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

Abstract

The invention designs, optimizes and provides a safe and controllable chimeric antigen receptor loaded with a suicide gene and a macrophage for expressing the chimeric antigen receptor by applying a gene editing technology. The chimeric antigen receptor comprises a single-chain variable region, a hinge region, a transmembrane region and an intracellular signal region of a targeted carcino-embryonic antigen (CEA). The chimeric antigen receptor-Hoxb8 macrophage CAR-Hoxb8-M has the advantages that the chimeric antigen receptor-Hoxb8 macrophage CAR-Hoxb8-M has efficient wettability, the phagocytic ability and antigen presentation ability are enhanced, and the chimeric antigen receptor-Hoxb8 macrophage CAR-Hoxb8-M is expected to become a new strategy for solid tumor cell immunotherapy. The constructed stem progenitor cells are rapid in proliferation, can be massively obtained in a short time, can be cryopreserved for a long time, and are short in macrophage induction cycle, simple and convenient, the macrophages efficiently express CAR and are stable and uniform, small molecule compounds regulate the progenitor cells and macrophage fate, safety and controllability are achieved, and the anti-colorectal cancer treatment effect is achieved. The CAR-Hoxb8-M is combined with an immune checkpoint inhibitor, so that tumor growth can be further inhibited, and the survival rate can be increased.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a chimeric antigen receptor targeting CEA carcinoembryonic antigen, a macrophage expressing the chimeric antigen receptor, and applications thereof. Background Art

[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, chimeric antigen receptor expression of different immune cells (such as CAR-NK, CAR-iNKT, etc.) has been developed, and attempts have been made to overcome the difficulties in the treatment of solid tumors. However, due to factors such as the dense interstitial tissue of solid tumors and the harsh immunosuppressive tumor microenvironment, immune cells such as CAR-T cannot infiltrate or are irreversibly exhausted in the tumor microenvironment, resulting in poor treatment effects and severe limitations in the treatment of clinical solid tumors. In response to the difficulties in immunotherapy of solid tumors, researchers have developed highly infiltrating macrophages as a new treatment strategy. As an emerging adoptive cell immunotherapy technology, chimeric antigen receptor macrophage immunotherapy (CAR-Macrophage, CAR-M) uses monocytes / macrophages to construct CAR, providing a possible treatment option for conquering solid tumors.

[0003] Macrophages, abundant immune cells in the tumor microenvironment, are an important part of the innate immune system. Macrophages themselves have many innate advantages, such as more effective infiltration into tumor tissue sites. In the microenvironment of solid tumors, macrophages are the immune cells with the highest infiltration ratio. Studies have found that tumor cells or other cells in tumor tissues recruit macrophage infiltration by releasing chemokines such as CCL2, CXCL12, and CSF1. As well as the phagocytic function of macrophages and the ability of macrophages to engulf immunogenic dead tumor cells and present tumor antigens to T cells to exert anti-tumor immune effects. Because of this advantage, macrophages are becoming a new choice for engineering transformation of immune cells. At the same time, macrophages have strong plasticity, variable anti-tumor effects in the tumor microenvironment, and macrophages can reside for a long time. CAR-M cells can specifically aggregate to solid tumors, penetrate the dense interstitial tissue around the tumor, and kill tumor cells. However, there are the following problems with cell therapy using macrophages:

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

[0005] 2. Safe and reliable immune cell sources: Similar to cell therapies such as CAR-T, the traditional way of deriving immune cells is to transform the patient's own immune cells in vitro and then infuse them for treatment. This method faces problems such as difficulty in obtaining immune cells, limited sources, low separation efficiency, large individual differences, and the inability to guarantee the quality of immune cells, resulting in expensive treatment.

[0006] 3. Macrophages are terminally differentiated cell populations and are not easy to genetically modify: viral transfection of primary monocytes / macrophages is inefficient;

[0007] 4. The editing efficiency of macrophages in vivo is low, and in vitro modification followed by reinfusion therapy will ensure a more effective treatment.

