Acute myelogenous leukemia treatment medicine of targeted protein ADGRE2 and application of acute myelogenous leukemia treatment medicine

Abstract

The invention discloses a protein ADGRE2-targeted acute myelogenous leukemia treatment medicine and application, and relates to the technical field of biomedicine. The invention provides an application of a drug targeting protein ADGRE2 in preparation of a drug for preventing and / or treating acute myelogenous leukemia. The protein ADGRE2 is highly specifically expressed on the surface of an acute myelogenous leukemia cell membrane, and the targeted protein ADGRE2 can inhibit the proliferation ability and migration ability of acute myelogenous leukemia cells and promote the apoptosis of the acute myelogenous leukemia cells. Meanwhile, the protein ADGRE2 can be used as an effective drug target for CAR-T cell therapy, guides the development of novel CAR-T cells, and is expected to promote the novel target CAR-T cell therapy for acute myelogenous leukemia to make a major breakthrough.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a therapeutic agent and application for acute myeloid leukemia targeting the protein ADGRE2. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease originating from hematopoietic stem cells. It is characterized by abnormal proliferation and differentiation arrest of myeloid progenitor cells, which accumulate in large numbers in the bone marrow and peripheral blood, leading to suppression of normal hematopoietic function. Most cases are acute and severe, with a poor prognosis; without timely treatment, it can often be life-threatening.

[0003] Chimeric antigen receptor T (CAR-T) cell therapy is a revolutionary adoptive immunotherapy technology that has achieved good clinical efficacy and safety in hematological malignancies such as acute B-lymphoblastic leukemia, B-cell non-Hodgkin's lymphoma, and multiple myeloma. However, in AML, the most common type of acute leukemia in adults, CAR-T cell therapy has developed relatively slowly. Existing CAR-T cells targeting various targets, such as CD123, CD33, CD117, CD44v6, CLL-1, Siglec-6, and FLT3, have failed to achieve breakthrough progress due to off-target effects or low efficacy. The main reason for this is the lack of tumor antigens specifically expressed on the surface of AML cell membranes as effective therapeutic targets for CAR-T cells, which limits the development and application of CAR-T cell therapy in AML.

[0004] In addition, cytarabine (Ara-C) is a first-line chemotherapy drug for the treatment of AML and is widely used in induction remission and consolidation therapy regimens. However, clinical practice has shown that using Ara-C alone to treat AML often faces problems such as unstable efficacy, high relapse rate, and increased drug tolerance. Therefore, it is of great significance to study a new treatment regimen for AML. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects and deficiencies of the prior art and to provide the application of a drug targeting the protein adhesion G protein-coupled receptor E2 (ADGRE2) in the preparation of drugs for the prevention and / or treatment of AML.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention protects the use of a drug targeting the protein ADGRE2 in the preparation of a preventive and / or therapeutic medicament for AML.

[0008] Preferably, the drug targeting the ADGRE2 protein includes reagents that inhibit ADGRE2 gene expression and / or reagents that inhibit ADGRE2 protein expression.

[0009] This invention protects a combination therapy for treating AML, the combination therapy comprising a protein ADGRE2 inhibitor and an AML chemotherapy drug.

[0010] Preferably, the AML chemotherapy drug is Ara-C.

[0011] This invention protects the use of a combination drug in the preparation of a preventive and / or therapeutic drug for AML.

[0012] This invention protects the use of CAR-T cells targeting the protein ADGRE2 in the preparation of drugs for the prevention and / or treatment of AML.

[0013] This invention protects a detection kit for screening AML, including a protein ADGRE2 detection reagent.

[0014] This invention protects the use of a protein ADGRE2 detection reagent in the preparation of reagents for screening and / or diagnosing AML.

[0015] Preferably, the detection method for the protein ADGRE2 includes at least one of immunofluorescence, flow cytometry, and ELISA.

