Screening Kit for Populations Susceptible to or with Poor Prognosis of Acute Myeloid Leukemia

Abstract

The present invention relates to the field of biomedical technologies and provides a screening kit for populations susceptible to acute myeloid leukemia or with poor prognosis. The kit includes an antibody for detecting the level of LILRB4 protein, CD3 + CD4 + T cell count, and an antibody for CD3 + CD4 ‑ T cell count. By detecting the level of LILRB4 protein, CD3 + CD4 + T cell count, and CD3 + CD4 ‑ T cell count, the present invention determines whether a population with acute myeloid leukemia is susceptible to or has a poor prognosis due to carrying the G allele at the rs1048801 locus of the LILRB4 gene, and has advantages such as cost savings, simple operation, and short detection cycle.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and particularly relates to a screening kit for susceptible or poor prognosis populations of acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a highly aggressive hematological malignancy. Although there have been some advancements in existing immunotherapies, the efficacy is limited. For example, immune checkpoint inhibitors (ICIs) targeting the PD-1 / PD-L1 and CTLA-4 pathways are significantly less effective in myeloid malignancies than in solid tumors; new targets such as LAG-3, TIGIT, BTLA, VISTA, CD47, and TIM-3 limit the activity of immune cells through non-redundant mechanisms and show potential in preclinical and early clinical studies, but their efficacy is limited due to the heterogeneity of the tumor microenvironment and complex immunosuppressive mechanisms in acute myeloid leukemia.

[0003] Notably, in recent years, the high expression of leukocyte immunoglobulin-like receptor B subfamily member 4 (LILRB4, also known as ILT3 or LIR-5) in acute myeloid leukemia and its immunosuppressive function have made it a potential immunotherapy target. LILRB4 plays a dual role in monocytic acute myeloid leukemia (M4 and M5 subtypes in the FAB classification): on the one hand, it inhibits T cell activity through phosphorylation of the ITIM motif (Y412 / Y422), and on the other hand, it promotes the infiltration of leukemia cells.

[0004] As an emerging immune checkpoint molecule, the functional and clinical significance of the genetic polymorphism of LILRB4 in acute myeloid leukemia has been less studied. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a screening kit for susceptible or poor prognosis populations of acute myeloid leukemia, aiming to solve the problems mentioned in the background art.

[0006] On the one hand, the present invention provides a screening kit for susceptible or poor prognosis populations of acute myeloid leukemia, and the kit includes an antibody for detecting the level of LILRB4 protein, the number of cells and an antibody for the number of cells.

[0007] Furthermore, the antibody for detecting the level of LILRB4 protein includes at least one of anti-human LILRB4-APC or anti-human LILRB4-PE.

[0008] Furthermore, the detection of the number of cells and Antibodies for cells include CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, and CD4-FITC.

[0009] Furthermore, the kit detects the sample of the subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be people with poor prognosis of acute myeloid leukemia who carried the G allele at the rs1048801 site of the LILRB4 gene.

[0010] Furthermore, the kit detects the sample of the subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be susceptible to acute myeloid leukemia carrying the GG genotype at the rs1048801 site of the LILRB4 gene.

[0011] Furthermore, the sample of the subject is a bone marrow sample or a peripheral blood sample.

[0012] In a second aspect, the present invention also provides the use of the rs1048801 site in the preparation of an auxiliary diagnosis or prognosis evaluation reagent for acute myeloid leukemia, wherein the reagent is used to detect the genotype of the rs1048801 site of the LILRB4 gene in a sample.

[0013] In a third aspect, the present invention further provides the use of the rs1048801 site in the preparation of a drug for treating acute myeloid leukemia, wherein the drug is a blocker that specifically blocks the G allele regulatory signal pathway at the rs1048801 site of the LILRB4 gene.

[0014] The present invention has the following beneficial effects:

[0015] (1) By detecting the level of LILRB4 protein, Cell number and The number of cells can be used to determine whether the population is susceptible to acute myeloid leukemia or has a poor prognosis if it carries the GG genotype or G allele at the rs1048801 site of the LILRB4 gene, screen high-risk patients, guide early intervention, and gain more treatment time for patients; and more accurately determine the prognosis of patients, formulate personalized treatment plans for patients, and achieve precision medicine. Compared with the genotype detection of the rs1048801 site of the LILRB4 gene, it has the advantages of cost saving, simple operation, and short detection cycle; and it can detect the level of LILRB4 protein, Cell number and The cell count can be carried out with conventional laboratory equipment, without the need for a professional gene sequencing platform, and the results can be issued quickly.

