An engineered tumor cell-derived microparticle targeting succinic acid, its preparation method and application

Abstract

The present invention discloses a kind of tumor cell-derived microparticles engineered to target succinate, including tumor cell-derived microparticles and succinate receptors loaded on the surface of their plasma membrane; also discloses a preparation method thereof, comprising the following steps: using lentivirus to infect cells, and performing monoclonal screening, selecting a cell line with a succinate receptor expression level of more than 5 times that of β-actin, and obtaining engineered cells; inducing apoptosis of the engineered cells and releasing cell-derived microparticles, collecting the supernatant, performing differential centrifugation, and taking the precipitate to obtain the engineered tumor cell-derived microparticles targeting succinate; and its use in the preparation of drugs for preventing and / or treating cancer. On the one hand, the invention can inhibit the succinate-succinate receptor signals of various cells in the tumor microenvironment, and on the other hand, when delivering succinate into macrophages, reverse the polarization of tumor-associated macrophages, improve the tumor immune microenvironment, and have a good therapeutic effect on a variety of individual tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an engineered succinic acid-targeted tumor cell-derived microparticle, a preparation method, and an application thereof. Background Art

[0002] Succinate is a metabolite of the tricarboxylic acid cycle and accumulates in the tumor microenvironment of certain tumors. In addition, succinate can also serve as an important signaling molecule, activating its downstream signaling pathway by binding to the succinate receptor (a G protein-coupled receptor). Succinate receptors are widely distributed in the human body and are expressed in different types of cells, including hepatic stellate cells, kidney and intestinal epithelial cells, macrophages, and monocyte-derived dendritic cells. In the tumor microenvironment, succinate receptors are mainly present on the surface of tumor cells and tumor-associated macrophages and play different functions, promoting tumor cell proliferation and migration.

[0003] Succinate receptor signaling in macrophages can promote the polarization of tumor-associated macrophages toward the M2 type, further producing a tumor-promoting effect. In summary, the succinate-succinate receptor axis plays a role in promoting tumor development in the tumor microenvironment.

[0004] There are two currently discussed approaches to targeting the succinate-succinate receptor axis. One is to use blocking antibodies to block the succinate receptor, but the production of blocking antibodies is expensive and time-consuming. The other is to deliver siRNA targeting the succinate receptor to reduce succinate receptor expression in cells. However, the targeting and safety of this approach in vivo are unclear. Furthermore, both approaches can only inhibit succinate receptor signaling to a limited extent, resulting in poor results. Summary of the Invention

[0005] The purpose of the present invention is to provide an engineered tumor cell-derived microparticle targeting succinic acid and its preparation method and application, so as to solve the technical problem in the prior art that succinic acid is not easily inhibited in the tumor microenvironment.

[0006] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0007] The present invention provides an engineered tumor cell-derived microparticle targeting succinic acid, comprising the tumor cell-derived microparticle and a succinic acid receptor loaded on the plasma membrane surface thereof.

[0008] As a preferred embodiment of the present invention, in the parent cell, the mRNA expression level of the succinate receptor is more than 5 times the expression level of β-actin.

[0009] As a preferred embodiment of the present invention, the particle size of the tumor cell-derived microparticles is 50-500 nm, preferably 100-300 nm, and more preferably 120-250 nm.

[0010] The present invention also provides a method for preparing engineered succinic acid-targeted tumor cell-derived microparticles, comprising the following steps:

[0011] S100, using lentivirus to infect cells and perform monoclonal screening to select cell lines expressing succinate receptors at levels more than 5 times that of β-actin to obtain engineered cells;

[0012] S200, inducing apoptosis of the engineered cells and releasing cell-derived microparticles, collecting the supernatant, performing differential centrifugation, and taking the precipitate to obtain the engineered tumor cell-derived microparticles targeting succinate.

[0013] As a preferred solution of the present invention, in S100,

[0014] The cells are solid tumor cells;

[0015] The steps for infecting cells were as follows: the multiplicity of infection (MOI) was 10, and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 h;

[0016] The cells were suspended in physiological saline and placed at 37°C for 20-30 hours; or 2 The cells were irradiated with ultraviolet light for 0.5-1.5 h.

