Construction method and application of CAR-M cell based on ICAM-1 targeting ligand gene

Abstract

The invention relates to the technical field of biological medicine, and discloses a construction method and application of a CAR-M cell based on an ICAM-1 targeting ligand gene, a transfer plasmid containing the ICAM-1 targeting ligand gene, a VSV-G envelope plasmid and a lentivirus packaging auxiliary vector are transfected to an HEK293T packaging cell, after transfection is finished, a clear virus solution is obtained through collection and culture, and after concentration, the CAR-M cell is obtained. The preparation method comprises the following steps of: adding the mCherry-ICAM-1 lentivirus suspension into a culture medium; tHP-1 cells are transduced by using the virus, and then the transduced THP-1 cells are sequentially subjected to antibiotic screening and monoclonal cell screening, so that CAR-M cells are obtained. According to the invention, the ICAM-1 extracellular domain is integrated into the extracellular antigen binding region of the CAR, so that the CAR-M cell is successfully constructed, and the macrophage is endowed with the capability of directionally recognizing and eliminating iRBCs.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to a construction method of CAR-M cells based on an ICAM-1 targeting ligand gene and application thereof. BACKGROUND

[0002] Malaria is an ancient and serious infectious and parasitic disease caused by Plasmodium infection, mainly transmitted through female Anopheles bites, and a small number of blood transfusions or mother-to-child transmission. The pathogen includes five pathogenic Plasmodium: P. falciparum (the most lethal), P. vivax, P. ovale, P. malariae and P. knowlesi (zoonotic, rare). The life cycle of Plasmodium is complex, which needs to go through two hosts, human and female Anopheles, including asexual reproduction (in the human body) and sexual reproduction (in the body of Anopheles), with sporozoites as the infection stage and gametocytes as the transmission stage. The development in the human body (intermediate host, mainly asexual reproduction) mainly has an extra-erythrocytic stage (extracellular stage) and an intra-erythrocytic stage (intracellular stage). Among them, the intra-erythrocytic stage is the main stage of Plasmodium leading to clinical symptoms. After Plasmodium enters the human body through Anopheles bites, it develops in the liver cells (extracellular stage), and then invades red blood cells to multiply (intracellular stage), leading to the rupture of red blood cells to release merozoites and metabolites, and causing clinical symptoms. The acute hazards mainly include periodic chills, high fever (body temperature can reach above 40℃), headache, fatigue, anemia (red blood cell destruction) and splenomegaly (monocyte-macrophage system activation). P. falciparum can adhere to the microvascular endothelium, leading to cerebral malaria (coma, convulsions, mortality rate of 15%-20%), acute renal failure, pulmonary edema, severe hypoglycemia and other complications, which is the main cause of death from malaria. The chronic hazards mainly include P. vivax and P. ovale due to the existence of "dormant" (in liver cells), which can cause recurrence (months to years); long-term infection affects the growth and development of children, and pregnant women infection can cause miscarriage, premature birth and low birth weight infants (newborn mortality rate increases by 2 times).

[0003] Drug is still the main measure for the treatment of malaria patients. At present, anti-malarial drugs are mainly designed based on different stages of the life cycle of Plasmodium, which can be divided into the following categories: (1) Red inner period killing drugs (control symptoms): artemisinin (first-line core): artesunate, dihydroartemisinin, etc., high efficiency, rapid effect, through the production of free radicals to destroy the membrane structure of Plasmodium, combined with other drugs (artemisinin combination therapy, ACTs) can reduce drug resistance. Quinoline: chloroquine (traditional drug, withdrawn from the first-line of malignant malaria due to drug resistance), quinine (intravenous drug for severe malaria), piperaquine (ACTs compatible drug). Anti-folic acid: pyrimethamine + sulfadoxine (compound, inhibit Plasmodium folate synthesis, due to drug resistance, only used for prevention in some areas). (2) Red outside period / gametocyte killing drugs: primaquine, used to cure vivax malaria / ovate malaria (kill dormant children) and block transmission (kill gametocytes), but need to pay attention to the risk of hemolysis in G6PD deficiency. (3) Drug resistance challenge: drug resistance of malignant malaria to chloroquine, sulfadoxine- pyrimethamine has spread globally; since 2010, Southeast Asia (Cambodia, Thailand) has appeared partial resistance to artemisinin (ACTs to clear Plasmodium speed slows down), which needs to be addressed by optimizing ACTs compatibility (such as adding long-acting drugs).

