An engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment and its preparation method

Abstract

The present invention belongs to the technical field of drug targeting carriers, and more specifically, relates to an engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment and a preparation method thereof. It includes microparticles produced by apoptosis of macrophages that overexpress tumor antigens, and small molecule effective ingredients of drugs that can reverse polarize M2 tumor-associated macrophages to M1 type encapsulated by the microparticles; the surface of the microparticles is also modified with M2 macrophage targeting molecules. The drug-loaded microparticles provided by the present invention are more conducive to being taken up by M2 tumor-associated macrophages, improving the reverse polarization effect of reverse polarization drugs on M2 tumor-associated macrophages, improving the tumor microenvironment, and utilizing the reverse polarized macrophages to process and present the tumor antigens carried by the microparticles, stimulating the activation of antigen-specific CD8T cells and the proliferation and differentiation of stem-like CD8T cells, thereby achieving an inhibitory effect on tumor types expressing the same antigens.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug targeting carriers, and more specifically, relates to an engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment and a preparation method thereof. Background Art

[0002] Tumor-associated macrophages (TAMs) account for about 50% of the mass of solid tumors, are an important component of the tumor microenvironment, and are one of the important causes of immunosuppression in the tumor microenvironment. A large number of studies have shown that M2 tumor-associated macrophages can promote tumor growth, angiogenesis, metastasis, drug resistance and immunosuppression, and play a pro-tumor role. In contrast, M1 tumor-associated macrophages have stronger tumor cell phagocytosis ability, can secrete pro-inflammatory cytokines such as IL-12 and TNF-α, release reactive oxygen species, and effectively kill tumor cells. At the same time, M1 tumor-associated macrophages can effectively present antigens to activate Th1 immune responses and play an important role in anti-tumor immune responses. Therefore, it is an important way to reverse the polarization of M2 tumor-associated macrophages to M1 and restore their anti-tumor ability in tumor immunotherapy.

[0003] Nanomaterials have obvious advantages in targeted drug delivery due to their unique physical and chemical properties and targeted modification. Compared with the poor biocompatibility of artificially synthesized nano drug carriers, the use of cells or cell-derived vesicles as drug carriers has attracted widespread attention. Macrophages are induced by various signals such as cytokines and chemokines in the tumor microenvironment in vivo and can be recruited to tumor tissues. Therefore, macrophages as drug carriers have natural tumor tissue targeting and deep penetration capabilities. However, the drugs loaded by macrophages will affect the activity of macrophages, resulting in a decrease in their drug delivery efficiency. Moreover, macrophages recruited to tumor tissues may be transformed into M2 tumor-associated macrophages under the induction of the tumor microenvironment, which has the risk of promoting tumor growth. Therefore, it is very necessary to find drug carriers that replace macrophages.

[0004] At present, cell-derived extracellular vesicles have attracted attention as drug carriers. This natural biological source of vesicles has the characteristics of low immunogenicity, excellent long-circulation characteristics in vivo and high cell targeting. As an important type of extracellular vesicles, microparticles (MPs) are produced when cells trigger changes in the skeleton under the cell membrane under the stimulation of relevant signals, causing the cell membrane to bulge outward locally, wrap the cell contents, and release them to the outside of the cell in the form of vesicles. Its particle size is 100-1000nm. Macrophage-derived drug-loaded microparticles can be taken up by macrophages, effectively deliver small molecule drugs, and reverse the polarity of macrophages. However, this macrophage drug-loaded microparticle cannot effectively target and reverse polarize M2 macrophages, affecting its immunotherapy effect on tumors.

[0005] Patent CN 109893515A discloses a macrophage drug-loaded microparticle preparation and its preparation method, which uses macrophage-derived microparticles modified with mannose as a drug-load system to enhance the targeting of M2 macrophages and reverse polarize M2 macrophages to M1 macrophages. However, it only improves the tumor microenvironment, does not fully utilize the antigen presentation function of macrophages, and has limited tumor treatment effects. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides an engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment and a preparation method thereof, which solves the technical problems of the prior art drugs such as off-target effects, toxic side effects on the body, and the inability of tumor-associated macrophages to effectively stimulate CD8 T cell antigen-specific activation and induce T cell exhaustion.

[0007] To achieve the above objectives, the present invention provides an engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment, comprising microparticles produced by apoptosis of macrophages overexpressing tumor antigens, and small molecule active ingredients of drugs encapsulated by the microparticles, wherein the small molecule active ingredients of drugs are small molecule drugs that can reverse polarize M2 tumor-associated macrophages to M1; the surface of the microparticles is also modified with M2 macrophage targeting molecules.

[0008] Preferably, the tumor antigen is overexpressed by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing; the macrophage is one of circulating monocytes derived from human peripheral blood, a human mononuclear cell line, mouse bone marrow-derived macrophages and a mouse monocyte / macrophage cell line.

[0009] Preferably, the mass ratio of the microparticles, the small molecule active ingredient of the drug and the M2 macrophage targeting molecule is 1000:(30-60):(2-5).

[0010] Preferably, the tumor antigens include one or more of cancer testicular antigen, liver cancer antigen AFP, melanoma antigen, prostate-specific antigen PSA, prostate-specific antigen PAP and tumor neoantigens. Optionally, the tumor neoantigens include one or more of Actn4, Adpgk, Ap3d1, Tubb3, Dag1, Eef2, Tnpo3, Tubb3, Reps1, Cpne1 and Cpsf3l.

[0011] Preferably, the small molecule active ingredient of the drug is an immune agonist small molecule drug, a metabolic small molecule drug or other small molecule drug with reverse polarization M2 as M1, the immune agonist small molecule drug is one or more of STING agonist CDN, TLR agonist R848, R837 and poly (I: C) and AMPK activator; the metabolic small molecule drug is one or more of aromatic hydrocarbon receptor AhR inhibitor and glutamine transporter inhibitor; the other small molecule drugs with reverse polarization M2 as M1 include one or more of histamine receptor inhibitor and aspirin;

[0012] The M2 macrophage targeting molecule is one or more of DSPE-PEG-M2pep, SR-B1 targeting peptide and DSPE-PEG-Man.

[0013] Preferably, the particle size of the engineered macrophage drug-loaded microparticles is 300-500 nm.

[0014] According to another aspect of the present invention, a method for preparing the engineered macrophage drug-loaded microparticle preparation is provided, comprising the following steps:

[0015] S1: preparing engineered macrophages that stably overexpress the tumor antigen through genetic engineering;

[0016] S2: after inducing apoptosis of the engineered macrophages stably overexpressing the tumor antigen by ultraviolet irradiation, the engineered macrophages are mixed and incubated with a small molecule active ingredient of a drug, wherein the small molecule active ingredient of the drug is a small molecule drug that can reverse polarize M2 tumor-associated macrophages to M1, and drug-loaded microparticles overexpressing the tumor antigen are collected;

[0017] S3: incubating the drug-loaded microparticles overexpressing the tumor antigen with M2 macrophage targeting molecules, exchanging the M2 macrophage targeting molecules to the membrane surface of the drug-loaded microparticles through membrane phospholipid exchange, and collecting the engineered macrophage drug-loaded microparticle preparation.

[0018] Preferably, in step S1, engineered macrophages stably overexpressing the tumor antigen are prepared by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing.

[0019] Further preferably, a lentivirus overexpressing the tumor antigen is first prepared by lentivirus transfection, and then the lentivirus overexpressing the tumor antigen is used to transfect macrophages to screen engineered macrophages stably overexpressing the tumor antigen; the content ratio of the macrophages to the lentivirus overexpressing the tumor antigen is 5×10 5 pcs:(3~6)×10 6 PFU.

[0020] Preferably, in step S3, the mass ratio of the drug-loaded microparticles overexpressing the tumor antigen to the M2 macrophage targeting molecule is (10-100):1; in steps S2 and S3, the collection conditions are: collection at a centrifugal force of 500-20000g at 4°C.

[0021] According to another aspect of the present invention, a drug for treating tumors is provided, which comprises the engineered macrophage drug-loaded microparticle preparation.

[0022] Further preferably, the drug for treating tumors, including the engineered macrophage drug-loaded microparticle preparation, also includes an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is one or more of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody and VISTA antibody.

