Preparation method and application of bacterial biomimetic vesicles for reprogramming macrophages

Abstract

The present invention discloses a preparation method and application of a bacterial bionic vesicle for reprogramming macrophages. The preparation of the bacterial bionic vesicle is to first fuse the bacterial outer membrane anchor protein with the IFN-γ gene, and then use high-pressure homogenization technology to drive genetically engineered bacteria to extrude from the cracks and self-assemble into bacterial bionic vesicles modified with IFN-γ surface. In vitro, the bacterial bionic vesicles of the present invention can promote the reprogramming of M1 macrophages into M2 macrophages, and release nitric oxide to directly kill tumor cells. In vivo, the bacterial bionic vesicles can efficiently enrich in the tumor area by virtue of their nanosize, reprogram the tumor-promoting M2TAMs to the tumor-killing M1TAMs and coordinate adaptive immunity to jointly inhibit tumor growth. In short, the bacterial bionic vesicles of reprogramming macrophages of the present invention are a powerful immunomodulator, which significantly inhibits tumor growth in a variety of tumor models and is of great significance in the immunotherapy of tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and immunology, and particularly relates to a preparation method and application of bacterial bionic vesicles for reprogramming macrophages. Background Art

[0002] Macrophages are an important component of the mononuclear phagocytic system and play a variety of important roles in host defense: (1) clearing pathogens, (2) secreting cytokines, enzymes, and other factors that form inflammatory responses, and (3) acting as antigen-presenting cells to present antigens to T cells and regulate the acquired immune system. The immune system recognizes and eliminates pathogenic microorganisms and tumor cells, thereby preventing the progression of many diseases. Among all immune cells, macrophages are the most plastic. As a complex cellular ecosystem, there is a close relationship between infiltrating tumor-associated macrophages (TAMs) and tumor cells in the tumor microenvironment (TME). On the one hand, tumor cells release inflammatory mediators, including some cytokines and chemokine family members, which act on TAMs and reprogram the functions of TAMs. On the other hand, reprogrammed TAMs release immune-related effector molecules with different functions, resulting in the host's immune response to tumors (pro-tumor or anti-tumor). Therefore, based on TAM function, TAMs are simplified into pro-inflammatory and anti-tumor M1 TAMs and anti-inflammatory and pro-tumor M2 TAMs.

[0003] It is worth noting that M2 TAMs are the most abundant innate immune cells in the TME. The immunosuppressive molecules released by M2 TAMs support the function of regulatory T cells (Treg) and inhibit the infiltration and activity of the adaptive immune system, which explains why most TMEs are immunosuppressive. In addition, the abundance of M2 TAMs is associated with poor prognosis in various human cancers, including glioblastoma, breast cancer, head and neck cancer, renal cancer, and lymphoma. Based on the strong plasticity of TAMs in the TME and the potential anti-tumor ability of M1 TAMs. Therefore, reprogramming M2 TAMs to M1 TAMs is a more attractive strategy for tumor immunotherapy. Activating macrophage polarization to the M1 phenotype through pattern recognition receptors (PRRs) has become a basic strategy, and toll-like receptor (TLR) agonists (such as R848) exert potential anti-tumor effects by inducing macrophage reprogramming to the M1 phenotype. Some of these TLR agonists have been evaluated in clinical trials, including R837 (TLR7 agonist), 852A (TLR7 agonist), and IMO-2055 (TLR9 ​​agonist). Unfortunately, the efficacy of R837 and IMO-2055 was not satisfactory. In the R837 clinical trial, only 2 of 10 breast cancer patients achieved partial remission, while in the IMO-2055 clinical trial, only 5 of 33 patients (15%) who were evaluable for efficacy achieved partial remission.

[0004] Therefore, how to efficiently reprogram M2 TAMs into M1 TAMs to enhance anti-tumor immunity is a problem that needs to be solved at present. Summary of the invention

[0005] In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide a preparation method and application of bacterial bionic vesicles for reprogramming macrophages, which greatly increases the loading capacity on the surface of the vesicles and reduces the endotoxin content. The bacterial bionic vesicles for reprogramming macrophages can be used to efficiently reprogram M2 macrophages into M1 macrophages, thereby inducing a strong anti-tumor immune response.