[0017] As a preferred solution of the present invention, in S100,

[0018] The lentivirus carries a succinate overexpression gene;

[0019] The preparation method of the lentivirus is a three-plasmid co-transfection system, and the specific conditions are as follows:

[0020] HEK293T cells were transfected with a plasmid carrying a succinate overexpression gene and cultured at 37°C, 5% CO2 for 72 h. The viral particles were concentrated by ultracentrifugation before the viral supernatant was harvested.

[0021] The plasmid carrying the succinate overexpression gene is a plasmid in which the expression of the succinate receptor gene is driven by a strong promoter;

[0022] The plasmid is a cytomegalovirus promoter or an elongation factor 1-α promoter.

[0023] As a preferred embodiment of the present invention, in S200, the step of inducing apoptosis of the engineered cells is as follows: suspending the engineered cells in physiological saline and placing them at 37° C. for 20-30 hours;

[0024] The conditions of the differential centrifugation are as follows: 1000-50000 g gradient centrifugation for 1-1.5 h, temperature is 4°C.

[0025] The present invention also provides the use of engineered succinic acid-targeted tumor cell-derived microparticles in the preparation of drugs for preventing and / or treating cancer.

[0026] As a preferred embodiment of the present invention, the drug comprises engineered tumor cell-derived microparticles targeting succinic acid and a pharmaceutically acceptable carrier.

[0027] As a preferred embodiment of the present invention, the cancer includes liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, and thyroid cancer.

[0028] As a preferred embodiment of the present invention, engineered tumor cell-derived microparticles targeting succinic acid are used in combination with PD-1 antibodies to enhance the reactivity of PD-1 antibodies and improve the efficacy of immunotherapy.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The microparticles provided by the present invention are endogenous substances of the body. Due to the "homing effect", they can better target tumor tissues. After binding to the succinate receptor, they can, on the one hand, effectively bind to succinate in the tumor microenvironment and inhibit the tumor-promoting effect mediated by succinate. On the other hand, they can achieve the effect of delivering endogenous succinate into macrophages. After delivery into the macrophages, they can inhibit the succinate-succinate receptor axis in the macrophages, inhibit the M2 polarization signal, increase the level of succinate in the macrophages, induce M1 polarization, improve the tumor immune microenvironment, and have a good therapeutic effect on various individual tumors.

[0031] The preparation method provided by the present invention is simple, and compared with chemically synthesized nanocarriers or other immune drugs, it is safer, has a richer source, is easier to obtain, targets most tumors, has good universality, is easy to produce, and is highly efficient.

[0032] Compared with unmodified microparticles, the engineered succinate-targeted tumor cell-derived microparticles provided by the present invention can be used to prepare drugs for preventing and / or treating various cancers. When used in combination with PD-1 antibodies, they can enhance the reactivity of PD-1 antibodies and improve the efficacy of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0034] Figure 1 Provides a schematic diagram of the large-scale expression of succinate receptor mRNA for Example 1 of the present invention;

[0035] Figure 2 Provided is an image of the cell membrane of an engineered tumor cell being developed by a red fluorescent secondary antibody for Example 1 of the present invention;

[0036] Figure 3 Provides a particle size diagram of engineered succinate-targeted tumor cell-derived microparticles for Example 1 of the present invention;

[0037] Figure 4 Provides a morphological diagram of tumor cell-derived microparticles engineered to target succinate for Example 1 of the present invention;

[0038] Figure 5 Provides a distribution diagram of microparticles with high expression of succinate receptors in the extracted microparticles for Example 1 of the present invention;

[0039] Figure 6 A schematic diagram of the ability of engineered succinate-targeted tumor cell-derived microparticles to bind succinate in the environment is provided for Example 1 of the present invention;

[0040] Figure 7 A schematic diagram of the situation in which engineered succinate-targeted tumor cell-derived microparticles reverse the polarization of M2 macrophages is provided for Verification Example 1 of the present invention;

[0041] Figure 8 Provided for the verification example 2 of the present invention are tumor morphology images of the mouse breast cancer 4T1 subcutaneous tumor model after treatment;

