Mononuclear / macrophage of targeted carcino-embryonic antigen (CEA) chimeric antigen receptor as well as construction method and application of mononuclear / macrophage
By carrying out CEA-CAR gene editing and transformation of monocytes, monocytes of CEA-CAR chimeric antigen receptor were constructed, and the drug resistance and inefficiency of EGFR mutant advanced NSCLC to existing treatment methods was solved, and precise immunotherapy and effective anti-tumor efficacy were achieved for this type of tumor.
Patent Information
- Application Number
- CN202411916689.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-21
AI Technical Summary
Patients with advanced non-small cell lung cancer (NSCLC) have resistant to tyrosine kinase inhibitors (TKIs) and have low therapeutic effects on immune checkpoint blockers (ICB). Existing cell therapy such as CAR-T or CAR-NK is poor in the treatment of solid tumors, and there are limited strategies to directly enhance the anti-tumor function of macrophages.
By targeting carcinoembryonic antigen (CEA) chimeric antigen receptor (CAR) gene editing and modification of monocytes, monocytes of CEA-CAR chimeric antigen receptor are constructed, which enhances their tumor targeting, phagocytosis and antigen presentation capabilities, and improves the immunosuppressive microenvironment of advanced NSCLC in EGFR mutant type.
Accurate immunotherapy for advanced NSCLC of EGFR mutant type was achieved, enhancing the tumor targeting and anti-tumor function of monocytes, improving the immunosuppressive state of the tumor microenvironment, and achieving effective anti-tumor efficacy.
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Figure CN119662550A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a monocyte / macrophage targeting carcinoembryonic antigen CEA chimeric antigen receptor and a construction method and application thereof, belonging to the technical field of protein expression. Background Art
[0002] In the past decade, chimeric antigen receptors (CARs) have been used in T cells and natural killer (NK) cells to enhance their anti-tumor effects, making important progress in the field of cancer immunotherapy. CARs are used to edit macrophages to make them anti-tumor effector cells and improve their phagocytic ability. Given the innate anti-tumor immune response ability of macrophages, engineering macrophages using gene editing technology may become an effective strategy for tumor immunotherapy.
[0003] There is a lack of treatment strategies for patients with advanced non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. Patients with NSCLC with EGFR mutations can initially be treated with tyrosine kinase inhibitors (TKIs). However, patients with EGFR mutations are resistant to TKIs due to secondary EGFR mutations or EGFR-independent mechanisms. Clinical treatment options after EGFR-TKI resistance are very limited, anti-PD-1 / PD-L1 inhibitors have little therapeutic effect on patients, and the objective response rate of immune checkpoint blockers (ICBs) is usually less than 5%. Faced with this dilemma, we intend to use adoptive cell immunotherapy technology to provide a possible treatment option for conquering NSCLC with EGFR mutations.
[0004] Therefore, it is necessary to develop new and effective immune cell therapy strategies for EGFR mutant advanced NSCLC. Cell therapy for EGFR mutant advanced NSCLC by engineering chimeric antigen receptor gene editing in monocytes / macrophages may be an effective strategy to achieve precision immunotherapy.
[0005] Defects and shortcomings of the existing technology:
[0006] EGFR mutant advanced NSCLC is prone to TKI resistance and is insensitive to immune checkpoint inhibitors. Cell therapy drugs such as CAR-T or CAR-NK have poor effects on solid tumors. Currently, the main way to treat TAMs in the tumor microenvironment is to change the number of TAMs, such as inhibiting macrophage chemotaxis to solid tumors, reducing the number of macrophages, inducing M2 macrophages to repolarize to M1, or promoting TAM reprogramming to M1. There are limited strategies to directly enhance the innate anti-tumor function of macrophages. Summary of the invention
[0007] The object of the present invention is to provide a monocyte / macrophage targeting carcinoembryonic antigen CEA chimeric antigen receptor.