[0016] (2)Specifically block the regulatory signaling pathway of the G allele at the rs1048801 locus of the LILRB4 gene using a blocker, reduce the clonogenic ability of acute myeloid leukemia cells, and restore cell function and extend the survival period of patients with acute myeloid leukemia. Brief Description of the Drawings

[0017] By referring to the following drawings, the exemplary embodiments of the present invention can be more fully understood:

[0018] Figure 1 It is a result diagram showing that the LILRB4 rs1048801 SNP (single nucleotide polymorphism) in Example 1 of the present invention significantly affects AML susceptibility; wherein:

[0019] Figure 1 A in

[0020] Figure 1 is a schematic diagram of the LILRB4 gene located in the region of chromosome 19q13.4;

[0021] Figure 2 is a correlation diagram between the polymorphism of the LILRB4 gene and acute myeloid leukemia susceptibility in Example 1 of the present invention.

[0022] Figure 3 is the association between the genetic model at the rs1048801 locus and the demographic and clinicopathological parameters of acute myeloid leukemia susceptibility in Example 2; wherein the forest plot shows the association between the genetic model at the rs1048801 locus (based on additive, dominant, and recessive models) and the age (≤60 years and >60 years), gender (male / female), and FAB classification (other subtypes, M4 + M5 subtypes) of patients with acute myeloid leukemia (AML). The Odds Ratios (95%CI) indicate that there is a 95% probability that the true value falls within the current confidence interval, and the P-value represents the probability of observing the sample data or more extreme data on the premise that the null hypothesis (usually the null hypothesis, H0) is true.

[0023] Figure 4 is a result diagram showing the correlation between the G allele at the rs1048801 locus and high expression of LILRB4 and poor prognosis in Example 3; wherein:

[0024] Figure 4In A, the LILRB4 mRNA expression of acute myeloid leukemia patients with different LILRB4 SNP genotypes was detected by RT-qPCR. ns indicates no significance, ** indicates P value < 0.01, and *** indicates P < 0.001;

[0025] Figure 4 In B, it is a Kaplan-Meier analysis graph showing the correlation between the genotype at the rs1048801 locus of the LILRB4 gene and the overall survival of acute myeloid leukemia patients;

[0026] Figure 4 In C, it is a Kaplan-Meier analysis graph showing the relationship between the LILRB4 mRNA level and the overall survival of patients based on the TCGA database (n = 139).

[0027] Figure 5 It is the result graph of Example 4 demonstrating the effect of the G allele at the rs1048801 locus on the growth of acute myeloid leukemia cells by using MTS cell proliferation and soft agar colony formation assays; Among them:

[0028] Figure 5 In A, it is the result graph of detecting the viability of acute myeloid leukemia cells (AA, AG, and GG genotypes at the rs1048801 locus) by the MTS method. * indicates P < 0.05, and ** indicates P < 0.01;

[0029] Figure 5 In B, it is a representative image of soft agar colony formation of acute myeloid leukemia cells (AA, AG, and GG genotypes at the rs1048801 locus) under an inverted microscope. 100x indicates a scale bar of 20 μm, and 200x indicates a scale bar of 10 μm;

[0030] Figure 5 In C, it is the statistics of the colony numbers of acute myeloid leukemia cells (AA, AG, and GG genotypes at the rs1048801 locus) (>50 cells / field of view). ** indicates P < 0.01, and *** indicates P < 0.001.