[0004] Reviewing the experience of Plasmodium developing resistance to previous first-line anti-malarial drugs (such as quinine, chloroquine, etc.) and current first-line anti-malarial drugs (artemisinin), even if new drugs are successfully developed and used for anti-malarial treatment, under the pressure of drug selection, Plasmodium will still undergo genetic mutations. Therefore, the problem of drug resistance is still difficult to avoid in the foreseeable future. Therefore, exploring new treatment methods to solve the problem of widespread drug resistance of existing drugs is a problem that must be faced and solved in malaria prevention and control.

[0005] Chimeric antigen receptors (CARs) are genetically engineered synthetic receptors. Their mediated immunotherapy has shown promising applications in the treatment of AIDS, systemic lupus erythematosus, type 1 diabetes, cardiac fibrosis, malignant hematopoietic tumors, HER2-positive tumors, and glioblastoma. Chimeric antigen receptor T-cell (CAR-T) therapy exhibits specific recognition and high-efficiency killing in the treatment of infectious diseases and hematologic malignancies, but it has significant limitations in the treatment of solid tumors. The number of T cells gradually decreases due to tumor infiltration in the immunosuppressive microenvironment, failing to achieve the expected therapeutic effect. Subsequently, based on this approach, CAR-M and CAR-NK therapies have been gradually developed, with CAR-M therapy showing great potential and compensating for the shortcomings of CAR-T cells. Macrophages play an important role in innate immunity, possessing functions such as phagocytosis, polarization, and antigen presentation, and are abundant in the tumor microenvironment (TME). CAR-M cells can turn the disadvantages of CAR-T cells into advantages, suggesting that we can further explore new avenues for malaria treatment by endowing macrophages with targeting functions and then utilizing their phagocytic properties. However, currently, there are no literature or patent reports on the application of CAR-M in the field of malaria treatment.

[0006] Therefore, providing a method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene and its application has important practical significance. Summary of the Invention

[0007] In view of this, the present invention proposes a method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene and its application, aiming to solve at least one of the current background technical problems.

[0008] This invention proposes a CAR-M cell based on the ICAM-1 targeting ligand gene, wherein the extracellular antigen-binding region of the CAR-M cell includes the ICAM-1 targeting ligand gene; The DNA sequence of the ICAM-1 targeting ligand gene is shown in SEQ ID No. 1.

[0009] This invention also provides a method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene as described in the above technical solution, comprising the following steps: The transfer plasmid containing the ICAM-1 targeting ligand gene, the VSV-G envelope plasmid, and the lentiviral packaging helper vector were co-transfected into HEK293T packaging cells. After transfection, the culture supernatant was collected, centrifuged, filtered, and a clear viral solution was obtained. The clarified viral solution was concentrated to obtain mCherry-ICAM-1 lentivirus suspension; THP-1 cells were transduced using the mCherry-ICAM-1 lentivirus, and then the transduced THP-1 cells were subjected to antibiotic screening and monoclonal cell screening to obtain CAR-M cells.

[0010] Preferably, the lentivirus packaging aid is: VB-VSV-G, VB-Rev, or VB-Gag / Pol.

[0011] Preferably, the transduction specifically refers to: The mCherry-ICAM-1 lentivirus suspension and Polybrene were mixed with THP-1 medium to obtain a mixed medium. THP-1 cells were first seeded in THP-1 medium and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then the medium was removed and the mixed medium was added, and the cells were cultured for another 48 hours. THP-1 medium was added at the 4th hour and replaced with fresh THP-1 medium at the 24th hour.

[0012] Preferably, the THP-1 medium is RPMI 1640 medium containing 10% FBS, 1% penicillin / streptomycin and 50 μmol / L β-mercaptoethanol.

[0013] Preferably, the multiplicity of infection (MOI) when transducing THP-1 cells using the mCherry-ICAM-1 lentivirus is 10.

[0014] Preferably, the antibiotic screening specifically involves: After transduction, the cell culture medium was replaced with THP-1 medium containing 600 μg / mL G418. Subsequently, the cells were passaged every 48 hours and replaced with THP-1 medium containing 300 μg / mL G418 for 3-5 days.

[0015] Preferably, the subculture ratio is 1:5.

