Application of CD132 in treatment of AML

Abstract

The invention discloses an application of CD132 in treatment of AML (acute myeloid leukemia). The CD132 is differentially expressed in the AML, the expression level of the CD132 is related to the malignant degree of the AML and the total survival rate of AML patients, and CD132 knock-down can inhibit the proliferation capacity of AML cells, influence the cell cycle of the AML cells and promote cell apoptosis of the AML cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to the application of biomarkers in the treatment of AML. BACKGROUND

[0002] AML (acute myeloid leukemia, AML) is the most common type of adult leukemia, which seriously endangers human health and life safety. The treatment of AML usually includes induction chemotherapy, consolidation chemotherapy and hematopoietic stem cell transplantation, etc. The purpose of induction chemotherapy is to make the patient achieve complete remission, i.e. the proportion of primitive cells in bone marrow is <5%, and clinical symptoms and signs disappear. Common chemotherapy regimens include cytarabine combined with anthracycline drugs, etc. For high-risk patients or relapsed refractory patients, hematopoietic stem cell transplantation may be an effective treatment option. In recent years, with the development of molecular biology and targeted therapy, some targeted drugs for specific gene mutations or fusion genes have also been gradually applied to the treatment of AML, such as FLT3 inhibitors, IDH1 / 2 inhibitors, etc.

[0003] The prognosis of AML varies with factors such as subtype, patient age, general condition, cytogenetic and molecular biological characteristics, etc. Overall, M3 type leukemia has a relatively good prognosis after timely and effective treatment; while patients with complex chromosomal abnormalities and certain gene mutations (such as FLT3-ITD high mutation) have a poor prognosis. Patients with older age and comorbidities also often have a poor prognosis, and the long-term survival rate of AML patients under 60 years old is 35-40%, while the long-term survival rate of patients 60 years old and above is only 5-15%. Acute granulocyte-monocyte / monocyte leukemia (including FAB classification, i.e. M4 and M5, accounting for 29.1% and 8.9% of AML, respectively) is an acute leukemia with malignant clonal proliferation of both granulocyte and monocyte lines, and its relapse rate is significantly higher than that of other AML subtypes, with the incidence of bone marrow and extramedullary relapse after hematopoietic stem cell transplantation being 34% and 50%, respectively. The 3-year disease-free survival rate of AML-M4 / 5 patients is only about 26%, so AML-M4 / 5 is the main group of relapsed refractory patients (Blood. 2018 Jul 26;132(4):362-8.; Cancer discovery. 2020 Apr;10(4):536-51). Improving the long-term survival rate of AML patients has been a clinical problem that needs to be solved urgently.

[0004] AML is a complex disease, and although some progress has been made in diagnosis, treatment and prognosis evaluation, there are still many deficiencies. Further research is needed in the future to develop more accurate diagnostic methods, effective treatment methods and reliable prognostic evaluation indicators to improve the survival rate and quality of life of patients. SUMMARY

[0005] To overcome the shortcomings of existing technologies, this invention provides a target capable of treating AML.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides the use of CD132 as a target in the preparation of medicaments for the treatment of AML.

[0007] Furthermore, the drug includes an inhibitor of CD132.

[0008] Furthermore, the inhibitor includes any substance that can reduce the expression of nucleic acid encoding CD132, reduce CD132 protein levels, or inhibit the activity of CD132.

[0009] Furthermore, the inhibitors include nucleic acid inhibitors, proteolytic enzymes, protein-binding molecules, or combinations thereof.

[0010] Furthermore, the nucleic acid inhibitors include interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA.

[0011] Furthermore, the nucleic acid inhibitor is selected from interfering RNA.

[0012] Furthermore, the nucleic acid inhibitor is shRNA.

[0013] Furthermore, the active sequence of the shRNA is shown in SEQ ID NO.1 and SEQ ID NO.4.

[0014] Furthermore, the sequences of the shRNA are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.6.

[0015] Furthermore, the nucleic acid inhibitor is gRNA.

[0016] Furthermore, the sequence of the gRNA is shown in SEQ ID NO.10.

[0017] Furthermore, the nucleic acid inhibitor includes gRNA and Cas9 protein.

[0018] Furthermore, the protein-binding molecule is selected from anti-CD132 antibodies or substances derived therefrom.

[0019] Furthermore, the anti-CD132 antibody is REGN7257, with the amino acid sequence shown in SEQ ID NO.11.

[0020] Furthermore, the AML includes M4 and / or M5 subtypes.

[0021] Furthermore, the drug inhibits the proliferation of AML cells, affects the cell cycle of AML cells, or promotes apoptosis of AML cells.

[0022] A second aspect of the invention provides a medicament for treating AML, the medicament comprising an inhibitor of CD132.

[0023] Furthermore, the inhibitor includes any substance that can reduce the expression of nucleic acid encoding CD132, reduce CD132 protein levels, or inhibit the activity of CD132.

[0024] Furthermore, the inhibitors include nucleic acid inhibitors, proteolytic enzymes, protein-binding molecules, or combinations thereof.

[0025] Furthermore, the nucleic acid inhibitors include interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA.

[0026] Furthermore, the nucleic acid inhibitor is selected from interfering RNA.

[0027] Furthermore, the nucleic acid inhibitor is shRNA.

[0028] Furthermore, the active sequence of the shRNA is shown in SEQ ID NO.1 and SEQ ID NO.4.

[0029] Furthermore, the shRNA is shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.6.

[0030] Furthermore, the nucleic acid inhibitor is gRNA.

[0031] Furthermore, the sequence of the gRNA is shown in SEQ ID NO.10.

[0032] Furthermore, the nucleic acid inhibitor includes gRNA and Cas9 protein.

[0033] Furthermore, the protein-binding molecule is selected from anti-CD132 antibodies or substances derived therefrom.

[0034] Furthermore, the anti-CD132 antibody is REGN7257, with the amino acid sequence shown in SEQ ID NO.11.

[0035] Furthermore, the derived material includes CAR-T cells made from antibodies against CD132.

[0036] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0037] Furthermore, the AML includes M4 and / or M5 subtypes.

[0038] A third aspect of the present invention provides the use of CD132 in screening candidate drugs for the treatment of AML.

[0039] Furthermore, the method for screening candidate drugs for the treatment of AML is as follows: treating a culture system expressing or containing the CD132 gene or its encoded protein with a screening substance; and detecting the expression or activity of the CD132 gene or its encoded protein in the system; wherein, when the screening substance inhibits the expression level or activity of the CD132 gene or its encoded protein, the screening substance is a candidate drug for the treatment of AML.