[0031] Figure 6 It is the result graph of the G allele at the rs1048801 locus of the LILRB4 gene inhibiting the cell proliferation of acute myeloid leukemia patients in Example 5; Among them:

[0032] Figure 6 In A, it is the result graph of detecting the expression intensity of LILRB4 in white blood cells and monocytic leukemia cells of different rs1048801 locus genotypes (GG, AG, AA) in acute myeloid leukemia (M5 subtype) by flow cytometry;

[0033] Figure 6 In which, B is the graph of the cell proportion results of individuals with different rs1048801 locus genotypes (GG, AG, AA) detected by flow cytometry in acute myeloid leukemia (M5 subtype); Graph of cell proportion;

[0034] Figure 6 In which, C is the statistical graph of the cell proportion of acute myeloid leukemia patients with different rs1048801 locus genotypes (GG, AG, AA). * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001; Statistical graph of cell proportion, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001;

[0035] Figure 6 In which, D is the co-culture of primary T cells (effector cells) and irradiated acute myeloid leukemia cells (target cells) (the ratios of effector cells to target cells are 1:1, 2:1, and 4:1 respectively, containing CD3 / CD28 activation magnetic beads and IL-2), and the cell morphology images are observed under an inverted microscope;

[0036] Figure 6 In which, E is the graph of the results of flow cytometry proliferation analysis after co-culture of CFSE-labeled T cells and leukemia cells with different rs1048801 locus genotypes (GG, AG, AA);

[0037] Figure 6 In which, F is the statistical graph of the number and subsets of T cells ( , ) in different genotype groups. ** indicates P<0.01, and *** indicates P<0.001. Specific implementation manners

[0038] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and do not limit the present invention.

[0040] In some embodiments, the present invention provides a screening kit for populations susceptible to or with poor prognosis of acute myeloid leukemia. The kit includes antibodies for detecting the level of LILRB4 protein, the number of cells and antibodies for the number of cells.

[0041] Specifically, the antibodies for detecting the level of LILRB4 protein include at least one of anti-human LILRB4-APC or anti-human LILRB4-PE.

[0042] Specifically, the detection of the number of cells and the antibodies for cells include CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, and CD4-FITC.

[0043] Specifically, the kit detects a sample from a subject. If the level of LILRB4 protein in the sample from the subject increases, the number of cells decreases, and there is no statistically significant difference in the number of cells, it is determined that the subject is a population with poor prognosis of acute myeloid leukemia carrying the G allele at the rs1048801 locus of the LILRB4 gene.

[0044] Specifically, the kit detects a sample from a subject. If the level of LILRB4 protein in the sample from the subject increases, the number of cells decreases, and there is no statistically significant difference in the number of cells, it is determined that the subject is a susceptible population of acute myeloid leukemia carrying the GG genotype at the rs1048801 locus of the LILRB4 gene.

[0045] Specifically, the sample from the subject is a bone marrow sample or a peripheral blood sample.

[0046] In some embodiments, the present invention further provides the use of the rs1048801 locus in the preparation of reagents for the auxiliary diagnosis or prognosis evaluation of acute myeloid leukemia, and the reagents are used to detect the genotype of the rs1048801 locus of the LILRB4 gene in a sample.

[0047] In some embodiments, the present invention further provides the use of the rs1048801 locus in the preparation of drugs for treating acute myeloid leukemia, and the drugs are blockers that specifically block the regulatory signaling pathway of the G allele at the rs1048801 locus of the LILRB4 gene.

[0048] Experimental materials:

[0049] (1) Experimental reagents: Red blood cell lysate and Ficoll-Paque density gradient centrifugation solution were purchased from Sigma-Aldrich; DNA extraction kit and RNAprep Pure Blood kit were purchased from Tiangen Biotech; PrimeScript™ Reverse Transcriptase reagent and TB Green Premix Ex Taq II were purchased from Takara Bio; MTS cell proliferation reagent was purchased from Promega; CD16 / 32 antibody, anti-human LILRB4-PE and anti-CD3-APC were purchased from BioLegend; anti-human CD45-APC-Cy™7, CD33-FITC, CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, CD4-FITC, CD8-PC5, CD45-APC (BD Biosciences), CD13-PerCP-Cy™5.5, anti-CD8-PE and anti-CD4-PE were purchased from BD Biosciences (Becton, Dickinson and Company), CD14-ECD was purchased from Beckman Coulter; anti-human LILRB4-APC was purchased from eBioscience; CFSE (carboxyfluorescein diacetate succinimidyl ester) cell dye and Dynabeads Human T-activator CD3 / CD28 were purchased from Thermo Fisher Scientific; recombinant human IL-2 (50 U / ml) was purchased from Novoprotein; isotype control antibodies (mouse IgG-APC, 1:400; rabbit IgG-PE, 1:100; mouse IgG-PE, 1:100); healthy human peripheral blood (No.: PB009-1-0).