[0016] Preferably, the monoclonal cell screening specifically involves: Collect cells after antibiotic selection, centrifuge, resuspend in THP-1 medium containing 300 μg / mL G418, and adjust the concentration to 2 × 10⁻⁶. 4 cells / mL, to obtain a cell suspension; Take a 96-well cell culture plate (8×12) and add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well, except for well A1 which is left untreated. Then add 200 μL of the cell suspension to well A1 and perform serial dilutions at a 1:2 ratio along the longitudinal direction (A1 to H1) and then along the horizontal direction (wells 1 to 12 of each row) at a 1:2 ratio. After dilution, add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well to bring the final volume of each well to 200 μL. Screen out wells containing only a single cell and mark them as monoclonal wells. Then incubate the cell culture plate at 37°C and 5% CO2 for 7–14 days.

[0017] The present invention also provides the application of CAR-M cells based on the ICAM-1 targeting ligand gene described in the above technical solution in the preparation of drugs to clear Plasmodium falciparum infection of red blood cells.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention creatively extends CAR-M cell therapy to the field of malaria treatment. It integrates the extracellular domain of ICAM-1 as a recognition module into the extracellular antigen-binding region of CAR cells, successfully constructing CAR-M cells capable of specifically targeting the interaction interface between the key pathogenic protein PfEMP1 of Plasmodium falciparum and its host receptor ICAM-1. This endows macrophages with the ability to directionally recognize and clear iRBCs, achieving targeted clearance of the pathogen. It overcomes the limitations of traditional CAR-T therapy, which suffers from insufficient infiltration in solid tumors or specific infection environments, and leverages the natural distribution and functional advantages of macrophages in the tissue microenvironment. Attached Figure Description

[0019] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 A vector map of a transfer plasmid vector containing the ICAM-1 targeting ligand gene provided in an embodiment of the present invention; Figure 2 This is a graph showing the RT-qPCR detection results provided in an embodiment of the present invention; Figure 3 This is a laser confocal microscope analysis diagram provided in an embodiment of the present invention; Figure 4 The flow cytometry results of CTL group cells, RBCs and iRBCs cultured separately provided in the embodiments of the present invention are shown in the figure. Figure 5 Flow cytometry results of RBCs co-cultured with CTL group cells and ICAM-1 group cells, respectively, provided in the embodiments of the present invention; Figure 6 The flow cytometry results of iRBCs co-cultured with CTL group cells and ICAM-1 group cells provided in the embodiments of the present invention are shown in the figure. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0021] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] This invention provides a CAR-M cell based on the ICAM-1 targeting ligand gene, wherein the extracellular antigen-binding region of the CAR-M cell includes the ICAM-1 targeting ligand gene; The DNA sequence of the ICAM-1 targeting ligand gene is shown in SEQ ID No. 1.

[0026] Specifically, SEQ ID No. 1 is:

[0027] This invention also provides a method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene as described in the above technical solution, comprising the following steps: The transfer plasmid containing the ICAM-1 targeting ligand gene, the VSV-G envelope plasmid, and the lentiviral packaging helper vector were co-transfected into HEK293T packaging cells. After transfection, the culture supernatant was collected, centrifuged, filtered, and a clear viral solution was obtained. The clarified viral fluid was concentrated to obtain a lentivirus suspension; THP-1 cells were transduced using the mCherry-ICAM-1 lentivirus, and then the transduced THP-1 cells were subjected to antibiotic screening and monoclonal cell screening to obtain CAR-M cells.

[0028] Specifically, the transfer plasmid vector containing the ICAM-1 targeting ligand gene was obtained by having the ICAM-1 targeting ligand gene sequence synthesized by Yunzhou Biotechnology (Guangzhou) Co., Ltd., and its vector map is shown in Table 1: Figure 1{hICAM-1[NM_000201.3404.5](22-212aa)} is the ICAM-1 targeting ligand gene.

[0029] Specifically, the VSV-G envelope plasmid and lentivirus packaging auxiliary vector were purchased directly from Yunzhou Biotechnology (Guangzhou) Co., Ltd.

[0030] Specifically, the clarified virus solution is concentrated using a polyethylene glycol precipitation method, which includes the following: (1) Preparation of clarified virus solution: The culture supernatant containing the virus is clarified to remove cells and debris. The clarification process includes centrifugation and / or filtration. Centrifugation is preferably carried out at 4°C with a centrifugal force of 3000–8000×g for 10–20 min. The filter pore size is preferably 0.45μm or 0.22μm to obtain the clarified virus solution.

