CN116087518A - MRD detection product - Google Patents
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
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- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70589—CD45
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
Abstract
The invention relates to the field of medicines, in particular to an MRD detection product, which comprises an MRD detection reagent, wherein the MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody, an anti-CD 138 antibody, an anti-CD 81 antibody, an anti-CD 117 antibody, an anti-CD 20 antibody, an anti-CD 200 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody or an anti-Lambda light chain antibody. The invention adopts a 2-tube 8-color scheme, can effectively set the gate ring to form CD 38-plasma cells and CD38+ plasma cells, covers the most complete plasma cell group, especially the indexes of CD229 and CD269, which are rarely adopted in the detection of the residual of the multiple myeloma at present, and can greatly reduce the false positive rate and the missed diagnosis probability.
Description
Technical Field
The invention relates to the field of medicines, in particular to an MRD detection product.
Background
The incidence of Multiple Myeloma (MM) is obviously increased, and the MM has become one of important diseases affecting human health, and is a blood tumor with the second incidence rate accounting for 10% of malignant tumors of the blood system. In the past decade, MM treatment regimens have been optimized, the efficacy has been significantly increased, and the rate of complete remission and survival have been increased, and current therapies, in addition to traditional chemotherapies, targeted drugs and cellular immunotherapy have become a new direction of development. The chimeric antigen receptor T cells (Chimeric Antigen Receptions Tcells, CAR-T) realize specific recognition of tumor-related antigens by genetically modifying the T cells, so that the effector T cells fully play an anti-tumor role, are important points of the current cellular immunotherapy, are science of treating the forefront of malignant tumors in the current medical field, have remarkable effects on the treatment of blood system diseases such as multiple myeloma and the like, remarkably improve the remission rate of refractory recurrence diseases which are ineffective in conventional treatment, and improve the prognosis of patients. However, for a number of reasons, this approach achieves a rate of long term remission of less than 50%, post-treatment attention monitoring, early detection of minimal residual disease (Minimal Residue Disease, MRD), early bridging transplantation or secondary CAR-T treatment is of great importance. Flow Cytometry (FCM) is a detection means capable of realizing quantitative analysis on single cells, has the advantages of rapidness, high precision, multiple parameters and the like, and is one of the most advanced cell quantitative analysis methods at present. FCM is widely applied in clinical and scientific research fields, and especially has wide and deep application in clinical in-vitro diagnosis, and is a gold standard for clinical diagnosis of malignant diseases such as leukemia, lymphoma and the like. Bone marrow MRD detection of MM patients by FCM is an important prognostic variable, and specific antibody compositions are used in combination with a multi-color flow cytometer to effectively evaluate prognosis, detect recurrence, and guide selection of treatment schemes, thus being an important clinical evaluation index of MM.
However, the correct selection of the detection antibody is the key to achieving the clinical evaluation, for example, the most common analysis scheme is that the plasma cell marker CD38 is gated by SSC or CD45 (CD 38/SSC or CD38/CD138 double parameters), but in patients treated with CD38 monoclonal antibody, CD138 monoclonal antibody or CD38-CAR-T, CD138-CAR-T cell infusion, gating by CD38 or CD138 is not applicable any more, because the plasma cells with normal or abnormal parts after treatment undergo clonal evolution, the surface CD38 antigen and CD138 antigen have been expressed lost or weakened, i.e. negative expression of the CD38 and CD138 antigen occurs, so no new effective scheme for gating the target plasma cells exists clinically. To solve this problem, researchers have made research studies including tembhre et al, which uses CD38 in combination with CD45/CD138/SSC to detect MRD after mab and CAR-T cell infusion therapy, but they still suffer from the following drawbacks: 1. the expression rate of CD138 in MM is 99.5%, in EDTA anticoagulation sample, thus the loop gate efficiency of plasma cell is affected, the expression rate of 2, CD38 in MM is >80%, on mononuclear cells, the CD38 expression is weakened in part of MM, all the loop gate efficiency of plasma cell is affected, and CD38 antigen is strongly expressed in precursor stage of B cell in the maturation process of normal B cell, and CD38 is strongly expressed in MM, thus the sensitivity of CD38 gating plasma cell is affected. 3. Patients not suitable for infusion therapy of CD38-CAR-T cells or CD38-CD138-CAR-T dual target cells and patients after CD38 mab and CD138 mab treatment.