[0040] Furthermore, the candidate drug inhibits the proliferation of AML cells, affects the cell cycle of AML cells, or promotes apoptosis of AML cells.

[0041] Furthermore, the AML is selected from subtypes including M4 and / or M5.

[0042] A fourth aspect of the present invention provides a method for screening candidate drugs for treating AML, the method comprising treating a culture system expressing or containing the CD132 gene or its encoded protein with a screening substance; and detecting the expression or activity of the CD132 gene or its encoded protein in the system; wherein, when the screening substance inhibits the expression level or activity of the CD132 gene or its encoded protein, the screening substance is a candidate drug for treating AML.

[0043] Furthermore, the method also includes a functional verification step for the candidate drug, such as verifying the effects of the candidate drug on AML cell morphology, cell proliferation, cell cycle, and cell apoptosis.

[0044] Furthermore, the candidate drug inhibits the proliferation of AML cells, affects the cell cycle of AML cells, or promotes apoptosis of AML cells.

[0045] Furthermore, the AML is selected from the M4 and / or M5 subtypes.

[0046] A fifth aspect of the present invention provides a computer-aided method for screening candidate drugs for the treatment of AML, the method comprising: Obtain the structure domain of CD132. Based on the spatial structure screening of CD132 domains, substances that regulate CD132 are selected as candidate drugs for the treatment of AML.

[0047] Furthermore, the method also includes a functional verification step for the candidate drug, such as verifying the effects of the candidate drug on AML cell morphology, cell proliferation, cell cycle, and cell apoptosis.

[0048] Furthermore, the AML is selected from the M4 and / or M5 subtypes.

[0049] The sixth aspect of the present invention provides the use of candidate drugs screened based on the method described in the fourth or fifth aspect of the present invention in the preparation of medicaments for treating AML.

[0050] The seventh aspect of this invention provides the application of CD132 in the construction of AML.

[0051] Furthermore, methods for constructing AML models include the application of a CD132 promoter.

[0052] Furthermore, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase CD132 protein levels, or promote the activity of CD132.

[0053] Furthermore, the promoter includes a CD132 overexpression system.

[0054] Furthermore, the CD132 overexpression system includes the selection of expression vectors, including plasmids, viral vectors, or transposon vectors, and is constructed using chemical transfection, physical transfection, or viral infection methods.

[0055] Furthermore, the plasmid includes pCDNA series vectors, the viral vector includes lentiviral vectors, adeno-associated virus (AAV) vectors or adenovirus vectors, and the transposon vectors include PiggyBac transposon vectors or Sleeping Beauty transposon vectors.

[0056] Furthermore, the viral vector is selected from lentiviral vectors.

[0057] Furthermore, the chemical transfection method includes liposome transfection and cationic polymer transfection, and the physical transfection method includes electroporation and gene gun transfection.

[0058] Furthermore, the AML includes M4 and / or M5 subtypes.

[0059] The eighth aspect of the present invention provides a method for constructing an AML model, the method comprising administering a reagent that regulates CD132.

[0060] Furthermore, the reagent includes a CD132 promoter.

[0061] Furthermore, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase CD132 protein levels, or promote the activity of CD132.

[0062] Furthermore, the promoter includes a CD132 overexpression system.

[0063] Furthermore, the AML model is a malignant model.

[0064] Furthermore, the malignant AML model includes one or more of the following: altered cell morphology, enhanced cell proliferation, cell cycle disorder, impaired cell differentiation, altered cell adhesion and migration, metabolic changes, and altered immune phenotype.

[0065] The ninth aspect of the present invention provides an AML cell model in which the cells overexpress CD132.

[0066] Furthermore, the AML cell model is constructed using the construction method described in the eighth aspect of this invention.

[0067] The tenth aspect of this invention provides the application of the AML cell model described in the ninth aspect of this invention in screening drugs for the treatment of AML or evaluating the efficacy of drug treatment for AML.

[0068] The eleventh aspect of the present invention provides a method for treating AML, the method comprising administering an effective amount of an inhibitor of CD132 to a subject.

[0069] The advantages and beneficial effects of this invention are as follows: This invention is the first to discover a novel target for the diagnosis, classification, prognostic assessment, and treatment of acute myeloid leukemia (AML) – CD132. This invention found that CD132 is significantly overexpressed in AML M4 and M5 subtypes. The expression level of CD132 is correlated with the malignancy of AML M4 / 5 subtypes and the overall survival rate of AML M4 / 5 subtype patients. CD132 knockdown can inhibit the proliferation of AML cells, affect the cell cycle of AML cells, and promote apoptosis of AML cells. This confirms the effectiveness of CD132 in the prevention, treatment, and / or diagnosis of AML. Furthermore, since this target is a membrane surface protein, it is more advantageous for targeted therapy of acute myeloid leukemia. Attached Figure Description