[0008] 5. CAR-T may induce the risk of a second primary tumor, and the same is true for CAR-M; at the same time, the release of pro-inflammatory factors such as IL-6 and TNF-α after macrophage activation may induce safety hazards such as cytokine storms. Summary of the invention

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

[0010] The technical idea of ​​this application is: Based on the current problems faced by CAR-M and the difficulties in clinical treatment, engineering optimization is carried out from the following aspects:

[0011] 1) Construct proliferative 4-hydroxytamoxifen-dependent expression of CAR ER-Hoxb8 progenitor cells. The stem progenitor cells have differentiation characteristics and can be continuously induced to differentiate into macrophages, thereby solving the problem of a safe and reliable source of macrophages;

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

[0013] 3) Currently, CAR-T therapy has the risk of cytokine storm and inducing second primary tumors. To avoid such risks in CAR-Hoxb8 macrophages, safety and controllability can be optimized by expressing the suicide gene iCas9.

[0014] To achieve the above objectives, this application adopts the following technical solutions:

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

[0016] SEQ ID No.1:

[0017] QVQLQQSGGGVVQPGRSLRLSCAASGFTLSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRLTGAPYYYYYGMDVWGRGTLVTVSS GGGGSGGGGSGGGGSTDVVMTQSPGTLSLSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR.

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

[0019] SEQ ID No.2:

[0020] VPRDCGCKPCICT.

[0021] Optionally, in the above-mentioned chimeric antigen receptor, the transmembrane region is a CD8α transmembrane region, and its amino acid sequence is shown in SEQ ID No.3.

[0022] SEQ ID No.3:

[0023] IWAPLAGICVALLLSLIITLI.

[0024] Optionally, in the above-mentioned chimeric antigen receptor, the intracellular signal region includes: a CD3ζ intracellular signal region, whose amino acid sequence is shown in SEQ ID No.4; or a FcRγ intracellular signal region, whose amino acid sequence is shown in SEQ ID No.5.

[0025] SEQ ID No.4:

[0026] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGG KQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTA TKDTYDALHMQTLAPR;

[0027] SEQ ID No.5:

[0028] KIHRLQREKKYNLEVPLVSEQGKKANSFQQVRSDGVYEEVTATASQTTPKEAPDGPRSSVGDCGPEQPEPLPPSDSTGAQTSQS。

[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 has the risk of cytokine storm and inducing a second primary tumor. To avoid such risks in the provided CAR-Hoxb8 macrophages, the inventors have 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 invention of the present application is to provide an expression vector, which carries the nucleotide encoding the chimeric antigen receptor.

[0038] Optionally, the above-mentioned expression vector is selected from at least one of a lentiviral expression vector, a retroviral expression vector or an adenoviral expression vector; preferably, it is a lentiviral expression vector.

[0039] The third invention of the present application is to provide a chimeric antigen receptor macrophage, wherein the macrophage expresses the chimeric antigen receptor.

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

[0041] The fourth inventive point of the present application is to provide the use 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. Source of macrophages: Extract mouse bone marrow-derived granulocyte-negative progenitor cells and human umbilical cord hematopoietic stem progenitor cells, transfect them with ER-Hoxb8 lentivirus, and then transfect them with CAR lentivirus to construct estrogen receptor-fused CAR Hoxb8 progenitor cells (CARER-Hoxb8 progenitor cells), which are 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 suicide gene iCas9 in CAR-M. After CAR-M exerts its therapeutic effect, AP1903 dimer can induce apoptosis of CAR-M cells in vivo and in vitro.

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

[0047] The present application provides a method for constructing a chimeric antigen receptor macrophage (CAR-M) that uses FcγRI, a typical signaling molecule of antibody-dependent cellular phagocytosis (ADCP) in macrophages, as an intracellular activation region and can target CEA targets and activate macrophages, and its application.

[0048] This type of chimeric antigen receptor macrophage is derived from the induced differentiation of normal Hoxb8 hematopoietic stem cells that proliferate in a 4-hydroxytamoxifen-dependent manner. It is safer than tumor-derived macrophages such as ThP1, easier to obtain than iPSC-derived macrophages, more convenient than the source of clinical patients' autologous immune cells, easier to transform in vitro, more economical, and more uniform in quality. It has the advantages of unlimited sources, long-term storage, safe and controllable, and efficient and rapid preparation. At the same time, the suicide gene iCas9 is also expressed in chimeric antigen receptor macrophages, and the use of AP1903 dimer can induce apoptosis of chimeric antigen receptor macrophages, ensuring that chimeric antigen receptor macrophages can be cleared in time after exerting their therapeutic effects.