More importantly, when a sample for residual monitoring after treatment is detected, a detection application form lacks detailed treatment history, such as traditional radiotherapy and chemotherapy or biological treatment, and biological treatment is specific to the scheme, so that due to the lack of the key information, a detector cannot adjust or optimize the detection scheme according to the specific treatment scheme at the first time, and the detection result is often false negative. In order to reduce the occurrence of false negative and improve the detection accuracy, as a inspector, a better detection scheme needs to be searched for to make up for the defects of the existing detection means and meet the clinical requirements. Although the expression of antigens such as CD56 on plasma cells is reported in the literature and can be used for detecting plasma cells, the conventional detection scheme generally does not bring a plurality of indexes into the residual detection scheme after the treatment of patients with multiple myeloma, different detection schemes of detection mechanisms are different, no unified detection scheme exists at present, the common detection scheme uses a combination of 4 to 8 antibodies for most, only plasma cell membrane antigens are detected, only plasma cell plasma light chains are detected, and the detection schemes cannot solve the problem of false negative.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide an MRD detection product for solving the problems of the prior art.
To achieve the above and other related objects, the present invention provides the use of an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody and an anti-CD 138 antibody for the preparation of a multiple myeloma Minimal Residual Disease (MRD) detection product.
The invention also provides an MRD detection product, wherein the MRD detection product comprises an MRD detection reagent, and the MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody and an anti-CD 138 antibody.
The invention also provides a screening method of the MRD therapeutic drug, which comprises the step of detecting an in-vitro sample by using the MRD detection product, wherein the in-vitro sample is from a subject after the drug candidate is acted.
As described above, the MRD detection product of the invention has the following beneficial effects: the invention adopts a 2-tube 8-color scheme, can effectively set the gate ring to form the CD 38-plasma cells and CD38+ plasma cells, covers plasma cell membrane antigens and cytoplasmic light chains, namely covers the most complete plasma cell groups, especially the indexes of CD229 and CD269, which are rarely adopted in the detection of the residual of the multiple myeloma at present, and can greatly reduce the false positive rate and missed diagnosis probability.
Drawings
FIG. 1 is a graph showing the results of normal plasma cell analysis;
the strategy of setting the door for the pipe A is as follows: p1: a debonding cell gate P1; p2: setting living cell gate to obtain single living cell. Setting each haemocyte gate using CD45/SSC antibodies in the P2 gate; the P2 internal multi-marker combination gates to observe plasma cells, lymphocytes, monocytes and granulocytes, and whether obvious tumor cells or abnormal cells exist; plasma cell gates were gated using CD45/CD38, CD38/CD138, SSC/CD38 antibodies (P8, P9, P11);
the B pipe is provided with the door strategy as follows: p1: a debonding cell gate P1; p2: setting a living cell gate to obtain a single living cell; setting each haemocyte gate using CD45/SSC antibodies in the P2 gate; the P2 internal multi-marker combination gates to observe plasma cells, lymphocytes, monocytes and granulocytes, and whether obvious tumor cells or abnormal cells exist; plasma cell gates were gated with CD45/CD38, CD38/CD229, SSC/CD38 antibodies (P8, P9, P10), B cell gates were gated with CD45/CD19 antibodies (CD19+Bclels) as internal controls, and cytoplasmic antibody staining was determined.
In the figure, the pipe A and the pipe B can be distinguished through the gating strategy.
FIG. 2 is a graph showing the results of MRD assays following conventional chemotherapy for patients with multiple myeloma.
FIG. 3 shows a graph of MRD detection results after treatment with CD269 mab from a patient with multiple myeloma.
FIG. 4 shows a graph of MRD detection results after treatment with CD38 mab from a patient with multiple myeloma.
FIG. 5 is a graph showing the detection results of MM-MRD samples detected by the conventional method.
FIG. 6 shows a graph of the detection results of MM-MRD samples detected by the method of the present invention.
Detailed Description
The present invention provides the use of anti-CD 38 antibodies, anti-CD 45 antibodies, anti-CD 229 antibodies, anti-CD 269 antibodies and anti-CD 138 antibodies in the preparation of a multiple myeloma Minimal Residual Disease (MRD) detection product.
CD38 is expressed in normal plasma cells at a significantly higher intensity than other hematopoietic cells, and in multiple myeloma CD38 is expressed strongly or partially weakly.
CD45 is generally expressed positively, with small numbers of CD45-/dim plasma cells present in normal bone marrow, and neoplastic plasma cells present CD45-/dim with small numbers of CD45+ subsets.
