in the magnified image on the right side, and the scales are 200nm (magnified) and 1 μm; j is the validation of binding of the Enucleated B16F10 cells to GFP-BDVs using flow cytometry.
(2) Ligation and characterization of BDVs-cGAMP with enucleated cells
500. Mu.g of the BDVs prepared above were first mixed with 100. Mu.g of 2'3' -cGAMP (Invivogen, tlrl-nacga 23) and incubated at 37℃for 2 hours to give BDVs-cGAMP.
To verify whether 2'3' -cGAMP (invitrogen, tlrl-nacga 23) was loaded onto BDVs and the release of 2'3' -cGAMP, loaded BDVs-cGAMP was cleaved with Triton-x100 and drug uv absorbance light detected at 254nm by a microplate reader to calculate the loaded drug content. The detection result is shown as a in fig. 3, and the maximum drug loading efficiency can reach 8.61%. The prepared BDVs-cGAMP were then placed in a 37℃water bath to detect drug release. As a result, as shown in FIG. 3 b, 2'3' -cGAMP in the outer membrane vesicles was substantially released at 48h with a release rate of about 67.44%. The above results demonstrate that the present invention successfully produces cGAMP-loaded BDVs (i.e., BDVs-cGAMP).
Then at the position containing 1X 10 6 40. Mu.M tetrA-Acylated N-azidoacetylgalactosamine (Ac) was added to the medium of individual/mL enucleated B16F10 cells 4 GalNAz, purchased at Thermo Fisher Scientific, cat No. 88905), for 72h. Next 20. Mu.M dibenzocyclooctyne-PEG was used 4 -N-hydroxysuccinimide ester (DBCO-PEG) 4 -NHS Ester) at room temperature 50. Mu.g BDVs-cGAMP 2h. Finally DBCO-PEG 4 BDVs addition with NHS ester treatmentThe azide-modified Enucleated B16F10 cells were incubated at room temperature for 2h to generate click chemistry to give the Enucleated B16F10-BDVs-cGAMP.
Taken together, the results show that the method of the present invention successfully couples the encumbered B16F10 with BDVs or BDVs-cGAMP to produce encumbered B16F10-BDVs and encumbered B16F10-BDVs-cGAMP.
Example 2: application of tumor vaccine in STING channel for activating antigen presenting cells
1. Isolation and culture of bone marrow derived dendritic cells and bone marrow derived macrophages
The isolation and culture of bone marrow derived dendritic cells (BMDCs) and Bone Marrow Derived Macrophages (BMDMs) of this example specifically includes the following steps:
(1) C57BL/6 mice (6 weeks old) were sacrificed by cervical dislocation, all femur and tibia were removed and the surrounding musculature was removed with scissors.
(2) The bones were transferred into a super clean bench, soaked in 70% alcohol for 5min for sterilization, and then washed with sterile PBS for 2 times.
(3) The bone was transferred to another new dish containing PBS, the two ends of the bone were cut off, and the needle of the syringe containing PBS was inserted into the bone marrow cavity and repeatedly washed out of the bone marrow until the bone was completely whitened. The bone marrow suspension was transferred to a centrifuge tube and small fragments and muscle tissue were filtered off with a 200 mesh nylon mesh.
(4) Centrifuge at 1200rpm for 5 minutes and discard the supernatant. The harvested cells were resuspended in erythrocyte lysis buffer and incubated for 10 minutes at room temperature to eliminate erythrocytes. Adjusting the cell concentration to 1×10 6 Each mL was added to a 6-well plate and cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, 10ng/mL IL-4 and 20ng/mL GM-CSF, after 24 hours all floating cells were removed and the culture was continued with addition of fresh medium.
(5) Half of the medium was changed every 2 days, and cells that were not adherent and half adherent were collected at day 8 to obtain BMDCs. For BMDMs, cultures were performed with DMEM complete medium supplemented with 10ng/mL M-CSF, and finally the fully adherent cells were collected for subsequent experiments.
