Safe and controllable chimeric antigen receptor loaded with suicide gene, macrophages expressing same and application thereof

Abstract

The application uses gene editing technology to design and optimize a safe and controllable chimeric antigen receptor loaded with a suicide gene, and a macrophage expressing the same, the chimeric antigen receptor comprising a single-chain variable region targeting carcinoembryonic antigen (CEA), a hinge region, a transmembrane region, and an intracellular signaling region. The application has the following advantages: the chimeric antigen receptor-Hoxb8 macrophage CAR-Hoxb8-M has high infiltration, enhanced phagocytosis and antigen presentation capacity, and is expected to become a new strategy for the immunotherapy of solid tumor cells. The constructed stem progenitor cells proliferate rapidly, can be obtained in large quantities in a short time, and can be frozen for long-term preservation. The induction of macrophages is short, simple and convenient, the macrophages efficiently express CAR, are stable and uniform, small molecule compounds regulate the fate of progenitor cells and macrophages, are safe and controllable, and have an anti-colorectal cancer treatment effect. The CAR-Hoxb8-M combined with an immune checkpoint inhibitor can further inhibit tumor growth and improve survival rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a chimeric antigen receptor targeting CEA carcinoembryonic antigen, macrophages expressing the same and applications. BACKGROUND

[0002] In recent years, immune cell therapy represented by chimeric antigen receptor T cells (CAR-T) has achieved remarkable therapeutic effects in the treatment of hematological tumors. On this basis, different immune cells have developed chimeric antigen receptor expression (such as CAR-NK, CAR-iNKT, etc.), and tried to tackle the treatment of solid tumors. However, due to the dense stroma of solid tumors and the poor immunosuppressive tumor microenvironment and other factors, CAR-T and other immune cells cannot infiltrate or appear irreversible exhaustion in the tumor microenvironment, resulting in poor treatment effect and serious limitation in clinical application of solid tumor treatment. In view of the difficulty of immunotherapy for solid tumors, researchers have developed strongly infiltrating macrophages as a new treatment strategy. Chimeric antigen receptor macrophage immunotherapy (CAR-Macrophage, CAR-M) as a newly developed adoptive cell immunotherapy technology, uses monocytes / macrophages to construct CAR, providing a possible treatment for solid tumors.

[0003] Macrophages, abundant immune cells in the tumor microenvironment, are an important part of the innate immune system. Macrophages have many innate advantages, such as more efficient infiltration into tumor tissue sites. Macrophages are the most infiltrating immune cells in the solid tumor microenvironment. Studies have found that tumor cells or other cells in tumor tissue recruit macrophages to infiltrate by releasing CCL2, CXCL12, CSF1 and other chemotactic factors. In addition, macrophages have phagocytic function and can phagocytose immunogenic dead tumor cells and present tumor antigens to T cells, playing an anti-tumor immune role. Because of these advantages, macrophages are becoming a new choice for immune cell engineering. At the same time, macrophages have strong plasticity, and their anti-tumor effect is variable in the tumor microenvironment, and macrophages can stay for a long time. CAR-M cells can specifically aggregate to solid tumors and penetrate the dense stroma around the tumor to kill tumor cells. However, cell therapy with macrophages has the following problems:

[0004] 1. Variable anti-tumor phenotype: M1 macrophages with anti-tumor function are easily affected by the tumor microenvironment and polarized to M2 phenotype, thereby promoting tumor progression;

[0005] 2. Safe and reliable immune cell source: Similar to CAR-T cell therapy, traditional immune cell source methods, such as in vitro modification and reinfusion therapy of immune cells from patient autologous source, have problems such as difficulty in obtaining immune cells, limited source, low separation efficiency, large individual difference, and inability to guarantee the quality of immune cells, resulting in high treatment cost;

[0006] 3. Macrophages belong to terminally differentiated cell populations and are not easy to be genetically modified: the efficiency of virus transfection of primary monocyte / macrophages is low;

[0007] 4. Low efficiency of in vivo editing of macrophages, in vitro modification and reinfusion therapy can ensure better treatment effect;

[0008] 5. CAR-T may induce the risk of second primary tumor, and CAR-M also does so; at the same time, activated macrophages release pro-inflammatory factors such as IL-6 and TNF-alpha, which may induce cytokine storm and other safety hazards. SUMMARY

[0009] In view of the above technical limitations, the present application provides a chimeric antigen receptor targeting CEA carcinoembryonic antigen, macrophages expressing the same and applications thereof, which overcomes the deficiencies and defects mentioned in the background art.

