CARs targeting human HER2, CAR genes and their recombinant vectors, CAR-M cells and their preparation methods and applications
By designing CARs that target human HER2 and optimizing the CAR-M cell culture medium, the problem of insufficient killing ability of CAR-M cells in the treatment of solid tumors was solved, and efficient tumor cell targeting and killing effects were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-09
- Publication Date
- 2026-04-03
AI Technical Summary
Existing CAR-M cells have low killing capacity in the treatment of solid tumors and are difficult to effectively infiltrate the tumor interior. They are limited by the immunosuppression of the tumor microenvironment, which affects the treatment effect.
The design of a CAR targeting human HER2 includes a leader peptide, an extracellular recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain. The FcγR family transmembrane region is used to activate macrophage phagocytic signals, and the CAR-M cells are prepared by combining with a specific intracellular signaling domain. The killing ability is enhanced by using a specific cell culture medium.
It improves the targeting and phagocytic activity of CAR-M cells to tumor cells, significantly enhances the killing ability of tumor cells, overcomes the immunosuppression of the tumor microenvironment, and achieves effective tumor treatment.
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Figure CN115746149B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of immunotherapy, specifically to a chimeric antigen receptor and its application, a CAR targeting human HER2, a CAR gene and its recombinant vector, CAR-M cells and their preparation methods and applications. Background Technology
[0002] With the development of tumor immunology theory and technology, adoptive cell therapy (ACT) in biotherapy has been widely used in clinical treatment. This method involves delivering autologous or allogeneic immune effector cells activated in vitro to the patient. Subsequently, the immune effector cells can specifically recognize and kill cancer cells and mutated cells in the patient's body, thereby achieving the goal of treating tumors.
[0003] In recent years, chimeric antigen receptor-T cells (CAR-T) have been widely used in adoptive immunotherapy. CAR-T cells are produced by genetically modifying T cells with chimeric antigen receptors (CARs), then expanding them in vitro before reinfusing them into the patient. This method has shown significant efficacy in treating hematological malignancies. However, in the clinical treatment of solid tumors, the extracellular matrix of solid tumor cells can hinder CAR-T cell infiltration; furthermore, solid tumors create an immunosuppressive tumor microenvironment, thus impacting CAR-T cell immunotherapy.
[0004] To address the challenges of immunotherapy for solid tumors, chimeric antigen receptor-macrophage (CAR-M) immunotherapy utilizes monocytes / macrophages to construct CARs. Compared to CAR-T, CAR-M can directly kill tumor cells; it can also improve the tumor immune microenvironment in solid tumors; and CAR-M more easily infiltrates the tumor, synergistically infiltrating the tumor with other immune cells. However, current research on CAR-M is limited, and macrophages have the limitation of not proliferating, resulting in a lower tumor-killing ability of CAR-M, which greatly restricts its application. Summary of the Invention
[0005] To enhance the killing ability of CAR-M cells, this application provides a CAR targeting human HER2, the CAR gene and its recombinant vector, CAR-M cells and their preparation methods and applications.
[0006] Firstly, this application provides a CAR targeting human HER2.
[0007] A CAR targeting human HER2 employs the following technical solution:
[0008] A CAR targeting human HER2 includes a leader peptide, an extracellular recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain;
[0009] The extracellular recognition region is a HER2 single-chain antibody that binds to tumor antigens; the transmembrane region is any one of FcγRⅠ, FcγRⅡ, FcγRⅢ, FCER1G, and CD36 that can activate macrophage phagocytic signals; the intracellular signaling domain is the P2 domain with an amino acid sequence as shown in SEQ ID NO.5.
[0010] Preferably, the leader peptide is a signal peptide SP.
[0011] Preferably, the hinge area is selected from either CD8 or CD28 hinge.
[0012] Optionally, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.6.
[0013] This application selects the P2 domain, as shown in SEQ ID NO.5, as the intracellular signaling domain and assembles and links it with the leader peptide, extracellular recognition region, hinge region, and transmembrane region. The resulting chimeric antigen receptor can effectively infect macrophages, and the obtained CAR-M cells can exert efficient targeting, phagocytosis, and killing effects on tumor cells.
