Biomarker for multiple myeloma

Abstract

The present invention provides a method and the like for predicting prognosis of multiple myeloma using, as a novel biomarker, IL5RA of extracellular vesicles derived from a biological sample of a subject.

Description

Biomarkers for Multiple Myeloma

[0001] The present invention relates to methods, compositions, kits, etc. for predicting the prognosis, diagnosing, and evaluating the condition of multiple myeloma using novel biomarkers for multiple myeloma.

[0002] Multiple myeloma (MM) is a malignant tumor of plasma cells, with an incidence rate of 6 per 100,000 people, and 4,500 people die annually in Japan. In recent years, with the introduction of new drugs such as immunomodulatory drugs, proteasome inhibitors, and anti-CD38 monoclonal antibodies, the prognosis of newly diagnosed (ND) multiple myeloma patients has been improving. For example, there is a report that when autologous peripheral blood stem cell transplantation and lenalidomide maintenance therapy were performed following bortezomib, lenalidomide, and dexamethasone, the progression-free survival (PFS) was 67.5 months. For patients ineligible for transplantation, incorporating daratumumab, an anti-CD38 monoclonal antibody, into the front-line treatment regimen has been reported to improve both the progression-free survival and the overall survival (OS). However, multiple myeloma is still an incurable disease, and the majority of patients experience disease progression several times during the course.

[0003] Currently, the diagnosis and prognosis prediction of multiple myeloma rely on bone marrow examination. However, bone marrow examination is invasive, a significant burden on patients, and there is also a possibility that the spatial heterogeneity cannot be accurately captured. Therefore, a less invasive approach for the diagnosis and prognosis prediction of multiple myeloma is needed.

[0004] For example, Ghermezi et al. reported that serum soluble BCMA (B-cell maturation antigen) levels were significantly elevated in multiple myeloma patients compared to healthy individuals, and that serum soluble BCMA can serve as a biomarker for predicting the prognosis of multiple myeloma (Non-Patent Literature 1). However, there have been no previous reports of elevated BCMA expression in extracellular vesicles (EVs) in multiple myeloma patients. Furthermore, there is still a need for useful biomarkers for multiple myeloma.

[0005] Haematologica 2017;102(4):785-795

[0006] The present invention aims to provide a novel biomarker for multiple myeloma.

[0007] As a result of diligent research by the inventors, it was found that IL5RA (Interleukin-5 Receptor Subunit Alpha), an extracellular vesicle derived from bone marrow and serum, is useful as a biomarker for predicting the prognosis and diagnosing multiple myeloma. Furthermore, it was found that LSR (Lipolysis-stimulated lipoprotein receptor) and BCMA, also derived from bone marrow and serum, are useful as biomarkers for diagnosing multiple myeloma. This invention was completed based on these findings.

[0008] In other words, the present invention is defined as follows: (1) A method for predicting the prognosis of multiple myeloma, comprising the step of detecting IL5RA in extracellular vesicles derived from a biological sample of a subject (hereinafter sometimes referred to as "the first method of the present invention"). (2) A composition for predicting the prognosis of multiple myeloma, comprising a reagent for detecting IL5RA in extracellular vesicles derived from a biological sample of a subject (hereinafter sometimes referred to as "the first composition of the present invention"). (3) A kit for predicting the prognosis of multiple myeloma, comprising a reagent for detecting IL5RA in extracellular vesicles derived from a biological sample of a subject (hereinafter sometimes referred to as "the first kit of the present invention").

[0009] [4] A method for diagnosing multiple myeloma, comprising the step of detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA from an extracellular vesicle derived from a biological sample of a subject (hereinafter sometimes referred to as "the second method of the present invention"). [5] A diagnostic composition for multiple myeloma, comprising a reagent for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA from an extracellular vesicle derived from a biological sample of a subject (hereinafter sometimes referred to as "the second composition of the present invention"). [6] A diagnostic kit for multiple myeloma, comprising a reagent for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA from an extracellular vesicle derived from a biological sample of a subject (hereinafter sometimes referred to as "the second kit of the present invention").

[0010] [7] The method according to [1] or [4] above, the composition according to [2] or [5] above, or the kit according to [3] or [6] above, wherein the biological sample is serum.

[0011] [8] A method for screening for a preventive or therapeutic agent for multiple myeloma, comprising the steps of: administering a candidate substance to a subject; detecting one or more extracellular vesicles derived from the subject that are selected from IL5RA, LSR, and BCMA; and selecting a target preventive or therapeutic agent for multiple myeloma using the obtained results as an indicator (hereinafter sometimes referred to as "the screening method of the present invention").

[0012] [9] A prophylactic or therapeutic agent for multiple myeloma comprising an active ingredient that targets one or two selected from IL5RA and LSR (hereinafter sometimes referred to as "the prophylactic or therapeutic agent of the present invention").

[0013] Other embodiments of the present invention include, for example, the following:

[10] A method for evaluating the state of multiple myeloma, comprising the step of detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject (hereinafter sometimes referred to as "the third method of the present invention").

[11] A composition for evaluating the state of multiple myeloma, comprising a reagent for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject (hereinafter sometimes referred to as "the third composition of the present invention").

[12] A kit for evaluating the state of multiple myeloma, comprising a reagent for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject (hereinafter sometimes referred to as "the third kit of the present invention").

[13] The method according to

[10] , the composition according to

[11] , or the kit according to

[12] , wherein the state comprises one or more selected from progression, severity, and treatment response.

[0014]

[14] The method according to

[10] or

[13] above, the composition according to

[11] or

[13] above, or the kit according to

[12] or

[13] above, wherein the biological sample is serum.

