Individualized drug screening method for leukemia based on CD45 monoclonal antibody enrichment

Abstract

The invention provides an individualized drug screening method for leukemia based on CD45 monoclonal antibody enrichment. The method comprises the following steps: collecting a bone marrow extract sample or a peripheral blood sample, separating to obtain a mononuclear cell, labeling the mononuclear cell from a patient by adopting a human CD45 antibody, enriching a leukemia cell population through the CD45 monoclonal antibody based on a CD45 / SSC gating method, adding a leukemia primary drug sensitivity detection culture medium, and detecting the cell activity inhibition condition after adding a corresponding drug to act. The leukemia cell population is enriched through the CD45 monoclonal antibody based on CD45 / SSC, the cells are subjected to drug sensitivity detection to screen out leukemia individualized drugs, interference of normal cells in different quantity proportions in a source sample on leukemia drug sensitivity is eliminated, and response of the source leukemia cells to the drugs can be reflected more truly and visually. The rapid and accurate leukemia individualized drug screening method has important significance for improving the treatment outcome of acute myelogenous leukemia.

Description

Technical Field

[0001] The present invention relates to the field of in vitro drug sensitivity detection of tumors, and in particular to a method for screening individualized drugs for leukemia based on CD45 monoclonal antibody enrichment, which is a novel method for rapidly and accurately screening individualized drugs for treating leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a heterogeneous disease with a large diversity of genetic abnormalities, accounting for about 15% to 20% of acute leukemia in children and adolescents and 85% of acute leukemia in adults, causing more than 10 million deaths in my country each year. The pathogenesis of acute myeloid leukemia is complex, and the existing WHO classification is far from meeting the needs of clinical precision diagnosis and treatment. The lack of precise stratification may lead to insufficient or excessive treatment. More importantly, nearly 80% of patients will relapse / refractory, and patients with relapse and refractory disease have no drugs available clinically, and ultimately have to face the outcome of death.

[0003] For personalized drug screening of clinical patients with acute myeloid leukemia, most of the existing methods are to test the drug sensitivity of all mononuclear cells in the patient's bone marrow or blood (Blood Adv.2020; 4(12):2768-2778, Cell Death Discov.2023; 9(1):435, Cell Death Discov.2023; 9(1):435), and will not test after sorting out the leukemia cells. Therefore, the accuracy of testing for samples with a low proportion of leukemia cells is not high. However, in clinical practice, the proportion of leukemia cells in patients with partial remission and relapse after treatment is relatively low. Therefore, it is urgent to propose accurate drug sensitivity testing methods for patient samples with a low proportion of leukemia cells, which is of great significance for screening personalized drugs for such patients and thus eliminating a small number of leukemia cells in the body.

[0004] At present, there is a lack of universal and effective means for enriching leukemia cells in patient-derived samples. Only a few literatures mention enriching leukemia samples with a low proportion and then testing the in vitro drug sensitivity of the samples. For example, Pemovska T. et al. enriched leukemia cells in a 20% acute myeloid leukemia sample by CD34 magnetic bead sorting method, and then performed drug sensitivity testing (Cancer Discovery. 2013; 3(12): 1416-1429); Lee, SHR et al. used CD19 magnetic bead sorting to enrich leukemia cells in samples with a low proportion in the process of detecting in vitro drug response of patient-derived acute lymphoblastic leukemia (Nature Medicine. 2023; 29(1): 170-179). In addition, Simoes C. et al. used multi-antibody combination multicolor flow cytometry sorting technology to enrich acute myeloid leukemia cells in different patient samples (Blood Adv. 2023; 7(1): 167-173). However, the above methods still have defects. For single-index magnetic bead positive sorting technology such as CD34, due to the high heterogeneity of acute myeloid leukemia, not all patients' leukemia cells are CD34 positive, and even the CD34 expression within the leukemia cell population of the same patient is very different; in addition, the method of sorting leukemia cells from blood based on multicolor flow cytometry is costly and it is difficult to obtain enough tumor cells from a large number of blood samples. Therefore, it is very necessary to find a universal and effective single antibody labeling method to enrich clinical patients' leukemia cells for personalized drug screening. Summary of the invention