[0070] Figure 1 These are differential expression plots of CD132 in AML. 1A shows the differential expression of CD132 in normal CD34+ cells and AML cells analyzed from the GEO database dataset. 1B shows the differential expression of CD132 in the AMLM4 / 5 subtype and healthy individuals in the flow cytometry training set. 1C shows the differential expression of CD132 in the AMLM4 / 5 subtype and healthy individuals in the flow cytometry validation set. 1D shows the differential expression of CD132 in the AMLM4 / 5 subtype and non-M4 / 5 subtype of AML in the flow cytometry training set. 1E shows the differential expression of CD132 in the AMLM4 / 5 subtype and non-M4 / 5 subtype of AML in the flow cytometry training set. Figure 2These are ROC curves for CD132 in diagnosing AML M4 / 5 subtypes; 2A shows the ROC curves for CD132 in the training set for diagnosing AML M4 / 5 subtypes and healthy individuals; 2B shows the ROC curves for CD132 in the validation set for diagnosing AML M4 / 5 subtypes and healthy individuals; 2C shows the ROC curves for CD132 in the training set for diagnosing AML M4 / 5 subtypes and AML non-M4 / 5 subtypes; 2D shows the ROC curves for CD132 in the validation set for diagnosing AML M4 / 5 subtypes and AML non-M4 / 5 subtypes. Figure 3 This is a correlation analysis diagram of WT1 and CD132; Figure 4 This is a prognostic prediction chart for CD132 in the AMLM4 / 5 subtype. 4A shows the differential expression of CD132 in different acute monocytic leukemia (FAB-M4 / 5) subtypes under the LSC17 (Nature 540, 433–437 (2016)) criteria, based on a dataset from the GEO database. 4B shows the correlation between CD132 and long-term overall survival in the AML M4 / 5 subtype, based on an analysis of the OHSU database. Figure 5 This is a diagram showing the differential colony formation of AML cells with CD132 high / low saturation. Figure 6 These are diagrams showing the effects of CD132 knockdown on cell function in human leukemia cell lines. 6A shows the effect on THP-1 cell proliferation; 6B shows the effect on THP-1 cell colony formation; 6C shows the effect on the THP-1 cell cycle; 6D shows the effect on THP-1 cell apoptosis; 6E shows the effect on Molm-13 cell proliferation; 6F shows the effect on Molm-13 cell colony formation; 6G shows the effect on the Molm-13 cell cycle; and 6H shows the effect on the OCI-AML3 cell cycle. Figure 7 This is a diagram showing the effect of CD132 overexpression on cell function in human leukemia cell lines. 7A shows the effect on THP-1 cell proliferation, and 7B shows the effect on THP-1 cell cycle. Figure 8 This is a graph showing the effect of CD132 antibody on the cell cycle of leukemia cells; Figure 9 The figure shows the experimental results of CD132 CAR T cells significantly killing CD132-positive tumor cells THP-1 (A) and U937 (B); Figure 10 This is a diagram showing the experimental results of CD132 CAR-T cells significantly killing CD132-positive AML primary tumor cells; Figure 11 The figure shows the experimental results showing that shRNA silencing of KASUMI1 had no effect on the cell cycle. Figure 12 The figure shows the experimental results showing that silencing KASUMI-1 with CRISPR / CAS9 has no effect on the cell cycle. Figure 13 This is a graph showing the experimental results of KG1 at the cell cycle level after silencing CD132; Figure 14 This is a graph showing the experimental results of CD132 monoclonal antibody failing to inhibit the proliferation of KASUMI1.

[0071] Note: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001). Detailed Implementation

[0072] The following provides definitions for some terms used in this specification. Unless otherwise stated, all technical and scientific terms used in this invention generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0073] This invention provides the application of CD132 as a target in the preparation of drugs for treating AML.

[0074] In this invention, CD132 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed CD132, as well as any form of CD132 derived from cell processing. The term encompasses naturally occurring variants of CD132 (e.g., splice variants or allelic variants). The term encompasses, for example, the CD132 gene, human CD132, and CD132 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, CD132 is a human gene with gene ID 3561.

[0075] In this invention, the term "treatment" can refer to therapeutic procedures or preventative measures where the goal is to prevent or slow (mitigate) an undesirable physical condition, impairment, or disease, or to achieve a beneficial or desired clinical outcome. In this invention, "treatment" can refer to both treatment and prevention. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief; reduction of the severity of a symptom, impairment, or disease; stabilization (i.e., non-exacerbation) of the state of a symptom, impairment, or disease; delaying the onset of a symptom, impairment, or disease or slowing its progression; improving the state of a symptom, impairment, or disease; and relief (whether partial or complete) (whether detectable or undetectable) or improvement or enhancement of a symptom, impairment, or disease. Treatment may include causing a clinically noticeable response without excessive side effects. Treatment also includes extended survival compared to the expected survival without treatment.

[0076] In this invention, the drug includes an inhibitor of CD132. An inhibitor is any substance that can inhibit the activity of the CD132 protein, inhibit the stability of the CD132 gene or protein, inhibit the expression level of CD132, inhibit the effective duration of CD132 protein, or inhibit the activity of CD132. As one embodiment of this invention, the "inhibitor" is a substance that inhibits the expression level of CD132.

[0077] In this invention, inhibitors include nucleic acid inhibitors, proteolytic enzymes, protein-binding molecules, and combinations thereof.

[0078] In some embodiments, the nucleic acid inhibitor is selected from: interfering molecules that target CD132 or its transcripts and are capable of inhibiting CD132 gene expression or transcription, including but not limited to shRNA, siRNA, ribozymes, antisense oligonucleotides, dsRNA, microRNA, zinc fingers, gRNA, or constructs that can express or form said shRNA, siRNA, ribozymes, antisense oligonucleotides, dsRNA, microRNA, zinc fingers, or gRNA. The protein inhibitor is selected from substances capable of inhibiting the CD132 protein. The proteolytic enzyme is selected from enzymes capable of catalyzing the hydrolysis of the CD132 protein. The protein-binding molecule is selected from substances that specifically bind to the CD132 protein, such as antibodies or ligands capable of inhibiting the activity of the CD132 protein.

[0079] In some embodiments, the nucleic acid inhibitor is selected from siRNA. siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinant RNA, as well as RNA modified to differ from native RNA by adding, deleting, substituting, and / or altering one or more nucleotides. Such alterations may include adding non-nucleotide substances, such as adding to the terminus of the siRNA or one or more internal nucleotides of the siRNA; modifications that make the siRNA resistant to nuclease digestion (e.g., using 2'...). (Replacement of ribonucleotides or modification of the sugar phosphate backbone); or replacement of one or more nucleotides in siRNA with deoxyribonucleotides.

[0080] In some embodiments, the nucleic acid inhibitor is selected from shRNA. shRNA is a non-coding small RNA molecule capable of forming hairpin structures. shRNA can inhibit gene expression through the RNA interference pathway. When shRNA is introduced into a cell, it is recognized by the intracellular nuclease Dicer and cleaved into small interfering RNA (siRNA) of approximately 21 nucleotides. The siRNA binds to a protein complex called the RNA-induced silencing complex (RISC). The Argonaute protein in RISC uses the antisense strand of the siRNA to recognize and bind to the target mRNA, and then degrades the target mRNA by cleavage or inhibition of translation, thereby inhibiting the expression of a specific gene. In a specific embodiment of the present invention, the nucleic acid inhibitor is selected from shRNA.

[0081] In some embodiments, the nucleic acid inhibitor is selected from gRNA. In some embodiments, the gRNA is a nucleotide complementary to the target DNA sequence that guides the Cas9 protein to a specific DNA site for double-strand DNA cleavage at the PAM sequence. Therefore, as a preferred embodiment, the nucleic acid inhibitor includes both gRNA and Cas9 protein.