[0006] In recent years, the delivery of immune stimulants through nanoparticles is an emerging approach that can regulate specific immune activation / inhibition pathways to enhance anti-tumor effects. As sensor cells of the immune system, the expression of PRRs on the surface of macrophages makes them highly sensitive to the invasion of pathogens (such as bacteria or bacterial derivatives). As bacterial derivatives, the nanoscale bacterial vesicles formed by Gram-negative bacteria carry pattern-associated molecular patterns (PAMPs) from donor bacterial pathogens, giving them the ability to activate multiple TLR signaling pathways, such as LPS and flagellin. The inventors have conducted research on this and found that bacterial outer membrane anchor proteins can be fused with expressed exogenous proteins and then stably anchored on the surface of the bacterial membrane; the present invention uses genetic engineering technology to fuse bacterial outer membrane anchor proteins with INF-γ and express them, and then prepares bacterial biomimetic vesicles (BBV) with a high load of INF-γ. Based on this, when further studying the efficacy of bacterial biomimetic vesicles with high INF-γ load, in vivo and in vitro experiments confirmed that bacterial biomimetic vesicles with high INF-γ load can reprogram M2 macrophages into M1 macrophages and synergize adaptive immunity to jointly inhibit tumor growth.

[0007] In view of this, the inventor provides the following solution of the present invention.

[0008] A first aspect of the present invention provides a bacterial biomimetic vesicle for reprogramming macrophages, wherein the preparation method of the bacterial biomimetic vesicle comprises the following steps:

[0009] (1) Preparation of recombinant plasmid:

[0010] According to the expression characteristics of Escherichia coli, the codons of the INF-γ gene and the bacterial outer membrane anchor protein gene sequences are optimized, and the codon-optimized INF-γ gene and the bacterial outer membrane anchor protein gene are inserted into the plasmid to obtain a recombinant plasmid;

[0011] The codon-optimized INF-γ gene sequence is shown in SEQ ID NO.1;

[0012] (2) Fusion expression of bacterial outer membrane anchor protein and INF-γ:

[0013] Inducing expression in competent E. coli cells transformed with the recombinant plasmid and collecting the E. coli expressing the recombinant protein;

[0014] (3) Preparation of INF-γBBV:

[0015] The Escherichia coli expressing the recombinant protein is resuspended in a buffer to obtain a bacterial suspension, and the bacterial suspension is treated by high-pressure homogenization. The bacteria are driven by ultrahigh pressure and squeezed through the cracks to form a membrane component carrying the recombinant protein. The suspension obtained after high-pressure homogenization is centrifuged to collect the supernatant, and the supernatant is ultracentrifuged to collect the precipitate to obtain a mixed precipitate of BBV and INF-γBBV;

[0016] The mixed precipitate of BBV and INF-γBBV is separated by chromatography column purification or density gradient centrifugation purification to obtain purified INF-γBBV.

[0017] Furthermore, in step (1), the bacterial outer membrane anchor protein includes ClyA, outer membrane protein A, outer membrane protein F and factor H binding protein.

[0018] Furthermore, in step (1), a linker sequence is contained between the INF-γ gene and the bacterial outer membrane anchor protein gene, and the amino acid sequence of the linker is shown in SEQ ID NO.2.

[0019] Furthermore, the plasmid is pThioHisA, and the codon-optimized INF-γ gene and the bacterial outer membrane anchor protein gene are inserted into the plasmid through the restriction sites Nde I and Kpn I.

[0020] Furthermore, the bacterial outer membrane anchored protein includes ClyA, and the codon-optimized ClyA gene sequence is shown in SEQ ID NO.3.

[0021] The second aspect of the present invention provides the use of the bacterial biomimetic vesicles of reprogrammed macrophages in in vitro reprogrammed macrophages, wherein the bacterial biomimetic vesicles of reprogrammed macrophages are added to anti-inflammatory, pro-tumor M2 macrophages and after co-incubation, the M2 macrophages are reprogrammed into pro-inflammatory, anti-tumor M1 macrophages.

[0022] Furthermore, the bacterial biomimetic vesicles were added to the cells with a cell number of 1×10 5 Up to 1×10 7 In the M2 macrophages with a volume of 100 μl, the final concentration is 0.1 μg to 1 μg vesicle protein / ml, and they are incubated for 0.5h to 48h, and the M2 macrophages are reprogrammed into M1 macrophages.

[0023] The third aspect of the present invention provides an in vitro reprogrammed anti-inflammatory and pro-tumor M2 macrophage, wherein the bacterial biomimetic vesicles of the reprogrammed macrophages are added to the anti-inflammatory and pro-tumor M2 macrophages and after co-incubation, the M2 macrophages are reprogrammed into pro-inflammatory and anti-tumor M1 macrophages.