[0015]

[15] A composition according to any one of [2], [5], [7],

[11] ,

[13] , and

[14] above, wherein the reagent is an antibody, or a kit according to any one of [3], [6], [7],

[12] ,

[13] , and

[14] above.

[0016]

[16] The method according to [1] or [7] above, further comprising the step of associating an elevated IL5RA value (level) with a high probability of a poor prognosis for the subject's multiple myeloma.

[17] The method according to [4] or [7] above, further comprising the step of associating an elevated 1 or 2 selected from the IL5RA value, LSR value, and BCMA value with a high probability of the subject having multiple myeloma.

[18] The method according to

[10] or

[14] above, further comprising the step of associating an elevated 1 or 2 selected from the IL5RA value, LSR value, and BCMA value with a high probability of the subject's multiple myeloma being in poor condition.

[0017]

[19] A method for diagnosing multiple myeloma, comprising the step of detecting one or more selected from ELAPOR1, IL5RA, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, BCMA, WDR74, LSR, TRAPPC14, TANGO2, ST6GAL1, TRAM2, and MZB1 in extracellular vesicles derived from the bone marrow of a subject.

[20] A diagnostic composition for multiple myeloma, comprising a reagent for detecting one or more selected from ELAPOR1, IL5RA, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, BCMA, WDR74, LSR, TRAPPC14, TANGO2, ST6GAL1, TRAM2, and MZB1 in extracellular vesicles derived from a biological sample of a subject.

[21] A diagnostic kit for multiple myeloma, comprising reagents for detecting one or more selected from ELAPOR1, IL5RA, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, BCMA, WDR74, LSR, TRAPPC14, TANGO2, ST6GAL1, TRAM2, and MZB1 in extracellular vesicles derived from a subject's biological sample.

[0018]

[22] A method for treating multiple myeloma, comprising the steps of diagnosing multiple myeloma, which includes detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject, and administering a multiple myeloma treatment drug to a subject diagnosed with multiple myeloma (hereinafter sometimes referred to as "the treatment method of the present invention").

[0019] Furthermore, other embodiments of the present invention include, for example, the following:

[23] Use of a reagent for detecting IL5RA in extracellular vesicles derived from a subject's biological sample for the production of a composition for predicting the prognosis of multiple myeloma; Use of a reagent for detecting IL5RA in extracellular vesicles derived from a subject's biological sample for the production of a kit for predicting the prognosis of multiple myeloma; A reagent for detecting IL5RA in extracellular vesicles derived from a subject's biological sample for use in predicting the prognosis of multiple myeloma.

[24] Use of reagents for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA derived from a subject's biological sample for the preparation of a diagnostic composition for multiple myeloma; Use of reagents for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA derived from a subject's biological sample for the preparation of a diagnostic kit for multiple myeloma; Reagents for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA derived from a subject's biological sample for use in the diagnosis of multiple myeloma.

[25] Use of reagents for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA from a subject's biological sample for the preparation of a composition for evaluating the status of multiple myeloma; Use of reagents for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA from a subject's biological sample for the preparation of a kit for evaluating the status of multiple myeloma; Reagents for detecting one or more extracellular vesicles selected from IL5RA, LSR, and BCMA from a subject's biological sample for use in evaluating the status of multiple myeloma.

[26] Use of a substance targeting 1 or 2 selected from IL5RA and LSR for the manufacture of a prophylactic or therapeutic agent for multiple myeloma; a substance targeting 1 or 2 selected from IL5RA and LSR for use in the prevention or treatment of multiple myeloma; a method for the prevention or treatment of multiple myeloma comprising administering a prophylactic or therapeutic effective amount of a substance targeting 1 or 2 selected from IL5RA and LSR to a patient in need of prevention or treatment.

[0020] According to the present invention, a method for predicting the prognosis of multiple myeloma can be provided, utilizing IL5RA from extracellular vesicles derived from a subject's biological sample as a novel biomarker. Furthermore, according to the present invention, a method for diagnosing multiple myeloma can be provided, utilizing one or more selected from IL5RA, LSR, and BCMA from extracellular vesicles derived from a subject's biological sample as novel biomarkers.

[0021] This figure outlines the experimental design for EV isolation from bone marrow and serum, as well as the subsequent proteomic analysis. In the figure, "MM" represents MM patients, and "Control" represents the control group (the same applies to the following figures). This figure shows the results of Western blotting evaluation of the expression of EV marker proteins CD9 and CD63 in isolated EVs. Data for serum-derived EVs MM14 and C29, and bone marrow-derived EVs MM4 and L4 are shown. This figure shows representative results of nanoparticle tracking analysis of isolated EVs. Data for bone marrow-derived EVs MM9 and L2, and serum-derived EVs MM9 and C26 are shown. This Venn diagram shows the number of proteins identified in bone marrow EVs from MM patients and controls by LC / MS analysis. This figure shows a volcano plot representing the difference in protein expression between bone marrow EVs from MM patients and controls. Proteins with significantly increased expression in MM patients (fold change ≥ 2 and p < 0.05) are shown as black dots, and proteins with significantly decreased expression in MM patients (fold change ≤ 0.5 and p < 0.05) are shown as dark gray dots. P-values ​​were calculated using the two-sided Welch's t-test. These are box plots showing the normalized expression levels of representative proteins detected only in bone marrow EVs of MM patients, or significantly elevated in those proteins. These are box plots of serum EV BCMA, serum EV IL5RA, and serum EV LSR comparing MM patients with controls. P-values ​​were calculated using the Mann-Whitne U test. These figures show ROC curves for each serum EV protein and serum total BCMA as predictors of progression-free survival. a) Serum EV BCMA, b) Serum EV IL5RA, c) Serum EV LSR, d) Serum soluble BCMA. These figures show Kaplan-Meier curves indicating progression-free survival in MM patients based on serum EV protein levels at diagnosis. a) Serum EV BCMA, b) Serum EV IL5RA, c) Serum EV LSR. These are box plots showing serum soluble BCMA concentrations in MM patients and controls. These figures show Kaplan-Meier curves indicating progression-free survival in MM patients based on serum soluble BCMA levels at diagnosis.This figure shows the results of evaluating the mRNA expression of TNFRSF17 (BCMA-encoding RNA) and IL5RA by RT-qPCR in CD138-positive bone marrow cells of MM patients, and comparing them with the expression in 48 different organs of normal human tissue. The relative expression of each target is 2. -ΔCT The data was normalized to GAPDH using the method. Error bars represent ±2 × SEM.

