Preparation method and application of engineered neutrophil liposome drug for enhancing anti-tumor immune effect

Abstract

The invention discloses a preparation method and application of an engineered neutrophil liposome drug for enhancing an anti-tumor immune effect, and finds that the combination of a neutrophil membrane liposome carrying CXCL9 and a CAR-T cell targeting B7-H3 has a synergistic treatment effect on solid tumors such as lung cancer for the first time. The invention provides a brand new effective drug combination strategy for the treatment of solid tumors such as lung cancer, and has important scientific significance and clinical application value in the technical field of development of drugs for treating solid tumors such as lung cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, it relates to a preparation method and application of an engineered neutrophil liposome drug for enhancing anti-tumor immune effects. Background Art

[0002] Currently, lung cancer remains one of the malignant tumors with the highest incidence and mortality rates, and the overall five-year survival rate for advanced lung cancer is only about 15%. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and nearly 75% of NSCLC patients are found to be in the middle and advanced stages at the time of diagnosis, without the opportunity for surgery. The preferred treatment option is platinum-based doublet chemotherapy, and the two-year survival rate is only 17% - 24%. Therefore, the existing treatment methods for lung cancer cannot meet the clinical needs. Exploring new treatment strategies for the clinical treatment of lung cancer has clear scientific value and clinical significance.

[0003] In recent years, CAR-T cell therapy has made breakthrough progress in hematological malignancies (such as leukemia and lymphoma). However, CAR-T therapy has not achieved satisfactory results in solid tumors (lung cancer). Due to its complex tumor microenvironment, solid tumors (lung cancer) result in insufficient infiltration of CAR-T cells, and thus CAR-T cells cannot exert their effector functions. Therefore, promoting the infiltration of CAR-T cells into lung cancer is the key to improving the efficacy of CAR-T therapy for solid tumors. Systemic application of cytokines can cause serious side effects, partly because of limited drug exposure in tumors and overstimulation of lymphocytes in healthy tissues. Therefore, a local tumor microenvironment delivery approach is essential. Summary of the Invention

[0004] In view of this, aiming at the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a preparation method and application of an engineered neutrophil liposome drug for enhancing anti-tumor immune effects.

[0005] The present invention creatively constructs a neutrophil membrane liposome carrying chemokine (CXCL9) (CXCL9-LNP), and combines it with CAR-T cells for the treatment of solid tumors such as lung cancer. In addition, the present invention unexpectedly discovers that the combination of the engineered neutrophil membrane liposome and CAR-T cells has a synergistic therapeutic effect on the treatment of solid tumors such as lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer. The present invention provides a new solution to solve the problem of difficult infiltration of CAR-T cells into solid tumors.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention purpose:

[0007] The first aspect of the present invention provides an anti-tumor pharmaceutical composition.

[0008] Furthermore, the pharmaceutical composition comprises engineered neutrophil membrane liposomes and CAR-T cells;

[0009] The engineered neutrophil membrane liposomes are neutrophil membrane liposomes carrying CXCL9;

[0010] The CAR in the CAR-T cells comprises a monoclonal antibody targeting B7-H3, a CD8α transmembrane region, a CD28 intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain;

[0011] The amino acid sequence of the heavy chain variable region of the monoclonal antibody targeting B7-H3 is as shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region of the monoclonal antibody targeting B7-H3 is as shown in SEQ ID NO:2;

[0012] The amino acid sequence of the CD8α transmembrane region is as shown in SEQ ID NO:3;

[0013] The amino acid sequence of the CD28 intracellular co-stimulatory domain is as shown in SEQ ID NO:4;

[0014] The amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:5.

[0015] Furthermore, the method for constructing the neutrophil membrane liposomes carrying CXCL9 comprises:

[0016] Introducing CXCL9 onto neutrophil membrane proteins by using a lentiviral transfection method to construct neutrophils with stable CXCL9-carrying cell membranes;

[0017] Subjecting the neutrophils with stable CXCL9-carrying cell membranes to cell disruption, membrane separation, and liposome formation respectively to obtain neutrophil membrane liposomes carrying CXCL9;

[0018] Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer.

[0019] Furthermore, the dosage ratio of the engineered neutrophil membrane liposomes to CAR-T cells is (0.16 mg - 0.32 mg) : (2×10 6 - 4×10 6 CAR-T cells).

[0020] In a specific embodiment of the present invention, the inventors of the present invention for the first time discovered that the combination of neutrophil membrane liposomes carrying CXCL9 and CAR-T cells targeting B7-H3 has a synergistic therapeutic effect on the treatment of solid tumors such as lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer, etc., and has no obvious toxic and side effects in vivo and has good biosafety.

[0021] In a specific embodiment of the present invention, the present invention verified the synergistic effect of the combination of the neutrophil membrane liposomes carrying CXCL9 and the CAR-T cells targeting B7-H3 on the treatment of solid tumors such as lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer, etc. by the Chou-Talalay method (Kim's formula).

[0022] The Kim's formula is mainly used for quantitative analysis of the therapeutic effect of drug combination. Specifically, the Kim's formula is Q = E a+b / (E a +E b -E a ×E b ), where E a+b is the inhibition rate of the combined drug, and E a and E b are the inhibition rates of the two drugs (drug a and drug b) used alone, respectively. When Q < 0.85, it is antagonistic; when 0.85 ≤ Q < 1.15, it is additive; when Q ≥ 1.15, it is synergistic). The verified Q = 1.25 > 1.15, that is, the combination of the neutrophil membrane liposomes carrying CXCL9 and the CAR-T cells targeting B7-H3 in the present invention has a synergistic therapeutic effect on the treatment of solid tumors such as lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer, etc.

[0023] In the present invention, the treatment refers to delaying the development of the disease, preventing the development of the disease and / or reducing the severity of the symptoms that will develop or are expected to develop. Therefore, the treatment includes improving the existing disease symptoms, preventing additional symptoms, improving or preventing the potential metabolic causes of the symptoms, inhibiting the disorder or disease, for example, preventing the development of the disorder or disease, alleviating the disorder or disease, regressing the disorder or disease, alleviating the conditions caused by the disease or disorder, or stopping the symptoms of the disease or disorder.

[0024] In some embodiments, the method of lentiviral transfection is used to introduce CXCL9 onto the neutrophil membrane protein, and the construction of neutrophils with stable CXCL9-carrying cell membranes specifically includes: isolation and culture of neutrophils, construction and packaging of lentiviral vectors, and lentiviral transfection of neutrophils.

[0025] In some embodiments, the construction and packaging of the lentiviral vector include: cloning the CXCL9 gene fragment into the lentiviral vector to construct a lentiviral vector plasmid expressing CXCL9. Through molecular biology techniques such as restriction digestion and ligation, the CXCL9 gene is correctly ligated to the regulatory elements of the lentiviral vector to ensure correct expression in cells. The constructed lentiviral vector plasmid is co-transfected with the packaging plasmid and the envelope plasmid into 293T cells. Using liposome transfection reagents, etc., the three plasmids are introduced into 293T cells, and the 293T cells are continuously cultured after transfection to carry out the packaging and production of lentivirus. At an appropriate time after transfection, the culture supernatant of 293T cells is collected, which contains the packaged lentiviral particles. The lentivirus is concentrated by methods such as ultracentrifugation and ultrafiltration to increase the virus titer for subsequent transfection experiments.

[0026] In some embodiments, the lentiviral transfection of neutrophils includes: before transfection, adjusting the neutrophils to an appropriate cell density, mixing the concentrated lentivirus with an appropriate amount of transfection reagent to form a lentivirus-transfection reagent complex. The role of the transfection reagent is to help the lentivirus enter neutrophils better. The lentivirus-transfection reagent complex is added to the neutrophil culture system and gently mixed to ensure full contact between the complex and the cells. Incubate to enable the lentivirus to infect neutrophils and integrate the CXCL9 gene into the cell genome.

[0027] In some embodiments, any suitable method can be used to break cells for cell disruption, such as the repeated freeze-thaw method (repeatedly freezing and thawing the cells in liquid nitrogen and a 37°C water bath several times), the ultrasonic disruption method (setting appropriate ultrasonic parameters such as power and time), or the mechanical homogenization method, etc., to rupture the cells and release the contents.

[0028] In some embodiments, the membrane separation includes centrifuging the disrupted cell suspension, such as first centrifuging to remove unbroken cells and large cell debris, and then transferring the supernatant to a new centrifuge tube for high-speed centrifugation to precipitate the cell membrane.

[0029] In some embodiments, the liposome formation includes resuspending the cell membrane precipitate with a suitable buffer (such as PBS), and reforming the cell membrane into a liposome structure by the extrusion method (using a liposome extruder and selecting a filter membrane with an appropriate pore size, such as 100 nm) or the ultrasonic method, etc., to obtain neutrophil membrane liposomes carrying CXCL9.

[0030] In some embodiments, a variety of methods can be employed to facilitate liposome formation, including but not limited to: the extrusion method and the sonication method. In a specific embodiment, the extrusion method refers to using a liposome extruder to extrude a mixed solution through polycarbonate membranes with different pore sizes (such as 100 nm, 200 nm, etc.) multiple times to obtain liposomes with uniform sizes. In a specific embodiment, the sonication method refers to using a sonicator to perform sonication on the mixed solution, and the sonication time and power are adjusted according to the actual situation to form stable liposomes.

[0031] In the present invention, there is no particular limitation on the specific construction method of the neutrophil cell membrane liposomes carrying CXCL9. Those skilled in the art can use conventional methods disclosed in the prior art to prepare the neutrophil cell membrane liposomes carrying CXCL9, and routinely adjust the parameters in the above methods according to the actual experimental situation, and these methods will all fall within the protection scope of the present invention.

[0032] The second aspect of the present invention provides an antitumor pharmaceutical preparation.

[0033] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the first aspect of the present invention;

[0034] Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer.

[0035] Furthermore, the dosage form of the pharmaceutical preparation is a non-parenteral dosage form or a parenteral dosage form.

[0036] Furthermore, the non-parenteral dosage form is an injectable dosage form, a respiratory dosage form, a cavity dosage form, a mucosal dosage form or a skin dosage form;

[0037] Optionally, the parenteral dosage form is a granule, a capsule, a solution, a tablet, a powder, a sustained-release agent, an emulsion, a suspension or a drop.