[0049] The present application prepares chimeric antigen receptor-Hoxb8 macrophages (CAR-Hoxb8-M), utilizes its efficient infiltration, enhances its phagocytic ability and antigen presentation ability, and makes CAR-Hoxb8-M a new strategy for cell immunotherapy of solid tumors.

[0050] The stem progenitor cells constructed in the present application proliferate rapidly, can be obtained in large quantities in a short period of time, and can be frozen for a long time. The induction cycle of macrophages is short and simple and convenient. The macrophages efficiently express CAR and are stable and uniform. Small molecule compounds regulate the fate of progenitor cells and macrophages, are safe and controllable, and have good anti-colorectal cancer therapeutic effects.

[0051] The chimeric antigen receptor-Hoxb8 macrophages prepared in the present application combined with immune checkpoint inhibitors (including PD-1, PD-L1, CD47, SIRPα, etc.) can further inhibit tumor growth and improve the survival rate of tumor mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Shown is the construction and identification of proliferative ER-Hoxb8 progenitor cells. Figure 1 A is a representative fluorescence microscopy image of 4-hydroxytamoxifen (4-OHT)-dependent proliferative ER-Hoxb8 progenitor cells. Figure 1 B is the expression of GFP in proliferative ER-Hoxb8 progenitor cells (where the horizontal axis is 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 ; The vertical coordinates are 0, 200K, 400K, 600K, 800K, 1.0M), Figure 1 C is the protein expression of Hoxb8 in proliferative ER-Hoxb8 progenitor cells. Figure 1 D is the proliferation detection of ER-Hoxb8 progenitor cells. Figure 1 E: Detection of 4-OHT dependence of proliferation of ER-Hoxb8 progenitor cells.

[0053] Figure 2 Shown is 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 is the flow cytometric analysis of Hoxb8-GFP and CAR-mCherry in proliferative CAR ER-Hoxb8 progenitor cells (where the horizontal axis is 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 is the flow cytometric analysis of the stemness marker CD117 in proliferating CAR ER-Hoxb8 progenitor cells (where the horizontal axis is 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 ; the vertical coordinates are 0, 50, 100, 150), Figure 2 D is the karyotype analysis of CAR ER-Hoxb8 progenitor cells. Figure 2 E is the detection of CAR mRNA expression in CAR ER-Hoxb8 progenitor cells, Figure 2 F is the proliferation detection of CAR ER-Hoxb8 progenitor cells.

[0054] Figure 3 Shown is the identification of constructing CAR Hoxb8 macrophages. Among them, Figure 3 A is the morphology and karyotype analysis of CAR Hoxb8 macrophages. Figure 3 B is the detection of F4 / 80 and CD11b markers in CAR Hoxb8 macrophages (where the horizontal axis is 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 3 C is the flow cytometry analysis of CAR-mCherry in CAR Hoxb8 macrophages. Figure 3 D is the detection of CAR mRNA expression in CAR Hoxb8 macrophages. Figure 3 E is the survival time detection of CAR Hoxb8 macrophages.

[0055] Figure 4 It shows the screening and construction of CEA-positive CRC cells. Figure 4 A is the flow cytometry detection of MC38 and CT26 cells overexpressing CEA (the horizontal axis is 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 is the detection of CEA mRNA expression in MC38CEA and CT26CEA cells, Figure 4 C is 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 CRC cells. Figure 4 E, Flow cytometric detection of CEA in human CRC cells.

[0056] Figure 5 It shows the phagocytic and cytotoxic effects of CAR Hoxb8 macrophages on CRC cells. Figure 5 A is the detection result of pro-inflammatory factors IL-6, IL-1β, IL-12 and TNF-α in the cell supernatant of CAR Hoxb8 macrophages and MC38CEA cells co-cultured for 48 hours. Figure 5 B shows the lysis effect of CAR Hoxb8 macrophages on MC38CEA and CT26CEA cells. Figure 5 C is the lysis effect of CAR Hoxb8 macrophages on human CRC cells. Figure 5 D is the phagocytosis of MC38CEA by CAR Hoxb8 macrophages (where the horizontal axis is -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 that CAR Hoxb8 macrophages effectively infiltrate the tumor. Figure 6 A is the survival time of CAR Hoxb8 macrophages in tumor mice detected by small animal in vivo imaging. Figure 6 B is the infiltration and enrichment of CARHoxb8 macrophages in tumor tissue detected by small animal in vivo imaging.