[0016] Preferably, the AML cell line for which the ADGRE2 protein is detected is selected from at least one of MOLM13, OCI-AML3, THP-1, and HL-60.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention identifies ADGRE2, a drug target highly specifically expressed on the AML cell membrane. The application of drugs targeting the ADGRE2 protein in the preparation of drugs for the prevention and / or treatment of AML, especially ADGRE2 protein inhibitors, can inhibit the proliferation and migration of AML cell lines, representing a new and effective treatment strategy for AML.

[0019] Furthermore, the combined use of the protein ADGRE2 inhibitor and Ara-C can promote apoptosis in AML cell lines.

[0020] Furthermore, the protein ADGRE2 can serve as an effective drug target for CAR-T cell therapy, guiding the development of novel CAR-T cells and potentially leading to a major breakthrough in novel target CAR-T cell therapy for AML. Attached Figure Description

[0021] Figure 1 This is a diagram showing the selection of AML cells by flow cytometry and sorting by magnetic beads in Example 1 of the present invention. Figure 1 A is a flow cytometry diagram showing different cell subpopulations in AML patients before sorting; Figure 1 B shows the CD34 levels in AML patients after magnetic bead sorting by flow cytometry. + Image showing the purity of primary tumor cells.

[0022] Figure 2 This is a membrane proteomics analysis diagram of Example 1 of the present invention. Figure 2 A is a heatmap showing the relative expression levels of several differentially expressed proteins in AML patients and healthy controls in samples from healthy controls and AML patients; Figure 2 B is an ADGRE2 radar chart, where the first circle represents differentially expressed proteins, sorted clockwise along the orange arrows from smallest to largest according to the P value; the second circle represents the Log2 conversion ratio, with pink indicating upregulation and light blue indicating downregulation, and the larger the dot, the greater the fold difference; the third circle represents the average quantitative level of the two groups.

[0023] Figure 3 This is a diagram showing the expression of ADGRE2 in AML cell lines in Example 2 of the present invention. Figure 3 AB: Flow cytometry detection of ADGRE2 high expression in 5 AML cell lines and its expression statistics; Figure 3 C: Immunofluorescence assay of ADGRE2 expression in AML cell lines; Figure 3 D: Gene transcriptional expression level of ADGRE2 in different AML cell lines.

[0024] Figure 4 This is a diagram showing the expression of ADGRE2 in various cells of the control group and AML patients in Example 2 of the present invention. Figure 4 A: Flow cytometry analysis of ADGRE2 membrane surface expression in different cell populations of iron deficiency anemia (IDA) patients and controls; Figure 4 B: Statistical chart of ADGRE2 expression ratio in each cell population.

[0025] Figure 5The above are heatmaps, waterfall plots, and bar charts showing ADGRE2 membrane surface expression in different cell types in the AML cohort of Example 2 of the present invention. The cells are sorted by the level of ADGRE2 expression in AML cells to show the expression differences among different cells.

[0026] Figure 6 These are heatmaps, waterfall plots, and bar charts showing ADGRE2 membrane surface expression in different cell types in the IDA cohort of Example 2 of this invention.

[0027] Figure 7 For the immunofluorescence detection of CD34 in AML patients in Example 2 of this invention + and CD34 - Diagram showing expression patterns on the cell membrane surface.

[0028] Figure 8 This is a diagram showing the expression of ADGRE2 in different tumors in the GEPIA database in Example 2 of the present invention.

[0029] Figure 9 This is a diagram showing the expression of ADGRE2 on the surface of primary tumor cells from AML patients in Example 2 of this invention. Figure 9 A: Expression of ADGRE2 in different cell subpopulations of AML patients; Figure 9 B: Expression of ADGRE2 in normal human bone marrow hematopoietic stem cells and different cell subsets; Figure 9 CL: ADGRE2 expression in specimen type, normal hematopoietic stem cells and AML patient tumor cells, FAB classification, chromosome karyotype, age, sex, gene, prognostic stratification, disease status, and response to initial standardized induction chemotherapy.

[0030] Figure 10 This is a diagram showing the expression of ADGRE2 in normal human tissues in Embodiment 2 of the present invention.