[0050] (2) Experimental primers: The primer sequences for DNA amplification are shown in Table 1; the primer sequences for real-time fluorescence quantitative PCR are shown in Table 2.

[0051] Table 1 Primer sequences for DNA amplification

[0052]

[0053] Table 2 Primer sequences for real-time fluorescence quantitative PCR

[0054]

[0055] Example 1: Collect clinical samples of acute myeloid leukemia to identify the effect of LILRB4 SNP (single nucleotide polymorphism) on the susceptibility of acute myeloid leukemia

[0056] To clarify the potential function of LILRB4 SNP in immune regulation, focus was placed on the Ig domain and ITIM domain of LILRB4 (responsible for ligand binding and inhibitory signal transduction, respectively). 151 newly diagnosed acute myeloid leukemia patients (acute myeloid leukemia group) and 203 healthy individuals (control group) were recruited from the First Affiliated Hospital of Nanchang University. All participants signed written informed consent forms in accordance with the Declaration of Helsinki and were approved by the hospital institutional ethics review board (Ethics review number: CDYFYYLK-01-038). Bone marrow (BM) aspiration fluid and peripheral blood (PB) samples were collected during the routine diagnosis of AML patients. The bone marrow aspiration fluid was treated with red blood cell lysate to remove red blood cells and enrich white blood cells. Peripheral blood samples were separated by Ficoll-Paque density gradient centrifugation to isolate mononuclear cells (MNCs, including lymphocytes and monocytes), genomic DNA was extracted from primary white blood cells using a DNA extraction kit, and amplified by PCR. The PCR products were sequenced by an ABI Prism 3100 genetic analyzer, and the sequences were aligned with the reference genome (GRCh38) using DNASTAR Lasergene v17.3 software.

[0057] The clinical characteristics of the 151 newly diagnosed acute myeloid leukemia patients and 203 healthy individuals recruited are shown in Table 3.

[0058] Table 3 Clinical characteristics of the populations in the acute myeloid leukemia group and the control group

[0059]

[0060] The results of the figure showing that the LILRB4 rs1048801 SNP significantly affects acute myeloid leukemia susceptibility are as Figure 1 shown. The results indicate that: the LILRB4 gene is located in the chromosomal region 19q13.4 (as shown by A in Figure 1 ); and three SNP loci, rs3050015, rs3745871, and rs1048801, were identified by Sanger sequencing and sequence alignment of clinical samples (as shown by B in Figure 1 ). Among them, the rs3050015 locus is a 7bp repeat sequence, the rs3745871 locus T>C is a synonymous mutation, while the rs1048801 locus A>G results in an arginine-to-glutamine substitution at the second tyrosine residue of the ITIM domain, but does not affect its phosphorylation.

[0061] The distribution characteristics of LILRB4 gene polymorphisms in acute myeloid leukemia and control populations (chi-square test) are shown in Table 4. The chi-square test results show that there are significant differences in the genotypes of the rs1048801 locus between the acute myeloid leukemia group and the control group.

[0062] Table 4 Distribution characteristics of LILRB4 gene polymorphisms in the populations of the acute myeloid leukemia group and the control group

[0063]

[0064] In Table 4, the data are expressed as n (%). Chi-square test was used for the statistics of the genotype distribution between the acute myeloid leukemia group and the control group; OR (95%CI) is the probability that the true value falls within the current confidence interval with 95% probability; the P value is the probability of observing the sample data or more extreme data on the premise that the null hypothesis (usually the null hypothesis, H0) is true.

[0065] The correlation between LILRB4 gene polymorphisms and susceptibility to acute myeloid leukemia is as Figure 2 shown. Among them, the data are expressed as n (%). The genotype distribution frequency of the control group population was tested for HWE (Hardy-Weinberg equilibrium). If P>0.01, it indicates that the control group conforms to the Hardy-Weinberg equilibrium; the P a value is the chi-square test of the distribution of LILRB4 single nucleotide polymorphisms between the acute myeloid leukemia group and the control group; the P value is to calculate the association between LILRB4 single nucleotide polymorphisms and susceptibility to acute myeloid leukemia using logistic regression analysis; the P b value was adjusted according to age and gender; AIC (Akaike information criterion) and BIC (Bayesian Information Criterion) were used for the selection of the optimal model; Crude OR(95%CI) is the odds ratio directly calculated by univariate analysis without adjusting for other confounding variables (such as age, gender, etc.); Adjusted OR (95%CI) b is the calculated odds ratio after adjustment for gender and age.