[0058] Figure 7 It shows the anti-tumor efficacy of CAR Hoxb8 macrophages. Figure 7 A is the fluorescence signal of small animal in vivo imaging after CAR Hoxb8 macrophages were used to treat tumor mice. Figure 7 B is Figure 7 Statistical analysis of fluorescence signals in A. Figure 7 C is the representative image of tumors in the treatment group and the control group. Figure 7 D is the tumor weight, Figure 7 E is the survival rate of tumor-bearing mice in the treatment group and the control group. Figure 7 F is the tumor growth curve after CAR Hoxb8 macrophage combined with Anti-PD-1 treatment, Figure 7 G is a representative picture of tumors in the CAR Hoxb8 macrophage combined with Anti-PD-1 treatment group and the control group. Figure 7 H is the tumor weight, Figure 7 I is the survival rate of tumor-bearing mice.

[0059] Figure 8 AP1903 induces apoptosis of iCas9-CAR Hoxb8 macrophages in vitro and in vivo. Figure 8 A is the cell survival rate of iCas9-CAR Hoxb8 macrophages after 6 hours of AP1903 treatment detected by CCK8. Figure 8 B is the cell apoptosis of iCas9-CAR Hoxb8 macrophages after AP1903 treatment for 6 hours (the horizontal axis is 10 0 , 101 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 ; The vertical coordinate is -10 2 , 0, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 ), Figure 8 C is the expression of apoptosis-related protein molecules in iCas9-CAR Hoxb8 macrophages after AP1903 treatment for 6 hours. Figure 8 D is the tumor growth curve of mice treated with iCas9-CAR Hoxb8 macrophages for MC38CEA tumors and AP1903. Figure 8 E is the iCas9-CAR Hoxb8 macrophage treatment of MC38CEA tumors. The proportion of iCas9-CAR Hoxb8 macrophages in peripheral blood, liver, lung and tumor of mice treated with AP1903 (the horizontal axis is -10 4 , 0, 10 4 , 10 5 , 10 6 ; The vertical coordinate is -10 3 , 0, 10 3 , 10 4 , 10 5 , 10 6 ).

[0060] Fig. 9 Shown is a flow chart for preparing safe and controllable chimeric antigen receptor-macrophages derived from proliferative stem / progenitor cells in one embodiment of the present application. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0062] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0063] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0064] Example 1

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

[0066] By extracting mouse bone marrow-derived Lin-progenitor cells and isolating human umbilical cord-derived hematopoietic stem / progenitor cells (CB-CD34+ cells), ER-Hoxb8 lentivirus was used for transfection, and flow sorting was used to obtain high-purity and proliferative ER-Hoxb8 progenitor cells. Proliferative ER-Hoxb8 progenitor cells were then transfected with CAR and iCas9-CAR lentiviruses, and high-purity and proliferative CAR ER-Hoxb8 progenitor cells and iCas9-CAR ER-Hoxb8 progenitor cells were obtained by flow 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. The anti-tumor activity of CAR Hoxb8 macrophages was verified in vitro and in vivo, and cell apoptosis was verified by treating iCas9-CAR Hoxb8 macrophages with AP1903.

[0067] The specific operations are:

[0068] 1. Construction of proliferative progenitor cells:

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

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

[0071] The cells were induced to differentiate into ER-Hoxb8 progenitor cells, CAR ER-Hoxb8 progenitor cells, and iCas9-CAR ER-Hoxb8 progenitor cells under the conditions of 20 ng / ml GM-CSF and 20 ng / ml M-CSF for 7 days to obtain CAR Hoxb8 macrophages and iCas9-CAR Hoxb8 macrophages.