[0031] (2) Constructing a polyethylene glycol precipitation system: Polyethylene glycol (PEG) and an inorganic salt are added to the clarified virus solution and mixed thoroughly to achieve a predetermined final concentration of PEG and promote the precipitation of virus particles; the molecular weight of the PEG is preferably PEG 6000 or PEG 8000; the final concentration of the PEG is preferably 8%–12% (w / v); the inorganic salt is preferably sodium chloride, and its final concentration is preferably 0.3–0.6 mol / L. PEG can be added by adding solid PEG or by adding PEG stock solution.

[0032] (3) Low temperature incubation to promote precipitation: The mixture after adding PEG and inorganic salt is placed under low temperature conditions to form a precipitate; the low temperature is preferably 4°C; the incubation time is preferably 4–16 h; the incubation method can be static or slow rolling mixing.

[0033] (4) Centrifugation to collect virus precipitate: After incubation, centrifuge the mixture to obtain virus precipitate; preferably at 4°C, centrifuge at 8000–12000×g for 20–60 min; discard the supernatant after centrifugation and retain the precipitate.

[0034] (5) Resuspending to obtain concentrated virus solution: Add buffer solution to the precipitate for resuspending to obtain concentrated virus solution; the buffer solution is preferably PBS, TNE or a buffer system compatible with downstream applications; the resuspending volume is determined according to the required concentration factor, preferably 1 / 10–1 / 50 of the original clarified virus solution volume.

[0035] (6) Removal of polyethylene glycol / liquid replacement treatment (optional): In order to reduce the impact of PEG and salt on subsequent applications, the concentrated virus solution can be subjected to PEG removal / liquid replacement treatment. The treatment methods include dialysis, ultrafiltration and / or gel filtration; after treatment, a concentrated virus solution suitable for subsequent purification, detection or formulation is obtained.

[0036] (6) Preservation (optional): The concentrated virus solution is aliquoted and stored; it can be stored at 4°C for a short period of time, or at -80°C for a long period of time after adding a protective agent, avoiding repeated freeze-thaw cycles.

[0037] This invention integrates the extracellular domain of ICAM-1 as a recognition module into the extracellular antigen-binding region of CAR, successfully constructing CAR-M cells that can specifically target the interaction interface between the key pathogenic protein PfEMP1 of Plasmodium falciparum and its host receptor ICAM-1, endowing macrophages with the ability to directionally recognize and clear iRBCs, thus achieving targeted clearance of pathogens.

[0038] In this invention, the lentivirus packaging aid carrier is: VB-VSV-G, VB-Rev, or VB-Gag / Pol.

[0039] In this invention, the transduction specifically refers to: The mCherry-ICAM-1 lentivirus suspension and Polybrene were mixed with THP-1 medium to obtain a mixed medium. THP-1 cells were first seeded in THP-1 medium and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then the medium was removed and the mixed medium was added, and the cells were cultured for another 48 hours. THP-1 medium was added at the 4th hour and replaced with fresh THP-1 medium at the 24th hour.

[0040] In this invention, the THP-1 medium is RPMI 1640 medium containing 10% FBS, 1% penicillin / streptomycin and 50 μmol / L β-mercaptoethanol.

[0041] In this invention, the multiplicity of infection (MOI) when transducing THP-1 cells using the mCherry-ICAM-1 lentivirus is 10.

[0042] In this invention, the antibiotic screening specifically involves: After transduction, the cell culture medium was replaced with THP-1 medium containing 600 μg / mL G418. Subsequently, the cells were passaged every 48 hours and replaced with THP-1 medium containing 300 μg / mL G418 for 3-5 days.

[0043] In this invention, the subculture ratio is 1:5.

[0044] In this invention, the monoclonal cell screening specifically involves: Collect cells after antibiotic selection, centrifuge, resuspend in THP-1 medium containing 300 μg / mL G418, and adjust the concentration to 2×10⁻⁶. 4 cells / mL, to obtain a cell suspension; Take a 96-well cell culture plate (8×12) and add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well, except for well A1 which is left untreated. Then add 200 μL of the cell suspension to well A1 and perform serial dilutions at a 1:2 ratio along the longitudinal direction (A1 to H1) and then along the horizontal direction (wells 1 to 12 of each row) at a 1:2 ratio. After dilution, add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well to bring the final volume of each well to 200 μL. Screen out wells containing only a single cell and mark them as monoclonal wells. Then incubate the cell culture plate at 37°C and 5% CO2 for 7–14 days.