[0010] The technical idea of the present application is as follows: based on the problems faced by current CAR-M and the difficulties in clinical treatment, the following aspects are optimized:

[0011] 1) Constructing proliferative 4-hydroxytamoxifen-dependent expression of CAR ER-Hoxb8 progenitor cells, which have differentiation characteristics and can be continuously induced to differentiate into macrophages, to solve the problem of safe and reliable macrophage source;

[0012] 2) Designing FcγRI intracellular activation domain for macrophages and specific elements targeting CEA positive tumors for CEA;

[0013] 3) Current CAR-T therapy has the risk of cytokine storm and inducing second primary tumor, and CAR-Hoxb8 macrophages are avoided to have such risk, and safety controllable optimization is performed by expressing suicide gene iCas9.

[0014] To achieve the above purpose, the following technical solutions are adopted in the present application:

[0015] The application point of the present application is to provide a chimeric antigen receptor, which comprises a single-chain variable region targeting CEA carcinoembryonic antigen, a hinge region, a transmembrane region and an intracellular signaling region; the amino acid sequence of the single-chain variable region targeting CEA carcinoembryonic antigen is SEQ ID No. 1.

[0016] SEQ ID No. 1:

[0017] QVQLQQSGGGVVQPGRSLRLSCAASGFTLSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRLTGAPYYYYYGMDVWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSTDVVMTQSPGTLSLSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR.

[0018] Optionally, the chimeric antigen receptor described above, the hinge region is an IgG1 hinge region, and the amino acid sequence is shown as SEQ ID No. 2.

[0019] SEQ ID No. 2:

[0020] VPRDCGCKPCICT.

[0021] Optionally, the chimeric antigen receptor described above, the transmembrane region is a CD8a transmembrane region, and the amino acid sequence is shown as SEQ ID No. 3.

[0022] SEQ ID No. 3:

[0023] IWAPLAGICVALLLSLIITLI.

[0024] Optionally, the chimeric antigen receptor described above, the intracellular signal region comprises: a CD3 zeta intracellular signal region, and the amino acid sequence is shown as SEQ ID No. 4; or a FcR gamma intracellular signal region, and the amino acid sequence is shown as SEQ ID No. 5.

[0025] SEQ ID No. 4:

[0026] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGG KQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTA TKDTYDALHMQTLAPR;

[0027] SEQ ID No. 5:

[0028] KIHRLQREKKYNLEVPLVSEQGKKANSFQQVRSDGVYEEVTATASQTTP KEAPDGPRSSVGDCGPEQPEPLPPSDSTGAQTSQS。

[0029] Optionally, the chimeric antigen receptor described above, wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID No.6 or SEQ ID No.7.

[0030] SEQ ID No.6 (Full-length CD3ζ-CAR sequence):

[0031] MASPLTRFLSLNLLLLGESIILGSGEAQVQLQQSGGGVVQPGRSLRLSCAASGFTLSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRLTGAPYYYYYGMDVWGRGTLVTVSSGGGGSGGGGSGGGGSTDVVMTQSPGTLSLSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRVPRDCGCKPCICTIWAPLAGICVALLLSLIITLIRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPREGRGSLLTCGDVEENPGPMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK;

[0032] SEQ ID No.7 (Full-length FcRγ-CAR):

[0033] .

[0034] The currently used CAR-T therapy carries the risk of cytokine storms and induction of a second primary tumor. To avoid such risks in the provided CAR-Hoxb8 macrophages, the inventors also optimized the safety and controllability by expressing the suicide gene iCas9.

[0035] The full-length sequence of iCas9-FcRγ-CAR is shown in SEQ ID No. 8:

[0036]

[0037] The second inventive point of this application is to provide an expression vector carrying nucleotides encoding the chimeric antigen receptor described above.

[0038] Optionally, the expression vector described above is selected from at least one of lentiviral expression vectors, retroviral expression vectors, or adenovirus expression vectors; preferably, it is a lentiviral expression vector.

[0039] The third inventive point of this application is to provide a chimeric antigen receptor macrophage, wherein the macrophage expresses the chimeric antigen receptor described above.

[0040] Optionally, the chimeric antigen receptor macrophages described above are derived from proliferating stem cells or proliferating progenitor cells.

[0041] The fourth inventive point of this application is to provide the application of the above-mentioned chimeric antigen receptor macrophages in the preparation of tumor therapeutic drugs, wherein the tumor is a CEA-positive tumor, preferably at least one of breast cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, and colorectal cancer, and most preferably colorectal cancer.

[0042] Differences from existing technologies:

[0043] 1. Macrophage origin: Granulonephric progenitor cells derived from mouse bone marrow and human umbilical cord hematopoietic stem and progenitor cells were extracted, transfected with ER-Hoxb8 lentivirus, and then transfected with CAR lentivirus to construct estrogen receptor fusion CAR Hoxb8 progenitor cells (CARER-Hoxb8 progenitor cells), which were then induced into CAR-Hoxb8 macrophages.