[0024] The fourth aspect of the present invention provides an application of the bacterial bionic vesicles for reprogramming macrophages in the preparation of anti-tumor drugs and vaccines.

[0025] Furthermore, the tumor is a solid tumor including cervical cancer, colon cancer, breast cancer and melanoma.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The outer membrane vesicles (OMV) naturally secreted by Gram-negative bacteria are easy to interact with or be absorbed by immune cells, and participate in regulating the immune system. OMVs carrying heterologous molecules obtained by genetic engineering are also widely used in tumor immunotherapy. However, the yield of OMVs and the limited heterologous molecule loading capacity limit their large-scale application. In addition, OMVs without reprogrammed macrophages may not be sufficient to reprogram macrophages after intravenous injection, and will also be accompanied by systemic toxicity caused by high doses. Compared with IFN-γOMV, the present invention uses high-pressure homogenization technology to drive the bacterial self-assembly of reprogrammed macrophages to form IFN-γBBV, which has a higher IFN-γ load (about 39.7% of total protein). In addition, the endotoxin content of IFN-γBBV is much lower than that of IFN-γOMV, which means that under the condition of intravenous injection of the same vesicle protein, IFN-γBBV is safer than IFN-γOMV, which is very important. Because excessive endotoxin intake will trigger inflammatory factor storms and fatal toxicity.

[0028] 2. As a derivative of bacteria, the IFN-γBBV of the present invention has good biocompatibility and nanostructure (30-150nm), and is easy to flow in the blood and lymphatic vessels. In the process of tumor immunotherapy, intravenous injection of IFN-γBBV is more likely to be enriched in the tumor area through the EPR effect to exert its effect. IFN-γBBV is loaded with TLR-activating PAMPs (such as LPS) and stimulatory factor IFN-γ at the same time, which can effectively stimulate macrophage polarization.

[0029] 3. Experiments have confirmed that in vitro, IFN-γBBV can reprogram M2 phenotype macrophages into M1 phenotype macrophages. Reprogrammed M1 phenotype macrophages release nitric oxide (NO) to directly kill tumor cells. As antigen presenting cells, reprogrammed M1 macrophages can effectively present tumor antigens to T cells through MHC molecules, triggering strong T cell immunity to jointly inhibit tumor growth.

[0030] 4. In tumor-bearing mice (TC-1 tumor, CT-26 tumor and 4T1 tumor), after intravenous injection, IFN-γBBV enriched in the tumor area can reprogram M2 TAMs into M1 TAMs, release NO to directly kill tumor cells, and synergize adaptive immunity to induce tumor antigen-specific CTL response. In fact, the anti-tumor effect of IFN-γBBV is applicable to the immunotherapy of various tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0032] Figure 1 This is a schematic diagram of the preparation process of IFN-γBBV described in Example 1 of the present invention.

[0033] Figure 2 It is a diagram of the verification and characterization results of IFN-γBBV of Example 2 of the present invention; wherein, Figure A is the SDS-PAGE and WB of BBV and IFN-γBBV; Figure B is the TME image and schematic diagram of BBV and IFN-γBBV from Escherichia coli BL21 (DE3); Figure C is the average particle size of BBV and IFN-γBBV.

[0034] Figure 3 This is a quantitative result diagram of the IFN-γBBV endotoxin content in Example 3 of the present invention.

[0035] Figure 4 The figure is a schematic diagram of the relevant results that IFN-γBBV can reprogram M2 BMDMs into M1BMDMs in vitro in Example 4 of the present invention; wherein, Figure A is a representative WB image of iNOS and CD206 after 36 hours of specified treatment of M2 BMDMs; Figure B is the NO production in the culture medium after different treatments; Figure C is the mRNA expression level of IL-12 after different treatments detected by RT-qPCR; Figure D is a schematic diagram of anti-tumor immunity after IFN-γBBV reprogramming of M2 BMDMs.

[0036] Figure 5 The figure is a schematic diagram of the results of IFN-γBBV being able to efficiently enrich in tumors in Example 5 of the present invention; wherein, Figure A is a representative fluorescence image of different organs and tissues in TC-1 tumor-bearing mice after intravenous injection of Cy7-labeled IFN-γBBV and Cy7-labeled IFN-γ 60 hours. Figure B is a quantitative analysis of the fluorescence intensity of Cy7 fluorescence signals in different organs.