[0038] In some embodiments, the injectable dosage form includes but is not limited to: various injections such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and intracavitary injection; the respiratory dosage form includes but is not limited to: sprays, aerosols, powder aerosols, etc.; the cavity dosage form includes but is not limited to: suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucosal dosage form includes but is not limited to: eye drops, nasal drops, ophthalmic ointments, gargles, sublingual tablets, adhesive tablets, film dressings, etc.; the skin dosage form includes but is not limited to: topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0039] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may further comprise a pharmaceutically acceptable carrier and / or excipient. Specifically, the pharmaceutically acceptable carrier and / or excipient include, but are not limited to: surfactants, humectants, adsorbent carriers, diluents, fillers, binders, lubricants, disintegrants.

[0040] In some embodiments, the diluents include, but are not limited to: sodium chloride, glucose, lactose, urea, starch, water, etc.

[0041] In some embodiments, the binders include, but are not limited to: dextrin, maltodextrin, sucrose, starch, pregelatinized starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and its salts, xanthan gum, hydroxypropyl cellulose, etc.

[0042] In some embodiments, the surfactants include, but are not limited to: sodium dodecyl sulfate, monoglyceryl stearate, cetyl alcohol, etc.

[0043] In some embodiments, the humectants include, but are not limited to: glycerol, starch, etc.

[0044] In some embodiments, the adsorbent carriers include, but are not limited to: lactose, starch, bentonite, saponite, etc.

[0045] In some embodiments, the lubricants include, but are not limited to: talc, calcium and magnesium stearate, zinc stearate, polyethylene glycol, polyoxyethylene monostearate, sucrose monolaurate, sodium dodecyl sulfate, etc.

[0046] In some embodiments, the fillers include, but are not limited to: xylitol, sorbitol, mannitol, maltose, glucose, lactose, sucrose, dextrin, starch, etc.

[0047] In some embodiments, the disintegrants include, but are not limited to: sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, cross-linked vinylpyrrolidone, cross-linked sodium carboxymethylcellulose, soybean polysaccharide, etc.

[0048] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may further comprise other drugs or reagents that can be used to treat solid tumors such as lung cancer or assist in the treatment of solid tumors such as lung cancer. There are no particular limitations on the other drugs or reagents, as long as they can produce the expected therapeutic or adjuvant therapeutic effects on solid tumors such as lung cancer, they will fall within the scope of protection of the present invention.

[0049] In some embodiments, the other drugs or reagents that can be used to treat solid tumors such as lung cancer or assist in the treatment of solid tumors such as lung cancer include, but are not limited to, chemotherapeutic drugs. Exemplarily, the chemotherapeutic drugs include, but are not limited to: platinum drugs (e.g., cisplatin, carboplatin, nedaplatin, lobaplatin), antimetabolic drugs (e.g., pemetrexed, gemcitabine), alkaloid drugs (e.g., vinorelbine, etoposide, paclitaxel, docetaxel), and antibiotic drugs (e.g., doxorubicin, epirubicin).

[0050] In some embodiments, the suitable dosage of the pharmaceutical composition or pharmaceutical preparation of the present invention can be prescribed in various ways according to factors such as the formulation method, administration method, age, weight, gender, morbidity, diet, administration time, administration route, excretion rate, and sensitivity of the patient. A skilled doctor can usually easily determine the prescription and the desired therapeutically effective dosage as long as it can produce the expected therapeutic effect on solid tumors such as lung cancer. Such dosages are within the protection scope of the present invention.

[0051] The third aspect of the present invention provides a method for constructing the neutrophil membrane liposome carrying CXCL9 described in the first aspect of the present invention.

[0052] Further, the method includes:

[0053] CXCL9 is introduced onto the neutrophil membrane protein by using the lentiviral transfection method to construct neutrophils with the cell membrane stably carrying CXCL9;

[0054] The neutrophils with the cell membrane stably carrying CXCL9 are respectively subjected to cell disruption, membrane separation, and liposome formation to obtain the neutrophil membrane liposome carrying CXCL9.

[0055] In the specific embodiments of the present invention, the construction of the neutrophils with the cell membrane stably carrying CXCL9 includes the following steps: preparation of neutrophils with the cell membrane stably carrying CXCL9 (dHL-60-CXCL9), and preparation of the neutrophil membrane liposome carrying CXCL9 (CXCL9-LNP).

[0056] Among them, the preparation of neutrophils stably carrying CXCL9 (dHL-60-CXCL9) in the cell membrane includes the following steps: Co-transfect 293T cells with pLV3-CMV-CXCL9 (human)-CD63 (human)-EGFP-Puro plasmid, pMD2.G plasmid, and psPAX2 plasmid for virus packaging. After 48 hours, collect the supernatant, and after centrifugation and filtration, infect HL-60 cells. After 48 hours of infection, add puromycin for screening. After continuous screening for 3 days, detect the CXCL9 / GFP expression effect of HL-60-CXCL9 cells by laser confocal microscopy, flow cytometry, and Western blot. The prepared cells are HL-60-CXCL9, and the differentiated cells are dHL-60-CXCL9.

[0057] Among them, the preparation of neutrophil cell membrane liposomes carrying CXCL9 (CXCL9-LNP) includes the following steps:

[0058] (1) Collect a large number of neutrophils dHL-60-CXCL9 cells, resuspend them with PBS, centrifuge at 1800 rpm for 5 minutes to remove the supernatant, and place about 2 - 3×10 7 cells in a -80 °C refrigerator for storage. (2) Take an appropriate amount of Membrane Protein Extraction Reagent A from Beyotime, add PMSF within a few minutes before use to make the final concentration of PMSF 1 mM. Add 1 mL of Membrane Protein Extraction Reagent A added with PMSF before use to dHL-60-CXCL9 cells, gently and fully suspend the cells, and place them on ice for 10 - 15 minutes. (3) Place the above samples in liquid nitrogen and a 37 °C water bath respectively; freeze-thaw repeatedly several times in sequence, and then take a small amount of the sample to detect the degree of cell lysis under a microscope. If the degree of cell lysis is less than 70%, the number of freeze-thaw cycles can be increased until the degree of cell lysis is greater than 70%. (4) Centrifuge at 4 °C and 700 g for 10 minutes, carefully collect the supernatant into a new centrifuge tube. Do not touch the precipitate when aspirating the supernatant, and about 30 - 50 μL of the supernatant can be left unaspirated to ensure a high purity of the aspirated supernatant. (5) Centrifuge at 4 °C and 14000 g for 30 minutes, the precipitate is cell membrane fragments, discard the supernatant, resuspend with 1 mL of PBS, and measure its concentration with NanoDrop. (6) Next, a series of explorations were carried out on the ultrasonic intensity of the ultrasonic instrument, and finally an ultrasonic intensity of 15% was selected, with 3 s on and 2 s off for a total of 5 minutes, which can ultrasonically break the neutrophil cell membrane into cell membrane liposomes with a particle size of 100 - 200 nm and uniform size, that is, neutrophil cell membrane liposomes carrying CXCL9 (CXCL9-LNP).

[0059] In addition, the present invention also provides a method for preparing neutrophil cell membrane liposomes (LNP), and the method comprises the following steps: collecting neutrophils, extracting the cell membranes of neutrophils, and forming neutrophil cell membrane liposomes.

[0060] In a specific embodiment of the present invention, the method for preparing the LNP comprises the following steps: (1) Extract a large number of neutrophils, resuspend them with PBS, centrifuge at 1800 rpm for 5 min, discard the supernatant, and store about 2 - 3×10 7 cells in a -80°C refrigerator. (2) Take an appropriate amount of Membrane Protein Extraction Reagent A from Beyotime, add PMSF within several minutes before use to make the final concentration of PMSF 1 mM. Add 1 mL of Membrane Protein Extraction Reagent A added with PMSF before use to the neutrophils, gently and fully suspend the cells, and place them on ice for 10 - 15 minutes. (3) Place the above sample in liquid nitrogen and a 37°C water bath; freeze-thaw repeatedly for several times in sequence, and then take a small amount of the sample to detect the degree of cell lysis under a microscope. If the degree of cell lysis is less than 70%, the number of freeze-thaw cycles can be increased until the degree of cell lysis is greater than 70%. (4) Centrifuge at 700 g for 10 minutes at 4°C, carefully collect the supernatant into a new centrifuge tube. Do not touch the precipitate when aspirating the supernatant, and about 30 - 50 μL of the supernatant can be left unaspirated to ensure a high purity of the aspirated supernatant. (5) Centrifuge at 14000 g for 30 minutes at 4°C, the precipitate is cell membrane fragments, discard the supernatant, resuspend with 1 mL of PBS, and measure its concentration with NanoDrop. (6) Next, a series of explorations were carried out on the ultrasonic intensity of the ultrasonic instrument, and finally an ultrasonic intensity of 15% was selected, with 3 s on and 2 s off for a total of 5 minutes, which can ultrasonically break the neutrophil cell membrane into neutrophil cell membrane liposomes with a particle size of 100 - 200 nm and uniform size.

[0061] The fourth aspect of the present invention provides a method for non-therapeutically inhibiting the growth and / or promoting the apoptosis of tumor cells or tumor organoids in vitro.

[0062] Furthermore, the method comprises: treating tumor cells or tumor organoids with the pharmaceutical composition described in the first aspect of the present invention or the pharmaceutical preparation described in the second aspect of the present invention;

[0063] Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer.

[0064] In a specific embodiment of the present invention, the present invention has experimentally demonstrated that the combination of neutrophil cell membrane liposomes carrying CXCL9 and CAR-T cells targeting B7-H3 has a synergistic therapeutic effect on the treatment of solid tumors such as lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer. Therefore, the pharmaceutical composition or pharmaceutical preparation can be used as an inhibitor for inhibiting the growth and proliferation of tumor cells or tumor organoids for non-therapeutic purposes in the scientific research field, for example, to more deeply study the growth and metabolic mechanisms or behaviors of tumor cell lines or tumor organoids, and to screen potential drugs that can be used to treat lung cancer, etc.