[0042] Figure 9 Provided for the verification example 2 of the present invention are statistical graphs of tumor volume and mouse survival rate after treatment in a mouse breast cancer 4T1 subcutaneous tumor model;

[0043] Figure 10 Provided for the verification example 2 of the present invention are tumor morphology images of the mouse breast cancer EMT-6 subcutaneous tumor model after treatment;

[0044] Figure 11 Provided for the verification example 2 of the present invention are statistical graphs of tumor volume and mouse survival rate after treatment in the mouse breast cancer EMT-6 subcutaneous tumor model;

[0045] Figure 12 Provided for the verification example 2 of the present invention, a statistical graph of ascites volume and mouse survival rate after treatment of a mouse liver cancer ascites model;

[0046] Figure 13 Provided for Verification Example 3 of the present invention are images of tumor morphology after immunotherapy of 4T1 mouse breast cancer subcutaneous tumor model with engineered succinate-targeted tumor cell-derived microparticles enhanced with anti-PD-1 antibodies;

[0047] Figure 14 Statistical graphs of tumor volume and mouse survival rate after immunotherapy of 4T1 mouse breast cancer subcutaneous tumor model with engineered succinate-targeted tumor cell-derived microparticles and enhanced anti-PD-1 antibodies are provided for Verification Example 3 of the present invention. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] The present invention provides an engineered tumor cell-derived microparticle targeting succinic acid, comprising the tumor cell-derived microparticle and a succinic acid receptor loaded on the plasma membrane surface thereof.

[0050] Microparticles are a type of extracellular vesicle that are produced by budding from the cell membrane and have a particle size distribution of mainly 50-500nm. According to the report in Reference 2, tumor-derived microparticles can induce tumor immune responses. In addition, microparticles are extremely excellent engineered carriers, and their safety, circulation stability, and targeting have been verified in multiple studies and clinical trials. In the present invention, "cell microparticles", "cell-derived microparticles", "cell-derived microparticles" and "cell-derived microparticles" are used interchangeably to refer to cell vesicles produced by apoptosis, which have not been engineered.

[0051] Microparticles can be derived from solid tumor cells, immune cells, and stem cells. Solid tumor cells include, but are not limited to, tumor cells in breast cancer, liver cancer, bladder cancer, bone cancer, brain cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, etc. Immune cells include T cells, B cells, natural killer cells, macrophages, dendritic cells, mast cells, neutrophils, and innate immune cells. Stem cells include embryonic stem cells, induced pluripotent stem cells, adult stem cells, and tissue-specific stem cells.

[0052] In the present invention, "succinate receptor" refers to a G protein-coupled receptor, also known as GPR91 or SUCNR1.

[0053] The engineered tumor cell-derived microparticles targeting succinate are obtained by "engineering" tumor cell-derived microparticles, which means that the succinate receptor gene is introduced into the recipient cells through lentiviral infection, and the succinate receptor protein is expressed on the surface of the receptor cell membrane, that is, the above-mentioned engineered tumor cell-derived microparticles targeting succinate.

[0054] Microparticles are endogenous substances. Compared with chemically synthesized nanocarriers or other immunotherapies, they are safer, more abundant, and more readily available. Tumor cell-derived microparticles are better able to target tumor tissue due to their "homing effect." Using these microparticles as engineered carriers can achieve tumor tissue targeting. Selecting appropriate parent cells can target the vast majority of tumors, demonstrating excellent universal applicability.

[0055] In a preferred embodiment of the present invention, succinate receptors are highly expressed in the above-mentioned engineered succinate-targeted tumor cell-derived microparticles.

[0056] However, the effects of extracellular succinate and intracellular succinate may be diametrically opposed. Studies have shown that the metabolic pattern of M1 macrophages changes, with significantly increased cytoplasmic succinate levels. When exogenous succinate is delivered into macrophages using succinate-loaded tumor cell-derived microparticles, succinate levels in the cytoplasm and nucleus increase, leading to succinylation of certain proteins and the induction of the macrophages into an M1 phenotype.

[0057] In the present invention, in particular, the cell-derived microparticles that highly express succinate receptors provided by the present invention can bind to succinate in the tumor microenvironment and deliver it to macrophages.