[0023] In general, the above technical solution conceived by the present invention has the following beneficial effects compared with the prior art:

[0024] (1) The present invention provides an engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment, including microparticles produced by apoptosis of macrophages overexpressing tumor antigens, and small molecule active ingredients of drugs encapsulated by the microparticles, wherein the small molecule active ingredients of drugs are small molecule drugs that can reverse polarize M2 tumor-associated macrophages to M1; and the surface of the microparticles is also modified with M2 macrophage targeting molecules. After the macrophage-derived microparticles expressing tumor antigens are modified with M2 macrophage targeting molecules as the drug preparation carrier of the present invention, the drug molecules as active ingredients can be enriched at the tumor site and taken up by M2 tumor-associated macrophages, while reducing the off-target effect of the drug and the toxic side effects on the body, the reverse polarization effect on M2 tumor-associated macrophages is improved, the tumor immune microenvironment is improved, and the killing effect on tumor cells is enhanced.

[0025] (2) The drug-loaded microparticles of the present invention are loaded with drugs that can reverse polarize M2 macrophages to M1 macrophages, and can reverse polarize M2 tumor-associated macrophages to M1 macrophages. The M1 macrophages obtained after reverse polarization are used to process and present tumor antigens carried by the microparticles. At the same time, the macrophages that exist in large numbers in tumor tissues are used as antigen presenting cells (APCs), which not only challenges the conventional method of using dendritic cells (DCs) as antigen presenting cells (APCs), but also gives full play to the antigen presentation function of macrophages, stimulates the antigen-specific activation of CD8 T cells, and thus enhances the specific killing effect on tumor cells.

[0026] (3) The drug-loaded microparticle preparation provided by the present invention is loaded with drugs that can reverse polarize M2 macrophages to M1 macrophages, and can reverse polarize M2 tumor-associated macrophages to M1 macrophages. At the same time, the macrophages present in large numbers in tumor tissues are used as APCs. The antigen presentation function of macrophages also enhances the infiltration and activation of antigen-specific CD8 T cells and antigen-specific stem-like CD8 T cells in tumor tissues, which is more conducive to achieving long-term immune surveillance of tumors.

[0027] (4) The present invention first obtains engineered macrophages that stably overexpress tumor antigens through genetic engineering, and then co-incubates with small molecule effective ingredients of drugs and M2 macrophage targeting molecules after ultraviolet irradiation apoptosis, and collects the engineered macrophage drug-loaded microparticles. This preparation method is simple and has certain versatility. The present invention selects tumor-associated antigen genes for specific tumor types as target genes, or sequences the whole gene of the patient's individualized tumor tissue or peripheral blood, screens personalized tumor neoantigen genes as target genes, and obtains macrophages that stably overexpress the corresponding personalized tumor neoantigens through genetic engineering, thereby achieving personalized tumor treatment.

[0028] (5) The present invention uses macrophage microparticles as carriers. On the basis of better circulation stability, immunogenicity and biosafety, in some embodiments, macrophages that stably express different tumor antigens are obtained by lentiviral transfection, thereby constructing a platform that can be used for personalized tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A AFP expression in macrophages after transfection with AFP-expressing lentivirus;

[0030] Figure 1B AFP expression in AFP-expressing macrophage-derived microparticles;

[0031] Figure 1C The particle size of drug-loaded microparticles carrying antigens and modified with targeting peptides;

[0032] Figure 1D The zeta potential of drug-loaded microparticles carrying antigens and modified with targeting peptides;

[0033] Figure 1E Electron microscopy of drug-loaded microparticles carrying antigens and modified with targeting peptides;

[0034] Figure 2A The uptake of PKH26-labeled microparticles by macrophages in different tissues;

[0035] Figure 2B The uptake of PKH26-labeled microparticles by different cells in tumor tissue;

[0036] Figure 3A The expression of M1-related protein CD80 after R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide reverse polarized M2 macrophages;

[0037] Figure 3B The expression of M2-related protein CD206 after R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide reverse polarized M2 macrophages;

[0038] Figure 3C After the reverse polarized M2 macrophages were co-incubated with CD8 T cells, AFP 212 The number of specific CD8 T cells;

[0039] Figure 3D After the reverse polarized M2 macrophages were co-incubated with CD8 T cells, AFP 212 Activation of specific CD8 T cells;

[0040] Figure 3E After the reverse polarized M2 macrophages were co-incubated with CD8 T cells loaded with R848 microparticles carrying AFP antigen and modified with M2 targeting peptide, CD8 T cells killed Hepa1-6 tumor cells;

[0041] Figure 3F After the reverse polarized M2 macrophages were co-incubated with CD8 T cells loaded with R848 microparticles carrying AFP antigen and modified with M2 targeting peptide, CD8 T cells killed B16-OVA tumor cells;

[0042] Figure 4A The R848-loaded microparticles carrying the Adpgk neoantigen peptide and modified with the M2 targeting peptide reversely polarized M2 macrophages and then expressed the M1-related protein CD80;

[0043] Figure 4BThe expression of M2-related protein CD206 after R848-loaded microparticles carrying Adpgk neoantigen peptide and modified with M2 targeting peptide reverse polarized M2 macrophages;

[0044] Figure 4C The number of Adpgk-specific CD8 T cells after co-incubation with CD8 T cells after reverse polarization of M2 macrophages by R848-loaded microparticles carrying Adpgk neoantigen peptide and modified with M2 targeting peptide;

[0045] Figure 4D Activation of Adpgk-specific CD8 T cells after co-incubation of M2 macrophages reversely polarized by R848-loaded microparticles carrying Adpgk neoantigen peptide and modified with M2 targeting peptide with CD8 T cells;

[0046] Figure 5A The MeAIB-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide reverse polarized M2 macrophages and then expressed the M1-related protein CD80;

[0047] Figure 5B The expression of M2-related protein CD206 after MeAIB-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide reverse polarized M2 macrophages;

[0048] Figure 5C After the reverse polarized M2 macrophages were co-cultured with CD8 T cells, AFP 212 The number of specific CD8 T cells;

[0049] Figure 5D After the reverse polarized M2 macrophages were co-cultured with CD8 T cells, AFP 212 Activation of specific CD8 T cells;

[0050] Figure 5E After co-incubation of M2 macrophages reversely polarized by MeAIB microparticles carrying AFP antigen and modified with M2 targeting peptide with CD8 T cells, CD8 T cells killed Hepa1-6 tumor cells;

[0051] Fig. 5F After co-incubation of M2 macrophages reversely polarized by MeAIB microparticles carrying AFP antigen and modified with M2 targeting peptide with CD8 T cells, CD8 T cells killed B16-OVA tumor cells;

[0052] Fig. 6AThis is a picture of the tumor-bearing liver after the R848-loaded microparticles carrying the AFP antigen and modified with the M2 targeting peptide were administered into the tail vein of mice bearing Hepa1-6 orthotopic liver cancer;

[0053] Figure 6B The weight of tumor tissue after the R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide were administered into the tail vein of Hepa1-6 orthotopic liver cancer-bearing mice;

[0054] Figure 6C The survival time of mice bearing Hepa1-6 orthotopic liver cancer after tail vein administration of R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide;

[0055] Fig. 7A The number of AFP antigen-specific CD8 T cells in tumor tissues after tail vein administration of R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide to mice bearing Hepa1-6 orthotopic liver cancer;

[0056] Figure 7B The number of AFP antigen-specific CD8 T cells secreting IFNγ in tumor tissues after tail vein administration of R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide to mice bearing Hepa1-6 orthotopic liver cancer;

[0057] Figure 7C The number of AFP antigen-specific stem-like CD8 T cells in tumor tissues of mice bearing Hepa1-6 orthotopic liver cancer after tail vein administration of R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide;

[0058] Fig.7D The number of terminally exhausted CD8 T cells secreting granzyme B in tumor tissues after tail vein administration of R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide to mice bearing Hepa1-6 orthotopic liver cancer;

[0059] Fig. 8A After the drug-loaded microparticles carrying antigens and modified with M2 targeting peptides were administered into the tail vein of Hepa1-6 orthotopic liver cancer-bearing mice, the serum alanine aminotransferase level of the mice was measured;

[0060] Figure 8B The level of lactate dehydrogenase in the serum of mice bearing Hepa1-6 orthotopic liver cancer was determined after the drug-loaded microparticles carrying antigens and modified with M2 targeting peptides were administered into the tail vein;

[0061] Figure 8C Blood urea nitrogen content in the serum of mice bearing Hepa1-6 orthotopic liver cancer after drug-loaded microparticles carrying antigens and modified with M2 targeting peptides were administered into the tail vein;