[0014] This application uses any one of FcγRⅠ, FcγRⅡ, FcγRⅢ, FCERT1G, and CD36 as the transmembrane region. The FcγR family can recognize and bind to the Fc fragment of Ig, effectively clearing IgG-Ag complexes and exhibiting strong immunomodulatory effects. FCERT1G contains a tyrosine-based immune receptor activation motif, capable of transmitting activation signals from immune receptors. CD36 is mainly expressed on the surface of monocytes / macrophages and can mediate phagocytosis. In other words, all five molecules can activate macrophages to generate phagocytic signals, promoting the phagocytic and killing capabilities of CAR-M against tumor cells.
[0015] Secondly, this application provides a CAR gene encoding the aforementioned CAR targeting human HER2.
[0016] Thirdly, this application provides a recombinant vector comprising the above-mentioned CAR targeting human HER2 or the above-mentioned CAR gene.
[0017] Fourthly, this application provides a CAR-M cell comprising the above-mentioned recombinant vector.
[0018] Fifthly, this application provides a method for preparing the above-mentioned CAR-M cells, which are obtained by introducing the recombinant vector into macrophages.
[0019] Preferably, the macrophages are mouse bone marrow-derived macrophages.
[0020] Preferably, the method for preparing CAR-M cells specifically includes the following steps:
[0021] The leader peptide, the extracellular recognition region, the hinge region, the transmembrane region, and the intracellular signaling domain are sequentially connected from the N-terminus to the C-terminus to obtain the CAR targeting human HER2.
[0022] The recombinant vector was constructed by recombining the CAR targeting human HER2 with a lentiviral vector.
[0023] The recombinant vector was introduced into the macrophages to obtain the CAR-M cells.
[0024] Preferably, the cell culture medium used in the process of introducing the recombinant vector into the macrophages includes a basal culture medium and the following components by weight percentage: 0.003-0.008 mg / mL sodium stearate, 0.007-0.013 mg / mL erythritol, and 0.16-0.24 mg / mL transferrin.
[0025] This application utilizes the aforementioned cell culture medium, which can effectively prevent the recombinant vector lentivirus from being phagocytosed by phagocytes, and facilitates the transfection of the recombinant vector into macrophages, thereby effectively enhancing the killing ability of CAR-M cells against target cells.
[0026] Sixthly, this application provides the use of the CAR targeting human HER2, the CAR gene, the recombinant vector, and the CAR-M cells in the preparation of drugs for treating tumors.
[0027] In summary, the technical solution of this application has the following effects:
[0028] This application screens the P2 domain as an intracellular signaling domain and assembles and connects it with the leader peptide, the extracellular recognition region of the human HER2 single-chain antibody, the hinge region, and the transmembrane region. The resulting CAR-M cells can exert efficient targeting, phagocytosis, and killing effects on tumor cells.
[0029] This application utilizes sodium stearate, erythritol, transferrin, and basal culture medium to obtain CAR-M cell culture medium, which can effectively enhance the killing ability of CAR-M cells against target cells. Attached Figure Description
[0030] Figure 1 This refers to the phagocytic capacity of different CAR-M cells against tumor cells in Experiment Example 1 of this application. Detailed Implementation
[0031] In the first aspect, this application provides a CAR targeting human HER2, including a leader peptide, an extracellular recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain;
[0032] Among them, the leader peptide is the signal peptide SP; the extracellular recognition region is a HER2 single-chain antibody that binds to tumor antigens; the hinge region is selected from either CD8 or CD28 hinge; the transmembrane region is any one of FcγRⅠ, FcγRⅡ, FcγRⅢ, FCER1G, or CD36 that can activate macrophage phagocytic signals; and the intracellular signal domain is the P2 domain with an amino acid sequence as shown in SEQ ID NO.5.
[0033] Specifically, the amino acid sequence of the CAR targeting human HER2 is shown in SEQ ID NO.6.
[0034] Secondly, this application provides a CAR gene encoding the aforementioned CAR targeting human HER2.
[0035] Thirdly, this application provides a recombinant vector including the aforementioned CAR gene.
[0036] Fourthly, this application provides a CAR-M cell comprising the above-mentioned recombinant vector.
[0037] Fifthly, this application provides a method for preparing the above-mentioned CAR-M cells, which are obtained by introducing the above-mentioned recombinant vector into macrophages.
[0038] Among them, the macrophages are mouse bone marrow-derived macrophages.