[0058] The delivery of exogenous succinate may elevate succinate levels in the tumor microenvironment and activate succinate receptor-mediated tumor-promoting signaling. Therefore, the present invention directly targets endogenous succinate in the tumor microenvironment and delivers it to macrophages. This can simultaneously inhibit succinate receptor signaling and mediate M1 polarization induced by intracellular protein succinylation. This, in turn, directly inhibits tumor proliferation and migration while improving the tumor immune microenvironment, presenting a promising new immunotherapy strategy.

[0059] The present invention can produce the following three effects: 1. Reduce the level of succinate in the tumor microenvironment, inhibit the tumor proliferation and migration signals mediated by the succinate-succinate receptor axis in tumor cells; 2. Inhibit the succinate-succinate receptor axis in macrophages, inhibit the tumor-promoting M2 polarization signal; 3. Increase the level of succinate in macrophages, inducing anti-tumor M1 polarization.

[0060] The phenotype and function of macrophages are not simply a binary opposition between M1 and M2, but are often in a dynamic state of flux and regulated by the environment in which they exist. In the tumor microenvironment, tumor-associated macrophages, influenced by multiple signals including succinate receptor signals, primarily exist with an M2-biased phenotype. M2-like tumor-associated macrophages can secrete cytokines such as IL-10, TGF-β, and VEGF to promote tumor development and progression. Tumor cell-derived microparticles engineered to target succinate can bind to succinate in the tumor microenvironment, resulting in a relative decrease in succinate levels without activating succinate receptor signals on tumor cells or macrophages.

[0061] Furthermore, because macrophages have a strong phagocytic effect on microparticles, they also ingest large amounts of succinate bound to the surface of tumor cell-derived microparticles engineered to target succinate during the phagocytic process. This leads to elevated succinate levels within macrophages, which in turn induces M1 polarization through succinylation of certain proteins and epigenetic modifications. M1-like tumor-associated macrophages can secrete cytokines such as TNF-α, IL-12, and IL-1β, enhancing anti-tumor immune responses. At the same time, M1-like tumor-associated macrophages can present tumor antigens to CD4+ T cells through upregulated MHC class II molecules, further promoting the activation and effector effects of CD8+ T cells.

[0062] It can be seen from this that choosing to use tumor cell-derived microparticles engineered to target succinate to bind to succinate in the tumor microenvironment can achieve two effects. One is to inhibit the succinate-succinate receptor signaling of various cells in the tumor microenvironment (mainly tumor cells and macrophages); the other is to deliver succinate into macrophages to achieve M1 polarization of macrophages (this process involves succinylation of proteins and epigenetic modification). Its effect is far greater than directly blocking the succinate receptor, so targeting succinate is a better choice.

[0063] Compared with unmodified microparticles, the engineered succinate-targeted tumor cell-derived microparticles provided by the present invention can reverse the polarization of tumor-associated macrophages, and have a significant therapeutic effect on a variety of mouse subcutaneous tumors and mouse tumor ascites models, thereby improving the survival of mice.

[0064] In addition, its combined use with PD-1 antibodies can enhance the responsiveness of PD-1 antibodies and improve the efficacy of immunotherapy.

[0065] In the present invention, the particle size of the cell-derived microparticles is 50-500 nm, preferably 100-300 nm, and more preferably 120-250 nm.

[0066] In the parental cells, the mRNA expression level of the succinate receptor is more than 5 times that of β-actin, thereby being able to more effectively reduce the level of succinate in the tumor microenvironment and inhibit the succinate-succinate receptor axis in tumor cells.

[0067] The present invention provides a method for preparing engineered succinate-targeted tumor cell-derived microparticles, which specifically comprises the following steps:

[0068] S100, using lentivirus to infect cells and perform monoclonal screening to select cell lines expressing succinate receptors at levels more than 5 times that of β-actin to obtain engineered cells;

[0069] S200, inducing apoptosis of the engineered cells and releasing cell-derived microparticles, collecting the supernatant, performing differential centrifugation, and taking the precipitate to obtain the engineered tumor cell-derived microparticles targeting succinate.