[0065] The fifth aspect of the present invention provides the following applications in any aspect:

[0066] (1) The application of the combination of the engineered neutrophil cell membrane liposomes and CAR-T cells described in the first aspect of the present invention in the preparation of a drug for treating tumors;

[0067] (2) The application of the combination of the engineered neutrophil cell membrane liposomes and CAR-T cells described in the first aspect of the present invention in the preparation of a pharmaceutical composition for treating tumors;

[0068] (3) The application of the combination of the engineered neutrophil cell membrane liposomes and CAR-T cells described in the first aspect of the present invention in the preparation of a pharmaceutical preparation for treating tumors;

[0069] (4) The application of the combination of the engineered neutrophil cell membrane liposomes and CAR-T cells described in the first aspect of the present invention in non-therapeutically inhibiting the growth of tumor cells or tumor organoids and / or promoting the apoptosis of tumor cells or tumor organoids in vitro.

[0070] Further, the dosage ratio of the engineered neutrophil cell membrane liposomes to CAR-T cells is (0.16 mg - 0.32 mg) : (2×10 6 -4×10 6 CAR-T cells);

[0071] Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer.

[0072] In addition, the present invention also provides a method for treating tumors, the method comprising: administering a therapeutically effective amount of the pharmaceutical composition described in the first aspect of the present invention or the pharmaceutical preparation described in the second aspect of the present invention to a subject in need;

[0073] Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer.

[0074] In some embodiments, the subject refers to mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. In a specific embodiment of the present invention, the subject is a human.

[0075] In some embodiments, the therapeutically effective amount refers to the amount of a drug, pharmaceutical composition, or pharmaceutical preparation that is effective in producing the desired prophylactic, alleviating, or therapeutic effect. Depending on factors such as the drug, pharmaceutical composition, or pharmaceutical preparation, the symptoms and their severity, the age of the treated mammal, etc., the amount of the drug, pharmaceutical composition, or pharmaceutical preparation of the present invention that reaches the effective amount will also vary. However, those of ordinary skill in the art can routinely determine the specific dosage according to the knowledge in the art they possess in combination with the content disclosed in the present invention. Doses that can produce the above effects are all within the protection scope of the present invention.

[0076] In some embodiments, the administration methods of the pharmaceutical composition or pharmaceutical preparation include, but are not limited to: intravenous injection, subcutaneous injection, intramuscular injection, rectal administration, nasal administration, ear administration, intramedullary administration, intra-articular administration, intrapleural administration, intra-arterial administration, oral administration, etc.

[0077] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0078] The present invention for the first time discovers that the combination of neutrophil cell membrane liposomes carrying CXCL9 and CAR-T cells targeting B7-H3 has a synergistic therapeutic effect on the treatment of solid tumors such as lung cancer, and has no obvious toxic side effects in vivo and has good biosafety. The inhibitory effect of this combined pharmaceutical composition on solid tumors such as lung cancer is significantly better than that of a single drug. The present invention provides a brand-new and effective drug combination strategy for the treatment of solid tumors such as lung cancer, and has important scientific significance and clinical application value in the technical field of the development of drugs for the treatment of solid tumors such as lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1Neutrophils infiltrate the lung cancer tissue microenvironment through the CXCR2-IL8 axis. Among them, Figure A: Detection of CXCR2 levels in mouse neutrophils; Figure B: CXCR2 levels in human neutrophils (dHL60); Figures C-E: Detection of IL8 levels in the supernatant after starvation stimulation of lung cancer cells by Elisa; Figure F: Schematic diagram of Transwell migration experiment; Figures G and H: Flow cytometry counting in the lower chamber of Transwell experiment; Figure I: Detection of the expression of CXCR2 in neutrophil membrane liposomes by Western blot; Figure J: A549-GFP tumor sphere experiment, PKH26 fluorescently labeled neutrophil membrane liposomes (LNP), and the CXCR2 inhibitor (SB225002) was added to the experimental group; n = 3, *P < 0.05, **P < 0.01, ***P < 0.001;

[0080] Figure 2 Neutrophil membrane liposomes successfully target lung cancer. Among them, Figures A-B: Detection of the change in fluorescence intensity of LNP after PKH26 staining by microplate reader; Figures C-E: Changes in fluorescence intensity of lung cancer tissue and mouse heart, liver, spleen, lung, and kidney at 24 h, 48 h, 72 h, and 96 h after intravenous injection of LNP-PKH26 into tumor-bearing C57 mice (LLC lung cancer cells);

[0081] Figure 3 Characterization and identification of engineered neutrophil membrane liposomes. Among them, Figure A: Confocal detection of GFP expression in HL-60-CXCL9 cells overexpressing CXCL9; Figure B: Flow cytometry detection of GFP expression in dHL-60-CXCL9; Figures C-E: Basic characterization and identification of the particle size, zeta potential, and electron microscopy of CXCL9-LNP; Figure F: Detection of GFP levels in CXCL9-LNP by microplate reader; Figure G: Detection of CXCL9 levels in CXCL9-LNP by Elisa; Figure H: Detection of CXCL9 expression in CXCL9-LNP by Western blot, and the data are expressed as mean ± SEM; ***P < 0.001;

[0082] Figure 4: Engineered neutrophil membrane liposomes successfully chemotax CAR-T cells without affecting the function of CAR-T cells. Among them, Figure A: Schematic diagram of Transwell; Figure B: Crystal violet staining of cells in the lower chamber of Transwell; Figure C: Flow cytometry count of cells in the lower chamber of Transwell; Figures D-E: CFSE proliferation assay of CAR-T cells after co-culture with LNP / CXCL9-LNP respectively; Figure F: Apoptosis index assay of CAR-T cells after co-culture with LNP / CXCL9-LNP respectively; Figure G: Detection of surface immunosuppressive receptors (Tim3, Lag3, PD-1) of CAR-T cells after co-culture with LNP / CXCL9-LNP respectively; Figures H-K: After CAR-T cells were co-cultured with LNP / CXCL9-LNP for 24 h, they were co-cultured with A549 cells for killing assay (E:T = 1:1 / 1:2). Data are presented as mean ± SEM; **p<0.01, ***p<0.001, ns, no statistical significance;

[0083] Figure 5 : Engineered neutrophil membrane liposomes do not affect the proliferation, migration, and invasion of lung cancer cells. Among them, Figures A-B: RTCA assay of the effect of LNP / CXCL9-LNP on the proliferation of A549 and H1299 cells; Figures C-D: Transwell assay of the effect of LNP / CXCL9-LNP on the migration and invasion of A549 and H1299 cells, n = 3; ns, no statistical significance;

[0084] Figure 6 : Engineered neutrophil membrane liposomes combined with CAR-T cells enhance the anti-lung cancer efficacy. Among them, Figure A: Schematic diagram of the NCG mouse subcutaneous lung cancer xenograft model and treatment protocol; Figure B: Solid figure of subcutaneous xenograft tumor; Figure C: Curve of subcutaneous xenograft tumor volume change; Figure D: Statistical chart of subcutaneous xenograft tumor weight; Figure E: Flow cytometry detection of the number of tumor-infiltrating T cells, n = 5; *P<0.05, **P<0.01, ***P<0.001, ns, no statistical significance;

[0085] Figure 7 : Engineered neutrophil membrane liposomes combined with CAR-T cell therapy have no obvious toxic and side effects. Among them, Figure A: Continuous monitoring of mouse body weight; Figure B: HE staining of mouse heart, liver, spleen, lung, and kidney, n = 5; ns: no statistical significance;

[0086] Figure 8 : Schematic diagram of the structure of CAR. Among them, LTR: Long terminal repeat sequence; scFv: Single-chain variable fragment;

[0087] Figure 9: Engineered CXCL9-LNP can recruit more CAR-T cells to effectively kill lung cancer, gastric cancer and colon cancer cells, among which, Figure A: Schematic diagram of Transwell killing experiment; Figure B: Staining of surviving tumor cells in the Transwell lower chamber; Figure C: Count statistics of surviving tumor cells in the Transwell lower chamber, data are expressed as mean ± SEM; ***p < 0.001, ns, not statistically significant. DETAILED DESCRIPTION

[0088] The present invention is further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0089] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0090] Example 1 Neutrophils infiltrate the lung cancer tissue microenvironment via the CXCR2-IL8 axis

[0091] 1. Experimental Materials

[0092] LLC cells, A549 cells, HL-60 cells and H1299 cells were purchased from Procell Life Science Co., Ltd., China. Anti-mouse Ly6G, anti-mouse CD11b, anti-mouse CXCR2: Biolegend, USA. Human and mouse IL8 detection kits: Jiangsu Enzyme Immunoassay. CXCR2 antagonist (SB225002): MCE. Percoll separation solution: GE Healthcare, USA. Transwell chamber (5μm): Corning, USA. Anti-CXCR2 antibody: Abcam. PKH26 red fluorescence cell membrane staining kit (D0030): Solarbio. Membrane protein extraction reagent: Shanghai Bio-Tech Biotechnology Co., Ltd.