[0005] The purpose of the present invention is to provide a personalized drug screening method for leukemia based on CD45 monoclonal antibody enrichment, which is achieved by the following steps: Bone marrow extracts or peripheral blood samples of leukemia patients are collected to isolate mononuclear cells, which are labeled with human CD45 antibody and then enriched by flow cytometry based on CD45 / SSC gating. Primary leukemia culture medium is added and the inhibition of cell activity is detected after the corresponding drugs are applied.

[0006] Furthermore, the novel detection method specifically comprises the following steps: Step 1, collect bone marrow extracts or peripheral blood samples from AML patients, including but not limited to patients at the initial stage, incomplete remission after treatment, and relapse after treatment; Step 2, using human lymphocyte separation fluid to separate mononuclear cells by density gradient centrifugation; Step 3, using human CD45 single antibody to label the separated cells, and using CD45 / SSC gating method combined with flow cytometry to enrich the immature cell population containing leukemia cells; Step 4, inoculating the enriched cells into a 384-well plate containing leukemia primary culture medium; Step 5, setting up a medication group, a control group and a blank group, and adding the test drug to the corresponding groups; Step 6: After 72 hours of drug action, the cell activity was detected using the adenosine triphosphate bioluminescence method.

[0007] Furthermore, in the above step 3, the antibody used is an anti-human CD45 antibody; flow cytometry is used for analysis, and based on the CD45 / SSC gating method, a population of immature cells with an abnormal proportion compared to a normal donor sample is identified, and the population is a leukemia cell-enriched population.

[0008] Further, in the above step 4, the basal culture medium of the leukemia primary culture medium is IMDM, which comprises, according to the final concentration, 20% (volume concentration) fetal bovine serum, 10U / mL penicillin, 10U / mL streptomycin, 1% (mass concentration) bovine serum albumin, 1mM β-mercaptoethanol, and 2mM L-glutamine, and the above components are dissolved in the basal culture medium;

[0009] Furthermore, in the above step 6, cell lysate and luciferase reaction reagent were added to react, and the luminescent signal was read by a microplate reader to indicate the cell activity of each drug administration group, and the cell activity inhibition rate was calculated, and the cell activity inhibition rate was (control group-drug administration group) / (control group-blank group)*100%.

[0010] This project used CD45 single antibody labeling combined with flow cytometry CD45 / SSC gating to find that compared with blood samples from normal donors, the CD45 / SSC population distribution and proportion of blood samples from leukemia patients were abnormal. Normal hematopoiesis in normal samples is distributed in proportion, including lymphocyte populations with strong CD45 signals and weak SSC signals (about 17%), granulocyte populations with medium CD45 signals and strong SSC signals (about 48%), monocyte populations with strong CD45 signals and medium SSC signals (about 6%), nucleated red blood cells with negative CD45 and weak SSC signals (about 6%), and normal immature B cells with low CD45 signals and low SSC signals (about 5%), while the CD45 / SSC population distribution and proportion of blood samples from leukemia patients were significantly different from those of normal samples. Furthermore, based on the CD45 / SSC gating method, there are certain differences in the distribution of abnormal cell populations in acute myeloid leukemia samples of different FAB classifications: the leukemia cell populations of M0 / M1 / M2 classifications have medium CD45 signals and weak SSC signals; the leukemia cell populations of M3 classifications have the characteristics of medium CD45 signals and strong SSC signals; the CD45 signals of M4 / M5 classifications are strong and the SSC signals are weak.

[0011] Furthermore, the research results of the present invention show that the proportion of leukemia cells contained in blood samples from different leukemia patients is different. The proportion of leukemia cells in some initial samples, samples that are not completely relieved after treatment, and samples that relapse after treatment is relatively low, as low as 10%.