[0070] The preparation method of the present invention can efficiently prepare engineered tumor cell-derived microparticles targeting succinic acid.

[0071] In a preferred embodiment, the method for preparing tumor cells that highly express succinate receptors uses lentiviral infection technology, preferably adding polybrene to enhance transfection efficiency, and the specific operating conditions are set as: the multiplicity of infection (MOI) is 10, and cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 hours to ensure that the viral particles fully infect the tumor cells and achieve efficient expression of the succinate receptor.

[0072] In a preferred embodiment, the method for preparing a lentivirus carrying a succinate overexpression gene is to use a three-plasmid co-transfection system, preferably using a calcium phosphate transfection method or a lipofectamine transfection method, and the specific conditions are: transfection is performed in HEK293T cells, and after transfection, the cells are cultured at 37°C and 5% CO2 for 72 hours, and ultracentrifugation is preferably performed to concentrate the viral particles before harvesting the viral supernatant.

[0073] In a preferred embodiment, the plasmid carrying the succinate overexpression gene is a plasmid in which the expression of the succinate receptor gene is driven by a strong promoter, preferably a CMV (cytomegalovirus) promoter or an EF1-α (elongation factor 1-α) promoter.

[0074] In a preferred embodiment, the tumor cell is a solid tumor cell. In some preferred embodiments, the solid tumor cell includes, but is not limited to, tumor cells from breast cancer, liver cancer, bladder cancer, bone cancer, brain cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, etc. In some specific embodiments, the tumor cell is a breast cancer cell.

[0075] The method of inducing tumor cell apoptosis is starvation treatment, preferably using physiological saline to starve the tumor cells. The specific conditions are: suspending the tumor cells in physiological saline and placing them at 37° C. for 20-30 hours, preferably 24 hours.

[0076] In a preferred embodiment, the number of tumor cells induced to apoptosis is 1×10 7 -1×10 8 Hours, you can get 1×10 10 -1×10 11 The cell-derived microparticles.

[0077] In a preferred embodiment, after inducing tumor cell apoptosis to release cell-derived microparticles, the microparticles are collected. The specific collection method can be: collecting the cell suspension, centrifuging at 500-1000g for 3-7 minutes, preferably at 800g for 5 minutes, collecting the supernatant, and gradient centrifuging the supernatant at 1000-50000g for 1-1.5 hours. The visible precipitate is the cell-derived microparticles.

[0078] In a preferred embodiment, the centrifugation conditions are: 1000-50000 g for 1-1.5 h, preferably 14000 g for 1 h, preferably in a low temperature environment (4° C.).

[0079] In a preferred embodiment, the centrifugation conditions are: 1000-50000 g for 1-1.5 h, preferably 14000 g for 1 h, preferably in a low temperature environment (4° C.).

[0080] The ability of the engineered succinate-targeted tumor cell-derived microparticles to bind succinate in the environment is 1×10 10 Each microparticle can bind 0.01-10 μmol of succinic acid.

[0081] The present invention also discloses the use of engineered succinic acid-targeted tumor cell-derived microparticles in the preparation of drugs for preventing and / or treating cancer.

[0082] The medicine comprises the engineered succinic acid-targeted tumor cell-derived microparticles prepared by the above preparation method and a pharmaceutically acceptable carrier.

[0083] Preferably, the cancer includes breast cancer, liver cancer, bladder cancer, bone cancer, brain cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer; more preferably, the cancer is breast cancer, liver cancer and colorectal cancer.

[0084] The present invention provides a method for preventing and / or treating cancer, which comprises administering an effective amount of the engineered succinate-targeted tumor cell-derived microparticles to an individual in need thereof; preferably, the cancer comprises breast cancer, liver cancer, bladder cancer, bone cancer, brain cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer; more preferably, the cancer is breast cancer, liver cancer and colorectal cancer.

[0085] The following examples further illustrate the above-mentioned engineered succinate-targeted tumor cell-derived microparticles.