[0037] Figure 6Figure 6 is a schematic diagram of the results of the anti-tumor effect of IFN-γBBV in TC-1 tumors in Example 6 of the present invention; wherein, Figure A is the experimental design for evaluating the anti-tumor immunity of IFN-γBBV in the TC-1 tumor model; Figure B is the tumor growth curves of different groups; Figure C is an anatomical image of the TC-1 tumor on day 33; Figure D is the statistical data of the tumor weight; Figures EH are respectively the proportion of infiltrating immune cells in the tumor analyzed by flow cytometry, including iNOS-active macrophages (CD11b + F4 / 80 + iNOS + ), M1 phenotype macrophages (CD11b + F4 / 80 + CD86 + ), M2 phenotype macrophages (CD11b + F4 / 80 + CD206 + ), CD8 T cells (CD3 + CD8 + ).

[0038] Figure 7 Figure 7 is a schematic diagram of the relevant results of the anti-tumor effect of IFN-γBBV on the CT26 tumor model in Example 7 of the present invention; wherein, Figure A is the experimental design for evaluating the anti-tumor immunity of IFN-γBBV in the CT26 tumor model; Figure B is the growth curve of each group of tumors; Figure C is the growth dynamics of individual tumors after different treatments; Figure D is the anatomical image of the CT26 tumor after different treatments on the 25th day; Figure E is the statistics of the tumor weight of each group; and Figure F is the spleen weight of each group.

[0039] Figure 8 Schematic diagram of the results of the anti-tumor effect of IFN-γBBV on the 4T1 orthotopic breast cancer model in Example 8 of the invention; wherein, Figure A is the experimental design for evaluating the anti-tumor immunity of IFN-γBBV in the 4T1 orthotopic breast cancer model; Figure B is the growth curve of each group of tumors; and Figure C is the statistics of the number of nodules of lung tumor metastasis. DETAILED DESCRIPTION

[0040] In the following examples, unless otherwise specified, the raw materials and experimental instruments involved are commercially available.

[0041] Raw materials, see Table 1.

[0042] Table 1

[0043]

[0044] Experimental instruments are shown in Table 2.

[0045] Table 2

[0046]

[0047]

[0048] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0049] Example 1 - Preparation of IFN-γBBV

[0050] like Figure 1 As shown, this embodiment provides a method for preparing bacterial biomimetic vesicle IFN-γBBV for reprogramming macrophages, and the preparation method is:

[0051] (1) Preparation of recombinant plasmid:

[0052] After removing the signal peptide (1-22 amino acids) of mouse IFN-γ, the mature IFN-γ region (23-155 amino acids) was connected to the C-terminus of ClyA through Linker (GGGGS), and codon optimization was performed according to the codon preference of Escherichia coli. Finally, the optimized DNA sequence was constructed on the plasmid pThioHisA through the restriction sites Nde I and Kpn I to obtain the recombinant plasmid pThioHisA-ClyA-IFN-γ.

[0053] Specifically, the amino acid sequence of the signal peptide is: MNATHCILALQLFLMAVSGCYC.

[0054] The amino acid sequence of the mature IFN-γ region is:

[0055] HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISF YLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQ RQAFNELIRVVHQLLPESSLRKRKRSRC.

[0056] Codon-optimized INF-γ gene sequence:

[0057] CATGGCACCGTGATTGAGAGCCTGGAAAGCCTGAACAACTATTTTAACAGCAGCGGCATTGATGTGGAAGAAAAAAGCCTGTTTCTGGATATTTGGCGCAACTGGCAGAAAGATGGCGATATGAAAATTCTGCAGAGTCAGATTATTAGCTTTTATCTGCGCCTGTTTGAAGTGCTGAAAGATAACCAAGCGATTAGCAA CAACATTAGCGTGATTGAAAGCCATCTGATTACCACCTTTTTTAGCAACAGCAAAGCGAAAAAAGATGCGTTTATGAGCATTGCGAAATTTGAAGTGAACAACCCGCAAGTGCAGCGCCAAGCGTTTAACGAACTGATTCGCGTGTGCATCAGCTGCTGCCGGAAAGCAGCCTGCGCAAACGCAAACGCAGCCGCTGC.