[0082] In some implementations, the nucleic acid inhibitor is selected from ribozymes, which are a class of RNAs that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific sequence-dependent manner. Ribozymes and their delivery methods are well known in the art (Hendry et al., BMC Chem. Biol., 4(1): 1(2004); Grassi et al., Curr. Pharm. Biotechnol., 5(4): 369). 386(2004); Bagheri et al., Curr. Mol. Med., 4(5): 489 506 (2004); Kashani Sabet M., Expert Opin. Biol. Ther., 4(11): 1749 1755 (2004), each incorporated herein by reference in its entirety. Ribozymes inhibit translation by cleaving target RNA, thereby preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory using methods known in the art and their structural modifications can be made to increase their stability and catalytic activity. Alternatively, the ribozyme gene can be introduced into the cell using gene delivery mechanisms known in the art.

[0083] In some embodiments, the nucleic acid inhibitor is selected from antisense oligonucleotides. Antisense oligonucleotides (antisense nucleic acid sequences) may include nucleotide sequences complementary to sense nucleic acids encoding proteins (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to CD132 mRNA). Antisense oligonucleotides and delivery methods are well known in the art (Goodchild, Curr. Opin. Mol. Ther., 6(2): 120). 128(2004); Clawson et al., Gene Ther., 11(17): 1331 Article 1341 (2004) is incorporated herein by reference in its entirety. Antisense oligonucleotides may be complementary to the full coding strand of the target sequence or only to a portion thereof. The length of an antisense oligonucleotide may, for example, be about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides.

[0084] In some embodiments, the inhibitor is selected from protein-binding molecules. Protein-binding molecules refer to any molecule that binds to a protein, including but not limited to small molecule compounds, peptides, proteins, antibodies, and substances derived therefrom such as CAR-T, ADCs (antibody-drug conjugates), AOCs (antibody-oligonucleotide conjugates), etc. In some embodiments, the protein-binding molecule is selected from antibodies. The antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In some embodiments, the antibody or a fragment thereof can be used to construct a recognition module in cell therapy, such as in chimeric antigen receptor T cells (CAR-T), wherein the antigen-binding domain is derived from the antibody or antibody fragment.

[0085] In one specific embodiment, the antibody is selected from monoclonal antibodies such as REGN7257.

[0086] In one specific embodiment, the antibody is selected from antibodies with an amino acid sequence as shown in SEQ ID NO.11.

[0087] In one specific embodiment, the protein-binding molecule is a CAR-T cell comprising an antibody targeting CD132, the amino acid sequence of which is shown in SEQ ID NO.11.

[0088] In some implementations, the inhibitor is selected from proteolytic enzymes. Proteolytic enzymes are a class of enzymes that catalyze the hydrolysis of proteins, including but not limited to serine proteases, thiol proteases, aspartic proteases, and metalloproteinases.

[0089] The medicament described in this invention can also be used in combination with other compounds for treating AML. These other compounds can be administered simultaneously with the main active ingredient (e.g., a CD132 inhibitor), even simultaneously in the same composition. Both can be administered sequentially (e.g., before or after) or simultaneously in the same formulation (i.e., together) or in different formulations (i.e., separately). Simultaneous administration in the same formulation is considered a single formulation, while simultaneous administration in different formulations is not considered a single formulation. Regarding the route of administration, the dosage of the other AML-treating compound and the main active ingredient (e.g., a CD132 inhibitor) can also differ.

[0090] In some embodiments, the medicament includes pharmaceutically acceptable excipients, including excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, flow aids, flavoring agents, solubilizers, etc. For administration, in addition to the active ingredient, the medicament of the present invention can preferably be formulated using at least one pharmaceutical carrier. When the composition is formulated as a liquid solution, it may contain at least one pharmaceutical carrier selected from: saline solution, sterile water, Ringer's solution, buffered saline, injectable albumin solution, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof. Other conventional additives, including antioxidants, buffers, antibacterial agents, etc., may be added if desired. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be further added to prepare injectable formulations (such as aqueous solutions, suspensions, or emulsions), pills, capsules, granules, or tablets.

[0091] As one implementation method, diluents include lactose, sodium chloride, glucose, urea, starch, water, etc.; binders include starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose and hydroxypropyl methylcellulose, etc.; surfactants include polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, hexadecyl alcohol, etc.; lubricants include zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium lauryl sulfate, etc.

[0092] The medicaments of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or through an implanted drug delivery device. The medicaments of this invention may contain any commonly used, non-toxic, pharmaceutically acceptable carriers, excipients, or formulations. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The medicaments of this invention can be delivered to the receptor via any route, provided the target tissue can be reached.

[0093] This invention provides the application of CD132 in screening candidate drugs for the treatment of AML. In some embodiments, the candidate drug can be any substance that modulates CD132, including but not limited to small molecule compounds, protein drugs, and nucleic acid drugs. The protein drugs include, but are not limited to, peptides and antibodies; the nucleic acid drugs include, but are not limited to, interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA.

[0094] The present invention provides a method for screening candidate drugs for the treatment of AML, the method comprising treating a culture system expressing or containing the CD132 gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the CD132 gene or its encoded protein in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CD132 gene or its encoded protein, the substance to be screened is a candidate drug for the treatment of AML.

[0095] In some implementations, the culture system includes (but is not limited to) cell systems, subcellular systems, solution systems, tissue systems, organ systems, or animal systems (such as animal models, preferably non-human mammalian animal models, such as mice, rabbits, sheep, monkeys, etc.).

[0096] This invention provides the application of CD132 in constructing AML models. In some embodiments, AML models are constructed by administering a CD132 promoter. The term "promoter" refers to any substance that can increase the expression of nucleic acid encoding CD132, increase CD132 protein levels, or promote CD132 activity, including CD132 overexpression systems or CD132 agonists.

[0097] In some implementation schemes, the CD132 overexpression system includes obtaining the CD132 gene through chemical synthesis, biological acquisition, or gene library screening, selecting an expression vector including a plasmid, viral vector, or transposon vector, and constructing it through chemical transfection, physical transfection, or viral infection.

[0098] The chemical synthesis method includes direct chemical synthesis of the CD132 gene or synthesis of multiple short fragments followed by splicing to form the CD132 gene. The biological acquisition method includes extracting total RNA, reverse transcribing it to obtain cDNA, and then using PCR technology to amplify the CD132 gene or directly extracting the CD132 gene from genomic DNA. The gene library screening method includes screening from genomic libraries and cDNA libraries.

[0099] The term "vector" refers to a tool that can carry a target gene into a host cell, where it replicates and is expressed. Common vectors include plasmids, viral vectors, and transposon vectors.