[0031] Figure 11 This is a verification diagram of ADGRE2 knockout in Embodiment 3 of the present invention. Figure 11 A: Graph showing ADGRE2 expression at the mRNA level as detected by PCR; Figure 11 BD: Flow cytometry and Western blotting to detect ADGRE2 protein expression and its statistical graph; Figure 11 E: Immunofluorescence assay of ADGRE2 protein expression.

[0032] Figure 12 This is a diagram showing the functional effects of ADGRE2 gene knockout on AML cells in Example 3 of the present invention. Figure 12 AB: Clonogenesis assay for ADGRE2 WT and ADGRE2 KO Cell line proliferation capacity and its statistical graph; Figure 12 CD: Cell migration assay to detect ADGRE2 WT and ADGRE2 KO Cell line migration ability and its statistical graph; Figure 12 EF: Senescence staining and its statistical graph; Figure 12 GH: Flow cytometry and its statistical plots for aging; Figure 12 IL: These are graphs and statistical diagrams showing the apoptosis and cell cycle status of the two cell lines after 15 days.

[0033] Figure 13 This is a response diagram of the ADGRE2 gene knockout to the chemotherapy drug Ara-C in Example 3 of the present invention. Figure 13 A: Flow cytometry verification of cell senescence induced 3 days after the addition of Ara-C; Figure 13 BC: Morphological staining detection and statistical graph of senescence staining 3 days after adding Ara-C; Figure 13 DE: Flow cytometry analysis of cell apoptosis and its statistical graph 3 days after Ara-C addition; Figure 13 FG: Flow cytometry analysis of cell cycle and its statistical graph 3 days after Ara-C addition; Figure 13 HK: Transwell assay and invasion assay to detect cell migration and invasion 3 days after Ara-C addition and statistical graphs. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.

[0035] Example 1

[0036] Membrane proteomics analysis:

[0037] ① Collect peripheral blood from AML patients and healthy individuals and extract peripheral blood mononuclear cells (PBMCs): Select 4 AML patients and 4 healthy individuals. Collect peripheral blood from them by venous blood collection. Dilute the peripheral blood with PBS and then use Ficoll separation buffer gradient centrifugation at 400g for 30 minutes to obtain PBMCs.

[0038] ② Sorting AML patients' CD34 + Primary tumor cells: CD34 magnetic beads were used to sort PBMCs from the above AML patients to obtain CD34 cells. + Primary AML tumor cells were sorted using flow cytometry, and the purity of the cells was verified (all >95%). The results are as follows: Figure 1 As shown.

[0039] ③ Membrane proteomics detection and database analysis: The sorted primary tumor cells or normal healthy human PBMCs were lysed, and membrane proteins were extracted for membrane proteomics detection and data analysis.

[0040] like Figure 2 As shown, the results indicate that ADGRE2 expression was significantly upregulated in the cell membrane protein of primary AML tumor cells compared with normal healthy human PBMCs. At the same time, the GEPIA database also showed that the transcriptional level of ADGRE2 gene expression was significantly upregulated in AML.

[0041] Example 2

[0042] 2.1 ADGRE2 expression in AML cell lines:

[0043] ① Flow cytometry detection of ADGRE2 expression: Isotype was used as an isotype control for the detection antibody. Four AML cell lines (MOLM13, HL-60, OCI-AML3, THP-1) and one chronic myeloid leukemia cell line (CML) (K562) were labeled with ADGRE2 antibody. The Isotype antibody was diluted 1:500; the ADGRE2 antibody was diluted 1:200. Flow cytometry was used to detect the intracellular and cell membrane surface expression of ADGRE2 in AML and CML cell lines and to detect the differences in these expression levels.

[0044] like Figure 3 A and Figure 3 As shown in Figure B, ADGRE2 was highly expressed (>92%) in the AML cell lines MOLM13, HL-60, OCI-AML3, and THP-1, while Isotype was lowly expressed (<1.68%). Furthermore, ADGRE2 expression was 39.33% in the CML cell line K562, while Isotype expression was 1.23%. ② Immunofluorescence detection of ADGRE2 expression: Using Isotype as an isotype control, five AML cell lines were labeled with ADGRE2 antibody. Immunofluorescence was observed using laser confocal microscopy to detect the intracellular and cell membrane surface expression of ADGRE2 in the AML cell lines and their differences.