[0044] 2. Selection of chimeric antigen receptor target CEA: Targeting CEA-positive tumors, such as colorectal cancer, but not limited to colorectal cancer.

[0045] 3. Safety and controllability of CAR-M: The expression of the suicide gene iCas9 in CAR-M, after CAR-M exerts its therapeutic effect, can induce apoptosis of CAR-M cells in vivo and in vitro through AP1903 dimer.

[0046] Compared with the prior art, this application has the following advantages:

[0047] This application provides a method for constructing chimeric antigen receptor macrophages (CAR-Ms) that utilize FcγRI, a typical signaling molecule of antibody-dependent phagocytosis (ADCP) in macrophages, as an intracellular activation region and can target CEA, and activate macrophages, as well as its application.

[0048] These chimeric antigen receptor macrophages are derived from the induced differentiation of normal Hoxb8 hematopoietic stem and progenitor cells that proliferate in a 4-hydroxytamoxifen-dependent manner. Compared to tumor-derived macrophages such as ThP1, they are safer; compared to iPSC-derived macrophages, they are simpler and more readily available; and compared to autologous immune cells from clinical patients, they are more convenient, easier to modify in vitro, more economical, and of more uniform quality. They also offer advantages such as unlimited source, long-term preservation, safety and controllability, and efficient and rapid preparation. Furthermore, these chimeric antigen receptor macrophages express the suicide gene iCas9, and apoptosis can be induced using the AP1903 dimer, ensuring that the chimeric antigen receptor macrophages are promptly eliminated after exerting their therapeutic effect.

[0049] This application prepares chimeric antigen receptor-Hoxb8 macrophages (CAR-Hoxb8-M), utilizing their high infiltrative capacity to enhance their phagocytic and antigen-presenting abilities, making CAR-Hoxb8-M a promising new strategy for cell immunotherapy of solid tumors.

[0050] The stem and progenitor cells constructed in this application proliferate rapidly, can be obtained in large quantities in a short time, and can be cryopreserved for a long time. The macrophage induction cycle is short, simple and convenient, and the macrophages express CAR efficiently, stably and uniformly. The small molecule compounds regulate the fate of progenitor cells and macrophages safely and controllably, and have good anti-colorectal cancer therapeutic effects.

[0051] The chimeric antigen receptor-Hoxb8 macrophage combined with immune checkpoint inhibitors (including PD-1, PD-L1, CD47, SIRPα, etc.) prepared in this application can further inhibit tumor growth and improve the survival rate of tumor-bearing mice. Attached Figure Description

[0052] Figure 1 This section shows the construction and identification of proliferating ER-Hoxb8 progenitor cells. Among them, Figure 1 Image A is a representative fluorescence microscopy image of 4-hydroxytamoxifen (4-OHT)-dependent proliferative ER-Hoxb8 progenitor cells. Figure 1 B represents the expression of GFP in proliferating ER-Hoxb8 progenitor cells (where the x-axis represents 10). 0 10 1 10 2 10 3 10 4 10 5 10 6 (The vertical axis is 0, 200K, 400K, 600K, 800K, 1.0M). Figure 1 C represents the protein expression of Hoxb8 in proliferating ER-Hoxb8 progenitor cells. Figure 1 D represents the proliferation assay of ER-Hoxb8 progenitor cells.Figure 1 E indicates that the proliferation of ER-Hoxb8 progenitor cells depends on the detection of 4-OHT.

[0053] Figure 2 This section shows the construction and identification of proliferative CAR ER-Hoxb8 progenitor cells. Among them, Figure 2 A is a schematic diagram of the CAR structure. Figure 2 B represents flow cytometry analysis of Hoxb8-GFP and CAR-mCherry in proliferating CAR ER-Hoxb8 progenitor cells (where the x-axis represents 10). 0 10 1 10 2 10 3 10 4 10 5 10 6 The vertical axis is 10. 0 10 1 10 2 10 3 10 4 10 5 10 6 ), Figure 2 C represents the flow cytometry analysis of CD117, a stem marker in proliferating CAR ER-Hoxb8 progenitor cells (where the x-axis represents 10). 0 10 1 10 2 10 3 10 4 10 5 10 6 (The vertical axis is 0, 50, 100, 150). Figure 2 D represents the karyotype analysis of CAR ER-Hoxb8 progenitor cells. Figure 2 E represents the detection of CAR mRNA expression in CAR ER-Hoxb8 progenitor cells. Figure 2 F represents the proliferation assay of CAR ER-Hoxb8 progenitor cells.