CD229 is expressed on normal plasma cells and T cells, is weakly expressed on B cell mononuclear cells, has obviously higher average fluorescence intensity expressed on abnormal plasma cells than normal plasma cells, can be used as a reliable index for plasma cell gating, and can be used for residual evaluation after CD38 and CD138 biotherapy.
CD269 is B Cell Maturation Antigen (BCMA), only expressed on the surface of germinal center B cells, malignant and normal plasma cells, is expressed in high expression and myeloma cell surface with relative specificity, has an expression rate of 70% -80% in patients with osteoma, and is a target point of multiple myeloma immunotherapy.
CD138 is expressed only in plasma cells and myeloma cells, a more specific plasma cell marker than CD38, and is expressed mostly in patients with multiple myeloma, as is CD138 in epithelial cells, mesenchymal cells and tumor cells in the non-hematopoietic system.
In certain embodiments of the present invention, any one or more of the following are also included in the MRD test product: anti-CD 81 antibodies, anti-CD 117 antibodies, anti-CD 20 antibodies, anti-CD 200 antibodies, anti-CD 56 antibodies, anti-CD 19 antibodies, anti-CD 27 antibodies, anti-Kappa light chain antibodies or anti-Lambda light chain antibodies.
CD117, CD20 and CD200 normal plasma cells are not expressed, but are partially expressed in neoplastic plasma cells.
CD56 is normally not expressed in normal plasma cells, often expressed in neoplastic plasma cells.
CD19 is expressed in most plasma cells, with lower intensity than B cells, and a partial subcellular population is expressed CD19 negative, with neoplastic plasma cells generally not expressing CD19.
CD27 is expressed in normal plasma cells, and CD27 is not expressed in patients with multiple myeloma, and is an important index for escaping from the immune system of the organism.
Kappa and Lambda are not expressed in normal and abnormal plasma cells basically, intracellular Kappa and Lambda antigens need to be detected when the clonality of the plasma cells is identified, malignant plasma cells are mostly monoclonal, and the normal plasma cells are expressed in a polyclonal manner. If the expression pattern is different from the normal expression pattern, the MRD is suspected to be malignant, and the MRD is diagnosed positive after the influence of other factors is eliminated.
The MRD detection product is an MRD diagnosis product. The MRD detection product is a product detected by using flow cytometry.
The invention also provides an MRD detection product, wherein the MRD detection product comprises an MRD detection reagent, and the MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody and an anti-CD 138 antibody.
In one embodiment, the MRD detection reagent further comprises any one or more of an anti-CD 81 antibody, an anti-CD 117 antibody, an anti-CD 20 antibody, an anti-CD 200 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody, or an anti-Lambda light chain antibody.
In one embodiment, the MRD detection reagent comprises a first MRD detection reagent comprising an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, and a second MRD detection reagent comprising an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 138 antibody.
The first MRD detection reagent further comprises any one or more of an anti-CD 269 antibody, an anti-CD 81 antibody, an anti-CD 117 antibody, an anti-CD 20 antibody, an anti-CD 200 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody or an anti-Lambda light chain antibody, and the second MRD detection reagent comprises the rest of the anti-CD 269 antibody, anti-CD 81 antibody, anti-CD 117 antibody, anti-CD 20 antibody, anti-CD 200 antibody, anti-CD 56 antibody, anti-CD 19 antibody, anti-CD 27 antibody, anti-Kappa light chain antibody or anti-Lambda light chain antibody except the antibodies contained in the first MRD detection reagent.
In one embodiment, the first MRD detection reagent further comprises five antibodies of anti-CD 269, anti-CD 81, anti-CD 117, anti-CD 20, anti-CD 200, anti-CD 56, anti-CD 19, anti-CD 27, anti-Kappa light chain or anti-Lambda light chain antibodies, and the second MRD detection reagent further comprises five antibodies of anti-CD 269, anti-CD 81, anti-CD 117, anti-CD 20, anti-CD 200, anti-CD 56, anti-CD 19, anti-CD 27, anti-Kappa light chain or anti-Lambda light chain antibodies other than the five antibodies contained in the first MRD detection reagent.
In one embodiment, the first MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody, an anti-CD 81 antibody, an anti-CD 117 antibody, an anti-CD 20 antibody, and an anti-CD 200 antibody.
In one embodiment, the second MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 138 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody, and an anti-Lambda light chain antibody.