[0058] Codon-optimized ClyA gene sequence:

[0059] CACTTCCGGCACCTCAAACAGGGTTTTTTTGCCATGGCGTTTCTGATATTCGTTGCAGGTGTTAATCATTTTTTTCGCCGCTTCTTTCAGCAGGCTCAGCATCAGATCATCATAATCCACATAAAAGCGGGTGGTTTCGGTTTCGGTTTTAATTTCGCCAATCGCCGCAATTTCGGTGGTCAGTTTCAGTTTCGCCGCATCAATATCTTTGTTCGCTTGTTTCACGGTGTTGCTCAGGGTGGTAAAAAAGTTCTGCACGCTTTTCAGTTTGTTTTTCAGTTCCGGAATCAGTTTGCCTTCCACCACCGCCGCGGCAATGCTATAGCTAATAATCAGGCCAAACGGGCCCGCCACCACGCCCGCCGCGGCGCCCGCATACGCTTCGCGGCGAATTTTATCCACTTGGCTCTGAAAATAGCTGCTTTTTTCGCTAAAATCGTTGGTCAGCTGACTATCCAGCGCCAGCAGTTTGCCGCTCGCGTTGTTAAAGCTCTGACTGCTCACCAGCAGGCTTTTCTGCGCTTCGTTCAGTTTGGTAATGCCATCATCCAGCACTTTAATCAGAATATCTTTCTGCGCGCTCGCTTTTTTTTCGTTATATTCATCAAACAGCAGAATATACGCCGCCAGCAGCTGCGTCGCCACGCCGCACCATTCATACACGGTCTGCGTCGCTTCAAAATATTTATCTTGGCTATCCATCAGCAGGGTTTTAATATCGCCCACCAGCACGCTCGCCGCTTGGCTATATTCTTGTTTAAAGCGGCTCAGTTCTTTAATGGTTTCATCAAAGGTCTGCCACGGAATCACTTGATCCAGATATTTGTTATACAGATCCAGCGCGCCATCCGCGGTTTCAATCGCGTTTTTCACCACTTCCACGGTTTTATCCGCCACAATTTCGGTCAT。

[0060] (2) Fusion expression of ClyA and INF-γ:

[0061] The recombinant plasmid pThioHisA-ClyA-IFN-γ was transformed into Escherichia coli competent cells. The specific operation was as follows: 100 μl of Escherichia coli (BL21 / DE3) competent cells were taken out from a -80°C refrigerator and thawed on ice; after the competent cells were thawed, 20 ng of the recombinant plasmid was added and mixed, and the mixture was placed on ice for 30 min; the competent cells were placed in a 42°C water bath for heat shock for 50 s, and then quickly returned to ice for 3 minutes, followed by adding 900 μl of LB culture medium and placed on a shaker at 200 rpm for 40 minutes; the transformed cells were evenly spread on a solid LB culture medium with ampicillin resistance and placed in a 37°C incubator for overnight culture.

[0062] A single clone was selected and inoculated in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) and cultured at 37°C with shaking at 220 rpm until OD600 reached 0.6. Then, 1 mM isopropyl β-D-1-mercaptogalactopyranoside (IPTG) was added to the culture and protein expression was induced at 30°C on a constant temperature shaker with rotation at 220 rpm for 16 hours. 200 μl was sampled before and after induction for electrophoresis identification. Then, the bacteria were collected by centrifugation (14000×g, 30 min, 4°C).

[0063] (3) Preparation of INF-γBBV:

[0064] The E. coli BL21 and E. coli BL21 expressing the ClyA-IFN-γ recombinant protein obtained in step (2) are resuspended in about 300 ml of HBSS buffer. The bacteria resuspended in the HBSS solution are passed through the high-pressure homogenizer crack three times under the conditions of 1200 bar and 4°C. The bacteria are driven and squeezed through the cracks by ultra-high pressure to form a membrane component carrying the recombinant protein. The sample passing through the cracks is centrifuged to precipitate the bacteria (10000×g, 30min, 4°C). The supernatant is collected and ultracentrifuged (100,000×g, 30min, 4°C) to obtain a mixed precipitate of BBV and IFN-γBBV. The mixed precipitate of BBV and IFN-γBBV is resuspended in HBSS, added to iodixanol, purified and separated by density gradient centrifugation (130,000×g, 4h, 4°C), and finally the sample layer is collected and a 0.22μm pore size filter is used to avoid contamination by bacteria or cell debris, and BBV and IFN-γBBV can be obtained respectively.

[0065] Example 2 - Identification of IFN-γBBV

[0066] In order to identify the proteins on BBV and IFN-γBBV prepared in Example 1, the purified BBV and IFN-γBBV in Example 1 were added to 5×SDS-PAGE loading buffer and heated to 100°C for 10 min. The proteins were then separated using a 12% SDS-PAGE gel and transferred to a PVDF membrane. Protein detection was performed using anti-IFN-γ rabbit polyclonal antibody (1:2000) as the primary antibody and enzyme-labeled anti-rabbit IgG (1:5000) as the secondary antibody. Finally, ImageLab (Version 6.0.1) software (BioRad) was used for imaging and analysis.