[0086] Example 1:

[0087] 1. Preparation of engineered tumor cell-derived microparticles targeting succinate

[0088] Collect 1x10 6 Total RNA was extracted from wild-type or succinate receptor-overexpressing tumor cells using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. One microgram of total RNA was reverse-transcribed into cDNA using reverse transcriptase. Real-time quantitative PCR reactions were performed using primers specific for the succinate receptor gene and primers for the internal reference gene β-actin.

[0089] The target gene Ct value was compared with its own internal reference Ct value to calculate the expression level of succinate receptor relative to the internal reference gene β-actin. 4 Wild-type or succinate receptor-overexpressing tumor cells were seeded onto confocal microscopy plates and incubated for 24 hours. The supernatant was discarded and the cells were fixed with paraformaldehyde for 30 minutes, followed by three washes with PBS. Anti-succinate receptor antibodies (NOVUS, NBP1-00861, 1:100) were then incubated at 4°C in the dark for 8 hours, followed by three washes with PBS. CY3 goat anti-rabbit fluorescent secondary antibody (1:100) was added and incubated at 4°C in the dark for 1 hour. Nuclei were labeled with DAPI (1:1000) and washed three times with PBS. The cells were then examined using a laser confocal microscope.

[0090] Figure 1This is the cell line with the highest expression of succinate receptor selected from twenty monoclonal cell lines. Compared with the wild-type EMT-6, its succinate receptor mRNA is expressed in large quantities.

[0091] Figure 2 The cell membrane of the engineered tumor cells was visualized by red fluorescent secondary antibody, showing that a large number of succinate receptors were expressed on the cell membrane.

[0092] The results are as follows Figure 1 and Figure 2 As shown, the succinate receptor is abundantly expressed in the engineered cells.

[0093] 2. Characterization: particle size, electron microscopy

[0094] Collect 1×10 8 Tumor cells were resuspended in physiological saline and plated into 15 cm cell culture dishes. After being placed in a cell culture incubator at 37°C for 24 h, the cells were collected and centrifuged at 800 g for 5 min. The supernatant was collected and centrifuged at 14,000 g for 2 min to remove cell debris. The supernatant was then centrifuged at 14,000 g for 1 h to collect tumor cell microparticles. The supernatant was discarded and the microparticles (visible precipitate) were resuspended in PBS and stored at 4°C.

[0095] The particle size of the microparticles was detected by nanoparticle tracking analysis (NTA), and the morphology of the microparticles was detected by electron microscopy.

[0096] The results are as follows Figures 3 and 4 Shown are the particle size and morphology of cell-derived microparticles.

[0097] Figure 3 The particle size of the cell-derived microparticles was detected by a nanoparticle tracking instrument. It can be seen that the particle size distribution of the cell-derived microparticles is relatively uniform, concentrated around 200 nm.

[0098] Figure 4 It is a morphological display of cell-derived microparticles observed by scanning electron microscopy.

[0099] 3. Receptor expression detection

[0100] Take 1×10 8 The engineered succinate-targeted tumor cell-derived microparticles were diluted to 100 μl with PBS, incubated with 1 μl of succinate receptor antibody (NOVUS, NBP1-00861, 1:100) at 4°C for 8 h, centrifuged at 14,000 g for 30 min, and washed three times.

[0101] Then, the cells were incubated with FITC goat anti-rabbit fluorescent secondary antibody (Abcam, ab6717, 1:100) and CD9 antibody (Abcam, ab82392, 1:100) at 4°C for 30 min, washed three times with PBS, diluted to the appropriate concentration, and detected using a nanoflow cytometer (Nanofcm U30E).

[0102] The results are as follows Figure 5 As shown, nearly 90% of the microparticles were double positive for succinate receptor and CD9, demonstrating the universal expression of succinate receptor on engineered microparticles, and their particle size distribution was consistent with the results of nanoparticle tracking.

[0103] 4. Detection of the binding capacity of succinic acid in the environment

[0104] 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 The engineered succinate-targeting tumor cell-derived microparticles prepared in step 1 were incubated at room temperature for 10 minutes and then centrifuged at 14,000 g for 30 minutes. The supernatant was diluted to 80% methanol by adding 100% methanol-water. The precipitate was lysed with 80% methanol-water, and the succinate levels in the sample were analyzed by liquid chromatography-tandem mass spectrometry.