[0022] (Multiple Myeloma) Multiple myeloma is a disease in which plasma cells, which differentiate from B cells (a type of lymphocyte in white blood cells), become cancerous and develop into myeloma cells, which then proliferate mainly in the bone marrow. Multiple myeloma is thought to progress through two stages: "monoclonal gammaglobulinemia of unknown significance (MGUS)" and "smoldering (asymptomatic) multiple myeloma (SMM)," before becoming "(symptomatic) multiple myeloma (MM)," which is symptomatic and requires treatment. The present invention may target multiple myeloma at any of these stages, but (symptomatic) multiple myeloma (MM) is preferred, and newly diagnosed multiple myeloma (NDMM) is more preferred. In addition, the "Revised International Staging Classification (R-ISS classification)" is generally used to classify multiple myeloma into stages I, II, or III, but the present invention may target multiple myeloma at any of these stages.

[0023] (Subjects) In the first method, composition, and kit of the present invention for predicting the prognosis of multiple myeloma, subjects are preferably patients with multiple myeloma, and more preferably newly diagnosed patients with multiple myeloma. In the second method, composition, and kit of the present invention for diagnosing multiple myeloma, subjects are preferably people with no history of multiple myeloma diagnosis or people who have completed treatment for multiple myeloma. In the third method, composition, and kit of the present invention for evaluating the status of multiple myeloma, subjects may be any of the following: people with no history of multiple myeloma diagnosis, patients with multiple myeloma, or people who have completed treatment for multiple myeloma.

[0024] (Extracellular vesicles derived from biological samples) The "extracellular vesicles derived from biological samples" used in the present invention are not particularly limited as long as they can be used in the present invention. However, since extracellular vesicles are known to be contained in bodily fluids such as bone marrow, blood (serum, plasma), saliva, and urine, the "biological sample" may be bodily fluids such as bone marrow, blood (serum, plasma), saliva, or urine, with bone marrow or serum being preferred. However, serum is more preferred as a biological sample because it can be obtained through routine blood tests, is less invasive to collect, and a sufficient amount can be obtained more easily.

[0025] Extracellular vesicles (EVs) are non-nuclear, lipid bilayer-enclosed particles released from cells that contain biomolecules such as proteins and nucleic acids. Extracellular vesicles are classified into exosomes, microvesicles, or apoptotic vesicles based on their production mechanisms, but in this invention, the term "extracellular vesicle" encompasses all of these. The method for obtaining extracellular vesicles from a biological sample is not particularly limited, but may include, for example, isolating, purifying, and / or concentrating extracellular vesicles from a biological sample by known methods, and these may be done using commercially available kits, for example. However, this does not apply if it is possible to detect biomarkers in extracellular vesicles in a biological sample without performing such processing.

[0026] (Detection of Biomarkers) In the first method, composition, and kit of the present invention, IL5RA is detected as a biomarker in extracellular vesicles derived from a subject's biological sample. In the second and third methods, compositions, and kits of the present invention, as well as the screening and treatment methods of the present invention, one or more selected from IL5RA, LSR, and BCMA are detected as biomarkers in extracellular vesicles derived from a subject's biological sample. That is, in the second and third methods, compositions, and kits of the present invention, as well as the screening and treatment methods of the present invention, only IL5RA may be detected as a biomarker in extracellular vesicles derived from a subject's biological sample, only LSR may be detected, only BCMA may be detected, IL5RA and LSR may be detected, IL5RA and BCMA may be detected, LSR and BCMA may be detected, or IL5RA, LSR, and BCMA may be detected.

[0027] Furthermore, in the first method, composition, and kit of the present invention, BCMA of extracellular vesicles derived from the subject's biological sample may be further detected as a biomarker, and serum soluble BCMA of the subject may be further detected. Furthermore, in the second and third methods, compositions, and kits of the present invention, and the screening method and treatment method of the present invention, serum soluble BCMA of the subject may be further detected as a biomarker. Specifically, the first method of the present invention may further include a step of detecting BCMA of extracellular vesicles derived from the subject's biological sample and / or a step of detecting serum soluble BCMA of the subject; the first composition and kit of the present invention may further include a reagent for detecting BCMA of extracellular vesicles derived from the subject's biological sample and / or a reagent for detecting serum soluble BCMA of the subject; the second and third methods of the present invention, and the screening method and treatment method of the present invention may further include a step of detecting serum soluble BCMA of the subject; and the second and third compositions and kits of the present invention may further include a reagent for detecting serum soluble BCMA of the subject.