[0100] As one implementation, the vector is selected from plasmids, and non-limiting examples of said plasmids include pQE-12, pUC-series, pCDNA series (e.g., pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1), pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, and pCAL. -n-EK, pESP-1, pOP13CAT, E-027pCAGKosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-restricted examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and various mammalian and avian cells is the multipurpose expression vector pCS2+.

[0101] In one embodiment, the vector is selected from viral vectors, such as retroviral vectors. These retroviral vectors include, but are not limited to, adenovirus vectors, lentiviral vectors, Sendai virus vectors, baculovirus vectors, Epstein-Barr virus vectors, multivaccinia virus vectors, vaccinia virus vectors, herpes simplex virus vectors, hybrid vectors, and adeno-associated virus (AAV) vectors. In a specific embodiment of the present invention, the vector is selected from lentiviral vectors.

[0102] This invention provides a method for constructing an AML model, wherein the AML model is a malignant model.

[0103] In this invention, the term "malignant model" refers to an experimental model used to study the mechanisms of malignant transformation of cells and the occurrence and development of tumors. Compared to conventional AML models, the malignant model exhibits the following changes in properties or characteristics: altered cell morphology, enhanced cell proliferation capacity, cell cycle disorder, impaired cell differentiation, altered cell adhesion and migration capacity, metabolic changes, and immune phenotype. In a specific embodiment of this invention, the AML malignant model is characterized by cell cycle disorder and enhanced cell proliferation capacity.

[0104] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0105] Example 1: Expression level of CD132 in AML cells 1. Experimental Methods 1) Database Analysis The DESeq package was used in R (version 4.3.2) to perform transcriptome differential analysis on AML cells and normal CD34+ cells in the GEO database dataset (GSE30029).

[0106] 2) Flow cytometry detection of CD132 and WT1 expression levels Bone marrow blood samples were collected from the subjects and divided into a training set and a validation set. The training set included 13 healthy donors, 16 AML patients with M0, M1, and M2 subtypes, and 21 AML patients with M4 and M5 subtypes. The validation set included 14 healthy donors, 16 AML patients with M0, M1, and M2 subtypes, and 22 AML patients with M4 and M5 subtypes.

[0107] The diagnostic criteria for AML are based on the WHO 2016 classification of hematopoietic and lymphoid tissue tumors: 1) ≥20% of peripheral blood or bone marrow blasts; 2) but when the patient is confirmed to have clonal recurrent cytogenetic abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and t(15;17)(q22;q12).

[0108] Preparation of primary AML patient / healthy donor bone marrow cells: Approximately 3 mL of bone marrow blood was obtained from the posterior superior iliac spine and anticoagulated using EDTA or heparin tubes. Mononuclear cells were extracted from the bone marrow blood using density gradient centrifugation; the mononuclear cells were placed in PBS; labeled with CD132-PE and AML surface antigens CD117-FITC / CD34-APC / CD33-BV421 (antibodies purchased from BD Biosciences or Biolegend); incubated for 15 min; washed twice with PBS; flow cytometry was used to analyze the expression level of CD132 on the surface of CD34+ cells and AML cells, and RT-qPCR was used to analyze the expression level of WT1, followed by ROC curve analysis.

[0109] 3) Use the Pearson correlation coefficient analysis method to analyze the association between WT1 and CD132 of all samples.

[0110] 2. Experimental Results 1) Analysis using the dataset (GSE30029) from the GEO database yielded the following results: Figure 1 As shown in Figure A, the expression level of CD132 in the transcriptome of AML cells was significantly higher than that in normal CD34+ cells, and the difference was statistically significant (P=0.0004).

[0111] 2) Flow cytometry was used to measure the expression level of CD132, and the results are as follows: Figure 1 As shown in B-1E, CD132 expression was significantly higher in AML-M4 / 5 subtypes than in healthy donors and AML patients without M4 / 5 subtypes (M0 / M1 / M2 subtypes).

[0112] The ROC curve analysis results are as follows: Figure 2 As shown, CD132 can effectively distinguish between M4 / 5 subtypes and healthy subjects (training set: AUC 0.8168, sensitivity 100%, specificity 66.67%, cutoff value 42.82; validation set: AUC 0.7727, sensitivity 100%, specificity 59.09%, cutoff value 50.9), as well as distinguish between M4 / 5 subtypes and non-M4 / 5 patients (training set: AUC 0.7783, sensitivity 87.5%, specificity 61.9%, cutoff value 48; validation set: AUC 0.7642, sensitivity 81.25%, specificity 59.09%, cutoff value 49.85).

[0113] 3) Results of correlation analysis, such as Figure 3 As shown, WT1 and CD132 are not related.

[0114] Example 2: Application of CD132 in predicting AML cell prognosis 1. Experimental Methods 1) In the GEO database dataset (GSE14468), the CD132 expression of acute monocytic leukemia (FAB-M4 / 5) subtypes was analyzed according to the LSC17 scoring criteria (Nature. 2016 Dec 15;540(7633):433-437.); 2) Survival analysis of the BeatAML acute myeloid leukemia dataset was performed using logrank in the OHSU database.

[0115] 2. Experimental Results 1) Analysis using the dataset (GSE14468) from the GEO database yielded the following results: Figure 4 As shown in Figure A, the expression level of CD132 in the top 50 samples with the strongest stemness was significantly higher than that in the top 50 samples with the weakest stemness (P=0.0077), suggesting that CD132 is associated with the degree of stemness in the AML M4 / 5 subtype.

[0116] 2) Survival analysis was performed using the acute leukemia dataset from the OHSU database. Landmark survival analysis was conducted with the period from diagnosis to 12 months of follow-up as the endpoint. The results are as follows: Figure 4 As shown in Figure B, the dashed line represents the overall survival rate before and after 12 months of follow-up from diagnosis. After 12 months, there was a significant difference in survival between patients with high CD132 and low CD132. High CD132 expression was significantly associated with the long-term overall survival of AML (FAB-M4 / 5) patients. The overall survival of patients with high CD132 expression was significantly lower than that of AML (FAB-M4 / 5) patients with low CD132 expression. Therefore, CD132 can be used to predict the prognosis of AML (FAB-M4 / 5) patients.