[0062] Fig.8D Changes in the body weight of mice bearing Hepa1-6 orthotopic liver cancer during tail vein administration of drug-loaded microparticles carrying antigens and modified with M2 targeting peptides;

[0063] Fig. 9A This is a picture of the tumor-bearing liver of mice bearing Hepa1-6 orthotopic liver cancer after treatment with R848 microparticles carrying AFP antigen and modified with M2 targeting peptide combined with PD-1 antibody;

[0064] Fig. 9B The weight of tumor tissue in mice bearing Hepa1-6 orthotopic liver cancer treated with R848 microparticles carrying AFP antigen and modified with M2 targeting peptide combined with PD-1 antibody;

[0065] Fig. 9C The survival rate of mice bearing Hepa1-6 orthotopic liver cancer after treatment with R848 microparticles carrying AFP antigen and modified with M2 targeting peptide combined with PD-1 antibody. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0067] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0068] The present invention provides an engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment, comprising microparticles produced by apoptosis of macrophages that overexpress tumor antigens, and small molecule active ingredients of drugs encapsulated by the microparticles, wherein the small molecule active ingredients of drugs are small molecule drugs that can reverse polarize M2 tumor-associated macrophages to M1; the surface of the microparticles is also modified with M2 macrophage targeting molecules.

[0069] In some embodiments, the tumor antigen is overexpressed by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing; the macrophage is one of circulating monocytes derived from human peripheral blood, a human mononuclear cell line, mouse bone marrow-derived macrophages and a mouse monocyte / macrophage cell line.

[0070] In some embodiments, the mass ratio of the microparticles, the small molecule active ingredient of the drug and the M2 macrophage targeting molecule is 1000:(30-60):(2-5).

[0071] The engineered macrophages stably overexpressing tumor antigens of the present invention, where the tumor antigens can be medically recognized or tumor-associated antigens and tumor-specific antigens that can be identified by whole genome sequencing and exome sequencing. In some embodiments, the tumor-associated antigens are cancer testis antigens (including but not limited to NY-ESO-1), liver cancer antigens AFP, melanoma antigens (including but not limited to MAGE family, MART-1 and gp100) and prostate-specific antigens PSA and PAP. One or more. In some embodiments, the tumor antigens also include tumor neoantigens, wherein tumor neoantigens are antigenic peptides produced by tumor cell gene mutations that can be identified by genome sequencing technology, including one or more of Actn4, Adpgk, Ap3d1, Tubb3, Dag1, Eef2, Tnpo3, Tubb3, Reps1, Cpne1 and Cpsf3l. The small molecule active ingredient of the drug of the present invention is a small molecule drug that can reverse polarize M2-type tumor-associated macrophages to M1-type. In some embodiments, the small molecule active ingredient of the drug is an immune agonist small molecule drug, a metabolic small molecule or other small molecule drug with the function of reversing polarization M2 to M1. The immune agonist small molecule drug is one or more of STING agonist CDN, TLR agonist R848, R837 and poly(I:C) and AMPK activator; the metabolic small molecule drug is one or more of aromatic hydrocarbon receptor AhR inhibitor and glutamine transporter inhibitor; the other drugs include one or more of histamine receptor inhibitor and aspirin.

[0072] In some embodiments, the M2 macrophage targeting molecule is one or more of DSPE-PEG-M2pep, SR-B1 targeting peptide and DSPE-PEG-Man.

[0073] In some embodiments, the engineered macrophage drug-loaded microparticle preparation has a particle size of 300 to 500 nm. The drug-loaded microparticle preparation is nanosized, which is more conducive to the enrichment of the drug-loaded microparticle preparation at the tumor site, more effectively targeting and reverse polarization of M2 tumor-associated macrophages, restoring macrophages to tumor killing, improving the tumor suppressive immune microenvironment, effectively killing tumor cells, and will not cause any damage to normal tissues, avoiding the toxic side effects of using exogenous materials as carriers on the body.

[0074] The present invention also provides a method for preparing the engineered macrophage drug-loaded microparticle preparation, comprising the following steps:

[0075] S1: preparing engineered macrophages that stably overexpress the tumor antigen through genetic engineering;

[0076] S2: after inducing apoptosis of the engineered macrophages stably overexpressing the tumor antigen by ultraviolet irradiation, the engineered macrophages are mixed and incubated with a small molecule active ingredient of a drug, wherein the small molecule active ingredient of the drug is a small molecule drug that can reverse polarize M2 tumor-associated macrophages to M1, and drug-loaded microparticles overexpressing the tumor antigen are collected;

[0077] S3: incubating the drug-loaded microparticles overexpressing the tumor antigen with M2 macrophage targeting molecules, exchanging the M2 macrophage targeting molecules to the membrane surface of the drug-loaded microparticles through membrane phospholipid exchange, and collecting the engineered macrophage drug-loaded microparticle preparation.

[0078] In some embodiments, in step S1, engineered macrophages stably overexpressing the tumor antigen are prepared by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing. Preferably, a lentivirus overexpressing the tumor antigen is first prepared by lentiviral transfection, and then the lentivirus expressing the tumor antigen is used to transfect macrophages to screen for engineered macrophages stably overexpressing the tumor antigen.

[0079] In some embodiments, HEK 293T cells are transfected with a lentiviral packaging plasmid, a lentiviral envelope plasmid, and a vector plasmid carrying a tumor antigen gene as a target gene, and lentivirus overexpressing the tumor antigen is collected; wherein the lentiviral packaging plasmid is one or more of psPAX2, pMDLg / pRRE, and pRSV / Rev; the lentiviral envelope plasmid is one or more of pMD2.G-VSVG and pCMV-VSV-G; the vector plasmid carrying a tumor antigen gene as a target gene, wherein the tumor antigen gene is prepared from one or more tumor-associated antigens and tumor-specific antigens that are medically recognized or can be identified by whole genome sequencing and exome sequencing, including cancer testicular antigens (including NY-ESO-1), liver cancer antigens AFP, melanoma antigens (including gp100, Trp2, and MART1)), prostate-specific antigens PSA, and PAP. In some embodiments, the tumor antigen gene can also be prepared by tumor neoantigens, wherein the tumor neoantigens include one or more of Actn4, Adpgk, Ap3d1, Tubb3, Dag1, Eef2, Tnpo3, Tubb3, Reps1, Cpne1 and Cpsf3l.

[0080] In some embodiments, the vector plasmid carrying the tumor antigen gene as the target gene, wherein the tumor antigen gene can also be a personalized tumor neoantigen gene prepared by whole-genome sequencing of personalized tumor tissue and peripheral blood of cancer patients. Engineered macrophages that overexpress the patient's personalized tumor neoantigens and further construct engineered macrophage drug-loaded microparticle preparations, thereby achieving personalized treatment for patients.

[0081] In some embodiments, in step S1, the mass ratio of the lentiviral packaging plasmid, the lentiviral envelope plasmid, and the vector plasmid carrying the target gene is regulated to obtain a high titer lentivirus. In some embodiments, the lentiviral packaging plasmid, the lentiviral envelope plasmid, and the vector plasmid carrying the tumor antigen gene as the target gene are transfected into HEK293T cells at a mass ratio of 3:1:4.

[0082] In some embodiments, in step S1, the specific operation of collecting the lentivirus overexpressing the tumor antigen is: mixing the lentivirus packaging plasmid, the lentivirus envelope plasmid and the vector plasmid carrying the tumor antigen gene as the target gene in a mass ratio of 3:1:4, and culturing them with HEK293T cells in a sterile incubator at 37°C and 5% CO2 for 72 hours, and at 4°C, 8×10 4 The mixture was centrifuged at a centrifugal force of 1000 g for 2 h, and the precipitate was collected to obtain the lentivirus overexpressing the tumor antigen.

[0083] In some embodiments, in step S1, the ratio of the content of the macrophages to the content of the lentivirus expressing the tumor antigen is 5×10 5 pcs:(3~6)×10 6 PFU.

[0084] In some embodiments, in step S1, the specific operation of obtaining the engineered macrophages that stably overexpress the tumor antigen is: 5×10 5 RAW264.7 cells were inoculated into a cell culture plate and cultured in a sterile incubator at 37°C and 5% CO2. During the culture period, the old culture medium was discarded and supplemented with new culture medium. The type of culture medium was changed according to the culture conditions. Finally, the culture medium was replaced with a complete culture medium containing the screening agent and replaced every two days. Cells that survived for 10 days of continuous culture were engineered macrophages that stably overexpressed the tumor antigen.