[0066] The results showed that after adjustment for confounding factors such as gender and age, multivariate logistic regression analysis revealed that rs3050015 (adjusted OR = 1.02, 95% CI = 0.539 - 1.930, P = 0.951) and rs3745871 (adjusted OR = 1.125, 95% CI = 0.578 - 2.154, P = 0.745) were not significantly associated with the risk of acute myeloid leukemia; while rs1048801 showed significant associations in multiple genetic models. In the co-dominant model, the GG genotype at the rs1048801 locus was significantly associated with an increased risk of acute myeloid leukemia compared with the AA genotype at the rs1048801 locus (adjusted OR = 4.996, 95% CI = 2.645 - 9.403, P < 0.001); in the dominant model, the risk of carriers of the G allele (AG + GG) at the rs1048801 locus was twice that of rs1048801 AA homozygotes (adjusted OR = 2.140, 95% CI = 1.374 - 3.335, P < 0.001); in the recessive model, the GG genotype at the rs1048801 locus had the highest risk compared with the AA + AG genotypes at the rs1048801 locus (adjusted OR = 4.054, 95% CI = 2.273 - 7.230, P < 0.001).

[0067] The prediction results of SIFT (Sorting Intolerant From Tolerant) are shown in Table 5, and the results indicate that rs1048801 A>G is a harmful mutation.

[0068] Table 5 SIFT prediction results

[0069]

[0070] In summary, the GG genotype at the rs1048801 locus of the LILRB4 gene significantly increases the susceptibility to acute myeloid leukemia.

[0071] Example 2: Analyze the association between rs1048801 and demographic and disease factors of susceptibility to acute myeloid leukemia through genetic model analysis:

[0072] The association results between the rs1048801 genetic model and demographic and clinicopathological parameters of susceptibility to acute myeloid leukemia are as Figure 3As shown in the figure. The results showed the association between the rs1048801 genetic model and the gender, age, and disease classification of patients with acute myeloid leukemia. In the additive model, the GG genotype of rs1048801 significantly increased the risk of acute myeloid leukemia in each subgroup: age > 60 years (OR = 6.706, 95% CI: 2.097 - 21.445), female (OR = 7.667, 95% CI: 2.841 - 17.575), M4 + M5 subtypes (OR = 5.438, 95% CI: 2.653 - 11.147). In the dominant model, the patients carrying the AG + GG genotype had a significantly higher risk than AA in patients with age ≤ 60 years (OR = 2.355, 95% CI: 1.354 - 4.009), female (OR = 2.994, 95% CI: 0.920 - 5.564), M4 + M5 subtypes (OR = 2.285, 95% CI: 1.337 - 3.906). In the recessive model, the GG genotype was significantly higher than the AA + AG genotype in patients with age > 60 years (OR = 6.405, 95% CI: 2.615 - 18.953), female (OR = 4.800, 95% CI: 2.228 - 10.828), M4 + M5 subtypes (OR = 4.417, 95% CI: 2.317 - 8.433).

[0073] In summary, the GG genotype at the rs1048801 locus of the LILRB4 gene significantly increases the susceptibility to acute myeloid leukemia, especially in women and the elderly population with a higher risk, and is more susceptible to M4 and M5 subtype leukemias.

[0074] Example 3: Verification of the correlation between the G allele at the rs1048801 locus and the mRNA expression and poor prognosis of LILRB4 by RT-qPCR (reverse transcription quantitative polymerase chain reaction) method

[0075] The results of the correlation between the G allele at the rs1048801 locus and the high expression of LILRB4 and poor prognosis are as Figure 4 shown.

[0076] The LILRB4 mRNA expression in acute myeloid leukemia patients with different LILRB4 SNP genotypes (rs3050015, rs3745871, rs1048801) was detected by RT-qPCR as shown in A of Figure 4 ; only single-site mutations were analyzed in each group: wild type / deletion type (n = 10, 5 cases in each group), TT / TC / CC (n = 15, 5 cases in each group), AA / AG / GG (n = 15, 5 cases in each group). The results showed that the LILRB4 mRNA expression in individuals carrying the G allele at the rs1048801 locus was significantly increased.