[0054] Figure 3 This is shown as the identification of CAR Hoxb8 macrophages. Among them, Figure 3 A represents the morphological and karyotype analysis of CAR Hoxb8 macrophages. Figure 3 B represents the detection of F4 / 80 and CD11b markers in CAR Hoxb8 macrophages (where the x-axis represents 10). 0 10 1 10 2 10 3 10 4 10 5 10 6The vertical axis is 10. 0 10 1 10 2 10 3 10 4 10 5 10 6 ), Figure 3 C represents the flow cytometry analysis of CAR-mCherry in CAR Hoxb8 macrophages. Figure 3 D represents the detection of CAR mRNA expression in CAR Hoxb8 macrophages. Figure 3 E represents the survival time of CAR Hoxb8 macrophages.

[0055] Figure 4 The screening and construction of CEA-positive CRC cells were shown. Figure 4 A represents the flow cytometry detection of CEA overexpression in MC38 and CT26 cells (where the x-axis represents 10). 0 10 1 10 2 10 3 10 4 10 5 10 6 The vertical axis is 10. 0 10 1 10 2 10 3 10 4 10 5 10 6 ), Figure 4 B represents the detection of CEA mRNA expression in MC38CEA and CT26CEA cells. Figure 4 C represents the detection of CEA content in the supernatant of MC38CEA and CT26CEA cells. Figure 4 D is the detection of CEA mRNA expression in human-derived CRC cells. Figure 4 E is the flow cytometry detection of CEA in human CRC cells.

[0056] Figure 5 This demonstrates the phagocytic and cytotoxic effects of CAR Hoxb8 macrophages on CRC cells. Among them, Figure 5 A shows the results of detecting pro-inflammatory factors IL-6, IL-1β, IL-12, and TNF-α in the supernatant of CAR Hoxb8 macrophages and MC38CEA cells after 48 hours of co-incubation. Figure 5 B represents the lytic effect of CAR Hoxb8 macrophages on MC38CEA and CT26CEA cells. Figure 5 C represents the lytic effect of CAR Hoxb8 macrophages on human CRC cells.Figure 5 D represents the phagocytic activity of CAR Hoxb8 macrophages on MC38CEA (where the x-axis represents -10). 4 0, 10 4 10 5 10 6 The vertical axis is 10. 0 10 1 10 2 10 3 10 4 10 5 10 6 ).

[0057] Figure 6 The results show effective tumor infiltration by CAR Hoxb8 macrophages. Figure 6 A represents the survival time of CAR Hoxb8 macrophages in tumor-bearing mice as detected by in vivo imaging in small animals. Figure 6 B represents the infiltration and enrichment of CARHoxb8 macrophages in tumor tissue using in vivo imaging in small animals.

[0058] Figure 7 This demonstrates the anti-tumor efficacy of CAR Hoxb8 macrophages. Among them, Figure 7 A represents the fluorescence signal detected by in vivo imaging in small animals after CAR Hoxb8 macrophage treatment of tumor-bearing mice. Figure 7 B is Figure 7 Statistical analysis of fluorescence signals in A Figure 7 C shows representative tumor images from the treatment and control groups. Figure 7 D represents the tumor weight. Figure 7 E represents the survival rate of tumor-bearing mice in the treatment and control groups. Figure 7 F represents the tumor growth curve after CAR Hoxb8 macrophage combined with Anti-PD-1 treatment. Figure 7 G represents representative tumor images from the CAR Hoxb8 macrophage combined with Anti-PD-1 treatment group and the control group. Figure 7 H represents the tumor weight. Figure 8 I represents the survival rate of mice with tumors.

[0059] Figure 8 This demonstrates that AP1903 induces apoptosis in iCas9-CAR Hoxb8 macrophages both in vitro and in vivo. Figure 8 A represents the cell viability of iCas9-CAR Hoxb8 macrophages treated with AP1903 for 6 hours, as determined by CCK8 assay. Figure 8 B represents apoptosis in iCas9-CAR Hoxb8 macrophages 6 hours after treatment with AP1903 (where the x-axis represents 10). 0 101 10 2 10 3 10 4 10 5 10 6 The vertical axis is -10. 2 0, 10 2 10 3 10 4 10 5 10 6 ), Figure 8 C represents the expression of apoptosis-related proteins in iCas9-CAR Hoxb8 macrophages after 6 hours of AP1903 treatment. Figure 8 D represents the tumor growth curve in mice treated with iCas9-CAR Hoxb8 macrophages for MC38CEA tumors, after administration of AP1903. Figure 9 E represents the proportion of iCas9-CAR Hoxb8 macrophages in peripheral blood, liver, lung, and tumors of mice treated with AP1903 after iCas9-CAR Hoxb8 macrophage therapy for MC38CEA tumors (where the x-axis represents -10). 4 0, 10 4 10 5 10 6 The vertical axis is -10. 3 0, 10 3 10 4 10 5 10 6 ).