In one embodiment, the MRD test product further comprises any one or more of the following: erythrocyte lysate, buffer or membrane breaker. The buffer is, for example, a buffer commonly used in flow cytometry such as PBS and physiological saline.
The red blood cell lysate or the membrane breaker is commercially available.
The invention also provides a screening method of the MRD therapeutic drug, which comprises the step of detecting an in-vitro sample by using the MRD detection product, wherein the in-vitro sample is from a subject after the drug candidate is acted.
Specifically, the method for screening the MRD therapeutic agent is to detect a sample derived from a subject after the action of the MRD therapeutic agent, so as to observe the effectiveness of the MRD therapeutic agent.
The screening method is to carry out scheme design and result judgment according to the following modes: markers of normal non-expression or loss of normal expression are obtained by monitoring the phenotype of abnormal plasma cells, i.e., tumor cells, abnormally.
The isolated sample is a bone marrow or peripheral blood sample of a patient with multiple myeloma after treatment. The treatment method is selected from conventional chemotherapy, CD269 mAb treatment, CD38 mAb treatment, CD138 Car-T treatment or CD38 Car-T treatment. The residual normal plasma cells in the sample to be detected are small in quantity, and the residual normal plasma cells in the detection process can be used as the internal quality control of the detection.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
EXAMPLE 1 flow cytometry detection of multiple myeloma residual disease cells
Experimental principle:
incubating the antibody marked with fluorescein with single cell suspension at room temperature to combine the fluorescent antibody with corresponding antigen on the surface of the cell; the single cell suspension forms single cell liquid flow under the wrapping of sheath liquid and flows through the detection window; the laser generated by the instrument excites the fluorescein marked on the antibody so as to emit light with a specific wavelength; the instrument detects the light with different wavelengths respectively and converts the light into an electric signal; the electrical signal may be analyzed after processing by a computer system.
Experimental instrument, reagent:
BD CantoII flow cytometer, CD45 percp-CY5.5, CD117-BV510, CD81-FITC, CD27-BV510, CD200-APC, CD269-PE, CD38-PE-CY7, CD138-BV421, CD56-PE, CD38-FITC, CD229-BV421, CD20-APC-CY7, reagent manufacturers 1 to 7 are Biolegend, accession numbers 304028, 313219, 349504, 302835, 329208, 357504, 356608, respectively; antibodies 8 to 13 are BD company products, under the numbers 562935, 8308891, 9217298, 744901, 560734, respectively; CD19-PE-CY7 is a Beckmann product, product number: IM3628; kappa-APC was Dako product, cat: c0222; cLambda-APC-CY7 is a Cynogons product, cat#: CYT-LAC750; the erythrocyte lysate and the membrane breaker are all products of BD company, and the product numbers are 70-LSB0 and 641776 respectively. And (3) specimen:
1) Peripheral blood of healthy human (containing normal plasma cells);
2) Peripheral blood specimens following traditional chemotherapy of patients with multiple myeloma;
3) Peripheral blood specimens of patients with multiple myeloma after CD269 mab treatment;
4) Peripheral blood specimens of patients with multiple myeloma after CD38 mab treatment.
The experimental steps are as follows:
1. taking corresponding number of special flow tubes, numbering and marking specimen numbers
2. The corresponding antibodies are added into each tube, the dosage of the antibodies is added according to the instruction of the use of the antibodies, and the antibodies are added to the bottom of the tube, and the fluorescent labeling sequence of the antibodies is as follows:
a pipe A: CD81/CD269/CD38/CD45/CD229/CD117/CD20/CD200
And B, pipe B: CD38/CD56/CD19/CD45/CD138/CD27/cLambda/cKappa
3. Adding samples according to the sample count, and adding antibodies according to the sequence.
4. Shaking, mixing, and incubating for 20 minutes at room temperature in dark place.
5. Adding 1-3ml 10 Xlysate, shaking, mixing, and performing light-shielding cleavage for 10 min. Centrifugation was performed at 1500rpm for 5 minutes, and the supernatant was discarded.
6. 1-3ml PBS buffer was added, mixed by gentle shaking, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. Tube1 was resuspended with PAS and taken to the machine.
Adding 2ml PBS into the B tube, mixing, centrifuging to remove supernatant, adding membrane breaker a solution, adding 100 mu LPBMC into each tube, and incubating for 15min at room temperature.