[0045] like Figure 3 C and Figure 3 As shown in D, ADGRE2 is highly expressed in AML cell lines (SKM-1, NOMO-1, MV4-11, KG-1A, and MOLM13), and the gene transcriptional expression level of DGRE2 is high in AML cell lines, especially in MOLM13 where the gene transcriptional expression is as high as 30% or more.

[0046] 2.2 ADGRE2 expression in AML patients:

[0047] ① 100 μL of whole blood or bone marrow from AML patients was selected to label ADGRE2. Isotype was used as an antibody isotype control: 1 μL of Isotype antibody, 1 μL of ADGRE antibody, 10 μL of CD45-PC7 antibody, 1 μL of CD117-APC antibody, and 1 μL of CD34-FITC antibody. Flow cytometry was used to detect the expression of ADGRE2 in primary tumor cells and other blood cells (including neutrophils, monocytes, and lymphocytes) in AML patients and statistical analysis was performed. At the same time, the clinical significance of the cell membrane surface expression level of ADGRE2 in the treatment response and survival prognosis of AML patients was understood.

[0048] ② For AML patients with a large number of leukemia cells infiltrating the peripheral circulation, peripheral blood was collected and PBMCs (almost all of which were primary tumor cells) were extracted using Ficoll separation solution. CD34 cells were then sorted using magnetic beads to obtain CD34-positive cells. + and CD34 - Cell populations were observed using laser confocal microscopy to detect and compare ADGRE2 levels at CD34. + Leukemia stem cells and CD34 - Expression patterns and differences in leukemia cells.

[0049] 2.3 Expression of ADGRE2 in normal human blood cells and tissues:

[0050] ① 100 μL of peripheral blood or bone marrow fluid from IDA patients was selected to label ADGRE2. Isotype was used as an antibody isotype control: 1 μL of Isotype antibody, 1 μL of ADGRE antibody, 10 μL of CD45-PC7 antibody, 1 μL of CD117-APC antibody, and 1 μL of CD34-FITC antibody. The expression of ADGRE2 in normal human hematopoietic stem cells and other blood cells (including neutrophils, monocytes, and lymphocytes) was detected by flow cytometry and statistical analysis was performed.

[0051] ② Immunohistochemistry was used to detect the expression of ADGRE2 in normal human tissues: Normal human tissue microarrays were purchased, and Isotype was used as the antibody isotype control to detect the expression of ADGRE2 in normal human tissues (including: lungs, liver, pancreas, kidneys, skin, and lymph nodes).

[0052] like Figure 4-8The expression of ADGRE2 in various cells of IDA control group patients and AML patients is shown. The expression level of ADGRE2 in various cells of AML patients is much higher than that in normal healthy individuals.

[0053] like Figure 9 The table shows the expression of ADGRE2 on the surface of primary tumor cells in AML patients. The analysis included ADGRE2 expression in different cell subpopulations of AML patients, specimen type, normal hematopoietic stem cells versus AML patient tumor cells, FAB classification, chromosome karyotype, age, sex, gene type, prognostic stratification, disease status, and response to initial standardized induction chemotherapy. The clinical baseline characteristics of AML patients with ADGRE2 expression are shown in Table 1. Patients in the high ADGRE2 expression group had a poorer prognosis and shorter survival time.

[0054] Table 1

[0055]

[0056]

[0057] like Figure 10 The ADGRE2 expression in normal human tissues (lung, liver, pancreas, kidney, skin, lymph nodes) shown is negative in all of them.

[0058] Example 3

[0059] 3.1 Validation of ADGRE2 gene knockout:

[0060] ① The expression of the ADGRE2 gene in the MOLM-13 cell line derived from AML patients was knocked out using CRISPR-Cas9 technology;

[0061] ② Detection of ADGRE2 gene in wild-type MOLM-13 cell line (ADGRE2) using qRT-PCR WT ) and ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO ADGRE2 mRNA expression levels and their differences in )

[0062] ③ Western blot, flow cytometry, and immunofluorescence detection of the ADGRE2 gene wild-type MOLM-13 cell line (ADGRE2 WT ) and ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO The expression levels and differences of ADGRE2 protein in the samples were as follows: Figure 11 As shown.