[0093] 2. Isolation of Mouse Bone Marrow Neutrophils

[0094] (1) Autoclave surgical scissors and forceps in advance, pre-cool calcium- and magnesium-free HBSS, and prepare 20 mL and 1 mL syringes, 60 mm culture dishes. All the following operations are carried out in a laminar flow hood. (2) Euthanize the mouse, spray an appropriate amount of 70% alcohol to wet the fur, cut open the back skin with scissors, take out its femur and tibia, remove the surface muscle completely, and place them in a small dish containing pre-cooled HBSS for standby. (3) Cut off both ends of the bone with scissors. Hold the bone with forceps in the left hand, and use a syringe containing 10 mL HBSS in the right hand to wash the bone marrow into a 15 mL centrifuge tube. Then mix the solution evenly with a pipette gun, filter it through a 40 μm filter into a new 50 mL centrifuge tube to remove impurities. (4) Centrifuge at 1800 rpm for 5 min. At the same time, prepare 1× red blood cell lysis buffer and 62% Percoll separation solution at a dosage of 3 mL per mouse. Take a sterile 15 mL tube, add 9 mL ddH2O and 1 mL 10× red blood cell lysis stock solution, and shake well. Add 9 mL Percoll stock solution and 1 mL 10× PBS into a sterile 15 mL tube, shake well to obtain 100% Percoll. Then take 6.2 mL 100% Percoll and mix it with 3.8 mL 1× PBS and shake well to obtain 62% Percoll. Mark the tube clearly and store it in the dark at 4°C. (5) Remove the supernatant of the centrifuged cells, add 3 mL 1× red blood cell lysis buffer to each tube, shake well, and incubate in the dark at room temperature for 6 - 8 min. During this period, the tube can be taken out and gently flicked on the tube wall to lyse the red blood cells completely. (6) Add HBSS with a volume three times that of the red blood cell lysis buffer to terminate red blood cell lysis. After shaking well, centrifuge at 1800 rpm for 5 min and discard the supernatant. (7) Add 3 mL calcium- and magnesium-free HBSS solution to resuspend the cell pellet and transfer it to a new 15 mL tube. Use a pipette gun to disperse it until there are no obvious clumps. Slowly add 3 mL 62% Percoll to the bottom of the cell suspension. Do not add it too fast, and obvious stratification can be seen. (8) Pre-cool a centrifuge at 4°C, balance the centrifuge tubes, set the acceleration and deceleration to 0, and centrifuge at 2500 rpm for 35 min. (9) After centrifugation, carefully discard the supernatant in the tube, add HBSS for washing, centrifuge at 1800 rpm for 5 min, discard the supernatant. The precipitate is mouse neutrophils. Add 1 mL HBSS to resuspend the cell pellet and count. (10) Take out 1×10 5 cells, centrifuge at 1800 rpm for 5 min, remove the supernatant, dilute anti-mouse Ly6G and anti-mouse CD11b to 1 μg / mL with PBS, add 50 μL antibody dilution to resuspend the cell pellet, shake well, incubate at 4°C for 30 min, wash and filter it into a flow cytometry tube, and detect the purity of neutrophils by flow cytometry. If the proportion of double-positive cells is greater than 90%, it can be used for subsequent experiments.

[0095] 3. Induction of human neutrophils (dHL-60)

[0096] (1) HL-60 cell culture; (2) Transfer the cell suspension into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fresh medium to resuspend the cells, and count the cells under a microscope using a hemocytometer; (3) Add 2.5×10 2 cells to a 25 cm 6 culture flask and supplement the medium to 10 mL; (4) Add 130 μL of DMSO (1.3% of the total volume), and mix well; (5) Place it in an incubator at 37°C and 5% CO2 for 5 - 7 days to become neutrophil-like cells (dHL-60).

[0097] 4. Flow cytometry detection of the expression of neutrophil IL8 receptor CXCR2

[0098] (1) Take 5×10 5 mouse bone marrow neutrophils, centrifuge at 1800 rpm for 5 min, discard the supernatant, dilute anti-mouse Ly6G, anti-mouse CD11b, and anti-mouse CXCR2 to 1 μg / mL with PBS, add 50 μL of antibody dilution to resuspend the cell pellet, shake well, incubate at 4°C for 30 min, wash and filter into a flow cytometry tube, and detect the expression of CXCR2 in neutrophils by machine. (2) For 5×10 5 human neutrophils (dHL-60), repeat the above steps with anti-human CXCR2.

[0099] 5. ELISA detection of tumor supernatant IL8

[0100] (1) Culture human lung cancer cells (A549, H1299) and mouse lung cancer cells (LLC). After 1:2 cell passage and adhesion, replace with serum-free medium and starve for 48 h. (2) After 48 h, collect the supernatants of lung cancer cells (A549, H1299, LLC) and store them at -80°C. (3) Detect according to the detection steps provided by the detection kit.

[0101] 6. Transwell migration assay

[0102] (1) Add 600 μL of PBS to a 24-well plate. After assembling a 5 μm chamber, add 200 μL of PBS to the chamber, place it in an incubator at 37°C for membrane equilibration for 1 h, and discard the PBS in the well. (2) Take an appropriate amount of neutrophils, wash them with PBS, centrifuge at 1800 rpm for 5 min, discard the supernatant, and resuspend the pellet with 1640 basal medium. (3) Set up groups: Control group: Add 100 μL of cell suspension (1×10 6Cells) + 100 μL of 1640 basal medium, and 600 μL of 1640 basal medium was added to the lower chamber; A549-CM / LLC-CM treatment group: 100 μL of cell suspension (1×10 6 Cells) + 100 μL of 1640 basal medium, and 600 μL of the supernatant after A549 / LLC cell starvation treatment was added to the lower chamber; CXCR2 antagonist (SB225002) group: 100 μL of cell suspension + 100 μL of 1640 basal medium (containing 4 μM SB225002) was added to the upper chamber, and 600 μL of the supernatant after A549 / LLC cell starvation treatment was added to the lower chamber. (4) Samples were added according to the corresponding groups and placed in an incubator at 37 °C for 40 min. (5) The upper chamber was taken out, and the cells in the lower chamber were collected and counted by flow cytometry.

[0103] 7. Preparation of neutrophil cell membrane lipid nanoparticles (Lipid Nanoparticle, LNP)

[0104] (1) A large number of neutrophils were extracted, resuspended in PBS, centrifuged at 1800 rpm for 5 min to remove the supernatant, and about 2 - 3×10 7 cells were placed in a -80 °C refrigerator for storage. (2) An appropriate amount of Beyotime membrane protein extraction reagent A was taken, and PMSF was added within a few minutes before use to make the final concentration of PMSF 1 mM. 1 mL of membrane protein extraction reagent A added with PMSF before use was added to the neutrophils, and the cells were gently and fully suspended, and placed on ice for 10 - 15 minutes. (3) The above samples were placed in liquid nitrogen and a 37 °C water bath; they were repeatedly frozen and thawed several times in sequence, and then a small amount of the sample was taken to detect the degree of cell breakage under a microscope. If the degree of cell breakage is less than 70%, the number of freeze-thaw cycles can be increased until the degree of cell breakage is greater than 70%. (4) Centrifuge at 4 °C and 700 g for 10 minutes, and carefully collect the supernatant into a new centrifuge tube. Do not touch the precipitate when aspirating the supernatant, and about 30 - 50 μL of the supernatant can be left unaspirated to ensure the high purity of the aspirated supernatant. (5) Centrifuge at 4 °C and 14000 g for 30 minutes, the precipitate is the cell membrane fragments, discard the supernatant, resuspend with 1 mL of PBS, and measure its concentration with NanoDrop. (6) Next, a series of explorations were carried out on the ultrasonic intensity of the ultrasonic instrument, and finally an ultrasonic intensity of 15% was selected, with 3 s on and 2 s off for a total of 5 minutes, which can ultrasonically break the neutrophil cell membrane into neutrophil cell membrane lipid nanoparticles (LNP) with a particle size of 100 - 200 nm and uniform size.

[0105] 8. PKH26 staining of neutrophil cell membrane lipid nanoparticles

[0106] (1) Add the extracted LNP to 1 mL of diluent C, and gently pipette to mix evenly to prepare a 2× suspension. (2) Immediately before staining, add 4 μL of PKH26 solution to 1 mL of diluent C, and mix well to prepare a 2× staining solution. (3) Quickly add 1 mL of 2× liposome suspension to 1 mL of 2× staining solution, and immediately mix evenly and quickly with a pipette gun. Incubate the mixed solution at 25 °C for 2 - 5 min, and gently invert the centrifuge tube regularly to ensure thorough mixing at 25 °C. (4) Add an equal volume of serum (2 mL) to terminate the staining reaction, and incubate for 1 min to bind the excess dye. (5) Centrifuge the stained liposomes at 14000 g for 30 min at 4 °C, and discard the supernatant. Resuspend with 1 mL of PBS and centrifuge at 14000 g for 30 min at 4 °C for one wash, and resuspend with 1 mL of PBS to obtain LNP-PKH26.

[0107] 9. Protein Blot (Western Blot) Experiment

[0108] (1) Cell culture and induction of activation were the same as before. Total proteins of each group were extracted by the above method. (2) The stock protein solutions of each group were diluted to the same concentration with ddH2O, mixed with protein loading buffer, and heated to boiling for 10 min to form protein loading solutions. (3) The protein molecular weight Marker was taken out from the -20 °C freezer and slowly melted on ice. (4) Assembly of the gel plate: Align the two gel plates (glass plates), fix them in the clamping plate on the horizontal tabletop and clamp tightly (make the bottom surfaces flush to prevent leakage of the gel), pad with a rubber gasket, vertically fix the clamping plate on the gel rack, and prepare for pouring the gel. (5) Preparation of the gel (first prepare the separating gel, and then prepare the stacking gel after the separating gel solidifies). (6) Pouring the gel: After the separating gel is prepared, mix well to avoid forming bubbles, and then immediately slowly add it to the gel plate along the glass plate wall with a pipette. Stop pouring when it reaches about 1 - 2 mm below the green frame, and then add isopropanol to cover it (to prevent the oxidation of the separating gel by oxygen in the air). Observe carefully. When a clear boundary line appears between the isopropanol and the separating gel, it indicates that the separating gel has solidified. Wash off the isopropanol, pour out the upper layer of ddH2O, and blot it dry with filter paper. Add the prepared stacking gel on top of the separating gel. When it reaches 1 - 2 mm from the top of the gel plate, quickly insert the comb vertically to avoid forming bubbles. After the stacking gel solidifies, remove the clamping plate. (7) Installation of the electrophoresis tank: Remove the gel plate, place it on both sides of the electrode plates of the electrophoresis tank with the small plate facing inward. Insert the electrode plates into the electrode clamping plates and clamp tightly. Place it in the electrophoresis tank. Hold the comb with both hands and gently pull it out. Pour the electrophoresis buffer into the electrophoresis tank, fill the inner tank completely, and the outer tank should exceed the electrode wires. Use a pipette to suck out the bubbles in the inner tank and blow open each loading well. (8) Loading and electrophoresis: Slowly add the Marker and protein loading solutions to the loading wells with a micropipette to avoid sample overflow. Cover the lid of the electrophoresis tank, insert the electrodes, and start electrophoresis. First, apply a constant voltage of 80 V until the bromophenol blue migrates to the boundary between the stacking gel and the separating gel, and then apply a constant voltage of 120 V until the bromophenol blue migrates to the bottom of the gel plate. (9) Transfer: After electrophoresis, turn off the power, take out the gel plate, cut the gel on the glass plate. First, pry off the small plate with a gel cutting plate, then cut off the stacking gel, and then select the cutting range according to the Marker and cut the gel into a size of 8 cm × 2 cm (the same as the NC membrane and filter paper). Immerse the NC membrane, thick filter paper, and gel in a petri dish containing wet transfer buffer for 30 min. Then place 1 layer of thick filter paper on the positive electrode plate (transparent plate) of the transfer tank, drive out the bubbles with a glass rod, place the NC membrane on it with the smooth side facing up, drive out the bubbles, then place the gel with the large surface facing up, cover it with another layer of thick filter paper, drive out the bubbles, cover the negative electrode plate, and start the transfer; apply a constant current of 0.2A, 120 min; (10) Blocking: After the transfer is completed, turn off the power supply, take out the NC membrane, place the NC membrane with the gel side up in a petri dish containing TBST, wash it on a horizontal shaker for 1 - 2 min to wash away the wet transfer solution on the NC membrane, pour out the TBST, add 5% skim milk blocking solution, and block it on a horizontal shaker at room temperature for 2 h; (11) Primary antibody incubation: Make the blocked NC membrane gel side fully contact with the diluted primary antibody, and slowly shake it overnight on a vertical shaker in a 4°C cold storage; (12) Secondary antibody incubation: After taking out the NC membrane, add TBST, wash it on a horizontal shaker 5 min × 3 times, add the diluted secondary antibody, and incubate it on a horizontal shaker at room temperature for 1 h; (13) Color development: Place the NC membrane in TBST, wash it on a horizontal shaker 4 times, 5 min each time, and scan the NC membrane with a Tanon ECL luminescence imaging system.