[0008] The technical solution adopted by the present invention is:
[0009] A monocyte / macrophage targeting a carcinoembryonic antigen CEA chimeric antigen receptor, characterized in that the monocyte / macrophage expresses a chimeric antigen receptor targeting the carcinoembryonic antigen CEA, the chimeric antigen receptor comprising an intracellular domain, a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen recognition domain scFv for binding to the antigen CEA, and its sequence is shown in SEQ ID No.1.
[0010] Preferably, the extracellular domain further comprises a signal peptide and a hinge region connected to both ends of the antigen recognition domain, the antigen recognition domain is connected to the transmembrane domain through the hinge region, the amino acid sequence of the signal peptide is shown in SEQ ID No.2, and the amino acid sequence of the hinge region is shown in SEQ ID No.3.
[0011] Preferably, the amino acid sequence of the transmembrane domain is shown as SEQ ID No.4.
[0012] Preferably, the amino acid sequence of the intracellular domain is shown as SEQ ID No.5.
[0013] Preferably, the intracellular domain is further connected to a cell-linking peptide, and the amino acid sequence of the cell-linking peptide is shown in SEQ ID No.6.
[0014] Preferably, the monocytes / macrophages are macrophages differentiated from ThP1 cells.
[0015] The present invention also discloses the use of the monocyte / macrophage targeting carcinoembryonic antigen CEA chimeric antigen receptor in the preparation of a drug for treating EGFR mutant advanced NSCLC.
[0016] The present invention also discloses a method for constructing the above-mentioned monocyte / macrophage targeting carcinoembryonic antigen CEA chimeric antigen receptor, the steps of which include:
[0017] (1) constructing and packaging a lentiviral vector of a chimeric antigen receptor targeting carcinoembryonic antigen (CEA);
[0018] (2) Induce ThP1 cells to differentiate into macrophages;
[0019] (3) Transfecting the lentiviral vector constructed in step (1) into differentiated macrophages to obtain CEA-CAR-ThP1 cells.
[0020] Preferably, in step (2), phorbol ester is used to induce ThP1 cells to differentiate into macrophages.
[0021] The present invention also discloses an antigen recognition domain scFv for binding to the antigen CEA, and the sequence thereof is shown as SEQ ID No.1.
[0022] Beneficial effects of the present invention:
[0023] The present invention performs CAR gene editing on monocytes / macrophages, and utilizes the good tissue infiltration and cytokine chemotaxis of monocytes / macrophages in solid tumors to achieve the treatment of EGFR mutant advanced NSCLC. And through CEA-CAR gene editing and transformation, monocytes / macrophages have stronger tumor targeting, phagocytic ability, and tumor antigen presentation ability, while improving the immunosuppressive microenvironment of EGFR mutant advanced NSCLC, achieving effective anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The CEA-CAR vector plasmid map (left) and CEA-CAR structure schematic (right).
[0025] Figure 2 The flow cytometry results of UTD-ThP1 cells (left) and CEA-CAR-ThP1 cells (right).
[0026] Figure 3 These are the results of identifying the polarization phenotype of UTD-ThP1 cells and CEA-CAR-ThP1 cells.
[0027] Figure 4 These are the results of phagocytic function gene expression detection in UTD-ThP1 cells and CEA-CAR-ThP1 cells.
[0028] Figure 5 These are the results of pro-inflammatory cytokine gene expression detection in UTD-ThP1 cells and CEA-CAR-ThP1 cells.
[0029] Figure 6 These are the results of the phagocytic function test of UTD-ThP1 cells and CEA-CAR-ThP1 cells on A549 cells.
[0030] Figure 7 These are the detection results of pro-inflammatory cytokines secreted by UTD-ThP1 cells and CEA-CAR-ThP1 cells after co-incubation with A549 cells.
[0031] Figure 8 These are the results of reactive oxygen species (ROS) detection after UTD-ThP1 cells and CEA-CAR-ThP1 cells were co-incubated with A549 cells.
[0032] Fig. 9These are the results of the anti-tumor function test of UTD-ThP1 cells and CEA-CAR-ThP1 cells on A549 cells. DETAILED DESCRIPTION
[0033] The present invention is further illustrated by examples below, but is not intended to be limiting of the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, and materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.