[0085] In some embodiments, step S2 specifically comprises: subjecting the engineered macrophages stably overexpressing the tumor antigen to 300 J / m 2The cells were irradiated under ultraviolet light for 1 hour to induce apoptosis, mixed with the small molecule active ingredient of the drug at a final concentration of 150-250 μg / mL, and cultured in a sterile cell culture incubator at 37°C and 5% CO2 for 20-30 hours. The cells were centrifuged at 600 g for 10 minutes to remove cell debris, the supernatant was collected, and the supernatant was discarded after further centrifugation at 18000 g for 30 minutes. The precipitate was resuspended with sterile PBS to collect the drug-loaded microparticles overexpressing the tumor antigen.

[0086] In some embodiments, in step S3, the mass ratio of the drug-loaded microparticles overexpressing the tumor antigen to the M2 macrophage targeting molecule is (10-100):1.

[0087] In some embodiments, in steps S2 and S3, microparticles are collected at a centrifugal force of 500-20000 g at 4°C.

[0088] The present invention also provides a drug for treating tumors, which includes the engineered macrophage drug-loaded microparticle preparation. The drug is administered intravenously or intratumorally, and its dosage form includes an injection or a freeze-dried powder.

[0089] In some embodiments, the drug for treating tumors includes the engineered macrophage drug-loaded microparticle preparation and also includes an immunosuppressive molecule, wherein the immune checkpoint inhibitor is one or more of PD-1 antibody, PD-L1 antibody, CTL1-4 antibody and VISTA antibody. When the engineered macrophage drug-loaded microparticle preparation and the immune checkpoint inhibitor are specifically used, the two are administered separately, and the immune checkpoint inhibitor can be administered at a dose of 5-10 mg / kg.

[0090] The term "microparticles" as used herein refers to particles released by apoptotic macrophages.

[0091] In some embodiments of the present invention, HEK293T cells are transfected with a lentiviral packaging plasmid, a lentiviral envelope plasmid, and a vector plasmid carrying a tumor-specific antigen gene as a target gene to obtain a lentivirus that stably overexpresses the tumor antigen, and macrophages are transfected with the lentivirus that overexpresses the tumor antigen to screen macrophages that stably overexpress the tumor antigen, and after ultraviolet irradiation apoptosis, they are co-incubated with a small molecule effective ingredient of the drug and an M2 macrophage targeting molecule to collect the engineered macrophage drug-loaded microparticles. In addition to enriching the engineered macrophage drug-loaded microparticles at the tumor site and being taken up by M2 tumor-associated macrophages, thereby improving the reverse polarization effect on M2 tumor-associated macrophages and improving the tumor microenvironment, the M1 macrophages obtained after reverse polarization are used to process and present the tumor-associated antigens or tumor-specific antigens carried by the microparticles, thereby enhancing the killing effect on tumor cells and achieving personalized tumor treatment.

[0092] The drug-loaded microparticles of the present invention utilize macrophages present in large quantities in tumor tissues as antigen presenting cells (APCs), which not only challenges the conventional method of utilizing dendritic cells (DCs) as antigen presenting cells (APCs), but also because the engineered macrophages stably overexpress tumor antigens, they can fully exert the antigen presentation function of macrophages, stimulate the antigen-specific activation of CD8 T cells, and thus enhance the specific killing effect on tumor cells; in addition, the antigen presentation function of macrophages also enhances the infiltration and activation of antigen-specific stem-like CD8 T in tumor tissues, which is more conducive to achieving long-term immune surveillance of tumors.

[0093] The following are examples:

[0094] Various cells, reagents and experimental animals used in the following examples:

[0095] Hepa1-6 cells were purchased from Wuhan Boster Bioengineering Co., Ltd.; C57BL / 6 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; mouse macrophage cell lines RAW264.7 and HEK293T cells were purchased from Shanghai Cell Bank.

[0096] Toll-like receptor 7 / 8 agonist resiquimod (R848) was purchased from InvivoGen; DSPE-PEG-Mal was purchased from Shanghai Pengshuo Biotechnology Co., Ltd., M2pep was purchased from Hefei Guopi Biotechnology Co., Ltd.; psPAX2 and pM2.GVSVG were from Addgenge; expression plasmids pCDH-CMV-MCS-EF1-Puro-AFP and pCDH-Adpgk-Zeocin were constructed by conventional molecular biology methods; puromycin was purchased from Saiguo Biotechnology Co., Ltd.; IL-2 and IL-4 were purchased from Peprotech Biotechnology Co., Ltd.; MeAIB was purchased from MedChemExpress; PKH26 fluorescent dye was purchased from Sigma; and LDH kit was purchased from Abcam.

[0097] The following are examples:

[0098] Example 1 Engineered macrophage drug-loaded microparticles for treating tumors with high alpha-fetoprotein (AFP) expression and preparation method thereof

[0099] 1. Experimental Materials and Reagents

[0100] Mouse macrophage cell line RAW264.7, HEK293T cells, Toll-like receptor 7 / 8 agonist resiquimod (R848), DSPE-PEG-M2pep, and the UV device belong to the biological safety cabinet.

[0101] 2. Experimental Procedure

[0102] 1) RNA of MEF cells was extracted and reverse transcribed to obtain cDNA; CDS fragments were obtained by PCR amplification using AFP restriction primers; CDS fragments and expression vector pCDH-CMV-MCS-EF1-Puro were double-digested; double-digested products were connected by DNA ligase, and the AFP expression vector pCDH-CMV-MCS-EF1-Puro-AFP was obtained by screening the puromycin resistance of the pCDH-CMV-MCS-EF1-Puro vector and sequencing and identification.

[0103] Plasmids psPAX2, pM2.GVSVG, and pCDH-CMV-MCS-EF1-Puro-AFP were mixed evenly at a mass ratio of 3:1:4 (total amount of 16 μg), added to an EP tube containing 1 mL of opti-MEM medium, and incubated at 37°C for 5 min; at the same time, 20 μL of PEI and 1 mL of opti-MEM medium were incubated at 37°C for 5 min; the above liquids were mixed evenly and incubated at 37°C for another 15 min, and then added to 2×10 6 HEK293T cells were cultured in a sterile incubator at 37°C and 5% CO2 for 72 h.

[0104] Use a pipette to take the HEK293T cell supernatant in a clean bench and filter it with a 0.45 μm sterile filter to remove cell debris. Transfer the filtered supernatant into a sterile ultracentrifuge tube and incubate at 4°C, 8 × 10 4 g for 2 h, and the precipitate was collected to obtain the lentivirus expressing AFP. 5 RAW264.7 cells were seeded in a 6-well plate. The next day, the old culture medium was discarded and 900 μL serum-free culture medium was added. 6 About PFU of AFP lentivirus was cultured in a sterile incubator at 37°C and 5% CO2 for 6 hours, and then 100 μL of fetal bovine serum was added and supplemented with 2 mL of complete culture medium. The cells were incubated in a sterile incubator at 37°C and 5% CO2 for 24 hours. The old culture medium containing the lentivirus was discarded, and 3 mL of fresh complete culture medium was added to continue the culture. After 24 hours, the culture medium was replaced with a complete culture medium containing puromycin at a final concentration of 2 μg / mL. Fresh complete culture medium containing puromycin was replaced every two days. Cells that survived after 10 days of continuous culture were RAW264.7. AFP Trypsin digestion of RAW264.7 AFP , the cell pellet was obtained by centrifugation, and the cells were lysed by RIPA, 1×10 4 Centrifuge at rpm for 10 min, take the supernatant to obtain protein precipitate, cook the sample at 100°C for 10 min, add 5× protein loading buffer, and detect AFP expression by western blot.AFP The cell samples were the experimental group, the RAW264.7 cell samples were the control group 1, and the RAW264.7 cell samples transfected with the empty plasmid lentivirus (RAW264.7 EV ) was the control group 2, and the Hepa1-6 cell sample was the positive control.