2. The STING pathway of the antigen presenting cells is activated by the enclocked B16F10-BDVs-cGAMP
BMDCs or BMDMs were plated in 6-well plates (1X 10) 6 Number/well) and placing BMDCs or BMDMs in the lower layer of a 3 μm transwell cell, and adding PBS, cGAMP (10 μg), and Envolved B16F10-BDVs-cGAMP, respectively, in the upper layer of the cell, wherein Envolved B16F10 and BMDCs or BMDMs are in accordance with 1:1, and the amount of BDVs added was 50. Mu.g and the amount of cGAMP was 10. Mu.g. After 24 hours incubation, the cell-under-chamber cells were collected for subsequent detection of WB STING pathway protein activation (p-IRF 3, p-TBK 1).
The detection results are shown in fig. 4 a and b, where a is the STING signal pathway WB detection result of bone marrow derived dendritic cell BMDC and b is the STING signal pathway WB detection result of bone marrow derived macrophage BMDM. The results show that BMDCs and BMDMs STING pathway proteins (p-IRF 3, p-TBK 1) are significantly elevated after the treatment with the encumbered B16F10-BDVs-cGAMP, i.e., the encumbered B16F10-BDVs-cGAMP is capable of activating the STING pathway of antigen presenting cells.
Example 3: application of tumor vaccine in promoting maturation and activation of DCs and inducing polarization of macrophages
After BMDCs or BMDMs were extracted by the method of example 2, the BMDCs or BMDMs were treated with PBS, cGAMP (10. Mu.g), and Enuced B16F10-BDVs-cGAMP, respectively, in an amount of 50. Mu.g. Marker expression was then detected using flow cytometry and PCR, respectively.
The detection results are shown in fig. 5, where a is the activation of the flow detection BMDC (n=4); b and c are the M1 (b) and M2 (c) polarizations (n=4) of the flow detection BMDM; d is PCR to detect the expression of BMDM polarized gene (n=3), E-B16-BDVs is the engeled B16F10-BDVs, E-B16-BDVs-cGAMP is the engeled B16F10-BDVs-cGAMP, ＊ P<0.05， ＊＊ P<0.01， ＊＊＊ P<0.001， ＊＊＊＊ P<0.0001。
the results showed that expression levels of the marker CD40 were significantly increased in BMDCs maturation activated following stimulation with Envolved B16F10-BDVs-cGAMP as detected by flow cytometry, and that M1 (CD 86, il12B, tnf) polarized markers were significantly increased and M2 (CD 206, arg1, il 10) polarized markers were significantly decreased in BMDMs following stimulation with Envolved B16F10-BDVs-cGAMP as detected by flow and PCR, indicating that Envolved B16F10-BDVs-cGAMP was able to promote DCs maturation activation and induce macrophage polarization.
Example 4: application of tumor vaccine combined immune checkpoint antibody aPD-1 in treatment of mouse melanoma
1. Construction of mouse melanoma model and drug efficacy evaluation
1X 10 to stably express luciferase (Lucifer enzyme) 6 The B16F10 cells were injected subcutaneously in the left flank of mice to establish a murine melanoma model. Mice were divided into 7 experimental groups:
g1: a PBS group;
and G2: cGAMP group;
and G3: an encumbered B16F10 group;
and G4: an set of Enucleated B16F 10-BDVs;
and G5: a PD-1 antibody group;
g6: the set of Enucleated B16F 10-BDVs-cGAMP;
and G7: the set of the engeled B16F10-BDVs-cGAMP+PD-1 antibodies.
Wherein, except PD-1 antibody (2.5 mg/kg) was injected by tail vein, the rest of the medicines were injected subcutaneously into the right flank of the mouse once every 3 days, and the total injection was 3 times (wherein the injection doses of cGAMP, enucleated B16F10 cells and BDVs were 100 μg, 1×10, respectively) 6 And 50 μg). The tumor size was measured with a vernier caliper, and the tumor weight and survival time of each group of mice were recorded to evaluate the efficacy of the drug.