[0117] Example 3: Effects of CD132 on AML cells 1. Experimental Methods 1) Bone marrow cell colony formation assay (1) Sorting CD132 high and CD132 low cells Primary leukemia patient / healthy donor bone marrow cells: Approximately 3 mL of bone marrow blood was obtained from the posterior superior iliac spine and anticoagulated using EDTA or heparin tubes. Mononuclear cells were extracted from the bone marrow blood using density gradient centrifugation; the mononuclear cells were placed in PBS; labeled with CD132-PE and AML surface antigens CD117-FITC / CD34-APC / CD33-BV421 (antibodies purchased from BD Biosciences or BioLegend); incubated for 15 min; washed twice with PBS; cells with CD117 / CD34 / CD33+ and simultaneously CD132 high / low were sorted using flow cytometry and received in 15 mL centrifuge tubes; CFU were then plated.

[0118] (2) Colony formation experiment Melt the stem cell culture medium (CFU assay) at room temperature (each 15 mL centrifuge tube contains 3 mL of stem cell culture medium); resuspend and count the cells, seeding 5000 cells / well; add 1.1 mL of stem cell culture medium to a 35 mm dish, repeating the assay. Cap the dish. Add water or PBS to a third dish; place the three dishes into a larger dish, cap, and incubate for 7-14 days; observe and count the cells under a microscope.

[0119] 2) Functional experiments after silencing CD132 in cell lines (1) Construction of CD132 silencing cell lines Human leukemia cell lines: THP-1, Molm-13, OCI-AML3; Based on the human CD132 sequence information in the GeneBank nucleotide sequence database, an active interfering sequence siRNA was designed, as shown in SEQ ID NO.1 and SEQ ID NO.4. Accordingly, shRNA sequences capable of forming this siRNA sequence were designed using conventional methods in the art. The shRNA sequence includes a top strand and a bottom strand, as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.6.

[0120] siRNA#1: 5'-CAGCTGGACTGAACAATCAGTGGAT-3' (SEQ ID NO. 1); shCD132#1:Top strand:GATCCGCAGCTGGACTGAACAATCAGTGGATCTCGAGATCCACTGATTGTTCAGTCCAGCTGTTTTTTG (SEQ ID NO.2); shCD132#1: Bottom strand:AATTCAAAAAACAGCTGGACTGAACAATCAGTGGATCTCGAGATCCACTGATTGTTCAGTCCAGCTGCG (SEQ ID NO.3); siRNA#2: 5'-CATTGGAGTGAATGGAGCCACCCAA-3' (SEQ ID NO.4); shCD132#2:Top strand:GATCCGCATTGGAGTGAATGGAGCCACCCAACTCGAGTTGGGTGGCTCCATTCACTCCAATGTTTTTTG (SEQ ID NO.5); shCD132#2: Bottom strand:AATTCAAAAAACATTGGAGTGAATGGAGCCACCCAACTCGAGTTGGGTGGCTCCATTCACTCCAATGCG (SEQ ID NO. 6); Control viral vector siRNA sequence: 5'-TTCTCCGAACGTGTCACGTAA-3' (SEQ ID NO.7); Control viral vector shRNA: Top strand: GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTC (SEQ ID NO.8); Control viral vector shRNA: Bottom strand: AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAACG (SEQ ID NO.9); gRNA 5'-CAAAACACTGAACCTCTGGG-3' (SEQ ID NO. 10); shScr was the negative control; CD132 cloning vector: pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO; TrueCut™ Cas9 protein v2 (Thermo Fisher Scientific Invitrogen™).

[0121] Cell knockout: Prepare a medium containing RPMI 1640 with 2% FBS, and resuspend the cells at 5 × 10⁶ cells / day. 5 / mL was placed in 6-well plates. Using the above lentiviral vector, after transfection for 16 h at 30 MOI, the cells were centrifuged at 1000 rpm for 3 min, and then incubated in 10% FBS complete medium for 48 h. After drug screening with puromycin for 48 h, the transfection efficiency of the cells was verified by RT-qPCR and flow cytometry.

[0122] Cell knockout: CD132 gRNA was co-incubated with TrueCut™ Cas9 protein v2 (Thermo Fisher Scientific Invitrogen™) at room temperature for 15 min to construct the ribonucleoprotein complex (RNP). The cell line was resuspended at 1×10⁻⁶ cells / mL using the electroporation buffer from the Celetrix Cell Regeneration Kit (HY-15559). 6 Mix the incubated RNP complex with the resuspended cell line at a concentration of / mL and transfer the mixture to an electroporation cuvette. Set the Celetrix electroporator to cell line mode and place the cuvette for electroporation. After electroporation, transfer the cell line to complete culture medium and culture for 72 hours. Perform T7E1 genome verification followed by functional experiments.

[0123] (2) Experiment on the proliferation capacity of CD132 silent human leukemia cell line Cell plating: Adjust cell concentration to 1×10⁻⁶ 5 / mL, 100 μL / well in a 96-well plate, 3 replicates; lay up multiple concentration gradients to create a standard curve; after culturing for 0-4 days according to experimental requirements, add 10 μL / well of CCK8; 4 hours later, measure the OD value at 450 nm using a microplate reader.

[0124] (3) CD132 silent human leukemia cell line colony formation experiment Melt the CFU (Cellular Fuminated Cell) assay medium at room temperature (each 15 mL centrifuge tube contains 3 mL of CFU medium); resuspend and count the cells, seeding 2000-5000 cells / well; add 1.1 mL of cell culture medium to a 35 mm dish, repeating the assay. Cap the dish. Add water or PBS to a third dish; place the three dishes into a larger 100 mm dish, cap, and incubate for 7-14 days; observe and count the cells under a microscope.

[0125] (4) Cell cycle assay of CD132 silent human leukemia cell line Prepare a culture medium containing RPMI 1640 with 2% FBS and 10 μg / mL Hoechst 33342. Per 1×10 6Cells were resuspended in 1 mL of culture medium containing Hoechst 33342; incubated at 37°C for 60 min; washed once with PBS containing 10 μg / mL Hoechst 33342; resuspended in 100 μL PBS; and flow cytometry was performed in the BV421 channel.

[0126] (5) Apoptosis experiment of CD132 silent human leukemia cell line Aspirate cells into flow cytometry tubes; wash once with PBS. Add 1 mL of PBS to each tube to resuspend cells and centrifuge (1500 rpm, 5 min); wash once with Annexin V binding buffer. Add 1 mL of Annexin V binding buffer to each tube to resuspend cells and centrifuge (1500 rpm, 5 min); add 150 μL of Annexin V binding buffer working solution per tube to resuspend cells into a single-cell suspension; add 2.5 μL of Annexin V-APC and 5 μL of 7-AAD to each tube; incubate in the dark for 15 min; analyze using the flow cytometry.