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

[0073] The identification methods included the survival time of CAR Hoxb8 macrophages or iCas9-CAR Hoxb8 macrophages, the stemness marker CD117, the macrophage markers F4 / 80 and CD11b, morphology, and the expression of CAR.

[0074] 4. Screening of human target cells with high expression of CEA and construction of mouse target cells with high expression of CEA:

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

[0076] 5. Detection of anti-tumor functional activity of CAR Hoxb8 macrophages in vitro:

[0077] In vitro anti-tumor function detection includes 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 with target cells, and the phagocytic function of CAR Hoxb8 macrophages on target cells and target cell apoptosis detection.

[0078] 6. Detection of the in vivo distribution and persistence of CAR Hoxb8 macrophages after transfusion:

[0079] DIR-labeled CAR Hoxb8 macrophages were injected back into mice through the tail vein, and the in vivo distribution was evaluated by detecting the enrichment of CAR Hoxb8 macrophages in the heart, liver, spleen, lung, kidney, and tumor using small animal in vivo imaging technology. Small animal in vivo imaging technology continuously monitors the fluorescence signal of CAR Hoxb8 macrophages in vivo to evaluate the in vivo persistence.

[0080] 7. Evaluation of the efficacy of CAR Hoxb8 macrophages in the treatment of CRC:

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

[0082] 8.AP1903-induced iCas9-CAR Hoxb8 macrophage apoptosis detection:

[0083] AP1903 was used in vitro to act on 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 for treatment. AP1903 was given at the same time, and the tumor growth curve was monitored and the proportion of iCas9-CAR Hoxb8 macrophages in various tissues was detected by flow cytometry to evaluate whether AP1903 induced apoptosis of iCas9-CAR Hoxb8 macrophages.

[0084] Example 2

[0085] 1. Cell Isolation and Cell Culture:

[0086] Female C57BL / 6J mice aged 6-8 weeks were euthanized, bone marrow cells were extracted, and 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), 10ng / ml murine IL-3 (Peprotech), 20ng / ml murine IL-6 (Peprotech), and 25ng / ml murine SCF (Peprotech). 48 hours later, ER-Hoxb8 lentivirus was transfected to obtain proliferative ER-Hoxb8 progenitor cells, which were cultured in Opti-MEM complete medium, containing Opti-MEM, 10% FBS, 1% PSG, 10ng / ml murine SCF, 30μM beta mercapto ethanol, and 1μM 4-OHT. (The results are shown in Figure 2). Figure 1 As shown in Figure 2, the proliferation of ER-Hoxb8 progenitor cells is dependent on 4-hydroxytamoxifen. Figure 1DE). Positive cells were purified by flow cytometry. CAR lentivirus was transfected on the basis of proliferative ER-Hoxb8 progenitor cells, and the culture conditions of proliferative CAR ER-Hoxb8 progenitor cells were consistent with those of ER-Hoxb8 progenitor cells. HEK293T cells (ATCC), MC38 cells (Cell Resource Center, Peking Union Medical College), MC38CEA cells, MC38CEALuciferase cells, SW620 cells (ATCC) and SW620Luciferase cells were cultured in high-glucose DMEM (Gibco), which contained 10% FBS (Gibco) and 1x penicillin-streptomycin (Pen-Strep; 10,000U / ml penicillin and 10,000μg / ml streptomycin; Gibco). CT26 cells (Type Culture Cell Bank of the Chinese Academy of Sciences), CT26CEA cells, and CT26CEALuciferase cells were cultured in RPMI1640 (Gibco), which contained 10% FBS (Gibco) and 1× Pen-Strep (10,000U / ml penicillin and 10,000μg / 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), HT29Luciferase cells, HCT116 cells (ATCC), and HCT116Luciferase cells were cultured in McCoy's 5A (Hyclone) medium supplemented with 10% bovine serum (Gibco) and 1×penp-strep. SW480 cells (ATCC) and SW480Luciferase cells were cultured in Leibovitz's L-15 (Hyclone) medium supplemented with 20% bovine serum (Gibco) and 1× penp-strep (results as shown in Figure 4 In the present application, cells modified with Hoxb8 and chimeric antigen receptor 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] Lipofectamine 2000 (Invitrogen) was used to transfect CAR vector plasmids, psPAX2, and pMD2.G 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 ER-Hoxb8 progenitor 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. (Results are shown in Figure 2. Figure 2 B).