[0045] The present invention also provides the application of CAR-M cells based on the ICAM-1 targeting ligand gene described in the above technical solution in the preparation of drugs to clear Plasmodium falciparum infection of red blood cells.

[0046] Example 1 S1. Preparation of lentivirus: The transfer plasmid containing the ICAM-1 targeting ligand gene, the VSV-G envelope plasmid, and the VB-VSV-G, VB-Rev, and VB-Gag / Pol helper vectors were co-transfected into HEK293T packaging cells. 48 hours after transfection, the culture supernatant containing virus particles was collected, cell debris was removed by centrifugation, and the clear virus solution was obtained by filtration. Finally, the clear virus solution was concentrated by polyethylene glycol precipitation to obtain mCherry-ICAM-1 lentivirus suspension. S2. Lentiviral transduction: After gently mixing the mCherry-ICAM-1 lentivirus suspension with a pipette, take 5 μL of the lentivirus suspension and add it to 0.5 mL of THP-1 medium, and at the same time add 0.5 μL of Polybrene at a concentration of 5 mg / mL, mix well to obtain a mixed medium.

[0047] THP-1 cells were seeded at a density of 5 × 10⁴ cells per well in 12-well plates and cultured for 24 h at 37°C with 5% CO₂ (each well containing 0.5 mL of THP-1 medium). The original medium was then removed and replaced with a mixed medium. After replacement, the plates were gently mixed using a figure-eight motion and then placed back into the cell culture incubator for further culture. Four hours later, 0.5 mL of fresh THP-1 medium was added to each well to bring the total volume to 1 mL. Twenty-four hours after transduction, the medium was aspirated and replaced with fresh THP-1 medium. Simultaneously, THP-1 cells were seeded into 12-well plates using the same method, but without the mixed medium for virus transduction; only THP-1 medium was used for culture as a blank control.

[0048] S2. Antibiotic Screening: 48 hours after transduction, the culture medium in the wells of the experimental group and the blank control group was replaced with THP-1 medium containing 600 μg / mL G418. Subculturing and medium changes were performed every 48 hours, using THP-1 medium containing 300 μg / mL G418 at a subculture ratio of 1:5. Screening continued for 3-5 days until the cells in the blank control group died completely and the cell number in the experimental group stabilized.

[0049] S3. Monoclonal cell screening: Collect the screened cells, centrifuge and resuspend them, and adjust the cell density to 2×10⁻⁶. 4 cells / mL, to obtain a cell suspension; Take a 96-well cell culture plate (8×12), add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well, except for well A1 which is left untreated. Then add 200 μL of the cell suspension to well A1, followed by a 1:2 serial dilution along the longitudinal direction (A1 to H1), and then a 1:2 serial dilution along the transverse direction (wells 1 to 12 of each row). After dilution, add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well to bring the final volume to 200 μL. Label and confirm wells containing only a single cell under a microscope. Incubate the plate at 37°C in a 5% CO2 incubator for 2-3 days, observing cell growth. After 7-14 days of culture, pick cells from the labeled monoclonal wells to obtain stable CAR-M cell lines based on the mCherry-ICAM-1 targeting ligand gene.

[0050] Comparative Example S1. Preparation of lentivirus: EGFP / mCherry dual fluorescent control lentivirus loaded with the ICAM-1 target ligand gene was purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd. S2. Lentiviral transduction: The EGFP / mCherry dual-fluorescent control lentivirus was thawed on ice. After thawing, the lentivirus suspension was gently mixed by pipetting. 5 μL of the lentivirus suspension was added to 0.5 mL of THP-1 medium, along with 0.5 μL of Polybrene at a concentration of 5 mg / mL. The mixture was thoroughly mixed to obtain the mixed medium.

[0051] THP-1 cells were spaced at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 12-well plates and incubated at 37°C with 5% CO2 for 24 hours (each well containing 0.5 mL of THP-1 medium). The original medium was then removed from the wells and replaced with a mixed medium. After replacement, the plate was gently mixed using a figure-eight motion and placed back in the cell culture incubator for further incubation. Four hours later, 0.5 mL of fresh THP-1 medium was added to each well, bringing the total volume to 1 mL. Twenty-four hours after transduction, the medium in the wells was aspirated and replaced with fresh THP-1 medium.

[0052] S2. Antibiotic Screening: 48 hours after transduction, the culture medium in the control group wells was replaced with THP-1 medium containing 2 μg / mL Puromycin. Subculture and medium changes (THP-1 medium containing 2 μg / mL Puromycin) were performed every 48 hours thereafter, with a subculture ratio of 1:5. Screening continued for 3-5 days.