[0060] Figure 1 The diagram shown is a flowchart illustrating the preparation of safe and controllable chimeric antigen receptor-macrophages derived from proliferating stem / progenitor cells, according to one embodiment of this application. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0063] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0064] Example 1

[0065] The source and acquisition of immune cells are key issues in adoptive immunotherapy. The preparation and application of safe and controllable chimeric antigen receptor-macrophages derived from proliferating stem / progenitor cells for the treatment of colorectal cancer specifically includes the following steps:

[0066] Lin-progenitor cells derived from mouse bone marrow and hematopoietic stem / progenitor cells (CB-CD34+ cells) derived from human umbilical cord were extracted and transfected with ER-Hoxb8 lentivirus. High-purity, proliferative ER-Hoxb8 progenitor cells were obtained by flow cytometry sorting. These proliferative ER-Hoxb8 progenitor cells were then transfected with CAR and iCas9-CAR lentiviruses, and high-purity, proliferative CAR ER-Hoxb8 progenitor cells and iCas9-CAR ER-Hoxb8 progenitor cells were obtained by flow cytometry sorting. 4-OHT was removed, and GM-CSF and M-CSF were added to induce CAR ER-Hoxb8 progenitor cells and iCas9-CAR ER-Hoxb8 progenitor cells into CAR Hoxb8 macrophages and iCas9-CAR Hoxb8 macrophages, respectively. The antitumor activity of CAR Hoxb8 macrophages was verified in vitro and in vivo, and apoptosis was verified by treating iCas9-CAR Hoxb8 macrophages with AP1903.

[0067] The specific steps are as follows:

[0068] 1. Construction of proliferative progenitor cells:

[0069] These include ER-Hoxb8 progenitor cells, CAR ER-Hoxb8 progenitor cells, and iCas9-CAR ER-Hoxb8 progenitor cells. The chimeric antigen receptor includes the CD8 signal peptide, the anti-CEA single-chain variable region, the IgG1 hinge region, the CD8 transmembrane region, and the intracellular region of FcγRI or CD3ζ.

[0070] 2. Induction of differentiation of CEA-targeting CAR-Hoxb8 macrophages:

[0071] Differentiation into ER-Hoxb8 progenitor cells, CAR ER-Hoxb8 progenitor cells, and iCas9-CAR ER-Hoxb8 progenitor cells was induced and treated for 7 days under 20 ng / ml GM-CSF and 20 ng / ml M-CSF conditions to obtain CAR Hoxb8 macrophages and iCas9-CAR Hoxb8 macrophages.

[0072] 3. Identification of CAR Hoxb8 macrophages and iCas9-CAR Hoxb8 macrophages:

[0073] Identification methods include survival time of CAR Hoxb8 macrophages or iCas9-CAR Hoxb8 macrophages, stem cell marker CD117, macrophage markers F4 / 80 and CD11b, morphology, and CAR expression.

[0074] 4. Screening of target cells with high CEA expression in humans and construction of target cells with high CEA expression in mice:

[0075] Target cells include HT29, SW620, SW480, Caco2, HCT116 cells, as well as MC38CEA and CT26CEA cells.

[0076] 5. Detection of in vitro antitumor functional activity of CAR Hoxb8 macrophages:

[0077] In vitro antitumor function assays included the effect of tumor antigen CEA on the phenotype of CAR Hoxb8 macrophages, the content of pro-inflammatory factors in the supernatant after co-incubation of CAR Hoxb8 macrophages and target cells, and the phagocytic function of CAR Hoxb8 macrophages on target cells and the detection of target cell apoptosis.

[0078] 6. In vivo distribution and persistence of CAR Hoxb8 macrophages after reinfusion:

[0079] DIR-labeled CAR Hoxb8 macrophages were injected into mice via tail vein injection. Small animal in vivo imaging was used to assess the in vivo distribution of CAR Hoxb8 macrophages by detecting their enrichment in the heart, liver, spleen, lung, kidney, and tumors. Small animal in vivo imaging was used to continuously monitor the fluorescence signal of CAR Hoxb8 macrophages in vivo to assess their persistence.

[0080] 7. Evaluation of the efficacy of CAR Hoxb8 macrophage therapy for CRC:

[0081] A tumor model was constructed using MC38CEA+ cells. The treatment was combined with CAR Hoxb8 macrophage infusion therapy and the immune checkpoint inhibitor Anti-PD-1 therapy. The treatment effect was evaluated by in vivo imaging technology, tumor growth curves, tumor weight, and mouse survival rate.