[0105] 2) Adjust RAW264.7 AFP The cell concentration was 5 × 10 6 5 mL of cells were placed in a 10 cm cell culture dish and exposed to 300 J / m 2 Irradiate under ultraviolet light for 1 hour. Place in a sterile cell culture incubator at 37°C and 5% CO2. After 24 hours, apoptotic macrophages are separated. Centrifuge at 600g for 10 minutes to remove cell debris, collect the supernatant, centrifuge at 18000g for 30 minutes, discard the supernatant, resuspend the precipitate with sterile PBS, and collect the microparticles carrying AFP antigen. AFP , the samples were prepared according to the method in 1) above, and the expression of AFP was detected by western blot. EV Microparticles MPs isolated from apoptotic cell supernatant, MPs EV They were used as control groups 1 and 2 respectively, and Hepa1-6 cell samples were used as positive control.

[0106] 3) Weigh 10 mg of M2pep and dissolve it in 0.5 mL of PBS with a pH of 8.0, weigh 8.8 mg of DSPE-PEG-Mal and dissolve it in 0.5 mL of N, N-dimethylformamide (DMF), mix the two, add PBS with a pH of 8.0 to 12 mL, and stir the reaction at room temperature for 4 hours. Use a dialysis bag with a molecular weight cutoff of 3000 Da to dialyze in PBS, and after 24 hours, freeze-dry the liquid in the dialysis bag to obtain the M2 macrophage targeting peptide (DSPE-PEG-M2pep).

[0107] 4) Use the method in 2) above to process RAW264.7 AFP The cells were irradiated with UV light, and R848 was added at a final concentration of 200 μg / mL after the irradiation. After mixing, the cells were placed in a sterile cell culture incubator at 37°C and 5% CO2. After 24 hours, apoptotic macrophages were separated according to the method in 2) to obtain microparticles (R848@MPs AFP) and DSPE-PEG-M2pep at a mass ratio of 50:1 were cultured in PBS solution at 4°C for 24 h. After centrifugation at 18000g for 30 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL PBS. After centrifugation at 18000g for 30 min, the precipitate was resuspended in sterile PBS, and the drug-loaded microparticles R848@M2pep-MPs modified with M2 targeting peptide and carrying AFP antigen were collected. AFP According to the above treatment, R848 was not added, and the macrophage microparticles collected from the supernatant of RAW264.7 were not incubated with DSPE-PEG-M2pep as MPs, which was used as control group 1; R848 was not added, and the macrophage microparticles collected from the supernatant of RAW264.7 were incubated with DSPE-PEG-M2pep as M2pep-MPs, which was used as control group 2; R848 was not added, and RAW264.7 was not incubated with DSPE-PEG-M2pep as M2pep-MPs. AFP The macrophage microparticles collected from the supernatant were not incubated with DSPE-PEG-M2pep to form MPs. AFP , as control group 3; without adding R848, RAW264.7 AFP The macrophage microparticles collected from the supernatant were incubated with DSPE-PEG-M2pep to obtain M2pep-MPs. AFP , as control group 4; R848 was added, and the macrophage microparticles collected from the supernatant of RAW264.7 were not incubated with DSPE-PEG-M2pep to form R848@MPs, as control group 5; R848 was added, and the macrophage microparticles collected from the supernatant of RAW264.7 were incubated with DSPE-PEG-M2pep to form R848@M2pep-MPs, as control group 6; R848 was added, and RAW264.7 AFP The macrophage microparticles collected from the supernatant without incubation with DSPE-PEG-M2pep are R848@MPs AFP , as the control group7.

[0108] 5) The obtained microparticles were dissolved in PBS, and the particle size and zeta potential distribution were detected using a nanoparticle size and zeta potential instrument with a 633nm He-Ne laser. The specific setting conditions were: temperature of 25°C, and equilibrium time of 120s. At the same time, the copper mesh with the carbon film facing up was placed on a disposable PE glove, and a certain concentration of microparticles was dripped on the surface of the copper mesh, and balanced for 5 minutes. It was then fixed with 4% paraformaldehyde for 10 minutes, stained with phosphotungstic acid for 5 minutes, washed with PBS, and the liquid on the surface of the copper mesh was dried with filter paper, and the copper mesh was placed on the filter paper and left to stand overnight. The sample morphology was observed using TEM.

[0109] 3. Experimental results

[0110] The experimental results show that RAW264.7 AFPcell( Figure 1A ) and MPs AFP ( Figure 1B ) successfully expressed AFP antigen, and the average particle size distribution of various microparticles was about 400nm ( Figure 1C ), zeta potential is about -19mV ( Figure 1D ), indicating that the microparticles carrying antigens, DSPE-PEG-M2pep modification and loading of R848 do not substantially change the particle size and zeta potential of the microparticles. TEM images show ( Figure 1E ), the microparticles are irregular spherical in shape.

[0111] Example 2 Accumulation of microparticles in M2 tumor-associated macrophages

[0112] 1. Experimental Materials and Reagents

[0113] Hepa1-6 mouse hepatoma cells, PKH26 flow cytometry antibody, C57BL / 6 mice.

[0114] 2. Experimental Procedure

[0115] 1) Establishment of mouse Hepa1-6 orthotopic liver cancer model: 3×10 6 Hepa1-6 cells were used to construct a subcutaneous liver cancer model. When the tumor volume grew to about 500 mm 3 The tumor was peeled off and evenly cut into tumor masses of approximately 1 mm × 1 mm × 1 mm. Three to four tumor masses were inoculated into the liver of C57BL / 6 mice to establish a mouse Hepa1-6 orthotopic liver cancer tumor model.

[0116] 2) 15 days after model establishment, the tumor-bearing mice were randomly divided into 4 groups, with 4 mice in each group. AFP The experimental group was injected with 15 mg (protein amount) / kg of MPs into Hepa1-6 orthotopic liver cancer-bearing mice via the tail vein as the experimental group, the prepared PKH26-labeled MPs were injected with 15 mg (protein amount) / kg of MPs into Hepa1-6 orthotopic liver cancer-bearing mice via the tail vein as the control group 1, the prepared PKH26-labeled M2pep-MPs were injected with 15 mg (protein amount) / kg of MPs into Hepa1-6 orthotopic liver cancer-bearing mice via the tail vein as the control group 2, and the prepared PKH26-labeled MPs AFP The drug was injected into Hepa1-6 orthotopic liver cancer-bearing mice via the tail vein at a dose of 15 mg (protein amount) / kg as the control group 3.

[0117] 3) 24 hours after the tail vein injection, the mice in each group were killed, and the liver, spleen, lung, kidney and tumor were taken. The lung and tumor were cut as finely as possible with ophthalmic surgical scissors, digested in RPMI 1640 serum-free medium containing 0.08% collagenase I for 1 hour, squeezed through a metal screen, and then filtered twice with a 200-mesh nylon screen, lysed with red blood cell lysis buffer, and washed twice with PBS to obtain a single cell suspension. The liver, spleen and kidney were directly squeezed and ground with the inner core of a 10mL syringe, and then filtered twice with a 200-mesh screen, lysed with red blood cell lysis buffer, and washed twice with PBS to obtain a single cell suspension.

[0118] 4) Take 1×10 6 The fluorescent dye-labeled antibodies were added to each cell, and the fluorescence intensity of PKH26 in different cells was detected using CytoFLEXS flow cytometer. - For tumor cells, CD11b + F4 / 80 + Macrophages, CD11b + F4 / 80 + CD80 + M1 macrophages, CD11b + F4 / 80 + CD206 + M2 macrophages, CD45 + CD3 + For T cells, CD45 + F4 / 80 - CD11c + For dendritic cells, CD45 + CD11b + Gr1 + Myeloid-derived suppressor cells (MDSCs), CD45 + CD3 + CD4 + CD25 + Foxp3 + are regulatory T cells (Tregs).

[0119] 3. Experimental results

[0120] Depend on Figure 2A As shown in Figure 2, compared with liver Kupffer cells, spleen macrophages, lung macrophages and kidney macrophages, tumor-associated macrophages were able to take up more microparticles modified with targeting peptides; in tumor tissues ( Figure 2B ), compared with the microparticles without targeting peptide modification, the microparticles modified with targeting peptide were taken up more in M2 tumor-associated macrophages. This indicates that the microparticles modified with targeting peptide can effectively accumulate in the tumor site and have good targeting ability for M2 tumor-associated macrophages.

[0121] Example 3: Reprogramming of M2 macrophages by R848-loaded microparticles carrying AFP antigen and modified with targeting peptide

[0122] 1. Experimental Materials and Reagents

[0123] The RAW264.7 mouse macrophages and RAW264.7 expressing AFP antigen were used. AFP Mouse macrophages were the same as in Example 1, except for R848, IL-4, IL-2, CD8 T cell sorting kit, LDH kit, and fluorescently labeled flow cytometry antibodies.