[0028] IL5RA (Interleukin-5 Receptor Subunit Alpha) is one of the subunits of the IL5 (interleukin-5) receptor. IL5RA is sometimes called CD125 (Cluster of Differentiation 125). LSR (Lipolysis-stimulated lipoprotein receptor) is a receptor involved in the intracellular uptake of lipoproteins in lipid metabolism. BCMA (B-cell maturation antigen) is a transmembrane protein belonging to the TNFR superfamily, and is sometimes called TNFRSF17 (Tumor necrosis factor receptor superfamily member 17) or CD269 (Cluster of Differentiation 269). These IL5RA, LSR, and BCMA can be detected not only in the bone marrow of multiple myeloma patients but also in extracellular vesicles derived from serum. Therefore, by using these as biomarkers, serum can be used as a sample, which is less invasive during collection and can be obtained more easily in sufficient quantities.

[0029] Furthermore, when using extracellular vesicles derived from the subject's bone marrow, in addition to IL5RA, LSR, and BCMA, the following may be used: ELAPOR1 (Endosome / lysosome-associated apoptosis and autophagy regulator 1), ZNFX1 (NFX1-type zinc finger-containing protein 1), SLFN14 (Protein SLFN14), RNASEH2C (Ribonuclease H2 subunit C), TNFRSF10A (Tumor necrosis factor receptor superfamily member 10A), WDR74 (WD repeat-containing protein 74), TRAPPC14 (Trafficking protein particle complex subunit 14), TANGO2 (Transport and Golgi organization protein 2 homolog), ST6GAL1 (Beta-galactoside alpha-2,6-sialyltransferase 1), TRAM2 (Translocating chain-associated membrane protein 2), and MZB1 (Marginal zone B- and B1-cell-specific Proteins can also serve as biomarkers for diagnosing patients with multiple myeloma. Therefore, the second method of the present invention may include the step of detecting one or more selected from ELAPOR1, IL5RA, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, BCMA, WDR74, LSR, TRAPPC14, TANGO2, ST6GAL1, TRAM2, and MZB1 in extracellular vesicles derived from the bone marrow of a subject, and the second composition or kit of the present invention may include reagents for detecting one or more selected from ELAPOR1, IL5RA, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, BCMA, WDR74, LSR, TRAPPC14, TANGO2, ST6GAL1, TRAM2, and MZB1 in extracellular vesicles derived from the bone marrow of a subject.

[0030] The method for detecting the aforementioned biomarker proteins in extracellular vesicles derived from a subject's biological sample is not particularly limited and may be any known method for protein detection. For example, the biomarker may be detected by mass spectrometry after processing the extracellular vesicles as necessary, or by using a reagent for detecting biomarkers. The reagent for detecting biomarkers is not particularly limited, but may be an antibody, for example. That is, the reagent for detecting IL5RA, LSR, or BCMA as a biomarker may be, for example, an anti-IL5RA antibody, an anti-LSR antibody, or an anti-BCMA antibody. Furthermore, reagents for detecting ELAPOR1, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, WDR74, TRAPPC14, TANGO2, ST6GAL1, TRAM2, or MZB1 as biomarkers may include, for example, anti-ELAPOR1 antibody, anti-ZNFX1 antibody, anti-SLFN14 antibody, anti-RNASEH2C antibody, anti-TNFRSF10A antibody, anti-WDR74 antibody, anti-TRAPPC14 antibody, anti-TANGO2 antibody, anti-ST6GAL1 antibody, anti-TRAM2 antibody, or anti-MZB1 antibody. The reagents for detecting biomarkers may be commercially available or prepared by known methods. The method for detecting biomarkers using the reagents for detecting biomarkers is not particularly limited, but may include, for example, immunoassay methods such as ELISA (Enzyme-linked immunosorbent assay) or Western blotting.

[0031] Furthermore, detecting biomarkers in extracellular vesicles derived from a subject's biological sample may include, for example, one or more of the following steps, and may be detected by mass spectrometry or immunoassay, for example, any combination of these steps in any order: - A step of collecting a biological sample from a subject; - A step of obtaining extracellular vesicles from the biological sample collected from the subject; - A step of obtaining proteins and / or peptides from the extracellular vesicles; - A step of reacting the proteins and / or peptides obtained from the extracellular vesicles with a reagent for detecting the biomarker (primary reagent); - A step of capturing or immobilizing the extracellular vesicles on a solid surface; - A step of reacting proteins on the extracellular vesicle membrane with a reagent for detecting the biomarker (primary reagent); - A step of reacting a reagent for detecting the biomarker (primary reagent) with another reagent for detecting the reagent (secondary reagent); - A step of reacting a labeling substance conjugated to the primary or secondary reagent with a reagent for detecting the labeling substance; - A step of detecting the biomarker by detecting the labeling substance; - A step of quantifying the detected biomarker.

[0032] More specifically, when detecting biomarkers in extracellular vesicles derived from a subject's biological sample using the sandwich ELISA method, the following one or more steps may be included: - A step of isolating extracellular vesicles from the subject's biological sample; - A step of immobilizing an antibody against an extracellular vesicle surface marker molecule (e.g., CD9, CD63, CD81, etc.) onto a microplate; or a step of immobilizing Tim4 (T cell immunoglobulin and mucin domain-containing protein 4, a protein that specifically captures phosphatidylserine on the extracellular vesicle membrane) onto a microplate; - A step of reacting the extracellular vesicle surface marker molecule with the extracellular vesicle; or a step of reacting Tim4 with the extracellular vesicle; - A step of reacting a protein on the extracellular vesicle membrane with a biomarker detection antibody (primary antibody); - A step of reacting the biomarker detection antibody (primary antibody) with a secondary antibody; - A step of reacting an enzyme conjugated to the primary or secondary antibody with a substrate; - A process for detecting or quantifying a biomarker by detecting enzyme activity through absorbance measurement.