[0082] 8. Detection of AP1903-induced apoptosis in iCas9-CAR Hoxb8 macrophages:

[0083] In vitro, AP1903 was used to treat iCas9-CAR Hoxb8 macrophages, and the survival rate and apoptosis of iCas9-CAR Hoxb8 macrophages were detected. A tumor model of MC38CEA cells was constructed, and iCas9-CAR Hoxb8 macrophages were reinfused as treatment, while simultaneously receiving AP1903 treatment. Tumor growth curves were monitored, and the proportion of iCas9-CAR Hoxb8 macrophages in various tissues was detected by flow cytometry to assess whether AP1903 induced iCas9-CAR Hoxb8 macrophage apoptosis.

[0084] Example 2

[0085] 1. Cell isolation and cell culture:

[0086] Female C57BL / 6J mice aged 6-8 weeks were euthanized, and bone marrow cells were extracted. Lin-progenitor cells were obtained using a progenitor cell isolation kit (Direct Lineage Cell Depletion Kit, Miltenyi Biotec). Lin-progenitor cells were cultured in IMDM complete medium containing IMDM (Iscove's, Gibco), 15% FBS (Gibco), 1% pen / strep / glutamine (Gibco), 10 ng / ml murine IL-3 (Peprotech), 20 ng / ml murine IL-6 (Peprotech), and 25 ng / ml murine SCF (Peprotech). After 48 hours, the cells were transfected with ER-Hoxb8 lentivirus to obtain proliferating ER-Hoxb8 progenitor cells, which were then cultured in Opti-MEM complete medium containing Opti-MEM, 10% FBS, 1% PSG, 10 ng / ml murine SCF, 30 μM beta-mercaptoethanol, and 1 μM 4-OHT. (Results are as follows...) Figure 1 As shown, the proliferation of ER-Hoxb8 progenitor cells depends on 4-hydroxytamoxifen. Figure 4(See DE). Positive cells were purified by flow cytometry sorting. CAR lentivirus was then transfected into proliferating ER-Hoxb8 progenitor cells, and the culture conditions for the proliferating CAR ER-Hoxb8 progenitor cells were the same as those for the ER-Hoxb8 progenitor cells. HEK293T cells (ATCC), MC38 cells (Cell Resource Center of Peking Union Medical College), MC38CEA cells, MC38CEA Luciferase cells, SW620 cells (ATCC), and SW620 Luciferase cells were cultured in high-glucose DMEM (Gibco) containing 10% FBS (Gibco) and 1-fold penicillin-streptomycin (Pen-Strep; 10,000 U / ml penicillin and 10,000 μg / ml streptomycin; Gibco). CT26 cells (Chinese Academy of Sciences Type Culture Cell Bank), CT26CEA cells, and CT26CEA Luciferase cells were cultured in RPMI 1640 (Gibco) containing 10% FBS (Gibco) and 1× penicillin-strepmycin (Pen-Strep; 10,000 U / ml penicillin and 10,000 μg / ml streptomycin; Gibco). Caco2 cells (ATCC) and Caco2-Luciferase cells were cultured in MEM (containing NEAA, Hyclone) medium supplemented with 20% bovine serum (Gibco) and 1× penp-strep. HT29 cells (ATCC), HT29 Luciferase cells, HCT116 cells (ATCC), and HCT116 Luciferase cells were cultured in McCoy's 5A (Hyclone) medium supplemented with 10% bovine serum (Gibco) and 1× penp-strep. SW480 cells (ATCC) and SW480 Luciferase cells were cultured in Leibovitz's L-15 (Hyclone) medium supplemented with 20% bovine serum (Gibco) and 1× penp-strep (results as shown in the figure). Figure 2 (As shown). In this application, the cells modified with Hoxb8 and chimeric antigen receptors include mouse bone marrow-derived Lin-progenitor cells and human umbilical cord or bone marrow-derived stem / progenitor cells (CD34+ cells).

[0087] 2. Stable overexpression of CARs in ER-Hoxb8 progenitor cells:

[0088] CAR vector plasmids psPAX2 and pMD2.G were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). Culture medium containing lentivirus was collected at 24, 36, and 48 h post-transfection and purified by PEG8000. The lentivirus was then added to six-well plates containing ER-Hoxb8 progenitor cells. Fresh complete culture medium was added 6 hours after infection. Transfected cells were cultured for 72 h, followed by flow cytometry sorting and purification. (Results are shown below.) Figure 3 (as shown in B).

[0089] 3. CAR ER-Hoxb8 progenitor cells differentiate into macrophages:

[0090] Cultured CAR ER-Hoxb8 progenitor cells were washed twice with 1X PBS to remove 4-OHT, and then RPMI 1640 medium was supplemented with 20 ng / ml mouse M-CSF, 20 ng / ml mouse GM-CSF, 10% FBS, and 1% Pen-Strep-Glut. The medium was completely replaced on days three and five, and mature CAR-M cells were obtained on day seven. All recombinant factors were purchased from PeproTech. (Results are as follows...) Figure 9 (As shown in AB).