[0124] 2. Experimental Procedure

[0125] 1) 5×10 5 RAW264.7 cells were inoculated in a six-well cell plate, and 20 ng / mL IL-4 was added for stimulation and cultured in a sterile incubator at 37° C. and 5% CO 2 for 24 h to obtain M2 macrophages. The preparation method of drug-loaded microparticles was the same as in Example 1.

[0126] 2) Add drug-loaded microparticles to M2 macrophages and incubate them in a sterile incubator at 37°C and 5% CO2. After 24 hours, the cells were collected and labeled with flow cytometry fluorescent antibodies. The expression of M1 and M2 related proteins was detected by CytoFLEX S flow cytometer. R848-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide (R848@M2pep-MPs AFP ) were used as the experimental group, the untreated M2 macrophages were used as control group 1, the M2 macrophages treated with non-drug-loaded microparticles (MPs) without AFP antigen modification and M2 targeting peptide were used as control group 2; the M2 macrophages treated with non-drug-loaded microparticles (M2pep-MPs) without AFP antigen modification and M2 targeting peptide were used as control group 3; the non-drug-loaded microparticles (MPs) carrying AFP antigen and M2 targeting peptide were used as control group 4. AFP ) treated M2 macrophages as control group 4; non-drug-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide (M2pep-MPs AFP ) were used as control group 5; free R848 was used as control group 6; M2 macrophages treated with R848-loaded microparticles (R848@MPs) without AFP antigen modification and M2 targeting peptide were used as control group 7; M2 macrophages treated with R848-loaded microparticles (R848@M2pep-MPs) without AFP antigen modification and M2 targeting peptide were used as control group 8; M2 macrophages treated with R848-loaded microparticles (R848@MPs AFP ) were used as the control group9.

[0127] 3) Prepare mouse spleen single cell suspension according to the method in Example 2, separate mouse spleen single cell suspension with Mojosort mouse CD8 T cell sorting kit and Mojosort magnetic pole to obtain CD8 T cells, resuspend with RPMI1640 complete medium containing 20ng / mL IL-2, and inoculate in 6-well plates for use. The M2 macrophages treated with drugs in 2) above and CD8 T cells were co-incubated in a sterile incubator at 37°C and 5% CO2 for 5 days at a ratio of 1:1, labeled with flow cytometry fluorescent antibodies, and the number and activation of antigen-specific CD8 T cells were detected by CytoFLEX S flow cytometer.

[0128] After the activated CD8 T cells were co-incubated with Hepa1-6 at a ratio of 10:1 in a sterile incubator at 37°C and 5% CO2 for 6 hours, the LDH concentration in the supernatant was detected by the LDH kit to calculate the killing efficiency of CD8 T cells against tumor cells.

[0129] 3. Experimental results

[0130] like FIG. 3A to FIG. 3F As shown, compared with unloaded microparticles, free R848, drug-loaded microparticles without AFP antigen and not modified with targeting peptide, drug-loaded microparticles without AFP antigen but modified with targeting peptide, and microparticles carrying AFP antigen but not modified with targeting peptide, the drug-loaded microparticles carrying AFP antigen and modified with targeting peptide in the experimental group can significantly upregulate the M1-related protein CD80 ( Figure 3A ), down-regulating M2-related protein CD206 ( Figure 3B ) expression, reverse polarization of M2 macrophages to M1 type; at the same time, the M2 macrophages treated with drug-loaded microparticles in the experimental group can significantly promote the proliferation of AFP antigen-specific CD8 T cells ( Figure 3C ),activation( Figure 3D ), and activated CD8 T cells only expressed AFP antigen Hepa1-6 tumor cells ( Figure 3E ) but not B16-OVA tumor cells expressing OVA ( Figure 3F ) has a more specific killing effect. It shows that the drug-loaded microparticles carrying tumor antigens and modified with M2 targeting peptides provided by the present invention can effectively reverse polarize M2 macrophages, and at the same time use the reverse polarized macrophages as antigen presenting cells to process and present the tumor antigens carried by the microparticles, stimulate the antigen-specific activation of CD8 T cells, and specifically kill tumor cells expressing the same antigens.

[0131] Example 4: Reprogramming of M2 macrophages by R848-loaded microparticles carrying Adpgk neoantigen peptide and modified targeting peptide

[0132] 1. Experimental Materials and Reagents

[0133] The RAW264.7 mouse macrophages used were the same as those in Example 1, and the R848, IL-4, IL-2, and CD8 T cell sorting kit fluorescently labeled flow cytometry antibodies were the same as those in Example 3.

[0134] 2. Experimental Procedure

[0135] 1) The DNA sequence of the antigen peptide Adpgk was inferred from the amino acid sequence of the antigen peptide; primers were designed for circular PCR, and the gene sequence of Adpgk was integrated into the expression vector pCDH-Zeocin to obtain the expression vector pCDH-Adpgk-Zeocin; sequencing was performed and expression identification was performed in HEK293T cells.

[0136] RAW264.7 expressing Adpgk neoantigen Adpgk Mouse macrophage construction method: psPAX2, pM2.GVSVG, and pCDH-Adpgk-Zeocin plasmids were mixed at a mass ratio of 3:1:4 (total amount of 16 μg) and then transfected into HEK293T cells. Bleomycin with a final concentration of 100 μg / mL was used to select macrophage cell lines that stably expressed Adpgk. The remaining detailed steps were the same as in Example 1 to prepare RAW264.7 expressing the Adpgk neoantigen. Adpgk Mouse macrophages: The method for inducing M2 macrophages is the same as in Example 3, and the method for preparing drug-loaded microparticles is the same as in Example 1.

[0137] 2) The reverse polarization treatment and detection method of M2 macrophages are the same as in Example 3. R848-loaded microparticles carrying Adpgk neoantigen and modified with M2 targeting peptide (R848@M2pep-MPs Adpgk ) were used as the experimental group, the untreated M2 macrophages were used as control group 1, the M2 macrophages treated with non-drug-loaded microparticles (MPs) without Adpgk neoantigen and M2 targeting peptide modification were used as control group 2; the M2 macrophages treated with non-drug-loaded microparticles (M2pep-MPs) without Adpgk neoantigen and M2 targeting peptide modification were used as control group 3; the non-drug-loaded microparticles (MPs) carrying Adpgk neoantigen and M2 targeting peptide modification were used as control group 4. Adpgk ) treated M2 macrophages as control group 4; non-drug-loaded microparticles carrying Adpgk neoantigen and modified with M2 targeting peptide (M2pep-MPs Adpgk) were used as control group 5; free R848 was used as control group 6; M2 macrophages treated with R848-loaded microparticles (R848@MPs) without Adpgk neoantigen modification and M2 targeting peptide were used as control group 7; M2 macrophages treated with R848-loaded microparticles (R848@M2pep-MPs) without Adpgk neoantigen modification and M2 targeting peptide were used as control group 8; M2 macrophages treated with R848-loaded microparticles (R848@M2pep-MPs) without Adpgk neoantigen modification and M2 targeting peptide were used as control group 9. Adpgk ) were used as the control group9.

[0138] 3) The method of isolating mouse spleen CD8 T cells and stimulating antigen-specific activation is the same as in Example 3.

[0139] 4) The detection of specific killing of tumor cells by CD8 T cells activated in the above 3) is the same as in Example 3.

[0140] 3. Experimental results

[0141] like FIG. 4A to FIG. 4D As shown, compared with unloaded microparticles, free R848, drug-loaded microparticles without Adpgk neoantigen and without targeting peptide modification, drug-loaded microparticles without Adpgk neoantigen but modified with targeting peptide, and microparticles carrying Adpgk neoantigen but not modified with targeting peptide, the drug-loaded microparticles carrying Adpgk neoantigen and modified with targeting peptide in the experimental group can significantly upregulate M1-related protein CD80 ( Figure 4A ), down-regulating M2-related protein CD206 ( Figure 4B ) expression, reverse polarizing M2 macrophages to M1 phenotype; at the same time, M2 macrophages treated with drug-loaded microparticles carrying Adpgk neoantigens and modified with targeting peptides can present Adpgk neoantigens to CD8 T cells, promoting the proliferation of Adpgk neoantigen-specific CD8 T cells ( Figure 4C ),activation( Figure 4D ). This indicates that the drug-loaded microparticles provided in this embodiment can also carry tumor neoantigens and effectively reverse polarize M2 macrophages. At the same time, the reverse polarized macrophages are used as antigen presenting cells to present the tumor neoantigens carried by the microparticles to CD8 T cells, stimulating the antigen-specific activation of CD8 T cells.