The results are shown in fig. 6 as a-d, where a is the tumor growth curve (n=7) of melanoma mice from different treatment groups; b is the average tumor growth curve (n=7) for each group of tumor-bearing mice; c is the tumor weight (n=4) of melanoma mice from different treatment groups; d is the survival curve (n=10) of each group of tumor-bearing mice.
The results show that after the treatment of the Enuceated B16F10-BDVs-cGAMP, the tumor volume of the mice is obviously reduced, the tumor weight is obviously reduced, and the survival period of the mice is obviously prolonged.
2. Effect of Enuceated B16F10-BDVs-cGAMP on immune cells in tumor tissue
Melanoma tissues of the mice in the different treatment groups are collected and respectively prepared into single cell suspensions. Then detecting CD8 in tumor tissues of different treatment groups by using flow cytometry + T cell numbers and FCM was used to detect IFN- γ, gzmB, CD69 expression with CD8 as Gate.
The results of the assay are shown in FIG. 7, where a is CD8 of melanoma in mice of different treatment groups + T cell detection results, b is CD69 of melanoma of mice in different treatment groups + CD8 + T cell detection result, c is GzmB of melanoma of mice in different treatment groups + CD8 + T cell detection result, d is IFN-gamma of melanoma of mice in different treatment groups + CD8 + T cell detection results. The results show IFN-. Gamma.in mouse tumor tissue following treatment with Envolved B16F10-BDVs-cGAMP + CD8 + T、GzmB + CD8 + T、CD69 + CD8 + The number of T cells is obviously increased, which indicates that the Enuceated B16F10-BDVs-cGAMP can not only effectively inhibit the growth of melanoma, but also obviously improve the tumor immune microenvironment.
Example 5: application of tumor vaccine combined immune checkpoint antibody aPD-1 in preventing mouse melanoma
1. Preimmune mice and construction of mouse melanoma models
Mice were randomly assigned to 7 groups and immunized with PBS, cGAMP, encumbered B16F10-BDVs, PD-1 antibody, encumbered B16F10-BDVs-cGAMP, encumbered B16F10-BDVs-cGAMP+PD-1 antibody pairs (injection doses of cGAMP, encumbered B16F10 cells and BDVs were 100 μg, 1×10, respectively) first 21, 14 and 7 days prior to challenge with B16F10 cells 6 And 50 μg), wherein the remaining drug was injected subcutaneously into the right flank of the mouse except for the PD-1 antibody (2.5 mg/kg) by tail vein injection. After 7 days from the last immunization interval, 1X 10 will be 6 Injection of B16F10 cells into the left flank of miceA melanoma model was constructed partially subcutaneously. The tumor size was measured every other day with vernier calipers, and the tumor weight and tumor-free survival time of each group of mice were recorded, and the preventive effect of the drug was evaluated.
The results are shown in fig. 8 as a-c, where a is the tumor-free growth curve (n=7) for mice of the different treatment groups; b is the average tumor growth curve (n=7) for each group of tumor-bearing mice; c is the tumor weight (n=4) of melanoma mice from different treatment groups. The result shows that after the advanced preventive inoculation of the Enuceated B16F10-BDVs-cGAMP, the tumor volume of the mice is obviously reduced, the tumor weight is obviously reduced, and the tumor-free survival time of the mice is obviously prolonged.
2. Effect of Envolved B16F10-BDVs-cGAMP on memory T cells in tumor tissue
Melanoma tissues of mice from different treatment groups were collected on day 26 after injection of B16F10 melanoma cells and individually prepared as single cell suspensions. Detection of CD8 in tumor tissues of different treatment groups by flow cytometry + T cell number and CD8 as Gate, FCM was used to detect CD69 in mouse tumor tissue + CD8 + T cells, CD44 + CD69 + CD8 + T cells, CD8 + TEM(CD44 + CD62L - CD8 + T) expression.