[0033] (Predicting Prognosis) In this invention, "prognosis" means the outlook regarding the medical course of the disease. In this invention, the prognosis is not particularly limited, but may be, for example, progression-free survival. Progression-free survival is defined as the time from diagnosis to disease progression or death from any cause. In this invention, "predicting prognosis" means evaluating the likelihood of a good or bad prognosis. For example, this may be evaluating the likelihood of a longer or shorter progression-free survival.

[0034] In the present invention, predicting the prognosis of multiple myeloma may include, for example, associating an elevated IL5RA level in extracellular vesicles derived from a subject's biological sample with a high probability of a poor prognosis for the subject's multiple myeloma, or associating it with a poor prognosis. In this specification, "associating A with B" may mean "indicating that A is B." Furthermore, an elevated IL5RA level may mean, for example, that the IL5RA level is elevated compared to a reference value. The method for setting the reference value is not particularly limited, but for example, it may be set by drawing a receiver operating characteristic (ROC) curve. For example, as shown in the examples described later, if the IL5RA value of extracellular vesicles derived from the subject's serum is 1.40 fmol / mL or higher, 1.60 fmol / mL or higher, 1.80 fmol / mL or higher, 2.00 fmol / mL or higher, 2.20 fmol / mL or higher, 2.40 fmol / mL or higher, 2.60 fmol / mL or higher, 2.80 fmol / mL or higher, or 2.85 fmol / mL or higher in the serum, it may indicate that the subject has a high probability of having a poor prognosis for multiple myeloma, or that the prognosis is poor.

[0035] (Diagnosis) In the present invention, diagnosing multiple myeloma means evaluating the likelihood that a subject has multiple myeloma. In the present invention, diagnosing multiple myeloma may include, for example, associating a subject with multiple myeloma if one or more selected from the IL5RA, LSR, and BCMA values ​​of extracellular vesicles derived from a biological sample of the subject are elevated. An elevation of one or more selected from the IL5RA, LSR, and BCMA values ​​may, for example, mean that one or more selected from the IL5RA, LSR, and BCMA values ​​are elevated compared to their respective reference values. The method for setting each reference value is not particularly limited, but for example, it may be set from the IL5RA, LSR, or BCMA values ​​of multiple myeloma patients and / or controls. For example, as shown in the examples described later, if the FC (fold change) when comparing the IL5RA value of extracellular vesicles derived from the subject's serum with that of the control group is 1.1 or higher, 1.2 or higher, 1.3 or higher, or 1.33 or higher, it may indicate that the subject has a high probability of having multiple myeloma, or that the subject does have it, and / or if the FC when comparing the LSR value of extracellular vesicles derived from the subject's serum with that of the control group is 1.1 or higher, 1.2 or higher, 1.3 or higher, 1.4 or higher, 1.5 or higher, 1.6 or higher, or 1.63 or higher, it may indicate that the subject has a high probability of having multiple myeloma, or that the subject does have it, and / or if the FC (fold change) when comparing the BCMA value of extracellular vesicles derived from the subject's serum with that of the control group A change of 1.1 or higher, 1.2 or higher, 1.3 or higher, 1.4 or higher, 1.5 or higher, 2.0 or higher, 2.5 or higher, 3.0 or higher, 3.5 or higher, or 3.64 or higher may indicate that the subject is highly likely to have or has multiple myeloma.

[0036] Furthermore, in embodiments that also use ELAPOR1, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, WDR74, TRAPPC14, TANGO2, ST6GAL1, TRAM2, or MZB1 as biomarkers for diagnosing multiple myeloma, it may include, for example, that elevated levels of each of these biomarkers in extracellular vesicles derived from the subject's biological sample indicate a high probability that the subject has multiple myeloma, or that it is associated with the subject having multiple myeloma. Elevated levels of each biomarker may, for example, be elevated levels of each biomarker compared to each reference value. The method for setting each reference value is not particularly limited, but for example, it may be set from the biomarker values ​​of multiple myeloma patients and / or controls.

[0037] (Assessing the condition) In the present invention, assessing the condition of multiple myeloma includes, in addition to assessing the possibility of whether or not a person has multiple myeloma as described above, assessing the progression, severity, and / or treatment response of the multiple myeloma. In the present invention, assessing the condition of multiple myeloma may include, for example, if one or more selected from the IL5RA, LSR, and BCMA values ​​of extracellular vesicles derived from the subject's biological sample are elevated, it is highly likely that the subject's multiple myeloma condition is poor, or it may be associated with poor condition. Also, for example, if one or more selected from the IL5RA, LSR, and BCMA values ​​are elevated, it is highly likely that the subject's multiple myeloma is at a high stage of progression or severity, or it may be associated with high stage of progression or severity. Furthermore, if one or more selected from the IL5RA, LSR, and BCMA values ​​are significantly elevated, it is highly likely that the subject's multiple myeloma is untreatable or untreatable. Furthermore, if one or more selected from IL5RA, LSR, and BCMA values ​​are elevated, it may be associated with a high probability that the treatment will not be effective, or that the treatment will not be effective. Additionally, the progression of a subject's multiple myeloma may be evaluated by regularly monitoring the changes in one or more selected from IL5RA, LSR, and BCMA values. In particular, when serum is used as a biological sample, more frequent collection is possible, making such monitoring easier. For example, if one or more selected from IL5RA, LSR, and BCMA values ​​are lower than before, it may be associated with a high probability that the progression or severity of the subject's multiple myeloma is decreasing, or that the treatment is likely to be effective, or that the treatment is effective. An increase in one or more selected from IL5RA, LSR, and BCMA values ​​may, for example, mean that one or more selected from IL5RA, LSR, and BCMA values ​​are elevated compared to their respective reference values.There are no particular restrictions on how each reference value is set here; for example, it may be determined by drawing a receiver operating characteristic (ROC) curve, or it may be determined from the IL5RA, LSR, or BCMA values ​​of multiple myeloma patients and / or controls.