[0053] S3. Single-clonal cell screening: Collect the screened cells, centrifuge them, resuspend them in THP-1 medium containing 2 μg / mL Puromycin, and adjust the cell density to 2 × 10⁶ cells / mL. 4 cells / mL, to obtain a cell suspension; Take a 96-well cell culture plate (8×12), add 100 μL of THP-1 medium containing 2 μg / mL Puromycin to each well, except for well A1 which is left untreated. Then add 200 μL of the cell suspension to well A1, followed by a 1:2 serial dilution along the longitudinal direction (A1 to H1), and then a 1:2 serial dilution along the transverse direction (wells 1 to 12 of each row). After dilution, add 2 μg / mL THP-1 medium containing Puromycin to each well to bring the final volume to 200 μL. Label and confirm wells containing only a single cell under a microscope. Incubate the plate at 37°C in a 5% CO2 incubator for 2-3 days, observing cell growth. After 7-14 days of culture, pick cells from the labeled single-clone wells to obtain stable control (CTL) lines.

[0054] Test Example 1 (RT-qPCR Analysis) Test Groups: Cells from Example 1 that underwent antibiotic screening but not monoclonal cell screening were used as the ICAM-1 antibiotic screening group (CAR-M / CD54). ASG ); The stable CAR-M cell line based on the ICAM-1 targeting ligand gene prepared in Example 1 was used as the experimental group (CAR-M / CD54). SCL ); Pure THP-1 cells were used as a blank control group (THP-1). Cells from the comparative example that underwent antibiotic screening but not monoclonal cell screening were selected as the CTL antibiotic screening group (CAR-M / CTL). ASG ); The control group (CTL) stable strains prepared in the comparative example were used as the CTL stable group (CAR-M / CTL). SCL ); Test methods VeZol lysis buffer was added to the five cell groups mentioned above, and the cells were thoroughly lysed and mixed by pipetting. Total RNA was then extracted from each of the five groups, and genomic DNA contamination was removed. The purified RNA was reverse transcribed into cDNA. Using this cDNA as a template, it was mixed with specific primers targeting the ICAM-1 gene. The specific primers included CD54-forward and CD54-reverse, specifically CD54-forward: 5′-ATGCCCAGACATCTGTGTCC-3′; CD54-reverse: 5′-GGGGTCTCTATGCCCAACAA-3′. The reaction conditions were as follows: 95℃ for 5 min, 95℃ for 15 s, 58℃ for 30 s, 65℃ for 5 s, for 39 cycles, for RT-qPCR.

[0055] RT-qPCR test results as follows Figure 2 As shown, the ICAM-1 stable cell line (CAR-M / CD54) SCL The relative expression level of ICAM-1 in the group was the highest, showing a highly significant difference compared to the blank control group (THP-1) (P<0.0001). The ICAM-1 antibiotic screening group (CAR-M / CD54) showed the highest relative expression level. ASG The expression level of CAR-M / CD54 was the second highest, and it also showed a highly significant difference compared with the blank control group (THP-1) (P<0.0001). Furthermore, CAR-M / CD54... ASG With CAR-M / CD54 SCL The expression levels between the two groups also showed significant differences (P<0.0001).

[0056] Test Example 2 (Laser Confocal Microscopy Analysis) The stable CAR-M cell line (CAR-M / CD54) based on the ICAM-1 targeting ligand gene prepared in Example 1 was used. SCL ) and the control stable strain (CAR-M / CTL) prepared in comparison. SCL ) respectively with 5×10 per hole 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured overnight at 37°C with 5% CO2. After culture, cells were collected by centrifugation and washed three times with PBS. Then, 1 mL of 4% paraformaldehyde solution was added to each tube, and the cells were fixed at room temperature for 15 minutes. After fixation, the cells were washed three times again with PBS. After removing the PBS, 50 μL of anti-fluorescence quenching mounting medium containing DAPI was added to resuspend the cells, and the mixture was gently mixed. 20 μL of the cell suspension was dropped into the center of an adhesive slide, and a coverslip was carefully placed on top, avoiding air bubbles. Finally, the edges of the coverslip were sealed with acrylic resin, and the prepared slide was stored overnight at 4°C in the dark. All images were taken using a super-resolution laser confocal microscope (Olympus Corporation, FV3000RS) with integrated data processing software and a ×40 objective lens.