[0105] The results are as follows Figure 6 As shown, engineered succinate-targeted tumor cell-derived microparticles can bind succinate in the environment and reduce the concentration of succinate in the solution.

[0106] Example 1 illustrates that the engineered succinate-targeted tumor cell-derived microparticles prepared according to the method disclosed in the present invention have ubiquitous expression of succinate receptors on the engineered microparticles and can bind to succinate in the environment, thereby reducing the concentration of succinate in the solution.

[0107] The following is a series of validation examples to verify the functionality of engineered succinate-targeted tumor cell-derived microparticles.

[0108] Verification Example 1: Reversal of M2 macrophage polarization

[0109] Mouse bone marrow primary cells were obtained by separating the femur and tibia from C57BL / 6J mice. The bone marrow cells were rinsed out with PBS, and red blood cell lysis buffer was added and allowed to stand for 3 minutes. After neutralization with culture medium, the cells were centrifuged at 400g for 5 minutes. The cell pellet was resuspended and plated into 6-well plates. The cells were cultured with DMEM medium containing 20ng / ml M-CSF on the third day.

[0110] On the 5th day, 20ng / ml IL-4 was added to the culture medium, and after 24h of treatment, the cells became M2 bone marrow-derived macrophages.

[0111] Physiological saline, succinate, unmodified microparticles, tumor cell-derived microparticles engineered to target succinate, succinate plus unmodified microparticles, and succinate plus tumor cell-derived microparticles engineered to target succinate were added to each well of M2 macrophages. After 12 hours, cells were harvested and total RNA was extracted. Real-time quantitative PCR was performed after reverse transcription to calculate the relative expression levels of NOS2 and Arg1.

[0112] The results are as follows Figure 7 As shown, NOS2 expression was significantly increased, while Arg1 expression was significantly downregulated in the succinate + tumor cell-derived microparticles engineered to target succinate group. This indicates that tumor cell-derived microparticles engineered to target succinate can reverse the polarization of M2 macrophages in the presence of succinate, inducing them to become M1-like.

[0113] It can be seen from Verification Example 1 that the engineered succinate-targeted tumor cell-derived microparticles prepared in Example 1 can reverse the polarization of tumor-associated macrophages.

[0114] Verification Example 2: Significant therapeutic effects in various mouse subcutaneous tumor and mouse tumor ascites models

[0115] BALB / c mice were subcutaneously inoculated with 1×10 5 BALB / c mice were intraperitoneally injected with 1×10 4T1 or EMT-6 mouse breast cancer cells. 5 H22 mouse liver cancer cells were injected intravenously into the subcutaneous tumor model and intraperitoneally into the ascites model. One week later, treatment was initiated every two days for two weeks.

[0116] The mice were divided into three treatment groups, with 6 mice in each group, and the treatment methods within each group were the same: blank control group Con (100ul normal saline), unmodified microparticle group NC-MP (1x10 11 The engineered succinate-targeted tumor cell-derived microparticles eSRhe-MP (1x10 11 indivual).

[0117] The subcutaneous tumors of mice were observed and their volumes were measured.

[0118] The results are as follows Figures 8 to 12 As shown, it shows the therapeutic effect of tumor cell microparticles engineered to highly express succinate receptors on mouse tumor models.

[0119] Figure 8 、 9 , 10, 11 and 12 showed that after treatment, the tumor volume of mouse breast cancer 4T1 and EMT-6 subcutaneous tumor models was reduced and the survival period was prolonged.

[0120] Figures 8 to 12 It showed that the ascites volume of the mouse liver cancer ascites model was reduced after treatment and the survival time of the mice was prolonged after treatment.

[0121] Verification Example 2 shows that the results obtained in Example 1 indicate that the engineered tumor cell microparticles with high expression of succinate receptors have a good therapeutic effect on mouse breast cancer models and mouse liver cancer models, and improve the survival of mice.

[0122] Verification Example 3: Enhanced reactivity of PD-1 antibody after combination with PD-1 antibody

[0123] BALB / c mice were subcutaneously inoculated with 1×10 5 One week later, normal saline, unengineered microparticles, and the tumor cell-derived microparticles engineered to target succinate described in Example 1 were injected into the tail vein every 2 days, and PD-1 antibodies were injected into the tail vein every 4 days for two weeks.