[0039] The above-mentioned method for preparing CAR-M cells specifically includes the following steps:
[0040] By sequentially connecting the leader peptide, extracellular recognition region, hinge region, transmembrane region, and intracellular signaling domain from the N-terminus to the C-terminus, a CAR targeting human HER2 is obtained.
[0041] A recombinant vector was constructed by recombining a CAR targeting human HER2 with a lentiviral vector.
[0042] The recombinant vector was introduced into mouse bone marrow-derived macrophages to obtain CAR-M cells.
[0043] In the CAR-M cell preparation process, the CAR-M cell culture medium used is DMEM / F12 medium containing 0.003-0.008 mg / mL sodium stearate, 0.007-0.013 mg / mL erythritol, and 0.16-0.24 mg / mL transferrin.
[0044] Sixthly, this application provides the application of CARs targeting human HER2, CAR genes, recombinant vectors, and CAR-M cells in the preparation of drugs for treating tumors.
[0045] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application. Example Example 1
[0046] Example 1 provides a CAR recombinant vector, the preparation of which specifically includes the following steps:
[0047] Synthesis of chimeric antigen receptors: Through experimental screening and research, chimeric antigen receptor molecules targeting HER2 were assembled, including a leader peptide, extracellular recognition region, hinge region, transmembrane region, and intracellular signaling domain. The chimeric antigen receptor molecule was constructed sequentially from N-terminus to C-terminus, with the signal peptide SP (amino acid sequence shown in SEQ ID NO.1) as the leader peptide, HER2 scFv (amino acid sequence shown in SEQ ID NO.2) as the extracellular recognition region, CD28 hinge region (amino acid sequence shown in SEQ ID NO.3) as the hinge region, FcγRIA domain (amino acid sequence shown in SEQ ID NO.4) as the transmembrane region, and P2 domain (amino acid sequence shown in SEQ ID NO.5) as the intracellular signaling domain. The chimeric antigen receptor P2-CAR (amino acid sequence shown in SEQ ID NO.6, nucleotide sequence shown in SEQ ID NO.7) was synthesized.
[0048] Construction of the P2-CAR recombinant vector: The synthesized chimeric antigen receptor P2-CAR sequence was digested with Spe I / Xho I, and the pLVX-EF1α-AcGFP1-N1 vector was digested with Spe I / Xho I. The digested chimeric antigen receptor and vector were ligated with T4 ligase and transformed into competent E. coli (DH5α). After confirming the sequence was correct, the plasmid was extracted and purified using a plasmid purification kit (Qiagen) to obtain the P2-CAR recombinant vector for subsequent experiments. Example 2
[0049] Example 2 provides a P2-CAR-M cell. The preparation of P2-CAR-M cells specifically includes the following steps:
[0050] Preparation of P2-CAR lentivirus: 4 × 10 6HEK293T cells in logarithmic growth phase were evenly seeded in 6-well plates and cultured statically at 37°C with 5% CO2 until the cell confluence reached 70%-80%. Plasmids P2-CAR, psPAX2, and pMD2.G were added to centrifuge tubes containing Opti-MEM medium at a ratio of 3:1:1 and mixed well. The mixture was then incubated at room temperature. TurboFect Transfection Reagent was added and gently mixed by pipetting. The mixture was then incubated at room temperature.
[0051] The incubated mixture was slowly added dropwise to a 6-well plate seeded with 293T cells, while gently shaking the plate. After incubation at 37°C and 5% CO2 for 18-24 hours, the transfection mixture in each well was discarded. 3 mL of Advanced DMEM complete medium containing 10% fetal bovine serum was added, and the cell culture plate was returned to the incubator at 37°C and 5% CO2 for 48 hours. The viral supernatant was collected, centrifuged at 4000 rpm for 10 minutes, filtered through a 0.45 μm filter to remove cell debris, and then centrifuged at 25000 rpm for 2 hours at 4°C to obtain the P2-CAR lentivirus concentrate. The concentrate was aliquoted and stored at -80°C.