The B tube was centrifuged (5 min,1500 rpm) with 2ml PBS, the supernatant was removed, kappa and Lamnda were added, 100. Mu.L of breaker b was added, and the mixture was mixed well and protected from light for 15min.
9.B the tube was added with 2ml PBS, mixed well, centrifuged (5 min,1500 rpm), the supernatant removed, and 500. Mu.LPBS was added and the mixture was put on-line.
10. The treated cells were fixed with 0.5% formalin (300. Mu.L/tube) and stored at 2-8deg.C for 5 days.
11. Data were analyzed using BD FACSDive Software, first the area (a rea, a) and height (H) of forward angle light scattering (Forwa rd sca tter, FSC) were used to set the debonding cell gate (denoted P1). Cells within P1 are then shown, and living cell gates (denoted by P2) are set using FSC/Side Scatter (SSC) to give single living cells. Within a single living cell gate (P2 gate), each blood cell gate was first set up using CD45/SSC, and lymphocytes, monocytes, granulocytes were roughly observed, and whether obvious tumor cells or abnormal cells were present. Single living cell door (P2 door) in multi-marker combination door-to-door observation of plasma cells, especially monoclonal plasma cells: the A tube sample is selected from CD38/CD138, CD45/CD38 and SSC/CD38, and the B tube sample is selected from CD38/CD229, CD45/CD38/CD138, SSC/CD229, SSC/CD38, cKappa, cLambda and other antigens, and the like, and the antigens are jointly gated to distinguish plasma cells, so that the benign and malignant states can be judged through the phenotype of the plasma cells.
Experimental results:
as a result, as shown in FIG. 1, plasma cells expressed CD19, CD27st, CD38, CD45dim, CD81, CD138, CD229, and did not express CD20, CD56, CD117, CD200, cLambda, cKappa in polyclonal expression, and the specimens were MRD negative.
As a result, as shown in FIG. 2, the specimen was a specimen after conventional chemotherapy. Plasma cells expressed all CD20dim, CD27, CD38, CD45dim, CD56, CD138, clapda, and some expressed CD200, CD269dim, and did not express CD19, CD81, cKappa.
As a result, as shown in FIG. 3, plasma cells expressed CD27, CD38, CD45dim, CD138, CD229, cKappa, and some cells expressed CD20dim, CD81, and a few expressed CD19, and did not express CD56, cLambda. CD269 negative expression following CD269 treatment in this specimen.
The results are shown in FIG. 4, where plasma cells expressed all CD229, cKappa, part of CD27 and CD138 weakly, and CD19, CD38, CD45, CD56, CD117, CD81, CD20, CD200, CD269, cLambda were all expressed negatively. CD45/CD229, cKappa in this sample plays a critical role in capturing monoclonal plasma cells.
The scheme of the invention has the following advantages:
(1) The use of multiple marker combinations to gate can avoid missed diagnosis after CD38 and/or CD138 treatment due to reduced or lost expression of CD38 and/or CD 138. The protocol of the present invention found that cases negative for CD38, CD138, CD56, CD19, all expressing CD229 and cKappa or clapda, six markers could cover 100% of cases, covering the most complete plasma cell population;
(2) The combination of the markers helps to find special clones and weak clones besides preventing missed diagnosis, and although most tumors are single clones, a small number of tumors can have 2-3 clones at the same time, and small clones are usually easily ignored. The small clones have great significance for the future recurrence, phenotype change and even the evolution of main clones at a certain moment, whether secondary tumors are diagnosed or not, and revealing the development rule of tumorigenesis and targeted treatment;
(3) The combined gate is different from the traditional CD38 gate, and is not interfered by basophils, plasma cell-like dendritic cells, mast cells, granulocytes and the like, so that the influence of sodium citrate on the CD138 is avoided;
(4) The combination gate can not be influenced by biological targeting treatment. In the combined gate provided by the invention, CD229 is expressed on plasma cells, T cells and B cells, cytosol Kappa and Lambda are expressed in cells, targeting treatment developed by aiming at the antigens has predictable toxic and side effects, or the targeting cytosol antigen has great technical difficulty, is a mark which is difficult to select for targeting biological treatment such as CAR-T aiming at new targets in the future, is hardly influenced by the selection or change of a treatment scheme, is urgently needed for the targeting treatment of the multiple myeloma at present, and is suitable for MRD detection and analysis schemes widely applied and popularized.