[0067] like Figure 2 As shown in A, the protein content of ClyA-IFN-γ determined by Image Lab software accounted for 39.7% of the total protein in the vesicles, and the specificity of IFN-γ was also verified by WB. In addition, in order to identify the structure of IFN-γBBV, the morphology of IFN-γBBV was observed using a transmission electron microscope (TEM).

[0068] The results are as follows Figure 2 As shown in B, IFN-γBBV is a ring-shaped nanoparticle with a particle size of 30-150 nm. Figure 2 Center C shows that the average diameters of BBV and IFN-γBBV are 78.6 nm and 84.3 nm, respectively.

[0069] Example 3 - Comparative analysis of endotoxins of IFN-γBBV and IFN-γOMV

[0070] The preparation method of OMV and IFN-γ OMV is as follows:

[0071] The recombinant plasmid pThioHisA-ClyA-IFN-γ described in Example 1 was transformed into Escherichia coli BL21 competent cells, and after induced expression at 30°C for 16h, 5L of culture medium was centrifuged at 4°C and 8000g for 30min to remove bacteria, and the supernatant was passed through a 0.45μm vacuum filter. The filtrate was then concentrated using a filter with a 1000kDa molecular cutoff. The concentrated filtrate was ultracentrifuged at 120000g for 4h at 4°C to obtain OMV and IFN-γOMV. Iodixanol density gradient centrifugation was used for further purification to obtain OMV and IFN-γOMV, respectively.

[0072] For endotoxin detection of OMV, IFN-γOMV, BBV, and IFN-γBBV, Limulus amebocyte lysate (LAL) assay was performed using a commercial endotoxin detection kit according to the manufacturer’s instructions. Figure 3The results showed that at the same protein concentration, the amount of endotoxin on IFN-γBBV was reduced by about 18.8 times compared with IFN-γOMV, which was due to the increased amount of heterologous protein loading on BBV. In in vivo applications, the use of IFN-γBBV will significantly reduce the lethal toxicity caused by excessive endotoxin intake compared with IFN-γOMV.

[0073] Example 4 - Reprogramming of M2 macrophages into M1 macrophages in vitro

[0074] In this example, the IFN-γBBV prepared in Example 1 was used to reprogram macrophages in vitro. The specific method is as follows:

[0075] Bone marrow-derived macrophages (BMDMs) were extracted from the femur and tibia of 7-week-old C57BL / 6 mice and cultured for 7 days. Briefly, bone marrow cells were cultured in DMEM containing 10% fetal bovine serum and recombinant mouse macrophage colony stimulating factor (M-CSF, 10 ng / ml). On the third day, the culture medium was replaced with fresh culture medium containing M-CSF. After 3 days, BMDMs were collected and treated with 10 ng / ml of IL-4 to obtain M2 BMDMs.

[0076] Then, the IFN-γBBV prepared in Example 1 was added to 1×10 5 Up to 1×10 7 , in a volume of 100μl M2 BMDMs, the final concentration was 0.5μg vesicle protein / ml, and other control groups were set up including LPS (final concentration of 100ng / ml), IFN-γ (final concentration of 20ng / ml), LPS (final concentration of 100ng / ml) + IFN-γ (final concentration of 20ng / ml), BBV (final concentration of 0.3μg vesicle protein / ml), BBV + IFN-γ (final concentration of 20ng / ml). After 36h of co-incubation, samples were collected for WB and RT-qPCR analysis of changes in molecular markers of M1 macrophages and M2 macrophages.

[0077] like Figure 4 Figure A shows that IFN-γBBV can significantly upregulate the expression of iNOS, a marker of M1 macrophages, and downregulate the expression of CD206, a marker of M2 macrophages. Since the ability of macrophages to kill tumor cells is closely related to the activity of iNOS in macrophages, nitric oxide (NO) produced by iNOS metabolism of arginine is the main effector molecule for iNOS-active macrophages to destroy tumor cells. Therefore, this example uses Griess reagent to evaluate the release level of NO. Figure 4Figure B shows that IFN-γBBV can significantly enhance the level of NO released by macrophages, which is very important for the direct killing of tumor cells. In addition, the changes of pro-inflammatory cytokine IL-12 were detected by RT-qPCR ( Figure 4 In general, IFN-γBBV can reprogram M2 BMDMs into M1 BMDMs in vitro, upregulate the expression of iNOS, release NO to directly kill tumor cells, and promote M1 BMDMs to secrete proinflammatory cytokines to further enhance anti-tumor immunity ( Figure 4 (middle D).