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

[0090] The cultured CAR ER-Hoxb8 progenitor cells were washed twice with 1XPBS to remove 4-OHT, and then 20ng / ml mouse M-CSF, 20ng / ml mouse GM-CSF, 10% FBS, and 1% Pen-Strep-Glut were added to the RPMI1640 medium. The complete medium was replaced on the third and fifth days, and mature CAR-M was obtained on the seventh day. All recombinant factors were purchased from PeproTech. (Results are shown in Figure 3 AB).

[0091] The preparation process of safe and controllable chimeric antigen receptor-macrophages derived from proliferative progenitor cells is as follows Fig. 9 shown.

[0092] 4. Cell morphology and karyotype analysis:

[0093] To evaluate cell morphology and nuclei, progenitor cells or macrophages were fixed on slides, cell morphology was stained with Diff-Quick stain (Solarbio, G1540-3), nuclei were stained with Wright-Giemsa stain (NJJCBio, D010), and cells were imaged using a microscope. (Results are shown in Figure 2 D and Figure 3 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 based on the protocol in the instructions, and 2 μg of total RNA was reversed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Nanjing Novigene) instructions. Real-time PCR was performed in a 7500 detection system using SYBR-GreenI 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 are shown in Figure 2 E and Figure 3 D).

[0096] 6. Protein Blotting:

[0097] Cell samples were collected and lysed with RIPA buffer containing protease inhibitors (Beyotime, P0013K), and then protein was extracted. Protein concentration was determined using Pierce BCA protein detection kit (Thermo). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and then the samples were transferred to polyvinylidene fluoride 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). The 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 shown in Figure 1 C and Figure 8 C).

[0098] 7. Cytokine release experiment:

[0099] The cell-free supernatant of macrophages and tumor cells co-cultured was collected and the cytokine concentrations of IL-1β, IL-6, IL-12 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) according to the instructions of the kit supplier. The cytokine kits used for quantitative analysis were 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. (The results are shown in Figure 5 A).

[0100] 8. Cytotoxicity assay:

[0101] Target cells (stable tumor cell lines generated by Luciferase lentiviral transfection) were inoculated into a black 96-well ELISA plate at a density of 5000 cells / 100 μL / well. Effector cells were macrophages from different groups, and the ratios of effector cells to target (E:T) cells were added into the wells at 0:1, 1:1, 5:1, and 10:1. After incubation for 24 hours, the 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 Figure 5 BC).

[0102] 9. Phagocytosis experiment:

[0103] The cells were collected and single cell suspension was prepared, washed with PBS containing 1% BSA, and then labeled with antibodies for 20 minutes. Flow cytometry antibodies: APC anti-mouse CD117 (BioLegend, 105811), Brilliant Violet (BV) 711anti-mouse F4 / 80 (BioLegend, 123147), APC anti-mouse / human CD11b (BioLegend, 101212), APC anti-human CEA (BioLegend, 342308). All flow cytometry experiments were performed on a ThermoScientific flow cytometer and analyzed on FlowJo V10 software. (Results are shown in Figure 2. Figure 5 D).

[0104] 10. Detection of in vivo distribution and persistence of CAR Hoxb8 macrophages after transfusion:

[0105] To evaluate the distribution of macrophages in vivo, 1×106 MC38CEALuciferase cells were mixed in PBS and injected subcutaneously into 6- to 8-week-old female C57BL / 6J mice to establish a CRC tumor model. On the 7th day after tumor inoculation, 1×10 7 Macrophages labeled with DiRIodide (Cy7 DiC18, MedChemExpress) were imaged in vivo using an animal imaging system (IVIS Lumina XR) at the same time point on days 1, 6, 11, 16, and 21 after injection to capture the fluorescent signal at the tumor site (n = 3 per group). Mice were euthanized on day 28 after tumor inoculation. Heart, liver, spleen, lung, kidney, and tumor tissues were obtained for live animal imaging. (Results are shown in Figure 6 shown).