[0109] 10. Tumor sphere experiment

[0110] (1) Add 0.15 g of agarose to 10 mL of 1640 basal medium, autoclave it at 120°C for 20 minutes, and then cover each well of the 96-well plate with 50 mL of hot agarose solution and cool it to room temperature. (2) Inoculate 3000 A549-GFP cells of 200 μL into the agarose-covered 96-well plate and further incubate for 4 days. (3) After the formation of tumor spheres, change the well plate medium to serum-free 1640 medium, and change the CXCR2 antagonist (SB225002) treatment group to serum-free 1640 medium (containing 4 μM SB225002). (4) After 24 h, add 50 μL (0.8 mg / mL) of LNP-PKH26, and then observe it through a fluorescence microscope.

[0111] 11. Statistical analysis

[0112] All experimental data were statistically analyzed using GraphPad Prism 8 software and expressed as mean ± standard deviation (Mean ± SD). When comparing two groups of data, the t-test method was used; when comparing multiple groups of data, one-way ANOVA was used, and further Tukey test was used. A P value < 0.05 was used as the standard for significant difference.

[0113] 12. Experimental results

[0114] First, we collected mouse neutrophils and human neutrophils (dHL-60) to detect the expression of the IL8 receptor CXCR2 on neutrophils by flow cytometry. The flow cytometry results showed that neutrophils highly expressed CXCR2( Figure 1A, B). Next, the supernatants of A549, H1299, and LLC lung cancer cells after 48 hours of starvation were collected, and the level of IL8 secreted by lung cancer cells was detected using an Elisa kit. The data showed that compared with the supernatants of lung cancer cells cultured normally, lung cancer cells could secrete a large amount of IL8 after starvation stimulation. Figure 1 C-E).

[0115] To verify that lung cancer cells recruit neutrophils through IL8 / CXCR2, we placed neutrophils / neutrophils + CXCR2 antagonist (SB225002) in the upper chamber of a Transwell experiment and added the supernatant of starved lung cancer cells to the lower chamber. After 2 hours, the cells in the lower chamber were collected and accurately counted by flow cytometry. We found that the supernatant of starved lung cancer cells could chemotactically attract a large number of neutrophils. After introducing the CXCR2 antagonist (SB225002), the number of migrating neutrophils showed a significant decrease, indicating that lung cancer cells recruit neutrophils by releasing IL8. Figure 1 F-H).

[0116] Next, we prepared neutrophil membrane liposomes (LNP), and Western blot detection showed that the extracted neutrophil membrane liposomes expressed CXCR2. Figure 1 I). This indicates its ability to target lung cancer tissue. Therefore, we constructed a tumor sphere model of A549-GFP cells in vitro. After the spheres formed, the serum-free medium was replaced for starvation. After 24 hours, LNP stained with PKH26 was added, and the CXCR2 antagonist (SB225002) was additionally added to the experimental group. Observation with a fluorescence microscope showed that LNP could be recruited by lung cancer cell tumor spheres, and after introducing the CXCR2 antagonist, this phenomenon was significantly reduced, indicating that neutrophil membrane liposomes also target lung cancer tissue through the IL8 / CXCR2 pathway. Figure 1 J).

[0117] Example 2 Successful targeting of neutrophil membrane liposomes to lung cancer in mice

[0118] 1. Experimental materials

[0119] C57 mice: Animal Center of Xuzhou Medical University.

[0120] 2. Preparation of neutrophil membrane liposomes (LNP)

[0121] The preparation method of neutrophil membrane liposomes and PKH26 staining was the same as before.

[0122] 3. Establishment of subcutaneous transplanted tumors of mouse LLC lung cancer

[0123] (1) Tumor cell preparation: When the LLC cells grow to 80 - 90% confluence at the bottom of the dish, digest the cells with trypsin, resuspend the cells with PBS, centrifuge at 800 rpm for 5 min, discard the supernatant to obtain cell pellet, repeat the resuspension and centrifugation steps twice, dilute the cells with PBS to 3×10 6 / mL, prepare 120 μL for each tube, and keep it on ice for later use. (2) Mouse preparation: Prepare healthy adult 6-week-old C57 / 6 male mice in advance, remove the hair on the right leg to the lumbar and abdominal regions of the mice to fully expose the inoculation site. (3) Inoculation: Wipe the inoculation site of the mice with 75% alcohol cotton ball, shake the cell suspension thoroughly, draw 100 μL of cell suspension with a 1 mL insulin syringe, and keep the number of inoculated LLC cells at 3×10 5 / mouse, the inoculation site is subcutaneous in the right lumbar and abdominal region. After inoculation, an obvious skin bump can be seen.

[0124] 4. Experimental results

[0125] To further detect whether neutrophil membrane liposomes can successfully target the lung cancer site in vivo, we constructed a subcutaneous xenograft tumor model of LLC lung cancer cells in wild-type C57 mice. We first detected the change in the fluorescence intensity of LNP after PKH26 staining and found that the fluorescence intensity could remain relatively stable within 144 h ( Figure 2 A, B). Next, we intravenously injected LNP into tumor-bearing mice via the vein. After 24 h, the mice were dissected, and the enrichment of the tumor site and important tissues and organs was observed by in vivo imaging. It was found that neutrophil membrane lipids could target lung cancer tissues ( Figure 2 C, D). At the same time, we detected the change in fluorescence intensity at 48 h, 72 h, and 96 h, and the results showed that the enrichment of neutrophil membrane liposomes at the lung cancer site could remain relatively stable for a long time ( Figure 2 E, F).

[0126] Example 3 Characterization and Identification of Engineered Neutrophil Membrane Liposomes

[0127] 1. Experimental materials

[0128] Anti-CXCL9 antibody: Abclonal Company. CXCL9 recombinant protein: Proteintech Company, USA.

[0129] 2. Lentiviral transfection of HL-60 cells

[0130] The pLV3-CMV-CXCL9 (human)-CD63 (human)-EGFP-Puro plasmid (Miaoling Biology, P51183), pMD2.G plasmid (Addgene, #12259), and psPAX2 plasmid (Addgene #12260) were co-transfected into 293T cells for virus packaging. The supernatant was collected 48 h later, centrifuged and filtered, and then used to infect HL-60 cells. Puromycin was added for screening 48 h after infection. After continuous screening for 3 days, the expression of CXCL9 / GFP in HL-60-CXCL9 cells was detected by confocal laser scanning microscopy, flow cytometry, and Western blot, and the cells were prepared as HL-60-CXCL9.

[0131] Among them, the ratio of the pLV3-CMV-CXCL9 (human)-CD63 (human)-EGFP-Puro plasmid, pMD2.G plasmid, and psPAX2 plasmid was 3:2:3.

[0132] 3. Confocal detection

[0133] (1) Neutrophil lineage cells (dHL-60-CXCL9) on the 5th day of induction were diluted with HBSS to 1×10 5 / 100 μL, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded to obtain cell pellets. (2) The cell pellets were resuspended in 1640 medium. Glass slides coated with polylysine were placed at the bottom of a 24-well plate, and the cell suspension was added; then the plate was incubated in an incubator at 37 °C. (3) After 4 h, the supernatant of each group was discarded, and the glass slides were incubated with 100 μL of Sytoxgreen working solution. After 20 min of incubation at room temperature in the dark, the supernatant was discarded, and the glass slides were inverted and placed on the glass slides, and then sealed with nail polish for later confocal observation of the NETs structure. (4) Mouse neutrophils were isolated, and the conditioned medium of LLC cells was used as the experimental condition. The specific experimental methods and detection methods were as above.