[0078] Example 5 - Evaluation of the potential of IFN-γBBV in tumor immunotherapy

[0079] In order to evaluate the potential of IFN-γBBV prepared in Example 1 in tumor immunotherapy, the enrichment ability of IFN-γBBV in tumor tissue after intravenous injection was first evaluated. In this example, Cyanine7 (Cy7)-labeled IFN-γ and IFN-γBBV were intravenously injected into tumor-bearing mice (tumor volume was about 50 mm 3 After 60 hours, the fluorescence signal of Cy7 in major organs and tumor tissues was measured ( Figure 5 (A) Cy7 fluorescence quantitative analysis showed that obvious fluorescence signals could be observed in the liver and lung tissues of the IFN-γ and IFN-γBBV groups ( Figure 5 (B). However, IFN-γ showed only weak fluorescence signals in tumor tissues. In contrast, strong fluorescence signals were observed in tumor tissues with IFN-γBBV (approximately 20 times that of free IFN-γ). These results show that IFN-γBBV can be enriched in tumor areas in large quantities after intravenous injection, which is of great significance for reprogramming M2 TAMs.

[0080] Example 6 - Effect of IFN-γBBV in TC-1 tumor-bearing mouse model

[0081] This example uses the TC-1 tumor-bearing mouse model to evaluate the anti-tumor immune effect of IFN-γBBV prepared in Example 1. The specific method is as follows:

[0082] In the TC-1 tumor-bearing mouse model, when the tumor volume grows to about 50 mm 3 At the same time, IFN-γBBV (40μg) was injected intravenously. BBV was set as a control, and immunization was performed twice, with an interval of 2 days. The immunotherapy diagram is shown in Figure 6 As shown in A. The growth of tumor volume was monitored after IFN-γBBV injection ( Figure 6B), tumor tissue was collected on day 33. It can be clearly seen that IFN-γBBV can promote tumor regression and even completely eliminate established tumors ( Figure 6 (C, D).

[0083] To reveal the potential mechanism of this powerful anti-tumor effect, Ben Shili further analyzed the immune cells in tumor tissues using flow cytometry. By analyzing the iNOS-active macrophages (CD11b + F4 / 80 + iNOS + ) and M1 TAMs (CD11b + F4 / 80 + CD86 + ) ratio was significantly increased, which helps to inhibit the growth of tumor cells and the presentation of tumor antigens ( Figure 6 In addition, the immunosuppressive M2 TAMs (CD11b + F4 / 80 + CD206 + ) was significantly downregulated ( Figure 6 The increase in the M1 / M2 ratio is key to enhancing immunotherapy and promoting CTL infiltration in the TME, as CD8 T cells (CD3 + CD8 + ) was significantly upregulated in IFN-γBBV ( Figure 6 These results indicate that the enrichment of IFN-γBBV can effectively induce polarization of M1 TAM macrophages in tumor sites and promote T cell immunity, enhance tumor immunotherapy, and inhibit tumor growth.

[0084] In general, IFN-γBBV-mediated anti-tumor immunity is the result of both the innate and adaptive immune systems.

[0085] Example 7 - Effect of IFN-γBBV in the CT26 colon cancer model

[0086] In order to broaden the application of IFN-γBBV in different tumor models, this example uses the colon cancer CT26 model to evaluate the anti-tumor immune effect of IFN-γBBV prepared in Example 1. The specific method is as follows:

[0087] 1×10 5 CT26 tumor cells, and wait until the tumor volume grows to 50mm 3 In this example, 10 μg and 40 μg of IFN-γBBV were intravenously injected, and BBV was set as a control. The immunization scheme was as follows Figure 7 As shown in A.

[0088] It can be clearly seen that after injection of IFN-γBBV, tumor growth was gradually inhibited and regressed on day 16 ( Figure 7 In the IFN-γBBV (40 μg) group, 50% of the tumors did not continue to grow after regression ( Figure 7 C). However, this phenomenon was not found in IFN-γBBV (10 μg), indicating that IFN-γBBV-mediated anti-tumor regression is dose-dependent. Consistent with the trend in the TC-1 model, IFN-γBBV can significantly inhibit the growth of CT26 tumors ( Figure 7 D, E) while avoiding pathological splenomegaly ( Figure 7 Middle F).