[0106] 11. Evaluation of the efficacy of CAR Hoxb8 macrophages combined with immune checkpoint inhibitor Anti-PD-1 in the treatment of CRC:

[0107] 5×10 5 MC38CEALuciferase cells were mixed in PBS and injected into the axilla of 6- to 8-week-old female C57BL / 6J mice to establish a CRC tumor model. On the 7th day after tumor inoculation, 1×10 7 Macrophages and intraperitoneal injection of 10 mg / kg Anti-PD-1 on the 9th, 12th, and 15th days of tumor inoculation. Five mice in each group were imaged for live animals at the same time point on the 7th, 12th, 17th, 22nd, and 28th days of tumor inoculation to monitor tumor growth. D-Luciferin (Yeasen, 40902ES03) was injected into the mice intraperitoneally at a dose of 150 mg / kg, and live animal imaging was performed using an animal imaging system (IVIS Lumina seriesIII) to capture fluorescent signals. At the same time, the mice were weighed and the tumor size was measured on the day of macrophage injection and on the 1st, 5th, 9th, 13th, 17th, and 21st days after injection to determine the tumor volume. The mice were euthanized on the 28th day of tumor implantation, and the tumor tissue was obtained and weighed. At the same time, 5 mice in each group were taken for survival monitoring.

[0108] (Results such as Figure 7 shown).

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

[0110] Macrophages in different groups were counted at 1×10 4 / well were plated in a 96-well plate, and different concentrations (0, 0.1, 1, 10, 100 ng / ml) of AP1903 were added for 6 hours. 100 μl of CCK8 reagent was added to each well and incubated for 2 hours. The survival rate of macrophages was analyzed by microplate reader. 5 / well were plated in 6-well plates, and different concentrations (0, 1, 10 ng / ml) of AP1903 were added for 6 hours. The cells were collected and labeled with Annexin V for flow cytometry to analyze macrophage apoptosis. 6 MC38CEALuciferase cells were mixed in PBS and injected into the axilla of 6- to 8-week-old female C57BL / 6J mice to establish a CRC tumor model. On the 7th day after tumor inoculation, 1×10 7 Macrophages. 5 mg / kg AP1903 was administered on the 11th and 14th days after tumor inoculation, and the tumor growth curve was monitored. On the 18th day after tumor inoculation, the mice were euthanized, and peripheral blood, liver, lung and tumor were collected for flow cytometry to analyze the proportion of macrophages infused. (The results are shown in Figure 8 shown).

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

Claims

1. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a single-chain variable region targeting CEA carcinoembryonic antigen, a hinge region, a transmembrane region and an intracellular signal region; the amino acid sequence of the single-chain variable region targeting CEA carcinoembryonic antigen is shown in SEQ ID No.

1.

2. The chimeric antigen receptor according to claim 1, characterized in that The hinge region is an IgG1 hinge region, and its amino acid sequence is shown in SEQ ID No.

2.

3. The chimeric antigen receptor according to claim 1, characterized in that The transmembrane region is the CD8α transmembrane region, and its amino acid sequence is shown in SEQ ID No.

3.

4. The chimeric antigen receptor according to claim 1, characterized in that The intracellular signal region includes: a CD3ζ intracellular signal region, whose amino acid sequence is shown in SEQ ID No.4; or a FcRγ intracellular signal region, whose amino acid sequence is shown in SEQ ID No.

5.

5. The chimeric antigen receptor according to any one of claims 1 to 4, characterized in that The amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No.6 or SEQ ID No.

7.

6. An expression vector, characterized in that: The expression vector carries a nucleotide encoding the chimeric antigen receptor according to any one of claims 1-5.

7. The expression vector according to claim 6, characterized in that The expression vector is selected from at least one of a lentiviral expression vector, a retroviral expression vector or an adenoviral expression vector; preferably a lentiviral expression vector.

8. A chimeric antigen receptor macrophage, characterized in that The macrophages express the chimeric antigen receptor according to any one of claims 1 to 5.

9. The chimeric antigen receptor macrophage according to claim 8, characterized in that The macrophages are derived from proliferative stem cells or proliferative progenitor cells.

10. Use of the chimeric antigen receptor macrophage according to claim 8 or 9 in the preparation of a tumor therapeutic drug, 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.

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