[0134] 4. Preparation of CXCL9-LNP

[0135] (1) A large number of neutrophil lineage dHL-60-CXCL9 cells were collected, resuspended in PBS, centrifuged at 1800 rpm for 5 min to remove the supernatant, and about 2 - 3×10 7The cells were stored in a -80 °C refrigerator. (2) Appropriate amount of Biyuntian Membrane Protein Extraction Reagent A was taken, and PMSF was added within several minutes before use to make the final concentration of PMSF 1 mM. 1 mL of Membrane Protein Extraction Reagent A added with PMSF before use was added to dHL-60-CXCL9 cells, and the cells were gently and fully suspended, then placed on ice bath for 10 - 15 minutes. (3) The above samples were placed in liquid nitrogen and a 37 °C water bath respectively; they were repeatedly frozen and thawed several times in sequence, and then a small amount of the sample was taken to detect the degree of cell breakage under a microscope. If the degree of cell breakage was less than 70%, the number of freeze-thaw cycles could be increased until the degree of cell breakage was greater than 70%. (4) Centrifuge at 700 g for 10 minutes at 4 °C, and carefully collect the supernatant into a new centrifuge tube. Do not touch the precipitate when aspirating the supernatant, and about 30 - 50 μL of the supernatant can be left unaspirated to ensure the high purity of the aspirated supernatant. (5) Centrifuge at 14,000 g for 30 minutes at 4 °C, the precipitate was cell membrane fragments, discard the supernatant, resuspend with 1 mL of PBS, and measure its concentration with NanoDrop. (6) Next, a series of explorations were carried out on the ultrasonic intensity of the ultrasonic instrument, and finally an ultrasonic intensity of 15% was selected, with 3 s on and 2 s off for a total of 5 minutes, which could ultrasonically break the neutrophil cell membrane into cell membrane liposomes with a particle size of 100 - 200 nm and uniform size. (7) Use Malvern nanoZSE to measure its particle size and potential. Send the sample to the company for electron microscopy measurement.

[0136] 5. Detection of the level of CXCL9 carried by CXCL9-LNP by ELISA

[0137] (1) Prepare LNP / CXCL9-LNP. (2) Detect according to the detection steps provided by the CXCL9 ELISA kit (MM-5115H1, Enzyme Immunoassay Company).

[0138] 6. Detection of CXCL9 level by WB

[0139] Repeat the above operations, and select human CXCL9 recombinant protein as the positive control.

[0140] 7. Experimental results

[0141] We modified HL60 cells through genetic engineering technology, and GFP and CXCL9 were respectively linked to the membrane protein CD63 to construct a cell line with stable cell membrane carrying CXCL9 and GFP. We first detected the modified cells by confocal microscopy and found that GFP fluorescence was expressed on the membrane, indicating the successful construction of the cell line ( Figure 3 A). The induced neutrophils (dHL-60-CXCL9) were detected by flow cytometry and found to carry GFP fluorescence ( Figure 3 B). Then we prepared CXCL9-LNP by the method mentioned above, and first carried out basic characterization and identification of the liposomes by particle size, potential and electron microscopyFigure 3 C-E). The particle size is between 100 - 200 nm, the potential is negatively charged, and the electron microscope also shows that it basically conforms to the shape of liposomes. Next, we first performed indirect detection using a microplate reader and found that CXCL9-LNP carried GFP fluorescence. Finally, we also directly detected that the modified cell membrane liposomes carried CXCL9 through Elisa and WB Figure 3 G-H).

[0142] Example 4 Engineered neutrophil membrane liposomes successfully chemotax CAR-T cells without affecting the function of CAR-T cells 1. Experimental materials

[0143] Transwell chamber (5μm): Corning, USA. CXCL9 recombinant protein: Proteintech, USA. 2. Preparation of CAR-T cells (targeting B7-H3 CAR-T)

[0144] The method for preparing CAR-T cells in this study mainly referred to our previously published article (L. Shibao, Z. Miaomiao, W. Meng, W. Haiting, W. Han, M. Lijun, Z. Meng, L. Huizhong, Z. Junnian, M. Ping, W. Gang, B7-H3 specific CAR-T cells exhibit potent activity against prostate cancer, Cell Death Discov. 9(1)(2023)147). Based on the 8H9 clone, we synthesized the sequence of the B7-H3 single-chain antibody fragment (scFv) and constructed the second-generation B7-H3 chimeric antigen receptor (CAR), which contains B7-H3 scFv, CD8α transmembrane region, CD28 intracellular co-stimulatory domain, and CD3ζ intracellular signaling domain (as Figure 8 shown). Among them, the amino acid sequences and nucleotide sequences corresponding to the B7-H3 scFv, CD8α transmembrane region, CD28 intracellular co-stimulatory domain, and CD3ζ intracellular signaling domain are shown in Table 1 below.

[0145] Table 1 Sequence information of each component in CAR

[0146]

[0147] We used Ficoll density gradient centrifugation to isolate peripheral blood mononuclear cells (PBMCs) from the whole blood of healthy donors. The isolated PBMCs were seeded in 24-well plates pre-coated with 1 μg / mL of CD3 / CD28 antibodies and cultured at 37°C and 5% CO2 for 48 hours to activate T cells. Based on the lentiviral vector targeting B7H3 CAR constructed in our previous study, the virus was transduced into the activated PBMCs to generate B7H3-targeted CAR-T cells. To detect the CAR expression level of CAR-T cells, cells were stained with PE-labeled CAR antibody (Cat No: 11044-H07E-P, Sino Biological) and APC-CD3 antibody (Cat No: 981012, Clone SK7, BioLegend) or PerCP-Cy5.5-CD3 antibody (Cat No: 317336, Clone OKT7, BioLegend). The staining process was carried out for 30 minutes at 4°C in the dark, and CAR expression was analyzed by flow cytometry (FCM) after staining. Activated T cells without virus transduction were used as the control group for comparative analysis. All experiments were carried out strictly according to the standard operating procedures to ensure the reliability and repeatability of the data.

[0148] 3. Transwell Migration Assay

[0149] (1) The membrane equilibration was the same as above. (2) Take an appropriate amount of CAR-T cells, wash them with PBS, centrifuge at 1800 rpm for 5 min, discard the supernatant, resuspend the pellet with 1640 basal medium, and adjust the cell density to 1×10 7 / mL. (3) Set up groups: 1640 group: Add 100 μL of cell suspension + 100 μL of 1640 basal medium to the upper chamber of transwell, and add 550 μL of 1640 basal medium + 50 μL of PBS to the lower chamber; LNP group: Add 100 μL of cell suspension + 100 μL of 1640 basal medium to the upper chamber of transwell, and add 550 μL of 1640 basal medium + 50 μL of LNP (0.8 mg / mL) to the lower chamber; CXCL9-LNP group: Add 100 μL of cell suspension + 100 μL of 1640 basal medium to the upper chamber of transwell, and add 550 μL of 1640 basal medium + 50 μL of CXCL9-LNP (0.8 mg / mL) to the lower chamber; CXCL9 positive control group: Add 100 μL of cell suspension + 100 μL of 1640 basal medium to the upper chamber of transwell, and add 600 μL of 1640 basal medium (containing 10 ng / mL CXCL9) to the lower chamber. Add samples according to the corresponding groups and place them in an incubator at 37 °C for 2 h. (4) Take out the upper chamber, collect the cells in the lower chamber into a 1.5 mL EP tube, and centrifuge at 1800 rpm for 5 min. Discard the supernatant, add 20 μL of crystal violet to resuspend, and stain at room temperature for 10 min. Drop 5 μL of the staining solution on a glass slide, cover with a coverslip, observe under a microscope, and select 3 - 5 fields of view for photography. (5) Repeat the above experiment, collect the cells in the lower chamber and accurately count them by flow cytometry.

[0150] 4. CFSE cell proliferation assay

[0151] (1) Filter FBS serum through a 0.2 μm filter and prepare the working solution, store at 4 °C. (2) Take out the CFSE powder from -20 °C, centrifuge at 14000 rpm for 10 min, prepare the CFSE solution, and store at -20 °C in the dark. (3) Take 5×10 6 CAR-T cells in good condition and in the logarithmic growth phase, add 5 mL of 0.1% FBS and wash twice, centrifuge at 1800 rpm for 5 min, and discard the supernatant. (4) Add 100 μL of 1.5 μM CFSE working solution to every 1×10 6 cells, gently blow and mix well with a pipette, then incubate at room temperature in the dark for 15 min, and mix well by shaking every 5 min. Add an equal volume of FBS serum to terminate the incubation, and incubate in a water bath in the dark for 10 min. Add 5 mL of 2% FBS and wash twice, centrifuge at 1800 rpm for 5 min, discard the supernatant, resuspend with X-VIVO complete medium (containing IL-7, IL-15), and take out about 2×10 5The remaining cells were grouped and then returned to the incubator at 37°C for continued culture. (5) Group setting: Ctrl group: cultured in X-VIVO complete medium containing IL-7 and IL-15; LNP / CXCL9-LNP treatment group: 100 μL of LNP / CXCL9-LNP (0.8 mg / mL) was added respectively to the X-VIVO complete medium containing IL-7 and IL-15. (6) After adding 1 mL of PBS to wash the taken-out cells, they were resuspended with 400 μL of PBS, filtered through a 400-μm filter into a flow tube, and the CFSE fluorescence intensity of the cells at 0 h was detected by a flow cytometer. (7) Thereafter, 2×10 5 cells were taken out from the culture dish every 24 h, and the CFSE fluorescence intensity was detected by flow cytometry. The steps were as described above, and the CFSE flow voltage should not be changed during this process.

[0152] 5. Detection of CAR-T cell apoptosis

[0153] (1) CAR-T cells in the logarithmic growth phase were taken out, and the cell density was adjusted to 5×10 5 / mL with X-VIVO basal medium. (2) Group setting: Ctrl group: cultured by adding X-VIVO complete medium; LNP / CXCL9-LNP treatment group: cultured by adding X-VIVO complete medium containing 40 μg / mL of LNP / CXCL9-LNP. After 24 h and 48 h, the cells were collected into 1.5-mL EP tubes, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with 1 mL of PBS, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. This operation was repeated twice. The cells were resuspended in 300 μL of Binding buffer, 3 μL of Annexin V-FITC and 3 μL of PI dye were added, shaken evenly, and incubated in the dark at room temperature for 10 min. After filtering into a flow tube, it was detected by the flow cytometer.

[0154] 6. Detection of CAR-T cell exhaustion indicators

[0155] The above grouping was repeated to treat CAR-T cells, and the cell membrane surface immunosuppressive receptors (Tim3, Lag3, PD-1) were detected by flow cytometry on the machine.

[0156] 7. Co-culture killing experiment

[0157] (1) CAR-T cells in the logarithmic growth phase were taken out and co-cultured with LNP / CXCL9-LNP for 24 h respectively. (2) A549 lung cancer cells were labeled with GFP and co-cultured with the CAR-T cells treated with LNP / CXCL9-LNP for 24 h at the E:T ratios of 1:1 and 1:2 respectively. Subsequently, the ratio of CD3 to GFP cells was analyzed by flow cytometry.