Example 2 two methods for testing samples after treatment for residual disease of multiple myeloma
1) Detection of MM-MRD by conventional methods
The antibodies used were anti-CD 38 antibody, anti-CD 45 antibody, anti-CD 56 antibody, anti-CD 138 antibody, anti-CD 27 antibody, anti-CD 19 antibody, anti-Kappa light chain antibody, anti-Lambda light chain antibody, and the same procedure as in example 1 was followed.
As shown in fig. 5, plasma cells fully expressed CD38, CD138, CD27, CD19dim, did not express CD56, ckappa/clamda=0.7, were polyclonal expressed, and plasma cells did not see light chain restricted expression, resulting in MRD negativity.
2) Detection of MM-MRD by the method of the invention
The experimental procedure is the same as in example 1.
As a result, as shown in FIG. 6, 0.008% of abnormal plasma cells (shown in dark green) expressed CD200, CD27, CD38, CD138, CD117, CD229, cKappa, and partially expressed CD56, and did not express CD19, cLambda, and CD269.
In conclusion, the traditional method is detected as false negative, and abnormal cells are successfully captured by using the method provided by the invention, and the detection result is MRD positive.
The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.
Claims (11)
1. Use of an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody and an anti-CD 138 antibody in the preparation of an MRD detection product.
2. The use according to claim 1, wherein the MRD test product further comprises any one or more of the following: anti-CD 81 antibodies, anti-CD 117 antibodies, anti-CD 20 antibodies, anti-CD 200 antibodies, anti-CD 56 antibodies, anti-CD 19 antibodies, anti-CD 27 antibodies, anti-Kappa light chain antibodies or anti-Lambda light chain antibodies.
3. The use according to claim 1, wherein a first MRD detection reagent and a second MRD detection reagent are included in the MRD detection product, wherein the first MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, and/or wherein the second MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 138 antibody.
4. The use according to claim 3, wherein the first MRD detection reagent further comprises any one or more of an anti-CD 269 antibody, an anti-CD 81 antibody, an anti-CD 117 antibody, an anti-CD 20 antibody, an anti-CD 200 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody or an anti-Lambda light chain antibody, and the second MRD detection reagent comprises any one or more of an anti-CD 269 antibody, an anti-CD 81 antibody, an anti-CD 117 antibody, an anti-CD 20 antibody, an anti-CD 200 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody or an anti-Lambda light chain antibody other than the antibodies comprised in the first MRD detection reagent.
5. The use according to claim 3, wherein the first MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody, an anti-CD 81 antibody, an anti-CD 117 antibody, an anti-CD 20 antibody and an anti-CD 200 antibody, and/or the second MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 138 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody and an anti-Lambda light chain antibody.
6. An MRD test product comprising an MRD test agent comprising an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, an anti-CD 269 antibody, and an anti-CD 138 antibody.
7. The MRD test product of claim 6, wherein said MRD test reagents comprise a first MRD test reagent comprising an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 229 antibody, and a second MRD test reagent comprising an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 138 antibody.
8. The MRD test product of claim 7, wherein the first MRD test agent further comprises any one or more of anti-CD 269, anti-CD 81, anti-CD 117, anti-CD 20, anti-CD 200, anti-CD 56, anti-CD 19, anti-CD 27, anti-Kappa light chain, or anti-Lambda light chain antibodies, and the second MRD test agent comprises any one or more of anti-CD 269, anti-CD 81, anti-CD 117, anti-CD 20, anti-CD 200, anti-CD 56, anti-CD 19, anti-CD 27, anti-Kappa light chain, or anti-Lambda light chain antibodies other than those included in the first MRD test agent.
9. The MRD test product of claim 7, wherein said first MRD test reagent comprises anti-CD 38 antibodies, anti-CD 45 antibodies, anti-CD 229 antibodies, anti-CD 269 antibodies, anti-CD 81 antibodies, anti-CD 117 antibodies, anti-CD 20 antibodies, and anti-CD 200 antibodies; and/or, the second MRD detection reagent comprises an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 138 antibody, an anti-CD 56 antibody, an anti-CD 19 antibody, an anti-CD 27 antibody, an anti-Kappa light chain antibody and an anti-Lambda light chain antibody.
10. The MRD test product of claim 6, further comprising any one or more of the following: erythrocyte lysate, buffer or membrane breaker.
11. A method of screening for a therapeutic MRD drug, said method comprising detecting an ex vivo sample from a subject following administration of a candidate drug using a MRD test product according to any one of claims 6 to 10.
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