[0052] Preparation of P2-CAR-M cells: Mouse mononuclear macrophages J774 A1 were seeded into 6-well plates. The CAR-M cell culture medium used was: containing 0.005 mg / mL sodium stearate, 0.01 mg / mL erythritol, 0.2 mg / mL transferrin, and 1 L DMEM / F12 medium. When the cells reached a confluence of 70%-80%, P2-CAR lentiviral particles were added to the macrophage culture medium at a ratio of 9:1. After static incubation at 37℃ and 5% CO2 for 24 h, the medium was replaced with fresh CAR-M medium. After 72 h of infection, the macrophages were infected with lentiviral virus, and P2-CAR-M cells were obtained. Cell growth and morphological changes were observed, and cells were passaged into new culture flasks or dishes for expansion or cryopreservation. Comparative Example 1
[0053] Comparative Example 1 provides a CAR recombinant vector.
[0054] The difference between this comparative example and Example 1 is that the CD28 domain is used as the intracellular signaling domain. Comparative Example 2
[0055] Comparative Example 2 provides a CAR recombinant vector.
[0056] The difference between this comparative example and Example 1 is that the CD3ζ domain is used as the intracellular signal domain. Comparative Example 3
[0057] Comparative Example 3 provides a CAR-M cell.
[0058] The difference between this comparative example and Example 2 is that the chimeric antigen receptor used was prepared from Comparative Example 1. Comparative Example 4
[0059] Comparative Example 4 provides a CAR-M cell.
[0060] The difference between this comparative example and Example 2 is that the chimeric antigen receptor used was prepared from Comparative Example 2.
[0061] Performance testing
[0062] Experimental Example 1
[0063] This experiment used animal experiments to detect the inhibitory effect of P2-CAR-M cells in Example 2 and CAR-M cells in Comparative Examples 3-4 on HER2-positive gastric cancer, and to examine the phagocytic ability of CAR-M cells on tumor cells.
[0064] The detection method specifically includes the following steps: resuspending MKN45 gastric cancer cells in physiological saline and adjusting the viable cell concentration to 4 × 10⁻⁶. 7 Cells / mL were injected subcutaneously into the hind legs of 30 male C57 mice aged 6-8 weeks, with 100 μL of cell suspension per mouse. Tumor size was measured using a diaphragm, with a target size of 100 mm. 3 The experimental mice with tumors were used as a mouse HER2-positive gastric cancer model; the formula for calculating tumor volume was: tumor volume (mm). 3 = Tumor long diameter (mm) × Tumor short diameter 2 (mm) 2 ) × 0.5.
[0065] The successfully established mouse HER2-positive gastric cancer model was divided into four groups. Drugs were administered via tail vein injection. Three groups were given PBS 100 μL / mouse and live cell concentration of 1 × 10⁻⁶ cells / mouse, respectively. 6 In Example 2, 100 μL of P2-CAR-M cells were used per cell, at a concentration of 1 × 10⁻⁶. 6 In Comparative Example 3, 100 μL of CAR-M cells / mL were used, with a concentration of 1×10⁻⁶ cells / cell. 6 In Comparative Example 4, 100 μL of CAR-M cells were used per animal. Starting from day 7 of drug administration, tumor size was measured every 5 days using a caliper.
[0066] Test results: such as Figure 1 As shown.
[0067] pass Figure 1It was found that administering PBS to experimental mice resulted in an exponential increase in tumor size; administering CAR-M cells from Comparative Examples 3-4 to experimental mice controlled tumor growth to some extent, but the tumors still showed a growth trend; while administering P2-CAR-M cells from Example 2 to experimental mice kept the tumor size essentially unchanged, and tumor growth was significantly inhibited. These results indicate that the P2-CAR-M cells provided in this application have a good effect on inhibiting and killing tumor cells.
[0068] Example 3-13
[0069] The difference between the above embodiments and Embodiment 2 is that the content of each component in the CAR-M cell culture medium used is different. See Table 1 for details.
[0070] Table 1. Content of each component in the CAR-M cell culture medium in Examples 2-13
[0071]
[0072] Experimental Example 2
[0073] This experiment used cell biology to detect the killing ability of P2-CAR-M cells against target cells in Examples 2-13.
[0074] S1: Take density 1×10 6 Mouse mononuclear macrophage J774 A1 suspension was mixed with 15 μL of 1 mg / mL Calcein-AM and incubated at 37 °C and 5% CO2 for 30 min, with gentle mixing every 10 min. The cells were then centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cells were resuspended in whole culture medium. The process was repeated twice to obtain Calcein-AM labeled target cells.