[0063] 3.2 Functional validation after ADGRE2 gene knockout

[0064] ① Colony formation assay: The colony formation assay was used to detect the ADGRE2 gene in the wild-type MOLM-13 cell line (ADGRE2). WT ) and ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO The proliferative capacity and differences of )

[0065] ② Cell migration and invasion assay: Transwell assay was used to detect the ADGRE2 gene in the wild-type MOLM-13 cell line (ADGRE2). WT ) and ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO The migration and invasion capabilities of ( ) and their differences;

[0066] ③ Cell senescence detection: Direct morphological staining with β-galactosidase or flow cytometry analysis was used to detect the ADGRE2 gene in the wild-type MOLM-13 cell line (ADGRE2). WT ) and ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO The proportion of senescent cells and their differences;

[0067] ④ Apoptosis detection: Annexin V / PI double staining combined with flow cytometry was used to detect the ADGRE2 gene in the wild-type MOLM-13 cell line (ADGRE2). WT ) and ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO The proportion of apoptosis and its differences among cells;

[0068] ⑤ Cell cycle detection: The ADGRE2 gene in the wild-type MOLM-13 cell line (ADGRE2) was detected using flow cytometry PI method. WT ) and ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO The cell cycle and its differences.

[0069] The results are as follows Figure 12 As shown, the ADGRE2 gene knockout MOLM-13 cell line (ADGRE2 KO The proliferation capacity of cells decreases, their migration capacity declines, and the proportion of apoptotic cells increases.

[0070] 3.3 Response to chemotherapy drugs after ADGRE2 gene knockout:

[0071] ① Experimental grouping: The experiment was conducted using the MOLM13 cell line and the chemotherapy drug Ara-C, and the grouping was set as ADGRE2.WT ADGRE2 KO ADGRE2 WT +Ara-C、ADGRE2 KO +Ara-C;

[0072] ②After Ara-C treatment of the above cells for 3 days, the following were used to detect cell migration and invasion ability, cell senescence, cell cycle, and apoptosis, respectively, by Transwell and invasion assays, morphological staining and flow cytometry, and flow cytometry.

[0073] The results are as follows Figure 13 As shown, the addition of Ara-C to ADGRE2 gene knockout cell lines induced cell senescence, accelerated apoptosis, and inhibited cell migration and invasion.

[0074] The above embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of a drug targeting the protein ADGRE2 in the preparation of a drug for the prevention and / or treatment of acute myeloid leukemia.

2. The application according to claim 1, characterized in that: The drug targeting the membrane protein ADGRE2 includes reagents that inhibit ADGRE2 gene expression and / or reagents that inhibit ADGRE2 protein expression.

3. A combination drug for treating acute myeloid leukemia, characterized in that: The combined medications include an ADGRE2 protein inhibitor and chemotherapy drugs for acute myeloid leukemia.

4. The combination drug for treating acute myeloid leukemia according to claim 3, characterized in that: The chemotherapy drug for acute myeloid leukemia is cytarabine.

5. The use of the combined drug of claims 3-4 in the preparation of a drug for the prevention and / or treatment of acute myeloid leukemia.

6. The application of a CAR-T cell targeting the protein ADGRE2 in the preparation of drugs for the prevention and / or treatment of acute myeloid leukemia.

7. A test kit for screening acute myeloid leukemia, characterized in that: Including reagents for detecting protein ADGRE2.

8. The use of a protein ADGRE2 detection reagent in the preparation of reagents for screening and / or diagnosing acute myeloid leukemia.

9. The application according to claim 8, characterized in that: The detection methods for the protein ADGRE2 include at least one of immunofluorescence, flow cytometry, and ELISA.

10. The application according to claim 8, characterized in that: The AML cell lines used for the ADGRE2 protein assay were selected from at least one of MOLM13, OCI-AML3, THP-1, and HL-60.

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