[0038] (Reagents) The reagents used to detect biomarkers such as IL5RA in extracellular vesicles derived from the subject's biological sample are not particularly limited as long as they can specifically detect the target biomarker, and any substance may be used, for example, an antibody. That is, the reagents used to detect IL5RA, LSR, or BCMA as biomarkers may be, for example, an anti-IL5RA antibody, an anti-LSR antibody, or an anti-BCMA antibody. Furthermore, reagents for detecting ELAPOR1, ZNFX1, SLFN14, RNASEH2C, TNFRSF10A, WDR74, TRAPPC14, TANGO2, ST6GAL1, TRAM2, or MZB1 as biomarkers may include, for example, anti-ELAPOR1 antibody, anti-ZNFX1 antibody, anti-SLFN14 antibody, anti-RNASEH2C antibody, anti-TNFRSF10A antibody, anti-WDR74 antibody, anti-TRAPPC14 antibody, anti-TANGO2 antibody, anti-ST6GAL1 antibody, anti-TRAM2 antibody, or anti-MZB1 antibody. Reagents for detecting biomarkers may be commercially available or prepared by known methods.

[0039] (First Method of the Invention) The first method of the Invention is a method for predicting the prognosis of multiple myeloma and provides an indicator for predicting the prognosis of multiple myeloma in a subject. The first method of the Invention may include a method for collecting information (data) for predicting the prognosis of multiple myeloma, a method for creating information for predicting the prognosis of multiple myeloma, a method for providing information for predicting the prognosis of multiple myeloma, and a method for assisting in predicting the prognosis of multiple myeloma. The first method of the Invention is not particularly limited in that it includes a step of detecting IL5RA in extracellular vesicles derived from a biological sample of a subject. The first method of the Invention may further include one or more other steps. However, the first method of the Invention does not include a diagnostic act by a physician.

[0040] (First Composition of the Invention) The first composition of the Invention is a composition for predicting the prognosis of multiple myeloma, that is, a composition whose use is specified as "for predicting the prognosis of multiple myeloma." The first composition of the Invention may also be a composition for use in the first method of the Invention. The first composition of the Invention is not particularly limited as long as it contains a reagent for detecting IL5RA in extracellular vesicles derived from a biological sample of a subject. The first composition of the Invention may contain, for example, one or more arbitrary components other than the reagent described above. The arbitrary components are not particularly limited, but may include, for example, surfactants, pH adjusters, buffers, isotonic agents, chelating agents, preservatives, carriers, solvents, etc.

[0041] (First Kit of the Invention) The first kit of the Invention is a kit for predicting the prognosis of multiple myeloma, that is, a kit whose use is specified as "for predicting the prognosis of multiple myeloma." The first kit of the Invention may also be a kit for use in the first method of the Invention. The first kit of the Invention is not particularly limited as long as it includes a reagent for detecting IL5RA in extracellular vesicles derived from a subject's biological sample. The first kit of the Invention may include, for example, one or more components other than the reagents described above. The components are not particularly limited, but may include, for example, reagents other than the reagents described above (e.g., capture antibody, secondary antibody, labeling substance, chromogenic substrate, diluent, washing solution, reaction stop solution), container, equipment, instruction manual regarding the method or procedure of use, instruction manual for carrying out the method of the Invention, and other accompanying documents. The first kit of the Invention may be an immunoassay kit such as an ELISA kit or a Western blot kit.

[0042] (Second Method of the Present Invention) The second method of the present invention is a method for diagnosing multiple myeloma, and provides an index for diagnosing that a subject is suffering from multiple myeloma. The second method of the present invention may include a method for collecting information (data) for diagnosing multiple myeloma, a method for creating information for diagnosing multiple myeloma, a method for providing information for diagnosing multiple myeloma, a method for assisting in the diagnosis of multiple myeloma, and the like. The second method of the present invention is not particularly limited as long as it includes a step of detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject. The second method of the present invention may further include one or two or more other steps. However, the second method of the present invention does not include a diagnostic act by a doctor.

[0043] (Second Composition of the Present Invention) The second composition of the present invention is a composition for diagnosing multiple myeloma, that is, a composition specified for the use of "for diagnosing multiple myeloma". Further, the second composition of the present invention may be a composition for use in the second method of the present invention. The second composition of the present invention is not particularly limited as long as it includes a reagent for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject. Similar to the first composition of the present invention, the second composition of the present invention may include one or two or more arbitrary components other than the above-mentioned reagents.

[0044] (Second Kit of the Present Invention) The second kit of the present invention is a kit for diagnosing multiple myeloma, that is, a kit specified for the use of "for diagnosing multiple myeloma". Further, the second kit of the present invention may be a kit for use in the second method of the present invention. The second kit of the present invention is not particularly limited as long as it includes a reagent for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject. Similar to the first kit of the present invention, the second kit of the present invention may include any one or two or more configurations other than the above-mentioned reagents. The second kit of the present invention may be an immunoassay kit such as an ELISA kit or a Western blot kit, for example.

[0045] (The third method of the present invention) The third method of the present invention is a method for evaluating the state of multiple myeloma and provides an index for evaluating the state of multiple myeloma in a subject. The third method of the present invention may include a method for collecting information (data) for evaluating the state of multiple myeloma, a method for creating information for evaluating the state of multiple myeloma, a method for providing information for evaluating the state of multiple myeloma, a method for assisting the evaluation of the state of multiple myeloma, and the like. The third method of the present invention is not particularly limited as long as it includes a step of detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject. The third method of the present invention may further include one or more other steps. However, the third method of the present invention does not include a diagnostic act by a physician.