[0034] Example
[0035] (1) ThP1 cell culture: RPMI1640 medium, 10% fetal bovine serum, 1% penicillin and streptomycin; ThP1 cells were treated with 100 ng / ml phorbol esterase (PMA) for 6 hours to differentiate into macrophages. A549 cell culture: RPMI1640 medium, 10% fetal bovine serum, 1% penicillin and streptomycin.
[0036] (2) Preparation of CEA-CAR-ThP1 cells: Lipofectamine 2000 (Invitrogen) was used to transduce the CEA-CAR vector plasmid (plasmid map as shown in Figure 1 As shown in the figure on the left, the structure of CEA-CAR is as follows Figure 1 As shown in the right figure, where mCherry is a fluorescent marker gene), psPAX2 and pMD2.G were transfected into HEK293T cells. The culture medium containing lentivirus was collected at 24h, 36h and 48h after transfection, and concentrated and purified with PEG8000. The lentivirus was then added to a six-well plate in which ThP1 cells were cultured. Fresh complete medium was replaced 6 hours after infection. The transfected cells were cultured for 72h and then sorted and purified by flow cytometry. (The results are shown in Figure 2 As shown in the right figure, UTD-ThP1 refers to ThP1 cells that have not been transduced with CAR virus, and other treatments are the same as CEA-CAR-ThP1 cells).
[0037] (3) Identification of CEA-CAR-ThP1 cell polarization phenotype: M1 markers: iNOS, CD86, M2 markers: Arg1, Ym1. CEA-CAR-ThP1 cells were treated with 100 ng / ml recombinant protein CEA for 48 hours, and total RNA was extracted from CEA-CAR-ThP1 cells using Trio Reagent (Invitrogen, Carlsbad, CA, USA). Specific conditions and parameters were based on the protocol in the instruction manual, and 1 μg of total RNA was reversed into cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Nanjing Novigene). Real-time PCR was performed in a 7500 detection system using SYBR-Green I as a double-stranded DNA-specific binding dye. The mRNA of the target gene and the mRNA of the housekeeping gene β-actin were quantified in separate tubes. (Results see Figure 3 )
[0038] (4) Detection of phagocytic function genes and pro-inflammatory cytokine gene expression in CEA-CAR-ThP1 cells: phagocytic function genes: Rab11a, Atg5, Lamp2, pro-inflammatory cytokine genes: TNF-α, INF-γ, IL-12. CEA-CAR-ThP1 cells were treated with 100 ng / ml recombinant protein CEA for 48 hours, and total RNA was extracted from CEA-CAR-ThP1 cells using Trio Reagent (Invitrogen, Carlsbad, CA, USA). The operation steps are the same as step (3). (Results are shown in Figure 4-5 )
[0039] (5) Detection of the phagocytic function of CEA-CAR-ThP1 cells on A549 cells: A549 cells were labeled with 5,6-hydroxyfluorescein diacetate succinimidyl ester (CFSE), and then CEA-CAR-ThP1 cells and A549 cells were co-cultured at a ratio of 1:1 for 24 hours. The cells were collected, washed with PBS containing 1% BSA, and then labeled with antibodies for 20 minutes to detect the efficiency of CEA-CAR-ThP1 cells in phagocytizing A549 (CFSE fluorescent labeling). Flow cytometry antibodies: Brilliant Violet (BV) 711 anti-mouse F4 / 80 (BioLegend, 123147), APC anti-mouse / human CD11b (BioLegend, 101212). Flow cytometry experiments were performed on a Thermo Scientific flow cytometer and analyzed on FlowJo V10 software. (Results are shown in Figure 6 )
[0040] (6) Detection of pro-inflammatory cytokines secreted by CEA-CAR-ThP1 cells after co-incubation with A549 cells: CEA-CAR-ThP1 cells were co-incubated with A549 cells for 24 hours, and the levels of pro-inflammatory cytokines TNF-α and IL-12 were detected by Elisa method (Thermo Fisher, according to the specific protocol in the instruction manual). (Results are shown in Figure 7 )
[0041] (7) ROS detection after co-incubation of CEA-CAR-ThP1 cells with A549 cells: CEA-CAR-ThP1 cells were co-incubated with A549 cells for 24 hours, and the generated ROS were detected by adding H2DCFDA probe (Biyuntian, Shanghai, China) according to the instructions. (Results are shown in Figure 8 )