[0057] Test results are as follows Figure 3 As shown, under 400x magnification using a laser confocal microscope, the following fluorescence expression can be clearly observed: CAR-M / CTL SCL The group of cells simultaneously carried EGFP green fluorescence and mCherry red fluorescence; while CAR-M / CD54 SCL The cells in this group only carried mCherry red fluorescence. The nuclei of all cells were stained with DAPI, showing blue fluorescence.

[0058] Test Example 3 (CAR-M Phagocytosis Rate Detection) CAR-M cell activation: The stable CAR-M cell line (CAR-M / CD54) based on the ICAM-1 targeting ligand gene prepared in Example 1 was used. SCL Cells were kept in RPMI 1640 (GibcoLife Sciences, Thermo Fisher Scientific (China) Co., Ltd.) supplemented with 10% FBS and 300 μg / ml LG418 for 24 hours to differentiate into macrophages, and then the culture medium was changed and the cells were left to stand for up to 48 hours. Simultaneously, the control group stable strain (CAR-M / CTL) prepared in the comparative example was taken. SCLCells were maintained in RPMI 1640 (Gibco Life Sciences, Thermo Fisher Scientific (China) Co., Ltd.) supplemented with 10% FBS and 2 μg / ml Luromycin, and differentiated into macrophages in the presence of 10 ng / ml Phorbol 12-myristate 13-acetate (Phorbol myristate acetate, PMA) for 24 hours. The culture medium was then changed and the cells were left to stand for up to 48 hours.

[0059] Resuscitation and passage of Plasmodium falciparum: Remove the cryopreserved tubes and incubate at 37°C for 1-2 min. Transfer the infected red blood cells (iRBCs) to a 50 mL centrifuge tube containing Plasmodium falciparum culture medium. Add 0.2 times the total volume of red blood cells to 37°C preheated 12% NaCl dropwise while shaking, and let stand for 5 min. Then add 9 times the total volume of red blood cells to 37°C preheated 1.6% NaCl dropwise while shaking, and let stand for 5 min. Centrifuge at 2500 rpm for 5 min, discard the supernatant, add 37°C culture medium, transfer to T25 culture, add 10 mL of culture medium and RBCs to maintain an 8% cell volume, inject CO2 mixed gas, tighten the culture flask, and let it incubate. After resuscitation, change the culture medium every 1-2 days and add fresh RBCs once a week on average.

[0060] CAR-M phagocytosis rate experiment: The activated CAR-M cells were distributed at a density of 2 × 10⁶ cells per well. 6 Cells were seeded at a density of [number] cells per well in 6-well plates. Experimental groups included CAR-M / CTL [number]. SCL and CAR-M / CD54 SCL Single iRBC samples were stained using a cell membrane staining kit (Beyotime, C1995S): 500 μL of staining working solution was prepared, containing 1.25 μL of 400× DiD far-red fluorescent dye for cell membranes, 1.25 μL of 400× staining enhancer, and 497.5 μL of staining buffer. The iRBCs were placed in this working solution and incubated at 37°C in the dark for 20 minutes. Subsequently, the cells were centrifuged at 2000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were slowly resuspended in pre-warmed Cell Staining Buffer, followed by two washes. 5×10⁵ cells were then added to the solution. 7 DiD-labeled RBCs (normal red blood cells from healthy individuals) and iRBCs were added to CAR-M / CTL. SCL CAR-M / CD54 SCLCells were cultured for 1 hour in each group. Cells from each group were collected, centrifuged at 2000 rpm for 5 minutes at room temperature, and the supernatant was discarded. 100 μL of Cell Staining Buffer and 1 μL of CD11b fluorescent antibody were added, and the cells were incubated at room temperature in the dark for 20 minutes for staining. Then, 1 mL of Cell Staining Buffer was added, and the cells were centrifuged at 2000 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 200 μL of Cell Staining Buffer, filtered, and analyzed using a Cytoflex flow cytometer (Beckman Coulter). The final data were processed and analyzed using FlowJo 10.4 software. The phagocytic rate increase rate of the experimental group (ICAM-1) compared to the control group (CTL) was calculated as follows: Improvement rate (%) = (Experimental group - CTL) / CTL × 100% The test results are shown in Table 1 and Figures 4-6 As shown, the cell proportions in each group in this experiment are as follows: CAR-M / CTL SCL The RBCs group had a prevalence of 0.68%, the RBCs group had a prevalence of 0.20%, and the iRBCs group had no prevalence (0%). The prevalence of RBCs was compared with that of CAR-M / CTL. SCL and CAR-M / CD54 SCL When co-cultured, their phagocytosis rates were 0.55% and 0.24%, respectively. This is in contrast to CAR-M / CTL. SCL Group, indicating CAR-M / CD54 SCL The phagocytosis rate did not change significantly. This result indicates that the constructed CAR-M cells do not have a targeted phagocytic effect on normal erythrocytes.