[0127] 3) Functional experiments after cell line overexpression (1) Construction of overexpression cell lines: Human leukemia cell lines: THP-1, CD132 overexpression vector: pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO. Cell overexpression: Prepare a medium containing RPMI 1640 with 2% FBS, and resuspend the cells at 5×10⁻⁶. 5 / mL was placed in 6-well plates. Using the above lentiviral vector, after transfection for 16 h at 30 MOI, the cells were centrifuged at 1000 rpm for 3 min, and then incubated in 10% FBS complete medium for 48 h. After drug screening with puromycin for 48 h, the transfection efficiency of the cells was verified by RT-qPCR and flow cytometry.

[0128] (2) Experiment on the proliferation capacity of CD132-overexpressing human leukemia cell lines Cell plating: Adjust cell concentration to 1×10⁻⁶ 5 / mL, 100 μL / well in a 96-well plate, 3 replicates; lay up multiple concentration gradients to create a standard curve; after culturing for 0-4 days according to experimental requirements, add 10 μL / well of CCK8; 4 hours later, measure the OD value at 450 nm using a microplate reader.

[0129] (3) Cell cycle assay of CD132-overexpressing human leukemia cell lines Prepare a culture medium containing RPMI 1640 with 2% FBS and 10 μg / mL Hoechst 33342. Per 1×10 6 Cells were resuspended in 1 mL of culture medium containing Hoechst 33342; incubated at 37°C for 60 min; washed once with PBS containing 10 μg / mL Hoechst 33342; resuspended in 100 μL PBS; and flow cytometry was performed in the BV421 channel.

[0130] 4) Functional experiments after blocking CD132 activity Primary AML cells were formulated into a 1×10⁶ culture medium using IMDM complete medium. 6 Cell suspensions were mixed with 21.1 ng / mL of CD132 antibody REGN7257 and 500 IU / mL of IL-2, and placed in six-well plates. The plates were incubated at 37°C for 72 h. Culture medium containing IMDM with 2% FBS and 10 μg / mL Hoechst 33342 was prepared. Cells were cultured per 1×10⁻⁶ cells. 6 Cells were resuspended in 1 mL of Hoechst 33342 medium; incubated at 37°C for 60 min; washed once with PBS containing 10 μg / mL Hoechst 33342; resuspended in 100 μL PBS; and detected by flow cytometry in the BV421 channel.

[0131] CAR-T cells targeting CD132, containing antibodies with the amino acid sequence shown in SEQ ID NO. 11, were used to kill CD132-positive AML tumor cell lines Thrp-1 and U937. The specific steps are as follows: a. Count CD132 CAR-T cells and adjust the cell density to 5*102 5 Cells / mL.

[0132] b. Count the CD132-positive AML tumor cell lines Thrp-1 and U937. After staining the AML tumor cell lines with the membrane staining reagent Did, adjust their cell density to 5*102. 5 Cells / mL.

[0133] c. Add CD132 CAR-T effector cells and target cells to the well plate at a ratio of effector cells:target cells = 1:1.

[0134] d. After co-culturing for 24 or 48 hours, remove the cells and perform flow cytometry analysis.

[0135] CAR-T cells targeting CD132, containing an antibody with the amino acid sequence shown in SEQ ID NO. 11, are used to kill CD132-positive M4 AML primary tumor cells. The specific steps are as follows: a. Count CD132 CAR-T cells and adjust the cell density to 5*102 5 Cells / mL.

[0136] b. Count CD132-positive primary AML tumor cells. After staining the primary AML tumor cells with Cell Tracer BV421, adjust the cell density to 1*102. 5 Cells / mL.

[0137] c. Add CD132 CAR-T effector cells and target cells to the well plate at a ratio of effector cells:target cells = 5:1.

[0138] d. After co-culturing for 48 h, remove the cells into flow cytometry tubes, wash with PBS, add PE anti-CD132 flow cytometry antibody, incubate at room temperature for 15 min, wash again, and then perform flow cytometry analysis.

[0139] SEQ ID NO.11 CD132 scfv EVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWMKQRPGQGLEWIGHIYLGGGATNYAEKFRSKATLTADTSSSTAYMQLSSLTSEDSAVYYCTRSQPYYYGMDSWGQGTSVTVSSGG GGSGGGGSGGGGSDIQMTQTPSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYKSRLHSGVPSRFSGSGSGTEYSLTINNLEQEDFATYFCHQGHTIPFTFGSGTKLEIK.

[0140] SEQ ID NO.12 CD132 CAR-T full-length YPYDVPDYAYPYDVPDYAEVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWMKQRPGQGLEWIGHIYLGGGATNYAEKFRSKATLTADTSSSTAYMQLSSLTSEDSAVYYCTRSQPYYY GMDSWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTPSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYKSRLHSGVPSRFSGSGSGTEYSLTINNLEQEDFATYFCHQGH TIPFTFGSGTKLEIKFETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAAA.

[0141] 2. Experimental Results 1) Results of bone marrow cell colony formation assay: Figure 5 As shown, the colony formation of AML cells in CD132high was significantly higher than that in CD132low (n=5). 2) Effects of CD132 knockdown on cell function in human leukemia cell lines, such as Figure 6 As shown, knocking down CD132 in THP-1 and Molm-13 cells with shRNA significantly reduced the cell proliferation (6A, 6E) and colony formation (6B, 6F) of human leukemia cell lines, and significantly affected the cell cycle (6C, 6G). Eleven days after infection, knocked-down tumor cells exhibited a large number of apoptosis (6D). Knockdown of CD132 in OCI-AML3 cells with gRNA significantly weakened the cell cycle.

[0142] 3) Results of cell cycle and cell proliferation experiments of human leukemia cell lines after CD132 overexpression using lentiviruses are as follows: Figure 7 As shown in A and 7B, CD132 overexpression in human leukemia cell lines significantly enhances cell cycle and cell proliferation capacity.

[0143] 4) Cell cycle assay results of leukemia cell lines after CD132 blockade using the CD132 antibody REGN7257 are as follows: Figure 8 As shown, the CD132 antibody REGN7257 can significantly reduce the cell cycle of primary AML cells, and can be rescued after the simultaneous addition of CD132 ligand IL-2.

[0144] like Figure 9 As shown, compared with the Control T group, CD132 CAR-T cells significantly killed both CD132-positive tumor cells THP-1 and U937.

[0145] like Figure 10 As shown, compared with the Control T group, CD132 CAR-T cells significantly killed CD132-positive AML primary tumor cells.