[0075] S2: Using the P2-CAR-M cells provided in Examples 2-13 as effector cells, respectively, at a concentration of 3 × 10⁶ cells / cells... 4 Seeds were planted per well in 96-well cell culture plates, with 5 replicates per group, and incubated for 24 hours. Then, Calcein-AM labeled target cells were added at a ratio of effector cells to target cells of 4:1. The experimental group with target cells served as the blank group, and the experimental group with 2% Triton X-100+ target cells served as the control group.
[0076] S3: After culturing at 37℃ in a 5% CO2 incubator for 8 hours, D-luciferin and potassium salt substrate at a concentration of 150 μg / mL were added to a 96-well plate. After incubation at 37℃ in the dark for 10 minutes, the fluorescence intensity was collected and analyzed using a microplate reader. The killing ability of P2-CAR-M cells against target cells was calculated using the following formula: Target cell killing rate = (fluorescence intensity of experimental group - fluorescence intensity of blank group) / (fluorescence intensity of control group - fluorescence intensity of blank group) * 100%.
[0077] Test results are shown in Table 2.
[0078] Table 2 Results of the detection of the killing ability of P2-CAR-M cells against target cells in Examples 2-13
[0079]
[0080] Referring to Table 2, and comparing the test results of Examples 2-13, this application utilizes sodium stearate, erythritol, transferrin, and basal culture medium to obtain CAR-M cell culture medium. The CAR-M cells prepared from this medium exhibit a cell killing rate of over 77.5% against target cells. In other words, the CAR-M cell culture medium provided in this application can effectively enhance the killing ability of CAR-M cells against target cells.
[0081] Comparing the detection results of Examples 2-4, it was found that controlling the sodium stearate content in the CAR-M cell culture medium to be 0.003-0.008 mg / mL further enhanced the CAR-M cell killing ability against target cells. Comparing the detection results of Examples 2 and 5-6, it was found that controlling the erythritol content in the CAR-M cell culture medium to be 0.007-0.013 mg / mL further enhanced the CAR-M cell killing ability against target cells. Comparing the detection results of Examples 2 and 7-8, it was found that controlling the transferrin content in the CAR-M cell culture medium to be 0.16-0.24 mg / mL further enhanced the CAR-M cell killing ability against target cells. Therefore, this application controls the above three components to the corresponding content ranges described above.
[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A CAR targeting human HER2, characterized in that, The CAR consists of a leader peptide, an extracellular recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain, in that order from the N-terminus to the C-terminus. The leader peptide is a signal peptide SP with the amino acid sequence shown in SEQ ID NO.
1. The extracellular recognition region is the HER2 scFv amino acid sequence shown in SEQ ID NO.
2. The hinge region is the CD28 hinge region with the amino acid sequence shown in SEQ ID NO.
3. The transmembrane region is the FcγRIA domain as shown in SEQ ID NO.4, which has an amino acid sequence. The intracellular signaling domain is the P2 domain as shown in SEQ ID NO.
5. The amino acid sequence of the CAR targeting human HER2 is shown in SEQ ID NO.
6.
2. A CAR gene, characterized in that, The CAR gene encodes the CAR targeting human HER2 as described in claim 1.
3. A recombinant vector, characterized in that, Includes the CAR gene as described in claim 2.
4. A CAR-M cell, characterized in that, Includes the recombinant vector as described in claim 3.
5. The method for preparing CAR-M cells as described in claim 4, characterized in that, It is obtained by introducing the recombinant vector of claim 3 into macrophages.
6. The method for preparing CAR-M cells according to claim 5, characterized in that, The macrophages are mouse bone marrow-derived macrophages.
7. The method for preparing CAR-M cells according to claim 5, characterized in that, Specifically, the following steps are included: The leader peptide, the extracellular recognition region, the hinge region, the transmembrane region, and the intracellular signaling domain are sequentially connected from the N-terminus to the C-terminus to obtain the CAR targeting human HER2. The recombinant vector was constructed by recombining the CAR gene targeting human HER2 with a lentiviral vector. The recombinant vector was introduced into the macrophages to obtain the CAR-M cells.
8. The use of the CAR targeting human HER2 according to claim 1, the CAR gene according to claim 2, the recombinant vector according to claim 3, and the CAR-M cell according to claim 4 in the preparation of a drug for treating human HER2-targeted gastric cancer.
Citation Information
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