[0158] 8. Experimental Results

[0159] To study whether neutrophil membrane liposomes carrying chemokine CXCL9 can function and chemotax more CAR-T cells, we first conducted a Transwell experiment. In the lower chamber, we added neutrophil control LNP and CXCL9-LNP respectively, and added CXCL9 alone as a positive control. In the upper chamber, we added CAR-T cells. After 2 hours, we collected the cells that had migrated to the lower chamber. First, we observed under the microscope by crystal violet staining and found that the CXCL9-LNP group could chemotax more CAR-T cells. At the same time, we also accurately counted the cells in the lower chamber by flow cytometry, which further confirmed that the CXCL9-LNP group chemotaxed more CAR-T cells than the LNP group and had statistical significance ( Figure 4 A-C).

[0160] Next, we explored whether engineered neutrophil membrane liposomes affect the function of CAR-T cells. First, we stained CAR-T cells with CFSE and co-cultured them with LNP / CXCL9-LNP, and detected them at multiple time points. The results showed that LNP / CXCL9-LNP did not affect the proliferation of CAR-T cells ( Figure 4 D-E). Next, we detected the apoptosis index of CAR-T cells after co-culture with LNP / CXCL9-LNP and found that LNP / CXCL9-LNP did not induce apoptosis of CAR-T cells ( Figure 4 F). At the same time, we detected the immunosuppressive receptors (Tim3, Lag3, PD-1) on the surface of CAR-T cell membranes. The flow cytometry results indicated that LNP / CXCL9-LNP did not induce CAR-T cells to present an exhausted phenotype ( Figure 4 G). Finally, we conducted a co-culture killing experiment. After co-culturing CAR-T cells with LNP / CXCL9-LNP for 24 hours, we added them to lung cancer cells. The results showed that CAR-T cells treated with LNP / CXCL9-LNP had no effect on the ability to kill tumors under different effector-target ratios ( Figure 4 H-K). These results indicate that neutrophil membrane liposomes rich in chemokine CXCL9 can chemotax more CAR-T cells, and at the same time have no negative effects on the proliferation, apoptosis, exhaustion, and co-culture killing tumor ability of CAR-T cells.

[0161] Example 5 Engineered Neutrophil Membrane Liposomes Do Not Affect the Proliferation, Migration, and Invasion of Lung Cancer Cells

[0162] 1. Experimental Materials

[0163] RTCA Real-Time Cell Analyzer: Agilent Technologies, Inc., USA. Transwell Chamber (8μm): Corning Incorporated, USA.

[0164] 2. RTCA Experiment

[0165] (1) Add 50 μL of 1640 complete medium to the wells of E-Plate96 and place it on the RTCA instrument to start step1. (2) Prepare tumor cells: Take A549 cells in good growth state. The digestion and counting steps are the same as before. Adjust the cell density to 3×10 5 / mL with 1640 complete medium. Remove the E-Plate96 plate and add 50 μL of tumor cell suspension to each well. If the target cells are H1299, adjust the density to 2×10 5 / mL. (3) Place the E-Plate96 plate in a 37°C incubator and let it stand for 30 min, then place it on the RTCA instrument to start step2 and grow overnight. When the cell index reaches 1, terminate. (4) Remove the E-Plate96 plate. In the experimental group, add 50 μL of LNP / CXCL9-LNP (0.8 mg / mL) + 100 μL of 1640 complete medium to the detection wells. In the Blank group, add 50 μL of PBS + 100 μL of 1640 complete medium to the detection wells and put it back on the RTCA instrument to start step3, continuously monitoring the cell index. (5) Determine the termination time according to the cell index of each group and export the excel graph to analyze the killing effect.

[0166] 3. Transwell Migration and Invasion

[0167] (1) Membrane equilibration is the same as before. (2) Prepare tumor cells: Digest and count routinely, add PBS to wash, centrifuge at 800 rpm for 5 min, resuspend with 1640 basal medium, and adjust the cell density to 1×10 6 / mL. (3) Grouping: Blank group: Add 100 μL of cell suspension + 100 μL of 1640 basal medium to the upper chamber; LNP / CXCL9-LNP treatment group: Add 100 μL of cell suspension + 100 μL of 1640 basal medium containing LNP / CXCL9-LNP to the upper chamber. Add 600 μL of 1640 basal medium containing 20% FBS to the lower chamber. (4) Incubate in a 37 °C incubator for 12 h. Take out the transwell chamber, discard the liquid, place the chamber in 600 μL of methanol, and fix at room temperature for 20 min. (5) Transfer the chamber to PBS and wash twice, then place it in crystal violet and stain at room temperature for 20 min. (6) Take out the chamber, discard the crystal violet, rinse it in PBS, and gently wipe the inner membrane of the transwell chamber with a cotton swab. (7) Invert the transwell chamber, observe under a microscope, and select 3-5 fields of view for photography. (8) Invasion experiment: After the membrane is balanced, discard the liquid in the upper chamber, add 10% Matrigel, and incubate at 37 °C for 4 h. The subsequent steps are the same as above.

[0168] 4. Experimental Results

[0169] This example further explored the effect of neutrophil-derived cell membrane liposomes on lung cancer cells. The RTCA results showed that LNP / CXCL9-LNP did not promote the progression of lung cancer cells ( Figure 5 A-B). At the same time, the Transwell experiment showed that LNP / CXCL9-LNP also did not promote the migration and invasion of lung cancer cells. These results indicate that neutrophil cell membrane liposomes do not affect the proliferation, migration, and invasion of lung cancer cells ( Figure 5 C-D).

[0170] Example 6 Engineering of neutrophil cell membrane liposomes combined with CAR-T cells to enhance anti-tumor efficacy

[0171] 1. Experimental Materials

[0172] Severely immunodeficient NCG mice, 4-6 weeks old, male: Jiangsu Jicui Yakang Biotechnology Co., Ltd. Housed in the Animal Experiment Center of Xuzhou Medical University that meets the SPF level and has appropriate humidity and temperature. All animal experiments have passed the ethical review of the Ethics Committee of Xuzhou Medical University.

[0173] 2. Experiment on treating A549 subcutaneous tumor-bearing mice with engineered neutrophil cell membrane liposomes combined with CAR-T cells

[0174] (1) Prepare NCG male mice at 4 - 6 weeks of age and raise them in an SPF environment. After one week of acclimation to the environment, shave the hair on the right back of the mice. (2) Prepare A549 lung cancer cells. Collect the cells into a 15 mL tube, wash them twice with PBS, then centrifuge at 800 rpm for 5 min and discard the supernatant. Resuspend in 1 mL of PBS for counting and adjust the cell density to 5×10 7 / mL. Prepare 100 μL for injection into each mouse. (3) When the tumors grow to an appropriate volume, randomly divide the mice into four groups: PBS group, CAR-T group, CAR-T + LNP group, and CAR-T + CXCL9-LNP group. On Day 0 and Day 14, the third and fourth groups are intravenously injected with 200 μL (0.8 mg / mL) of LNP / CXCL9-LNP respectively. On Day 1, inject 4×10 6 CAR-T cells into the tail vein of each mouse. Continuously monitor the tumor volume and body weight of the mice during the treatment process. (4) On Day 24, euthanize the mice, take out the mouse organs and soak them in tissue fixative, prepare paraffin sections and stain them with HE. Take pictures and weigh the tumor tissues, grind part of the tumor tissues to prepare cell suspensions, and detect the number of CD3 + cells in the tumor by flow cytometry. Store the remaining tissues in tissue fixative and prepare paraffin sections.

[0175] 3. Experimental Results

[0176] To confirm whether the engineered fine particle cell membrane liposomes can enhance the anti-lung cancer efficacy in mice, we used NCG mice to establish a subcutaneous xenograft model of human lung cancer A549. On Day 0, first intravenously inject LNP / CXCL9-LNP (0.8 mg / mL), and 24 h later inject CAR-T cells (4×10 6 ). When the content of CAR-T cells in the mice reaches the peak in the second week, intravenously inject LNP / CXCL9-LNP for the second time. Finally, on Day 24, sacrifice the mice for dissection ( Figure 6 A). The research results show that the tumor volume and tumor weight in the CAR-T + CXCL9-LNP group are significantly lower than those in the other three groups ( Figure 6 B - D). Calculate Q according to the King's formula: Q = (Q = E a+b / (E a +E b -E a ×E b ) = 0.845 / (0 + 0.67373 + 0×0.67373) = 1.25. Among them, E a+b is the inhibition rate of the combined drug, E a and E bare the inhibition rates of the two drugs used alone. It can be seen that Q > 1.15, indicating that CXCL9-LNP and CAR-T cells have a synergistic therapeutic effect, that is, when the dosage ratio of CXCL9-LNP to CAR-T cells is (0.16 mg - 0.32 mg) : (2 × 10 6 - 4 × 10 6 CAR-T cells), the combination of the two has a synergistic therapeutic effect on the treatment of lung cancer. We analyzed the CAR-T cells in the tumor by flow cytometry and found that the infiltration of CAR-T cells in the tumor in the CAR-T + CXCL9-LNP group was the highest among the four groups ( Figure 6 E). In summary, these results indicate that neutrophil membrane liposomes rich in chemokine CXCL9 enhance the anti-lung cancer efficacy by promoting the infiltration of CAR-T cells into the tumor.

[0177] Example 7 Engineering neutrophil membrane liposomes combined with CAR-T cell therapy has no obvious toxic and side effects

[0178] 1. Experimental materials

[0179] Paraffin tissue sections of mouse heart, liver, spleen, lung and kidney: Wuhan Sevier Biotechnology Co., Ltd. Hematoxylin staining solution: Shanghai Beyotime Biotechnology Co., Ltd. Eosin staining solution: Tianjin Solomon Biotechnology Co., Ltd. Hematoxylin differentiation solution, hematoxylin blue-returning solution: Wuhan Sevier Biotechnology Co., Ltd.