[0089] Example 8 - Effect of IFN-γBBV in 4T1 orthotopic breast cancer model

[0090] TAMs contribute to the progression and dissemination of breast cancer, and strategies to target and reprogram TAMs have shown promising preclinical results. In fact, in breast cancer, macrophages play an important role in suppressing the anti-tumor activity of T cells.

[0091] Based on the above results, this example studies the therapeutic effect of IFN-γBBV prepared in Example 1 on the in situ 4T1 breast cancer model, which is closer to the natural physiological condition than the microenvironment of subcutaneous transplanted tumors. 4T1 is a "cold" tumor with low immunogenicity, which results in insufficient infiltration of activated T lymphocytes in the TME. Figure 8 As shown in A, first 1×10 5 4T1 tumor cells were injected into the left mammary pad of mice, and the tumor grew to 50 mm after 16 days. 3 Afterwards, 10 μg of IFN-γBBV was injected intravenously twice, and tumor growth was monitored.

[0092] Obviously, IFN-γBBV significantly inhibited tumor growth ( Figure 8 In addition, tumor metastasis to the lungs was also significantly inhibited ( Figure 8 Middle C). This data suggests that IFN-γBBV-induced reprogramming of M2 TAMs can also inhibit tumor growth and metastasis in the 4T1 orthotopic breast cancer model.

[0093] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A bacterial biomimetic vesicle for reprogramming macrophages, characterized in that: The preparation method of the bacterial biomimetic vesicle comprises the following steps: (1) Preparation of recombinant plasmid: According to the expression characteristics of Escherichia coli, the codons of the INF-γ gene and the bacterial outer membrane anchor protein gene sequences are optimized, and the codon-optimized INF-γ gene and the bacterial outer membrane anchor protein gene are inserted into the plasmid to obtain a recombinant plasmid; The codon-optimized INF-γ gene sequence is shown in SEQ ID NO.1; a Linker sequence is included between the INF-γ gene and the bacterial outer membrane anchor protein gene, and the amino acid sequence of the Linker is shown in SEQ ID NO.2; The plasmid is pThioHisA, and the codon-optimized INF-γ gene and the bacterial outer membrane anchor protein gene are inserted into the plasmid through the restriction sites Nde I and Kpn I; The bacterial outer membrane anchor protein includes ClyA, and the codon-optimized ClyA gene sequence is shown in SEQ ID NO.3; (2) Fusion expression of bacterial outer membrane anchor protein and INF-γ: Inducing expression in competent E. coli cells transformed with the recombinant plasmid and collecting the E. coli expressing the recombinant protein; (3) Preparation of INF-γBBV: The Escherichia coli expressing the recombinant protein is resuspended in a buffer to obtain a bacterial suspension, and the bacterial suspension is treated by high-pressure homogenization. The bacteria are driven by ultrahigh pressure and squeezed through the cracks to form a membrane component carrying the recombinant protein. The suspension obtained after high-pressure homogenization is centrifuged to collect the supernatant, and the supernatant is ultracentrifuged to collect the precipitate to obtain a mixed precipitate of BBV and INF-γBBV; The mixed precipitate of BBV and INF-γBBV is separated by chromatography column purification or density gradient centrifugation purification to obtain purified INF-γBBV.

2. A use of the bacterial biomimetic vesicles for reprogramming macrophages as claimed in claim 1 in reprogramming macrophages in vitro, characterized in that: The bacterial biomimetic vesicles for reprogramming macrophages as claimed in claim 1 are added to anti-inflammatory and tumor-promoting M2 macrophages and co-incubated, so that the M2 macrophages are reprogrammed into pro-inflammatory and anti-tumor M1 macrophages.

3. The use according to claim 2, characterized in that: The bacterial biomimetic vesicles as claimed in claim 1 are added to the cell number of 1×10 5 Up to 1×10 7 In the M2 macrophages with a volume of 100 μl, the final concentration is 0.1 μg to 1 μg vesicle protein / ml, and they are incubated for 0.5h to 48h, and the M2 macrophages are reprogrammed into M1 macrophages.

4. An in vitro reprogrammed pro-inflammatory and anti-tumor M1 macrophage, characterized in that: The bacterial biomimetic vesicles for reprogramming macrophages as claimed in claim 1 are added to anti-inflammatory and tumor-promoting M2 macrophages and co-incubated, so that the M2 macrophages are reprogrammed into pro-inflammatory and anti-tumor M1 macrophages.

5. Use of the bacterial biomimetic vesicles for reprogramming macrophages as claimed in claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor is a solid tumor.

6. The use according to claim 5, characterized in that: The solid tumor is cervical cancer, colon cancer or breast cancer.

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