[0042] (8) Detection of the anti-tumor function of CEA-CAR-ThP1 cells on A549 cells: Target cells A549 (stable tumor cell line produced by Luciferase lentiviral transfection) were inoculated in a black 96-well ELISA plate at a density of 5000 cells / 100 μL / well. Effector cells were UTD-ThP1 cells and CEA-CAR-ThP1 cells. The ratios of effector cells to target cells (E:T) were 0:1, 1:1, 5:1 and 10:1, respectively. After incubation for 24 hours, the cells were incubated according to ONE-Glo TM The luciferase assay system was used for detection on a Varioskan Flash luminometer (Thermo Scientific). The experimental method was based on the instructions of the kit supplier (Promega, E6110). The results are shown in Fig. 9 .
[0043] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.
Claims
1. A monocyte / macrophage targeting carcinoembryonic antigen (CEA) chimeric antigen receptor, characterized in that: The monocytes / macrophages express a chimeric antigen receptor targeting carcinoembryonic antigen CEA, wherein the chimeric antigen receptor comprises an intracellular domain, a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen recognition domain scFv for binding to the antigen CEA, and its sequence is shown in SEQ ID No.
1.
2. The monocyte / macrophage targeting carcinoembryonic antigen (CEA) chimeric antigen receptor according to claim 1, characterized in that: The extracellular domain also includes a signal peptide and a hinge region connected to both ends of the antigen recognition domain. The antigen recognition domain is connected to the transmembrane domain through the hinge region. The amino acid sequence of the signal peptide is shown in SEQ ID No.2, and the amino acid sequence of the hinge region is shown in SEQ ID No.
3.
3. The monocyte / macrophage targeting carcinoembryonic antigen (CEA) chimeric antigen receptor according to claim 2, characterized in that: The amino acid sequence of the transmembrane domain is shown in SEQ ID No.
4.
4. The monocyte / macrophage targeting carcinoembryonic antigen (CEA) chimeric antigen receptor according to claim 3, characterized in that: The amino acid sequence of the intracellular domain is shown in SEQ ID No.
5.
5. The monocyte / macrophage targeting carcinoembryonic antigen (CEA) chimeric antigen receptor according to claim 4, characterized in that: The intracellular domain is connected to a fluorescent label via a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID No.
6.
6. The monocyte / macrophage targeting carcinoembryonic antigen (CEA) chimeric antigen receptor according to any one of claims 1 to 5, characterized in that: The monocytes / macrophages are macrophages differentiated from ThP1 cells.
7. Use of the monocyte / macrophage targeting carcinoembryonic antigen (CEA) chimeric antigen receptor according to any one of claims 1 to 6 in the preparation of a drug for treating EGFR mutant advanced NSCLC.
8. The method for constructing monocytes / macrophages targeting carcinoembryonic antigen (CEA) chimeric antigen receptor according to any one of claims 1 to 6, characterized in that The steps include: (1) constructing and packaging a lentiviral vector of a chimeric antigen receptor targeting carcinoembryonic antigen (CEA); (2) Induce ThP1 cells to differentiate into macrophages; (3) Transfecting the lentiviral vector constructed in step (1) into differentiated macrophages to obtain CEA-CAR-ThP1 cells.
9. The construction method according to claim 8, characterized in that: In step (2), phorbol ester is used to induce ThP1 cells to differentiate into macrophages.
10. An antigen recognition domain scFv for binding to the antigen CEA, characterized in that Its sequence is shown in SEQ ID No.1.