[0142] Example 5: Reprogramming of M2 macrophages by MeAIB microparticles carrying the AFP antigen and the inhibitor of glutamine transporter (SNAT2)

[0143] 1. Experimental Materials and Reagents

[0144] The RAW264.7 mouse macrophages and RAW264.7 expressing AFP antigen were used.AFP Mouse macrophages, MeAIB, IL-4, IL-2, CD8 T cell sorting kit, LDH kit, and fluorescently labeled flow cytometry antibodies are the same as those in Example 3.

[0145] 2. Experimental Procedure

[0146] 1) R848 was replaced by MeAIB, and the other steps of the drug-loaded microparticle collection method were the same as those in Example 1. The incubation concentration of MeAIB was 50 nM.

[0147] 2) The method for inducing M2 macrophages is the same as in Example 3, the method for treating M2 macrophages for reverse polarization and the method for detecting M2 macrophages are the same as in Example 3, and the MeAIB-loaded microparticles (MeAIB@M2pep-MPs) carrying AFP antigen and modified with M2 targeting peptides are AFP ) were used as the experimental group, the untreated M2 macrophages were used as the control group 1, the M2 macrophages treated with non-drug-loaded microparticles (MPs) without AFP antigen modification and M2 targeting peptide were used as the control group 2; the M2 macrophages treated with non-drug-loaded microparticles (M2pep-MPs) without AFP antigen modification and M2 targeting peptide were used as the control group 3; the non-drug-loaded microparticles (MPs) carrying AFP antigen and M2 targeting peptide were used as the control group 4. AFP ) treated M2 macrophages as control group 4; non-drug-loaded microparticles carrying AFP antigen and modified with M2 targeting peptide (M2pep-MPs AFP ) were used as control group 5; free R848 was used as control group 6; M2 macrophages treated with MeAIB microparticles (MeAIB@MPs) without AFP antigen and M2 targeting peptide modification were used as control group 7; M2 macrophages treated with MeAIB microparticles (MeAIB@M2pep-MPs) without AFP antigen and M2 targeting peptide modification were used as control group 8; M2 macrophages treated with MeAIB microparticles (MeAIB@MPs AFP ) were used as the control group9.

[0148] 3) The mouse CD8 T cell isolation method, activation method and activation detection method are the same as those in Example 3.

[0149] 4) The killing of tumor cells by CD8 T cells activated in the above 3) and the detection method are the same as those in Example 3.

[0150] 3. Experimental results

[0151] like FIG. 5A to FIG. 5FAs shown, compared with unloaded microparticles, free MeAIB, drug-loaded microparticles without AFP antigen and not modified with targeting peptide, drug-loaded microparticles without AFP antigen but modified with targeting peptide, and microparticles carrying AFP antigen but not modified with targeting peptide, the drug-loaded microparticles carrying AFP antigen and modified with targeting peptide in the experimental group can significantly upregulate the M1-related protein CD80 ( Figure 5A ), down-regulating M2-related protein CD206 ( Figure 5B ) expression, reverse polarization of M2 macrophages to M1 phenotype; at the same time, M2 macrophages treated with drug-loaded microparticles carrying AFP antigen and modified with targeting peptides can significantly promote the increase of antigen-specific CD8 T cells ( Figure 5C ),activation( Figure 5D ), and activated CD8 T cells only expressed AFP antigen Hepa1-6 tumor cells ( Figure 5E ) but not B16-OVA tumor cells expressing OVA ( Fig. 5F ) has a more specific killing effect. It shows that the microparticles carrying tumor antigens and modified with M2 targeting peptides provided in this embodiment can carry various drugs for reverse polarization of M2 macrophages to achieve reverse polarization from M2 to M1, and at the same time, the reverse polarized macrophages are used as antigen presenting cells to process and present the tumor antigens carried by the microparticles, stimulate the antigen-specific activation of CD8 T cells, and kill tumor cells expressing the same antigens.

[0152] Example 6: Inhibitory effect of drug-loaded microparticles carrying AFP antigen on Hepa1-6 in situ liver cancer in mice

[0153] 1. Experimental Materials and Reagents

[0154] The RAW264.7 cells used were the same as those in Example 1, the Hepa1-6 mouse liver cancer cells, the C57BL / 6 mice were the same as those in Example 2, and the R848 mice were the same as those in Example 2.

[0155] 2. Experimental Procedure

[0156] 1) Microparticle collection is the same as in Example 1.

[0157] 2) The construction of mouse Hepa1-6 orthotopic liver cancer model was the same as in Example 2.

[0158] 3) Ten days after model establishment, the tumor-bearing mice were divided into 10 groups, with 13 mice in each group, and the tail veins were injected with PBS, MPs, M2pep-MPs, and MPs AFP 、M2pep-MPs AFP , R848, R848@MPs, R848@M2pep-MPs, R848@MPs AFP and R848@M2pep-MPs AFPThe PBS-treated group was injected with 100 μL PBS; the unloaded microparticles (MPs, M2pep-MPs, MPs AFP 、M2pep-MPs AFP ) was administered at a protein dosage of 15 mg / kg, dissolved in 100 μL PBS; the R848 treatment group was administered at a dosage of 0.5 mg / kg; the drug-loaded microparticles (R848@MPs, R848@M2pep-MPs, R848@MPs AFP and R848@M2pep-MPs AFP ) R848 was administered at a dose of 0.5 mg / kg, dissolved in 100 μL PBS for administration). The mice were administered once every 3 days for a total of 6 times, and the weight of the mice was measured at a fixed time every day. After the administration, 8 mice were randomly selected from each group to continue the survival experiment. The remaining mice were anesthetized and killed by cervical dislocation, and the tumor tissues were removed, weighed and photographed. R848@M2pep-MPs AFP The tumor-bearing mice treated with PBS, MPs, M2pep-MPs, and MPs AFP 、M2pep-MPs AFP , R848, R848@MPs, R848@M2pep-MPs, R848@MPs AFP The tumor-bearing mice treated were control groups 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively.

[0159] 3. Experimental results

[0160] like Fig. 6A Tumor images and Figure 6B Tumor weight, R848@M2pep-MPs AFP The growth of Hepa1-6 in situ tumors was significantly inhibited, and its tumor inhibition effect was significantly better than that of the other groups. Figure 6C ) also shows R848@M2pep-MPs AFP The survival time of tumor-bearing mice was significantly prolonged, which was significantly better than that of other control groups.

[0161] Example 7: Drug-loaded microparticles carrying AFP antigen significantly improve the number and quality of CD8 T cells in Hepa1-6 in situ liver cancer tissues in mice

[0162] 1. Experimental Materials and Reagents

[0163] The RAW264.7 cells and R848 cells used were the same as those in Example 1, and the Hepa1-6 mouse liver cancer cells and C57BL / 6 mice were the same as those in Example 2.

[0164] 2. Experimental Procedure

[0165] 1) Microparticle collection is the same as in Example 1.

[0166] 2) The construction of mouse Hepa1-6 orthotopic liver cancer model was the same as in Example 2.

[0167] 3) Model construction and drug administration were the same as in Example 6. After drug administration, 5 mice were randomly selected from each group and anesthetized and then killed by cervical dislocation. The tumor tissue was removed and prepared into a single cell suspension. The preparation method was the same as in Example 2. After fluorescent antibody labeling, the number and activation of antigen-specific CD8 T cells and the number and differentiation of stem-like CD8 T cells were detected by CytoFLEX S flow cytometer. CD45 + CD3 + CD8 + AFP 212 -MHC-I tetramer + AFP antigen-specific CD8 T cells, CD45 + CD3 + CD8 + AFP 212 -MHC-I tetramer + IFNγ + For activated AFP antigen-specific CD8 T cells, CD45 + CD3 + CD8 + AFP 212 -MHC-I tetramer + PD-1 + TCF-1 + Stem-like CD8 T cells, CD45 + CD3 + CD8 + AFP 212 -MHC-I tetramer + PD-1 + TCF-1 - Granzyme B + Differentiated stem-like CD8 T cells. R848@M2pep-MPs AFP The tumor-bearing mice treated with PBS, MPs, M2pep-MPs, and MPs AFP 、M2pep-MPs AFP , R848, R848@MPs, R848@M2pep-MPs, R848@MPs AFP The tumor-bearing mice treated were control groups 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively.