[0124] The mice were divided into five treatment groups, with 8 mice in each group, and the treatment methods within each group were the same: blank control group Con (100ul normal saline), unmodified microparticle group NC-MP (1x10 11 ), the tumor cell-derived microparticle group eSRhe-MP (1x10 11 The subcutaneous tumors of the mice were observed and their volumes were measured.

[0125] The results are as follows Figures 13 and 14 As shown, the therapeutic effect of PD-1 antibody combined with tumor cell microparticles engineered to highly express succinate receptors on mouse tumor models is shown.

[0126] Verification Example 3 shows that the combined use of the engineered succinate-targeted tumor cell-derived microparticles prepared in Example 1 and PD-1 antibodies can enhance the reactivity of PD-1 antibodies and improve the efficacy of immunotherapy.

[0127] Through the engineered succinate-targeted tumor cell-derived microparticles of this embodiment, as well as their preparation method and application, the succinate-succinate receptor signaling of various cells in the tumor microenvironment can be inhibited, endogenous succinate can be delivered into macrophages, and the polarization of tumor-associated macrophages can be reversed, which has a significant therapeutic effect on tumors. When used in combination with PD-1 antibodies, the reactivity of PD-1 antibodies can be enhanced, thereby improving the efficacy of immunotherapy.

[0128] The above embodiments are merely exemplary embodiments of the present application and are not intended to limit the scope of the present application. The scope of protection of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and scope of protection of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present application.

Claims

1. Use of engineered succinate-targeted tumor cell-derived microparticles in the preparation of a drug for preventing and / or treating cancer, characterized in that: The drugs include PD-1 antibodies and tumor cell microparticles engineered to highly express succinate receptors; The engineered tumor cell-derived microparticles targeting succinate include tumor cell-derived microparticles and succinate receptors loaded on the plasma membrane surface thereof; In the parent cells of the tumor cell-derived microparticles, the mRNA expression level of the succinate receptor is more than 5 times the expression level of β-actin; The particle size of the tumor cell-derived microparticles is 120-250 nm; The cancer is breast cancer.

2. The use according to claim 1, wherein the method for preparing the engineered tumor cell-derived microparticles targeting succinate comprises the following steps: S100, using lentivirus to infect cells and perform monoclonal screening to select cell lines expressing succinate receptors at levels more than 5 times that of β-actin to obtain engineered cells; S200, inducing apoptosis of the engineered cells and releasing cell-derived microparticles, collecting the supernatant, performing differential centrifugation, and taking the precipitate to obtain the engineered tumor cell-derived microparticles targeting succinate.

3. The application according to claim 2, wherein in S100, The cells are solid tumor cells; The steps for infecting cells were as follows: the multiplicity of infection (MOI) was 10, and the cells were cultured in a constant temperature incubator at 37°C and 5% CO for 48 h; The cells were suspended in physiological saline and incubated at 37°C for 20-30 hours; or the cells were irradiated with ultraviolet light at an irradiation intensity of 1.0 mW / cm² for 0.5-1.5 hours.

4. The application according to claim 2, wherein in S100, The lentivirus carries a succinate overexpression gene; in, The preparation method of the lentivirus is a three-plasmid co-transfection system, and the specific conditions are as follows: HEK293T cells were transfected with a plasmid carrying a succinate overexpression gene and cultured at 37°C, 5% CO2 for 72 h. The viral particles were concentrated by ultracentrifugation before the viral supernatant was harvested. The plasmid carrying the succinate overexpression gene is a plasmid in which the expression of the succinate receptor gene is driven by a strong promoter; The plasmid is a cytomegalovirus promoter or an elongation factor 1-α promoter.

5. The use according to claim 2, wherein in S200, the step of inducing apoptosis of the engineered cells is as follows: suspending the engineered cells in physiological saline and placing them at 37°C for 20-30 hours; The conditions of the differential centrifugation are as follows: 1000-50000 g gradient centrifugation for 1-1.5 h, temperature is 4°C.

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