[0077] The results of the survival analysis are shown in B and C of Figure 4 as follows. Among them, Figure 4 B in Figure 4 shows the correlation between the Kaplan-Meier analysis of the LILRB4 rs1048801 genotype and the overall survival of patients with acute myeloid leukemia (n = 151). In the AA group, n = 49; in the AG group, n = 55; in the GG group, n = 47. The P value was calculated by the log-rank test.

[0078] Example 4: Use the MTS cell proliferation and soft agar colony formation assays to demonstrate the effect of the G allele at the rs1048801 locus on the growth of acute myeloid leukemia cells

[0079] (1) MTS cell proliferation assay: Primary acute myeloid leukemia white blood cells (rs1048801 locus AA, AG, and GG genotypes) in the logarithmic growth phase were seeded in 96-well plates at a density of cells / well. After culturing for 24, 48, 72, 96, and 120 hours, 10 μL of MTS reagent was added to each well and incubated at 37 °C for 3 hours. The cell viability was calculated by measuring the absorbance at 490 nm.

[0080] The results of detecting the viability of acute myeloid leukemia cells (rs1048801 locus AA, AG, and GG genotypes) by the MTS method are shown in A of Figure 5 as follows. The results show that the absorbance (OD value) of acute myeloid leukemia cells with the GG and AG genotypes at the rs1048801 locus was significantly higher than that of acute myeloid leukemia cells with the AA genotype at the rs1048801 locus from the 3rd day, and the absorbance of acute myeloid leukemia cells with the GG genotype at the rs1048801 locus was the highest.

[0081] (2) Soft agar colony formation assay: Single cells ( cells) isolated from acute myeloid leukemia bone marrow / peripheral blood samples (rs1048801 locus AA, AG, and GG genotypes) were suspended in 0.5% agarose and plated on a bottom layer containing 0.8% agar (12-well plates). After culturing for 18 days, the cells were fixed with methanol and stained with 0.5% crystal violet. Representative images (5 per well) were collected using a Nikon Eclipse TS100 microscope.

[0082] Representative images of soft agar colony formation of acute myeloid leukemia cells (AA, AG, and GG genotypes at the rs1048801 locus) under an inverted microscope are as shown in Figure 5 B in Figure 5 C in

[0083] The results showed that the average clone diameters of cells with AG and GG genotypes at the rs1048801 locus were 40 ± 3 μm and 23 ± 4 μm, respectively, which were significantly larger than those of cells with the AA genotype at the rs1048801 locus (6 ± 2 μm); the average number of colonies with the GG genotype at the rs1048801 locus reached 817 ± 40 / well, which was significantly higher than those of the AG genotype and the AA genotype at the rs1048801 locus.

[0084] Example 5: Demonstrating the effect of the G allele at the rs1048801 locus on T cells in acute myeloid leukemia using flow cytometry and a mixed cell co-culture system

[0085] (1) Flow cytometry analysis: To reduce non-specific antibody binding, white blood cells were pre-blocked with anti-human CD16 / 32 antibody for 15 minutes, and then the following antibodies were added: anti-human LILRB4-APC, anti-human LILRB4-PE, CD45-APC-Cy™7, CD33-FITC, CD13-PerCP-Cy™5.5, CD14-ECD, CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, CD4-FITC, CD8-PC5, and CD45-APC, as well as isotype control antibodies (mouse IgG-APC, 1:400; rabbit IgG-PE, 1:100; mouse IgG-PE, 1:100). Cells were analyzed by a Calibur flow cytometer, and the data were processed using Flowjo software.

[0086] The LILRB4 expression intensities of white blood cells and monocytic leukemia cells with different rs1048801 locus genotypes (GG, AG, AA) in acute myeloid leukemia (M5 subtype) are as shown in A in Figure 6 ; the cell proportion results in individuals with different rs1048801 locus genotypes (GG, AG, AA) in acute myeloid leukemia (M5 subtype) are as shown in B in Figure 6 ; patients with acute myeloid leukemia with different rs1048801 genotypes (GG, AG, AA) The cell ratio statistics are as shown in Figure 6 C in . The results show that the GG genotype at the rs1048801 locus has the highest expression of LILRB4 in white blood cells and monocytic leukemia cells, and the AA genotype at the rs1048801 locus has the lowest expression, which is related to the decrease in the cell ratio of the GG or AG genotype at the rs1048801 locus.