The results are shown in FIG. 8, d-f, where d is CD8 of melanoma tissue from mice of different treatment groups + T cell detection results, e is CD44 of melanoma tissues of mice in different treatment groups + CD69 + CD8 + T cell detection results, f is CD44 of melanoma tissues of mice in different treatment groups + CD62L + CD8 + T cell detection results. The results indicate that CD69 in tumor tissue of mice after advanced prophylaxis with Enuceated B16F10-BDVs-cGAMP + CD8 + T cells, CD44 + CD69 + CD8 + T cells, CD8 + TEM(CD44 + CD62L - CD8 + The expression ratio of T) is significantly increased. The above results demonstrate that the encumbered B16F10-BDVs-cGAMP is effective in preventing melanoma and producing immunological memory.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A tumor vaccine based on enucleated melanoma cells, characterized in that: the tumor vaccine comprises:
a1 Removing the melanoma cells of the nucleus; the method comprises the steps of,
a2 A bacterial outer membrane vesicle coupled to the enucleated melanoma cells;
wherein the bacterial outer membrane vesicles are loaded with STING agonists.
2. The tumor vaccine of claim 1, wherein: the bacterial outer membrane vesicle is obtained by the induction expression and purification of an escherichia coli recombinant expression vector.
3. The tumor vaccine of claim 1, wherein: the STING agonist includes cGAMP or a cGAMP derivative.
4. The tumor vaccine of claim 1, wherein: the tumor vaccine also includes a PD-1 antibody.
5. A method of preparing a tumor vaccine according to any one of claims 1 to 4, wherein: the method comprises the following steps:
s1, preparing cell nucleus-removed melanoma cells, constructing recombinant bacteria with STING agonist synthesis capacity, and extracting bacterial outer membrane vesicles;
s2, mixing the bacterial outer membrane vesicle with a dibenzocyclooctyne coupling agent for reaction to obtain a dibenzocyclooctyne coupling modified bacterial outer membrane vesicle;
s3, mixing azido sugar analogues with the cell nucleus-removed melanoma cells to obtain azide-modified enucleated melanoma cells, and then mixing the dibenzocyclooctyne-coupled modified bacterial outer membrane vesicles with the azide-modified enucleated melanoma cells to react to obtain the product.
6. The method of manufacturing according to claim 5, wherein: in S1, the method for preparing the cell nucleus-removed melanoma cells comprises a density gradient centrifugation method.
7. The method of manufacturing according to claim 5, wherein: in S2, the dibenzocyclooctyne coupling agent comprises dibenzocyclooctyne-PEG 4 -N-hydroxysuccinimide ester;
and/or, in S3, the azido sugar analog comprises tetrA-Acylated N-azidoacetate galactosamine.
8. The method of manufacturing according to claim 5, wherein: s3, the temperature of the reaction is 15-37 ℃;
and/or the reaction time is 1-40 h.
9. Use of a tumor vaccine based on enucleated melanoma cells according to any one of claims 1 to 4 for the preparation of a product for the treatment and/or prevention of melanoma.
10. The use according to claim 9, characterized in that: the functions of the product include at least one of B1) to B6):
b1 Activating STING pathway of antigen presenting cells;
b2 Promoting maturation and activation of dendritic cells;
b3 Inducing macrophage polarization;
b4 Increasing IFN-gamma in tumor tissue + CD8 + T cells, gzmB + CD8 + T cells, CD69 + CD8 + A number of at least one T cell in the T cells;
b5 Increasing CD69 in tumor tissue + CD8 + T cells, CD44 + CD69 + CD8 + T cells, CD44 + CD62L - CD8 + The expression proportion of at least one T cell in the T cells;
b6 Reducing melanoma volume and/or weight.
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