[0146] Example 4: Effects of CD132 on non-M4 / M5 AML cells In the KASUMI-1 (M2 subtype) and KG1 cell lines, which are not M4 or M5 subtypes, KASUMI-1 was silenced using shRNA and CRISPR / Cas9, while KG1 was silenced using CRISPR / Cas9. However, silencing CD132 did not reduce the cell cycle level of KASUMI-1. Figure 11 To silence KASUMI1 with shRNA, Figure 12 Silencing KASUMI-1 with CRISPR / CAS9, and further enhancement of KG1 at the cell cycle level after silencing CD132 ( Figure 13 Further treatment of the KASUMI1 cell line with CD132 monoclonal antibody at concentration gradients of 0–10 μg / mL also failed to inhibit its proliferation. Figure 14 ).

[0147] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of CD132 as a target in the preparation of drugs for the treatment of AML; Preferably, the drug comprises an inhibitor of CD132; Preferably, the inhibitor comprises any substance that can reduce the expression of nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD132; Preferably, the inhibitor comprises nucleic acid inhibitors, proteolytic enzymes, protein-binding molecules, or combinations thereof; Preferably, the nucleic acid inhibitor includes interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA; Preferably, the nucleic acid inhibitor is selected from interfering RNA; Preferably, the interfering RNA is selected from shRNA, siRNA, and microRNA; Preferably, the nucleic acid inhibitor is shRNA; Preferably, the active sequence of the shRNA is shown in SEQ ID NO.1 and SEQ ID NO.4; Preferably, the nucleic acid inhibitor is selected from gRNA; Preferably, the sequence of the gRNA is shown in SEQ ID NO.10; Preferably, the nucleic acid inhibitor further includes Cas protein; Preferably, the protein-binding molecule includes an antibody against CD132 or a derivative thereof; Preferably, the antibody is REGN7257 and has the amino acid sequence shown in SEQ ID NO.11; Preferably, the AML is selected from the M4 and / or M5 subtypes.

2. A drug for treating AML, characterized in that, The drug includes an inhibitor of CD132; Preferably, the inhibitor comprises any substance that can reduce the expression of nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD132; Preferably, the inhibitor comprises nucleic acid inhibitors, proteolytic enzymes, protein-binding molecules, or combinations thereof; Preferably, the nucleic acid inhibitor is selected from interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA; Preferably, the interfering RNA is selected from shRNA, siRNA, and microRNA; Preferably, the nucleic acid inhibitor is shRNA; Preferably, the active sequence of the shRNA is shown in SEQ ID NO.1 and SEQ ID NO.4; The selected site is where the nucleic acid inhibitor is gRNA; Preferably, the sequence of the gRNA is shown in SEQ ID NO.10; Preferably, the nucleic acid inhibitor further includes Cas protein; Preferably, the protein-binding molecule includes an antibody against CD132 or a derivative thereof; Preferably, the antibody is REGN7257 and has the amino acid sequence shown in SEQ ID NO.11; Preferably, the derived substance comprises CAR-T cells made from an antibody against CD132; Preferably, the drug further includes pharmaceutically acceptable excipients; Preferably, the AML includes M4 and / or M5 subtypes.

3. Application of CD132 in screening candidate drugs for the treatment of AML; Preferably, the method for screening candidate drugs for the treatment of AML is as follows: treating a culture system expressing or containing the CD132 gene or its encoded protein with the substance to be screened; and detecting the expression or activity of the CD132 gene or its encoded protein in the system; wherein, When the substance to be screened inhibits the expression level or activity of the CD132 gene or the protein it encodes, the substance to be screened is a candidate drug for the treatment of AML. Preferably, the AML is selected from the M4 and / or M5 subtypes.

4. A method for screening candidate drugs for the treatment of AML, characterized in that, The method includes treating a culture system expressing or containing the CD132 gene or its encoded protein with a screening substance; and detecting the expression or activity of the CD132 gene or its encoded protein in the system; wherein, when the screening substance inhibits the expression level or activity of the CD132 gene or its encoded protein, the screening substance is a candidate drug for the treatment of AML. Preferably, the method further includes a functional verification step for the candidate drug; Preferably, the AML is selected from the M4 and / or M5 subtypes.

5. A computer-aided method for screening candidate drugs for the treatment of AML, characterized in that, The method includes: Obtain the structure domain of CD132. Based on the spatial structure screening of CD132 domains, substances that regulate CD132 are selected as candidate drugs for the treatment of AML. Preferably, the method further includes a functional verification step for the candidate drug; Preferably, the AML is selected from the M4 and / or M5 subtypes.

6. The use of candidate drugs screened based on the method of claim 4 or 5 in the preparation of medicaments for treating AML.

7. Application of CD132 in building AML models; Preferably, the method for constructing the AML model includes administering a promoter of CD132; Preferably, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase the level of CD132 protein, or promote the activity of CD132; Preferably, the promoter is selected from the CD132 overexpression system; Preferably, the CD132 overexpression system includes selecting an expression vector, including a plasmid, a viral vector, or a transposon vector, and constructing it by chemical transfection, physical transfection, or viral infection. Preferably, the plasmid includes a pCDNA series vector, the viral vector includes a lentiviral vector, an adeno-associated virus (AAV) vector, or an adenovirus vector, and the transposon vector includes a PiggyBac transposon vector or a Sleeping Beauty transposon vector. Preferably, the viral vector is selected from lentiviral vectors; Preferably, the chemical transfection method includes liposome transfection and cationic polymer transfection, and the physical transfection method includes electroporation and gene gun transfection. Preferably, the AML is selected from the M4 and / or M5 subtypes.

8. A method for constructing an AML model, characterized in that, The method includes administering a reagent that regulates CD132; Preferably, the reagent includes a CD132 promoter; Preferably, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase the level of CD132 protein, or promote the activity of CD132; Preferably, the promoter comprises a CD132 overexpression system; Preferably, the AML model is a malignant model; Preferably, the malignant AML model includes one or more of the following: altered cell morphology, enhanced cell proliferation, cell cycle disorder, impaired cell differentiation, altered cell adhesion and migration, metabolic changes, and altered immune phenotype.

9. An AML cell model, characterized in that, The cells overexpressed CD132; Preferably, the AML cell model is constructed using the construction method described in claim 8.

10. The use of the AML cell model of claim 9 in screening drugs for the treatment of AML or evaluating the efficacy of drug treatment for AML.

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