[0180] 2. HE staining

[0181] (1) Dewax and rehydrate the tissue sections of mouse heart, liver, spleen, lung and kidney: Dewax with xylene I (10 min) and xylene II (10 min); Rehydrate by removing xylene with 100% alcohol (5 min), 90% alcohol (5 min), 80% alcohol (5 min), 70% ethanol (5 min), and distilled water wash (5 min). (2) Stain with hematoxylin staining solution for 10 min, wash off the floating color with distilled water (1 - 3 s), decolorize with 1% hydrochloric acid alcohol (1 - 3 s), wash with water (10 - 30 s), return to blue with ammonia water (10 - 30 s), wash off the attached ammonia water with water (10 - 15 min), and stain with alcohol-soluble eosin for 30 s. (3) Dehydrate, clear and mount: Dehydrate step by step through 70% alcohol rinse (1 - 2 s), 80% alcohol (1 - 2 s), 90% ethanol (5 min), 100% ethanol I (5 min), 100% ethanol II (5 min); Remove alcohol and clear with xylene I (10 min) and xylene II (10 min); Mount with neutral gum and examine under the microscope.

[0182] 3. Experimental results

[0183] During the above animal experiments, we weighed the mice every 4 days. The results showed that there was no significant difference in the body weight of the mice in the CXCL9-LNP+CAR-T group compared with the other three groups at different time points ( Figure 7 A). Finally, we performed HE staining on the important tissues and organs of the mice and found that there were no obvious structural damages or obvious inflammatory infiltrations in the important tissues and organs of the mice in each group ( Figure 7 B). This indicates that the treatment with engineered neutrophil membrane liposomes combined with CAR-T cells has no obvious subacute toxic and side effects in vivo and has good biosafety.

[0184] Example 8 Engineered CXCL9-LNP can recruit more CAR-T cells to effectively kill lung cancer, gastric cancer and colon cancer cells

[0185] 1. Experimental materials

[0186] Transwell chamber (5μm): Corning, USA; Calcein-AM: Dojindo Molecular Technologies.

[0187] 2. Transwell migration and killing experiment

[0188] (1) Digest lung cancer cells A549, gastric cancer cells MKN45, and intestinal cancer cells SW480 with trypsin, wash them once with PBS, and resuspend the cells in complete medium; Place a polylysine-coated cell glass slide at the bottom of a 24-well plate, and inoculate lung cancer cells A549 (cell number is 1.0×10 5 ), gastric cancer cells MKN45 (cell number is 1.37×10 5 ), and intestinal cancer cells SW480 (cell number is 3.43×10 5 ), and put them into the incubator;

[0189] (2) After the tumor cells adhere to the wall for 24 hours, aspirate the culture medium, wash it once with PBS, and add 600 μL of serum-free 1640 medium respectively;

[0190] (3) Take appropriate amounts of Ctrl-T cells and CAR-T cells, wash them with PBS, centrifuge at 1800 rpm for 5 min, discard the supernatant, resuspend the precipitate with 1640 basal medium, and adjust the cell density to 1×10 7 / mL, and add 100 μL of the T cell suspension to the upper chamber respectively;

[0191] (4) Set up groups:

[0192] 1) Ctrl control group: Add 100 μL of 1640 medium + 100 μL of 1640 basal medium to the upper chamber of Transwell, and add the above-mentioned tumor cells + 550 μL of 1640 basal medium + 50 μL of PBS to the lower chamber;

[0193] 2) Ctrl-T + LNP group: Add 100 μL of Ctrl-T cell suspension + 100 μL of 1640 basal medium to the upper chamber of Transwell, and add the above-mentioned tumor cells + 550 μL of 1640 basal medium + 50 μL of LNP (0.8 mg / mL) to the lower chamber;

[0194] 3) Ctrl-T + CXCL9-LNP group: Add 100 μL of Ctrl-T cell suspension + 100 μL of 1640 basal medium to the upper chamber of Transwell, and add the above-mentioned tumor cells + 550 μL of 1640 basal medium + 50 μL of CXCL9-LNP (0.8 mg / mL) to the lower chamber;

[0195] 4) CAR-T + LNP group: Add 100 μL of CAR-T cell suspension + 100 μL of 1640 basal medium to the upper chamber of Transwell, and add the above-mentioned tumor cells + 550 μL of 1640 basal medium + 50 μL of LNP (0.8 mg / mL) to the lower chamber;

[0196] 5) CAR-T + CXCL9-LNP group: Add 100 μL of CAR-T cell suspension + 100 μL of 1640 basal medium to the upper chamber of Transwell, and add the above-mentioned tumor cells + 550 μL of 1640 basal medium + 50 μL of CXCL9-LNP (0.8 mg / mL) to the lower chamber;

[0197] (5) Add samples according to the corresponding groups and place them in an incubator at 37 °C for 2 h.

[0198] (6) Take out the upper chamber, add 60 μL of FBS to each well of the lower chamber, and place it in the incubator for 24 h;

[0199] (7) Aspirate the supernatant, wash twice with PBS, and add Calcein-AM for live cell staining for 20 min;

[0200] (8) Fix with paraformaldehyde for 10 min, wash three times with PBS, take out the cell slides, attach them to the glass slides, and take pictures with a fluorescence microscope;

[0201] (9) Count with Image J and statistically analyze the number of surviving tumor cells.

[0202] 3. Experimental results

[0203] To verify whether CXCL9-LNP can chemotactically recruit CAR-T cells to kill various solid tumor cells, we performed a Transwell killing experiment. As Figure 9 shown in A, lung cancer cells A549, gastric cancer cells MKN45, and intestinal cancer cells SW480 were seeded in the lower chamber respectively. After the CAR-T cells migrated from the upper chamber to the lower chamber and killed the tumor cells, the remaining viable tumor cells were labeled with Calcein-AM. The results showed that the number of viable tumor cells in the CXCL9-LNP group was significantly reduced compared with that in the control LNP group ( Figure 9 B-C), while Ctrl-T had no effect on the survival of tumor cells ( Figure 9 B-C). The above results indicate that CXCL9-LNP can recruit more CAR-T cells to effectively kill lung cancer, gastric cancer, and colon cancer cells.

Claims

1. A pharmaceutical composition for anti-tumor, characterized in that, The pharmaceutical composition comprises engineered neutrophil membrane liposomes and CAR-T cells; The engineered neutrophil membrane liposomes are neutrophil membrane liposomes carrying CXCL9; The CAR in the CAR-T cells comprises a monoclonal antibody targeting B7-H3, a CD8α transmembrane region, a CD28 intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain; The amino acid sequence of the heavy chain variable region of the monoclonal antibody targeting B7-H3 is as shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region of the monoclonal antibody targeting B7-H3 is as shown in SEQ ID NO:2; The amino acid sequence of the CD8α transmembrane region is as shown in SEQ ID NO:3; The amino acid sequence of the CD28 intracellular co-stimulatory domain is as shown in SEQ ID NO:4; The amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:

5.

2. The pharmaceutical composition according to claim 1, wherein The method for constructing the neutrophil membrane liposomes carrying CXCL9 comprises: Introducing CXCL9 onto neutrophil membrane proteins by lentiviral transfection to construct neutrophils with stable CXCL9-carrying cell membranes; Performing cell disruption, membrane separation, and liposome formation on the neutrophils with stable CXCL9-carrying cell membranes respectively to obtain neutrophil membrane liposomes carrying CXCL9; Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer, or kidney cancer.

3. The pharmaceutical composition according to claim 1, characterized in that, The dosage ratio of the engineered neutrophil cell membrane liposomes to CAR-T cells is (0.16 mg - 0.32 mg) : (2×10 6 - 4×10 6 CAR-T cells).

4. An antitumor pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 1-3; Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer, or kidney cancer.

5. The pharmaceutical preparation according to claim 4, wherein The dosage form of the pharmaceutical preparation is a non-parenteral dosage form or a parenteral dosage form.

6. The pharmaceutical preparation according to claim 5, characterized in that, The non-parenteral dosage form is an injection dosage form, a respiratory tract dosage form, a cavity dosage form, a mucosal dosage form, or a skin dosage form; Optionally, the parenteral dosage form is a granule, a capsule, a solution, a tablet, a powder, a sustained-release preparation, an emulsion, a suspension, or a drop.

7. A method for constructing a neutrophil cell membrane liposome carrying CXCL9 according to any one of claims 1-3, characterized in that, The method comprises: Introducing CXCL9 onto neutrophil membrane proteins by lentiviral transfection to construct neutrophils with stable CXCL9-carrying cell membranes; Performing cell disruption, membrane separation, and liposome formation on the neutrophils with stable CXCL9-carrying cell membranes respectively to obtain neutrophil membrane liposomes carrying CXCL9.

8. A method for non-therapeutically inhibiting the growth of tumor cells or tumor organoids and / or promoting the apoptosis of tumor cells or tumor organoids in vitro, characterized in that, The method comprises: treating tumor cells or tumor organoids with the pharmaceutical composition according to any one of claims 1-3 or the pharmaceutical preparation according to any one of claims 4-6; Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer, or kidney cancer.

9. Use in any of the following aspects: (1) Use of the combination of the engineered neutrophil membrane liposomes and CAR-T cells according to any one of claims 1-3 in the preparation of a drug for treating tumors; (2) Use of the combination of the engineered neutrophil cell membrane liposomes and CAR-T cells according to any one of claims 1-3 in the preparation of a pharmaceutical composition for treating tumors; (3) Use of the combination of the engineered neutrophil cell membrane liposomes and CAR-T cells according to any one of claims 1-3 in the preparation of a pharmaceutical preparation for treating tumors; (4) Use of the combination of the engineered neutrophil cell membrane liposomes and CAR-T cells according to any one of claims 1-3 in inhibiting the growth of tumor cells or tumor organoids and / or promoting the apoptosis of tumor cells or tumor organoids in vitro for non-therapeutic purposes.

10. The application according to claim 9, wherein, The dosage ratio of the engineered neutrophil membrane liposomes and CAR-T cells is (0.16 mg - 0.32 mg) : (2×10 6 - 4×10 6 CAR-T cells); Optionally, the tumor is lung cancer, gastric cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, liver cancer, ovarian cancer or kidney cancer.

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