[0012] Furthermore, the present invention conducted drug sensitivity tests on unsorted mixed cell samples and CD45 monoclonal antibody-enriched cell samples in parallel, and found that there were significant differences in the activity inhibition rates after the action of multiple drugs, indicating that normal hematopoietic cells in the unsorted cell samples have a great interference with the test results of drug sensitivity.

[0013] The present invention has the following beneficial effects:

[0014] The present invention is the first to enrich leukemia cells in patient-derived bone marrow or peripheral blood samples based on CD45 single antibody labeling, and perform drug sensitivity testing on these cells to screen out personalized drugs for clinical leukemia patients. Eliminating the interference of normal hematopoietic cells of different numbers and proportions in patient-derived samples on the drug sensitivity of leukemia cells can accurately reflect the true sensitivity of the patient's leukemia cells to drugs. The present invention is especially useful for samples with a low proportion of leukemia cells, including but not limited to samples from patients at some initial onset, incomplete remission after treatment, and relapse after treatment. It is beneficial to truly reflect the response of leukemia cells in patients to anti-tumor drugs, and then screen out personalized drugs for clinical leukemia patients, which can provide the possibility of further improving the clinical efficacy of leukemia patients and improving their prognosis and survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the flow cytometry results of the cell samples of a normal healthy donor bone marrow extract sample and a bone marrow extract sample of an acute myeloid leukemia patient before and after enrichment based on CD45 monoclonal antibody in Example 1 of the present invention.

[0016] Figure 2 This is the flow cytometry result of bone marrow extract samples of acute myeloid leukemia patients with different FAB typings based on CD45 / SSC gating method in Example 2 of the present invention. There are certain differences in the distribution of acute myeloid leukemia cell populations and CD45 / SSC signals of different FAB typings.

[0017] Figure 3 It is the proportion of leukemia cells in multiple bone marrow extract samples of acute myeloid leukemia patients detected based on the CD45 / SSC gating method in Example 3 of the present invention.

[0018] Figure 4 The results of drug sensitivity test of a leukemia cell sample enriched with CD45 monoclonal antibody and a non-leukemia cell sample remaining after enrichment, which were derived from an acute myeloid leukemia patient in Example 4 of the present invention, were compared based on the CD45 / SSC gating method.

[0019] Figure 5 Comparison of drug sensitivity test results of an unsorted mixed cell sample and a CD45 monoclonal antibody-enriched cell sample from an acute myeloid leukemia patient in Example 5 of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0021] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0022] Embodiment 1: The present invention provides a bone marrow extract sample from a normal healthy donor and a bone marrow extract sample from an acute myeloid leukemia patient based on flow cytometry results of cell samples before and after CD45 monoclonal antibody enrichment. The results show that compared with normal samples, the proportion of cell populations in samples from acute myeloid leukemia patients is abnormal, and the leukemia cell population has the characteristics of medium CD45 signal and weak SSC signal, accounting for about 37%; after CD45 monoclonal antibody enrichment by CD45 / SSC gating method, the proportion of leukemia cells increased significantly to about 87%.

[0023] Embodiment 2: The present invention provides flow cytometry results of bone marrow extract samples of acute myeloid leukemia patients with different FAB types based on CD45 / SSC gating method. The results show that there are certain differences in the distribution of acute myeloid leukemia cell populations and CD45 / SSC signals of different FAB types: the leukemia cell populations of M0 / M1 / M2 types have medium CD45 signals and weak SSC signals; the leukemia cell populations of M3 types have the characteristics of medium CD45 signals and strong SSC signals; the CD45 signals of M4 / M5 types are strong and the SSC signals are weak.

[0024] Embodiment 3: The present invention provides the proportion of leukemia cells in multiple bone marrow extract samples of acute myeloid leukemia patients detected based on CD45 / SSC gating method, including initial samples, samples with incomplete remission after treatment, and samples with relapse after treatment. The results show that the proportion of leukemia cells varies greatly between different samples, among which the proportion of tumor cells in some initial samples, samples with incomplete remission after treatment, and samples with relapse after treatment is low, as low as 10%.