[0046] (The third composition of the present invention) The third composition of the present invention is a composition for evaluating the state of multiple myeloma, that is, a composition for which the use of "for evaluating the state of multiple myeloma" is specified. Further, the third composition of the present invention may be a composition for use in the third method of the present invention. The third composition of the present invention is not particularly limited as long as it includes a reagent for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject. Similar to the first composition of the present invention, the third composition of the present invention may include one or more arbitrary components other than the above-mentioned reagents.

[0047] (Third Kit of the Invention) The third kit of the Invention is a kit for evaluating the state of multiple myeloma, that is, a kit whose use is specified as "for evaluating the state of multiple myeloma." The third kit of the Invention may also be a kit for use in the third method of the Invention. The third kit of the Invention is not particularly limited as long as it contains reagents for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a subject's biological sample. The third kit of the Invention may contain any one or more components other than the reagents described above, similar to the first kit of the Invention. The third kit of the Invention may be an immunoassay kit, such as an ELISA kit or a Western blot kit.

[0048] (Screening Method of the Present Invention) The screening method of the present invention is a method for screening for a preventive or therapeutic agent for multiple myeloma. The screening method of the present invention is not particularly limited in that it includes the following steps (a), (b), and (c), but may further include one or more other steps: (a) a step of administering a candidate substance to a subject; (b) a step of detecting one or more selected from IL5RA, LSR, and BCMA from extracellular vesicles derived from the subject; (c) a step of selecting a preventive or therapeutic agent for the target multiple myeloma using the obtained results as an indicator.

[0049] In the screening method of the present invention, the subjects are not particularly limited, but may include, for example, cultured cells, tissue models, and / or animals. More specifically, they may include cultured cells derived from multiple myeloma, tissue models of multiple myeloma, and animal models of multiple myeloma. As for animals, they may include, for example, mammals. However, the subjects may be subjects other than humans. The number of subjects is not particularly limited, but it is preferable that the number be sufficient for statistical testing, for example. The method of administration is not particularly limited, and administration may be performed in vivo or in vitro. The administration conditions and dosage may be set as appropriate. The candidate substances are not particularly limited, and may include, for example, proteins, peptides, nucleic acids, oligonucleotides, peptide nucleic acids, small molecule compounds, large molecule compounds, etc. The candidate substances may be formulated and administered as appropriate.

[0050] The screening method of the present invention is preferably carried out by providing a control group for comparison with a group administered with the candidate substance (administered group). The results obtained by comparing one or more detection values ​​selected from IL5RA, LSR, and BCMA in the administered group and the control group may be used as an indicator to select a prophylactic or therapeutic agent for the target multiple myeloma. For example, if, after administering a candidate substance to a model animal that has developed multiple myeloma, one or more detection values ​​selected from IL5RA, LSR, and BCMA are lower in the administered group than in the control group, the administered candidate substance may be selected as a therapeutic agent or a potential therapeutic agent. Alternatively, for example, if, after administering a candidate substance to a model animal that has already completed treatment for multiple myeloma, one or more detection values ​​selected from IL5RA, LSR, and BCMA are lower in the administered group than in the control group, the administered candidate substance may be selected as a prophylactic agent or a potential prophylactic agent. The prophylactic agent may be a drug that prevents the onset of multiple myeloma or a drug that prevents relapse.

[0051] (Preventive or therapeutic agent of the present invention) The preventive or therapeutic agent of the present invention is a preventive or therapeutic agent for multiple myeloma and is not particularly limited in that it contains an active ingredient that targets 1 or 2 selected from IL5RA and LSR. The preventive or therapeutic agent of the present invention may be an IL5RA-targeted agent for the prevention or treatment of multiple myeloma, or an LSR-targeted agent for the prevention or treatment of multiple myeloma, or both. The active ingredient that targets 1 or 2 selected from IL5RA and LSR is not particularly limited, but may be, for example, a substance that specifically binds to 1 or 2 selected from IL5RA and LSR (a binder), or a substance that specifically inhibits 1 or 2 selected from IL5RA and LSR (an inhibitor). Furthermore, the active ingredient is not particularly limited in any way, and may be, for example, a protein, peptide, nucleic acid, oligonucleotide, peptide nucleic acid, small molecule compound, large molecule compound, or a combination thereof, but may be, for example, an antibody. That is, the active ingredient may be, for example, 1 or 2 selected from anti-IL5RA antibody and anti-LSR antibody. For example, benralizumab, a humanized anti-IL5RA monoclonal antibody used in the treatment of bronchial asthma, is known as an anti-IL5RA antibody. The prophylactic or therapeutic agent of the present invention may be selected by the screening method of the present invention. The prophylactic or therapeutic agent of the present invention may further contain one or more optional components. The optional components are not particularly limited, but may be components commonly used in pharmaceuticals, such as surfactants, pH adjusters, buffers, isotonic agents, chelating agents, preservatives, excipients, stabilizers, binders, lubricants, osmotic pressure adjusters, colorants, disintegrants, carriers, solvents, etc. Furthermore, the dosage form of the prophylactic or therapeutic agent of the present invention is not particularly limited, and may be, for example, an injection, a tablet, a capsule, a granule, a powder, a liquid, etc.

[0052] (Therapeutic Method of the Present Invention) The therapeutic method of the present invention is a method for treating multiple myeloma. The therapeutic method of the present invention is not particularly limited in that it includes the following steps (x) and (y), but may further include one or more other steps: (x) A step of diagnosing multiple myeloma, comprising detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject; (y) A step of administering a multiple myeloma treatment drug to a subject diagnosed with multiple myeloma.