[0168] 3. Experimental results

[0169] like 7A to 7D As shown, R848@M2pep-MPs AFP Significantly promoted the proliferation of AFP antigen-specific CD8T cells in tumor tissues ( Fig. 7A ) and activation( Figure 7B ), while increasing the number of AFP antigen-specific stem-like CD8 T cells ( Figure 7C ) and terminally exhausted CD8 T cells secreting granzyme B ( Fig.7D ), description R848@M2pep-MPs AFP The improvement effect on the number and quality of CD8 T cells was significantly better than that of the other control groups.

[0170] Example 8: Biosafety of Drug-Loaded Microparticles

[0171] 1. Experimental Materials and Reagents

[0172] The RAW264.7 cells and R848 cells used were the same as those in Example 1, and the Hepa1-6 mouse liver cancer cells and C57BL / 6 mice were the same as those in Example 2.

[0173] 2. Experimental Procedure

[0174] 1) Microparticle collection is the same as in Example 1.

[0175] 2) The construction of mouse Hepa1-6 orthotopic liver cancer model was the same as in Example 2.

[0176] 3) Model construction and drug administration were the same as in Example 6. The body weight of mice was measured every day during the drug administration. After the drug administration, venous blood of mice in each group was collected and centrifuged to obtain serum, and the serum levels of alanine aminotransferase, lactate dehydrogenase, and blood urea nitrogen were detected. R848@M2pep-MPs AFP The tumor-bearing mice treated with PBS, MPs, M2pep-MPs, and MPs AFP 、M2pep-MPs AFP , R848, R848@MPs, R848@M2pep-MPs, R848@MPs AFP The tumor-bearing mice treated were control groups 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively.

[0177] 3. Experimental results

[0178] like FIG. 8A to FIG. 8D As shown, compared with the control group mice injected with PBS, mice injected with R848@M2pep-MPs AFP Alanine aminotransferase in mouse serum ( Fig. 8A ), lactate dehydrogenase ( Figure 8B )、blood urea nitrogen( Figure 8C ) content and mouse body weight ( Fig.8D ) showed no significant changes, indicating that R848@M2pep-MPs AFP Intravenous administration does not cause liver, myocardial and kidney toxicity, and the drug-loaded microparticles carrying antigens and modified with M2 targeting peptides have no toxic side effects on the body.

[0179] Example 9: Drug-loaded microparticles carrying AFP antigen enhance the anti-liver cancer effect of PD-1 antibody

[0180] 1. Experimental Materials and Reagents

[0181] RAW264.7 cells and R848 are the same as in Example 1, and Hepa1-6 mouse liver cancer cells and C57BL / 6 mice are the same as in Example 2.

[0182] 2. Experimental Procedure

[0183] 1) Microparticle collection is the same as in Example 1.

[0184] 2) The construction of mouse Hepa1-6 orthotopic liver cancer model was the same as in Example 2.

[0185] 3) Ten days after model establishment, tumor-bearing mice were divided equally into PBS, R848@M2pep-MPs AFP , PBS+PD-1 antibody and R848@M2pep-MPs AFP +PD-1 antibody, a total of 4 groups, 13 mice in each group. The PBS treatment group was injected with 100 μL PBS via tail vein, once every three days, for a total of 6 times; R848@M2pep-MPs AFP The treatment group was injected with tail vein (R848 dose was 0.5 mg / kg), once every three days, for a total of 6 times; the PBS+PD-1 antibody treatment group was injected with both separately, PBS was injected with tail vein (100 μL PBS) and PD-1 antibody was injected the next day (dose was 5 mg / kg), PBS was injected once every three days, for a total of 6 times, and PD-1 antibody was injected once every four days, for a total of 4 times; R848@M2pep-MPs AFP The +PD-1 antibody treatment group received both drugs alone, and R848@M2pep-MPs AFP The next day after tail vein injection (R848 dose of 0.5 mg / kg), PD-1 antibody (dose of 5 mg / kg) was injected, where R848@M2pep-MPs AFP The mice were injected once every three days for a total of 6 times, and the PD-1 antibody was injected once every four days for a total of 4 times. After the end of the administration, 8 mice were randomly selected from each group to continue the survival experiment. The remaining mice were anesthetized and killed by cervical dislocation, and the tumor tissue was removed, weighed and photographed. R848@M2pep-MPsAFP +PD-1 antibody as the experimental group, PBS, R848@M2pep-MPs AFP The tumor-bearing mice treated with PBS+PD-1 antibody were control groups 1, 2, and 3, respectively.

[0186] 3. Experimental results

[0187] like Fig. 9A Tumor images and Fig. 9B Tumor weight, PD-1 antibody + R848@M2pep-MPs AFP The growth of Hepa1-6 in situ tumors was significantly inhibited, and its tumor inhibition effect was significantly better than that of the other groups. Fig. 9C ) also showed that PD-1 antibody + R848@M2pep-MPs AFP The survival time of tumor-bearing mice was significantly prolonged, which was significantly better than that of other control groups.

[0188] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment, characterized in that: It includes microparticles produced by apoptosis of macrophages that overexpress liver cancer antigen AFP or tumor neoantigen Adpgk, the microparticles overexpress liver cancer antigen AFP or tumor neoantigen Adpgk, and small molecule active ingredients of drugs encapsulated by the microparticles, wherein the small molecule active ingredients of drugs are small molecule drugs that can reverse polarize M2 tumor-associated macrophages to M1; the surface of the microparticles is also modified with M2 macrophage targeting molecules; The small molecule active ingredient of the drug is an immune stimulatory small molecule drug, a metabolic small molecule drug or other small molecule drug with reverse polarization M2 being M1; the immune stimulatory small molecule drug is one or more of STING agonist CDN, TLR agonist R848, R837 and poly(I:C); the metabolic small molecule drug is one or more of aromatic hydrocarbon receptor AhR inhibitor and glutamine transporter inhibitor; the other small molecule drug with reverse polarization M2 being M1 is a histamine receptor inhibitor; The M2 macrophage targeting molecule is one or more of DSPE-PEG-M2pep, SR-B1 targeting peptide and DSPE-PEG-Man; The mass ratio of the microparticles, the small molecule effective ingredient of the drug and the M2 macrophage targeting molecule is 1000:(30-60):(2-5).

2. The engineered macrophage drug-loaded microparticle preparation according to claim 1, characterized in that: Tumor antigens are overexpressed by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing; the macrophages are one of circulating monocytes derived from human peripheral blood, human mononuclear cell lines, mouse bone marrow-derived macrophages and mouse mononuclear macrophage cell lines.

3. The engineered macrophage drug-loaded microparticle preparation according to claim 1, characterized in that: The particle size of the engineered macrophage drug-loaded microparticles is 300-500 nm.

4. A method for preparing the engineered macrophage drug-loaded microparticle preparation according to any one of claims 1 to 3, characterized in that: The steps include: S1: Genetically engineered macrophages that stably overexpress tumor antigens. S2: After inducing apoptosis of engineered macrophages that stably overexpress tumor antigens by ultraviolet irradiation, the macrophages are mixed and incubated with a small molecule active ingredient of the drug, wherein the small molecule active ingredient of the drug is a small molecule drug that can reverse polarize M2 tumor-associated macrophages to M1, and drug-loaded microparticles that overexpress tumor antigens are collected; S3: incubating the drug-loaded microparticles overexpressing tumor antigens with M2 macrophage targeting molecules, exchanging the M2 macrophage targeting molecules to the membrane surface of the drug-loaded microparticles through membrane phospholipid exchange, and collecting the engineered macrophage drug-loaded microparticle preparation.

5. The method for preparing the engineered macrophage drug-loaded microparticle preparation according to claim 4, characterized in that: In step S1, engineered macrophages that stably overexpress tumor antigens are prepared by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing.

6. The method for preparing the engineered macrophage drug-loaded microparticle preparation according to claim 4, characterized in that: In step S3, the mass ratio of the drug-loaded microparticles overexpressing tumor antigens to the M2 macrophage targeting molecule is (10-100): 1; In steps S2 and S3, the collection conditions are: collection at 4°C and a centrifugal force of 500-20000g.

7. A drug for treating liver cancer, characterized in that: It comprises the engineered macrophage drug-loaded microparticle preparation as described in any one of claims 1 to 3.

8. The drug according to claim 7, characterized in that The drug also includes an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is one or more of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, and a VISTA antibody.

Images