[0091] The preparation process of safe and controllable chimeric antigen receptor-macrophages derived from proliferating progenitor cells is as follows: Figure 2 As shown.

[0092] 4. Cell morphology and karyotype analysis:

[0093] To assess cell morphology and nuclei, progenitor cells or macrophages were fixed on slides. Cell morphology was stained with Diff-Quick stain (Solarbio, G1540-3), and nuclei were stained with Wright-Giemsa stain (NJJCBio, D010). Cells were then imaged using a microscope. (Results are shown below.) Figure 3 D and Figure 2 (As shown in A).

[0094] 5. Gene expression detection:

[0095] Total RNA was extracted from cells using Trio Reagent (Invitrogen, Carlsbad, CA, USA). Specific conditions and parameters were followed according to the manufacturer's instructions. 2 μg of total RNA was reverse-engineered into cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Nanjing Novizan). Real-time PCR was performed on a 7500 detection system using SYBR-GreenI as a double-stranded DNA-specific binding dye. The mRNA of the target gene and the housekeeping gene β-actin was quantified in separate tubes. (Results are shown below.) Figure 3 E and Figure 1 (as shown in D).

[0096] 6. Western blot:

[0097] Cell samples were collected and lysed with RIPA buffer (Beyotime, P0013K) containing protease inhibitors, followed by protein extraction. Protein concentration was determined using the Pierce BCA Protein Detection Kit (Thermo). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and samples were then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with TBS Tween 20 containing 5% milk. The blots were incubated with SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific, 34580). Blot signals were detected using a Tanon imager. Antibodies used: anti-Hoxb8 (Santa Cruz Biotechnology, sc-517156), anti-Caspase-3 (CST, 9662S), anti-Cleaved Caspase-3 (CST, 9654S), anti-Bcl2 (Abcam, ab182858), Bax (Abcam, ab32503), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (proteintech, HRP-60004). (Results are as follows) Figure 8 C and Figure 5 (as shown in C).

[0098] 7. Cytokine release assay:

[0099] Cell-free supernatant from co-culturing macrophages and tumor cells was collected, and the concentrations of cytokines IL-1β, IL-6, IL-12, and TNF-α were measured using enzyme-linked immunosorbent assay (ELISA) according to the kit supplier's instructions. The cytokine kits used for quantitative analysis were all purchased from Thermo Fisher Scientific: IL-1β, 88-7013-88; IL-6, 88-7064-88; IL-12, 88-7121-88; and TNF-α, 88-7324-88. (Results are as follows...) Figure 5 (As shown in A).

[0100] 8. Cytotoxicity assay:

[0101] Target cells (stable tumor cell lines transfected with Luciferase lentivirus) were seeded at a density of 5000 cells / 100 μL / well in black 96-well ELISA plates. Effector cells were macrophages from different groups, with effector cell to target (E:T) cell ratios of 0:1, 1:1, 5:1, and 10:1 added to the wells. After 24 hours of incubation, the cells were processed according to ONE-Glo... TM The luciferase assay was performed using a Varioskan Flash luminometer (Thermo Scientific). The experimental method followed the manufacturer's instructions (Promega, E6110). (Results are as follows...) Figure 5 (As shown in BC).

[0102] 9. Engulfment Experiment:

[0103] Cells were collected for testing, single-cell suspensions were prepared, washed with PBS containing 1% BSA, and then labeled with antibodies for 20 minutes. Flow cytometry antibodies included: APC anti-mouse CD117 (BioLegend, 105811), Brilliant Violet (BV) 711 anti-mouse F4 / 80 (BioLegend, 123147), APC anti-mouse / human CD11b (BioLegend, 101212), and APC anti-human CEA (BioLegend, 342308). All flow cytometry experiments were performed on a ThermoScientific flow cytometer and analyzed using FlowJo V10 software. (Results are as follows...) Figure 6 (as shown in D).

[0104] 10. In vivo distribution and persistence of CAR Hoxb8 macrophages after reinfusion:

[0105] To evaluate the distribution of macrophages in vivo, 1×106 MC38CEA Luciferase cells were mixed in PBS and subcutaneously injected into 6-8 week old female C57BL / 6J mice to establish a CRC tumor model. On day 7 post-tumor inoculation, 1×10⁻⁶ MC38CEA Luciferase cells were injected via the tail vein. 7 DiRIodide (Cy7 DiC18, MedChemExpress)-labeled macrophages were imaged in vivo using an IVIS Lumina XR system at the same time points on days 1, 6, 11, 16, and 21 after injection to capture fluorescence signals at the tumor site (n=3 per group). Mice were euthanized on day 28 after tumor inoculation. Heart, liver, spleen, lung, kidney, and tumor tissue were harvested for in vivo imaging. (Results are as follows...) Figure 7 (As shown).