[0061] When iRBCs are associated with CAR-M / CTL SCL and CAR-M / CD54 SCL When co-cultured separately, the proportions increased to 3.63% and 7.07%. This is compared to CAR-M / CTL. SCL Phagocytosis rate, CAR-M / CD54 SCL The phagocytic rate was increased by 94.77%. This result indicates that the constructed CAR-M cells have a targeted phagocytic effect on infected red blood cells.

[0062] Table 1. CAR-M phagocytosis rate

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A CAR-M cell based on the ICAM-1 targeting ligand gene, characterized in that, The extracellular antigen-binding region of the CAR-M cells includes the ICAM-1 targeting ligand gene; The DNA sequence of the ICAM-1 targeting ligand gene is shown in SEQ ID No.

1.

2. A method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene as described in claim 1, characterized in that, Includes the following steps: The transfer plasmid containing the ICAM-1 targeting ligand gene, the VSV-G envelope plasmid, and the lentiviral packaging helper vector were co-transfected into HEK293T packaging cells. After transfection, the culture supernatant was collected, centrifuged, filtered, and a clear viral solution was obtained. The clarified viral solution was concentrated to obtain mCherry-ICAM-1 lentivirus suspension; THP-1 cells were transduced using the mCherry-ICAM-1 lentivirus, and then the transduced THP-1 cells were sequentially screened for antibiotics and monoclonal cells to obtain CAR-M cells.

3. The method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene according to claim 2, characterized in that, The lentivirus packaging aids are: VB-VSV-G, VB-Rev, and VB-Gag / Pol.

4. The method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene according to claim 2, characterized in that, The transduction specifically refers to: The mCherry-ICAM-1 lentivirus suspension and Polybrene were mixed with THP-1 medium to obtain a mixed medium. THP-1 cells were first seeded in THP-1 medium and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then the medium was removed and the mixed medium was added, and the cells were cultured for another 48 hours. THP-1 medium was added at the 4th hour and replaced with fresh THP-1 medium at the 24th hour.

5. The method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene according to claim 4, characterized in that, The THP-1 medium was RPMI 1640 medium containing 10% FBS, 1% penicillin / streptomycin and 50 μmol / L β-mercaptoethanol.

6. The method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene according to claim 5, characterized in that, The multiplicity of infection (MOI) when transducing THP-1 cells using the mCherry-ICAM-1 lentivirus was 10.

7. The method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene according to claim 6, characterized in that, The antibiotic screening specifically involves: After transduction, the cell culture medium was replaced with THP-1 medium containing 600 μg / mL G418. Subsequently, the cells were passaged every 48 hours and replaced with THP-1 medium containing 300 μg / mL G418 for 3-5 days.

8. The method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene according to claim 7, characterized in that, The passage ratio for the subculture is 1:

5.

9. The method for constructing CAR-M cells based on the ICAM-1 targeting ligand gene according to claim 8, characterized in that, The monoclonal cell screening specifically involves: Collect cells after antibiotic selection, centrifuge, and resuspend in THP-1 medium containing 300 μg / ml LG418 to adjust the concentration to 2 × 10⁻⁶. 4 cells / mL, to obtain a cell suspension; Take a 96-well cell culture plate (8×12), add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well, except for well A1 which is left untreated. Then add 200 μL of the cell suspension to well A1. Then, perform serial dilutions at a ratio of 1:2 along the longitudinal direction (A1 to H1) and then serial dilutions at a ratio of 1:2 along the horizontal direction (wells 1 to 12 of each row). After dilution, add 100 μL of THP-1 medium containing 300 μg / mL G418 to each well to bring the final volume of each well to 200 μL. Screen out wells containing only a single cell and mark them as monoclonal wells. Then, incubate the cell culture plate at 37°C and 5% CO2 for 7-14 days.

10. The use of CAR-M cells based on the ICAM-1 targeting ligand gene as described in claim 1 in the preparation of drugs to clear Plasmodium falciparum infection of erythrocytes.

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