[0025] Embodiment 4: The present invention provides drug sensitivity test results of a leukemia cell sample and a non-leukemia cell sample remaining after enrichment based on CD45 / SSC gating method from an acute myeloid leukemia patient. The proportion of leukemia cells in the bone marrow extract of the patient was 25.2%, and we enriched it with CD45 single antibody. The results showed that compared with the remaining non-leukemia cell samples from the patient, brigatinib, temsirolimus, venetoclax, vorinostat, mitoxantrone, etoposide, fludarabine, idarubicin, cytarabine, actinomycin and other drugs had a large difference in the activity inhibition rate of leukemia cell samples enriched with CD45 single antibody, indicating that the responsiveness of non-leukemia cells to drugs is significantly different from that of leukemia cells, which will interfere with the test results of drug sensitivity.

[0026] Embodiment 5: The present invention provides drug sensitivity test results of an unsorted mixed cell sample from an acute myeloid leukemia patient and a cell sample enriched with CD45 monoclonal antibody based on the CD45 / SSC gating method. The proportion of leukemia cells in the patient's bone marrow extract was 71.1%. The drug sensitivity test results showed that compared with the unsorted mixed cell sample, the cell sample enriched with CD45 monoclonal antibody had a large difference in sensitivity to multiple drugs such as brigatinib, temsirolimus, venetoclax, vorinostat, mitoxantrone, etoposide, fludarabine, idarubicin, cytarabine, and actinomycin.

Claims

1. A method for screening personalized drugs for leukemia based on CD45 monoclonal antibody enrichment, characterized in that: Mononuclear cells are isolated by collecting bone marrow extracts or peripheral blood samples from leukemia patients, labeled with human CD45 antibody, and then enriched for leukemia cells by flow cytometry based on CD45 / SSC gating method; Add leukemia primary culture medium; detect cell activity inhibition after corresponding drug action, so as to screen out personalized drugs for leukemia patients.

2. The screening method according to claim 1, characterized in that This is accomplished by following these steps: (1) Collect bone marrow extracts or peripheral blood samples from AML patients; (2) Using human lymphocyte separation fluid, mononuclear cells were isolated by density gradient centrifugation; (3) Using human CD45 single antibody to label the isolated cells, the immature cell population containing leukemia cells was enriched based on CD45 / SSC gating method combined with flow cytometry sorting; (4) The enriched cells were inoculated into a 384-well plate containing leukemia primary culture medium; (5) Set up a medication group, a control group, and a blank group, and add the test drug to the corresponding groups; (6) After 72 hours of drug action, the cell activity was detected using the adenosine triphosphate bioluminescence method.

3. The screening method according to claim 1 or 2, characterized in that The extracts or samples collected in step (1) include those at the initial stage, incomplete remission after treatment, or relapse stage after treatment.

4. The screening method according to claim 1 or 2, characterized in that Step (3) After labeling with human CD45 monoclonal antibody, the cells are sorted by setting a gate based on CD45 / SSC and combining it with flow cytometry to enrich the leukemia cell population with abnormal CD45 / SSC signals.

5. The screening method according to claim 1 or 2, characterized in that The basal culture medium of the leukemia primary culture medium in step (4) is IMDM, which comprises, according to the final concentration, 20% fetal bovine serum, 10U / mL penicillin, 10U / mL streptomycin, 1% bovine serum albumin, 1mM β-mercaptoethanol, and 2mM L-glutamine, and the above components are dissolved in the basal culture medium.

6. The screening method according to claim 1 or 2, characterized in that In step (6), cell lysis solution and luciferase reaction reagent are added to react, and the luminescent signal is read by a microplate reader to indicate the cell activity of each drug administration group, and the cell activity inhibition rate is calculated. The cell activity inhibition rate is (control group-drug administration group) / (control group-blank group)*100%.

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