[0106] 11. Efficacy evaluation of CAR Hoxb8 macrophage combined with immune checkpoint inhibitor Anti-PD-1 therapy for CRC:

[0107] 5×10 5 MC38CEA Luciferase cells were mixed in PBS and injected under the armpit into 6-8 week old female C57BL / 6J mice to establish a CRC tumor model. On day 7 after tumor inoculation, 1×10⁻⁶ cells were injected via the tail vein. 7 Macrophages were injected intraperitoneally with 10 mg / kg Anti-PD-1 on days 9, 12, and 15 after tumor inoculation. Five mice from each group were used for in vivo imaging at the same time point on days 7, 12, 17, 22, and 28 after tumor inoculation to monitor tumor growth. D-Luciferin (Yeasen, 40902ES03) was injected intraperitoneally into mice at a dose of 150 mg / kg, and in vivo imaging was performed using an IVIS Lumina series III system to capture fluorescence signals. Mice were weighed and tumor size was measured on the day of macrophage injection and on days 1, 5, 9, 13, 17, and 21 after injection to determine tumor volume. Mice were euthanized on day 28 after tumor implantation, and tumor tissue was obtained and weighed. Survival rates were monitored in five mice from each group.

[0108] (Results are as follows) Figure 8 (As shown).

[0109] 12. Detection of apoptosis in iCas9-CAR Hoxb8 macrophages induced by the small molecule compound AP1903:

[0110] Macrophages from different groups were divided into groups of 1×10 4Macrophages were seeded into 96-well plates and treated with different concentrations (0, 0.1, 1, 10, 100 ng / ml) of AP1903 for 6 hours. Then, 100 μl of CCK8 reagent was added to each well, and the plates were incubated for 2 hours. Macrophage viability was analyzed using a microplate reader. Macrophages from different groups were cultured at 2 × 10⁻⁶ wells. 5 Cells were seeded in 6-well plates, and different concentrations (0, 1, 10 ng / ml) of AP1903 were added. After 6 hours of treatment, cells were collected and labeled with Annexin V for flow cytometry analysis to detect macrophage apoptosis. 1×10⁶ cells were then added to each well. 6 MC38CEA Luciferase cells were mixed in PBS and injected under the armpit into 6-8 week old female C57BL / 6J mice to establish a CRC tumor model. On day 7 after tumor inoculation, 1×10⁻⁶ cells were injected via the tail vein. 7 Macrophages. Mice were administered 5 mg / kg AP1903 on days 11 and 14 after tumor inoculation, and tumor growth curves were monitored. Mice were euthanized on day 18 after tumor inoculation, and peripheral blood, liver, lungs, and tumors were collected for flow cytometry analysis to determine the proportion of reinfused macrophages. (Results are as follows...) ​ (As shown).

[0111] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A chimeric antigen receptor, characterized in that, The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 6-SEQ ID No.

8. The chimeric antigen receptor includes a single-chain variable region, a hinge region, a transmembrane region, and an intracellular signaling region that target CEA carcinoembryonic antigen in a sequentially linked manner. The amino acid sequence of the single-chain variable region targeting CEA carcinoembryonic antigen is shown in SEQ ID No. 1; The hinge region is the IgG1 hinge region, and its amino acid sequence is shown in SEQ ID No. 2; The transmembrane region is the CD8α transmembrane region, and its amino acid sequence is shown in SEQ ID No. 3; The intracellular signaling region includes: the CD3ζ intracellular signaling region, the amino acid sequence of which is shown in SEQ ID No. 4; or the FcRγ intracellular signaling region, the amino acid sequence of which is shown in SEQ ID No.

5.

2. An expression carrier, characterized in that, The expression vector carries nucleotides encoding the chimeric antigen receptor of claim 1.

3. The expression vector according to claim 2, characterized in that, The expression vector is selected from at least one of lentiviral expression vectors, retroviral expression vectors, or adenovirus expression vectors.

4. The expression vector according to claim 2, characterized in that, The expression vector is a lentiviral expression vector.

5. A chimeric antigen receptor macrophage, characterized in that, The macrophages express the chimeric antigen receptor as described in claim 1.

6. The chimeric antigen receptor macrophage according to claim 5, characterized in that, The macrophages are derived from proliferating stem cells or proliferating progenitor cells.

7. The use of the chimeric antigen receptor macrophages according to claim 5 or 6 in the preparation of a tumor therapeutic drug, wherein the tumor is colorectal cancer.

Images