[0087] (2) Co-culture experiment of primary leukemia cells and T cells: In the contact co-culture system, T cells ( / well) derived from the peripheral blood of healthy donors were labeled with CFSE, and recombinant human IL-2 (50 U / ml) and CD3 / CD28 activation beads ( cells) were added. The leukemia cells irradiated (28 Gy) were mixed with the T cells at a specific effector-to-target ratio (E:T) in a U-bottom 96-well plate. After co-culture, the cell morphology was observed under an inverted microscope. After staining with anti-CD3-APC, anti-CD8-PE, and anti-CD4-PE, the proliferation was detected by flow cytometry (CFSE dilution method). Unstimulated T cells were used as negative controls, and bead-activated cells were used as positive controls.

[0088] The cell morphology images of primary T cells co-cultured with irradiated leukemia cells are shown in Figure 6 D in Figure 6 . The results of flow cytometry proliferation analysis of CFSE-labeled T cells co-cultured with leukemia cells of different genotypes are shown in Figure 6 E in . The statistical results of the number and subset ratio of T cells in different genotype groups by flow cytometry are shown in F in

[0089] . The results show that at the effector-to-target ratio of 1:1 to 4:1, the formation of T cell colonies with the GG or AG genotype at the rs1048801 locus is significantly inhibited. The CFSE dilution experiment confirmed that leukemia cells carrying the G allele at the rs1048801 locus can dose-dependently inhibit the proliferation of T cells, and the inhibition rate of the GG genotype at the rs1048801 locus reaches 2.6-fold. It is worth noting that the GG genotype at the rs1048801 locus mainly leads to a decrease in the number of cells, while there is no significant change in the number of

[0089] cells. In summary, the G allele at the rs1048801 locus of the LILRB4 gene specifically inhibits

[0090] cell proliferation by upregulating the expression of LILRB4 in monocytic leukemia cells. From the results analysis of Examples 1-5, the following conclusions are obtained in the present invention: By detecting the level of LILRB4 protein, The number of cells can be used to determine whether the population is susceptible to acute myeloid leukemia or has a poor prognosis if it carries the GG genotype or G allele at the rs1048801 site of the LILRB4 gene, screen high-risk patients, guide early intervention, and gain more treatment time for patients; and more accurately determine the prognosis of patients, formulate personalized treatment plans for patients, and achieve precision medicine. Compared with the genotype detection of the rs1048801 site of the LILRB4 gene, it has the advantages of cost saving, simple operation, and short detection cycle; and it can detect the level of LILRB4 protein, Cell number and The number of cells can be reduced, and conventional laboratory equipment can be used to conduct the test. No professional gene sequencing platform is required, and the results can be quickly obtained. In addition, the blocker is used to specifically block the G allele regulation signal pathway of the LILRB4 gene rs1048801 site, reduce the cloning ability of acute myeloid leukemia cells, and restore Cell function and prolong the survival of patients with acute myeloid leukemia.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody for detecting the level of LILRB4 protein, the number of cells and the application of the antibody for the number of cells in the preparation of a screening kit for populations susceptible to or with poor prognosis of acute myeloid leukemia, characterized in that: The kit detects a sample from a subject. If the LILRB4 protein level in the subject's sample increases, the number of cells decreases, and there is no statistically significant difference in the number of cells, it is determined that the subject is a population with poor prognosis of acute myeloid leukemia carrying the G allele at the rs1048801 locus of the LILRB4 gene.

2. The application according to claim 1, wherein: The antibodies for detecting the level of LILRB4 protein include at least one of anti-human LILRB4-APC or anti-human LILRB4-PE.

3. The application according to claim 2, characterized in that: The detection cell number and antibodies against cells include , and CD4-FITC.

4. The application according to claim 3, characterized in that: By detecting the sample of the subject, if the LILRB4 protein level in the sample of the subject increases, the number of cells decreases, and there is no statistically significant difference in the number of cells, it is determined that the subject is a susceptible population of acute myeloid leukemia carrying the GG genotype at the rs1048801 locus of the LILRB4 gene.

5. The application according to claim 4, wherein: The sample of the subject to be tested is a bone marrow sample or a peripheral blood sample.

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