[0053] In the treatment method of the present invention, the diagnosis in step x may be a diagnosis based on the indicators provided by the second method of the present invention described above, or a diagnosis further combining other diagnostic indicators for multiple myeloma. Furthermore, the therapeutic agent in step y may be the therapeutic agent of the present invention described above, another therapeutic agent, or a combination thereof.

[0054] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0055] 1. Methods 1-1. Patient and Sample Collection Patients diagnosed with multiple myeloma (MM) at the Cancer Institute Hospital of Japan between January 2022 and August 2024 were prospectively enrolled. Diagnosis was made according to the International Myeloma Working Group criteria (International Myeloma Working Group. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 121, 749-757 (2003).; Rajkumar SV, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol 15, e538-548 (2014).). Bone marrow and serum samples were collected at the time of diagnosis. Bone marrow samples from malignant lymphoma patients without bone marrow infiltration were used as controls for bone marrow analysis. For serum analysis, serum samples from healthy individuals who visited the Cancer Institute Hospital of Japan for cancer screening and had no history of cancer or prior diagnosis were used as controls. Clinicopathological information was obtained from electronic records and databases. All patients provided written informed consent. Procedures involving human subjects were conducted in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of the Japanese Foundation for Cancer Research.

[0056] 1-2. Sample Preparation A 2 mL sample of bone marrow aspirate was collected. The bone marrow aspirate was centrifuged at 3,000 rpm for 15 minutes, the supernatant was collected, and stored at -80°C until use. CD138-positive cells derived from bone marrow aspirate from MM patients were isolated from the bone marrow aspirate according to the manufacturer's instructions by magnetic activation cell sorting using an autoMACS Pro Separator (Miltenyi Biotec) with CD138 microbeads (#130-105-961 and #130-051-301, Miltenyi Biotec).

[0057] 1-3. Isolation of extracellular vesicles (EVs) from bone marrow supernatant or serum using MagCapture TM Exosomes (EVs) were isolated using the Exosome Isolation Kit PS Ver 2 (Fujifilm Wako Co., Ltd.) according to the manufacturer's instructions. Briefly, 200 μL of bone marrow supernatant or serum was thawed and diluted 1:5 with phosphate-buffered saline (PBS). Magnetic beads were added to the sample, and the mixture was incubated at room temperature for 60 minutes. The beads were then washed three times by placing the tube on a magnetic stand and removing the supernatant. EVs were eluted with 30 μL of sample buffer (25 mM Tris, 1% SDS, 4% glycerol, 2% 2-mercaptoethanol, 0.01% bromophenol blue) for subsequent protein digestion and LC / MS analysis, and with 30 μL of phase-transfer surfactant (PTS) buffer containing 12 mM sodium deoxycholate and 12 mM N-lauroyl sarcosinate for subsequent protein quantification and Western blot analysis.

[0058] 1-4. Western blot isolated EVs were lysed in SDS sample buffer. Proteins were separated on a 10% Bolt gel and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 4% Block ACE (#UKB80, Snow Brand Megmilk Co., Ltd.) and subsequently incubated overnight with mouse primary antibody against CD9 (SHI-EXO-M01, Cosmo Bio, dilution 1:2000) and mouse primary antibody against CD63 (SHI-EXO-M02, Cosmo Bio, dilution 1:2000). Afterward, incubation was performed with HRP-labeled anti-mouse IgG (#NA931-1ML, Cytiva, dilution 1:10000) and detected with Western Lightning ECL Pro (#NEL121001EA). Images are from ChemiDoc. TM The images were acquired using a Touch Imaging System (manufactured by BIORAD).

[0059] 1-5. Nanoparticle Tracking Analysis (NTA) EV samples obtained from bone marrow and serum were diluted 1:50 with PBS, and Nanoparticle Tracking Analysis (NTA) was performed using a ZetaView (Particle Metrix) with a laser wavelength of 488 nm and corresponding software ZetaView version 8.05.16 SP3 to measure size and particle concentration.

[0060] 1-6. Protein Digestion: Protein samples were dissolved in lysis buffer (10% SDS, 100mM TEAB, pH 8.5), reduced with 120mM TCEP at 55°C for 15 minutes, and then alkylated with 500mM iodoacetamide at room temperature in the dark for 45 minutes. Subsequently, the proteins were acidified with 27.5% phosphoric acid. The samples were transferred to an S-trap micro spin column (ProtiFi), centrifuged at 4,000×g for 30 seconds, and captured on a filter. The samples were washed twice with 90% methanol containing 100mM TEAB. The proteins were digested with trypsin / Lys-C Mix at 47°C for 2 hours. The peptides were eluted with 50mM TEAB, 0.2% formic acid, and 50% acetonitrile.

[0061] 1-7. Liquid Chromatography Mass Spectrometry Peptides were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) combined with an UltiMate 3000 RSLC nanosystem (Thermo Fisher Scientific). A two-step linear gradient was used, increasing the concentration of acetonitrile containing 0.1% formic acid from 2% to 30% over 110 minutes, and then from 30% to 95% over 2 minutes, at a flow rate of 250 nL / min for a total of 120 minutes. Full MS scans were acquired using FTMS with a resolution of 120,000 over the m / z range from 197.08 to 1500.00. The scan was performed with a compensation voltage of -60.00 V, an ion implantation time of 50.0 ms, a scan duration of 0.339 seconds, and a mass tolerance of 500 mmu. Subsequently, an MS / MS scan was performed using a data-dependent acquisition method in ITMS mode, with activation type HCD, compensation voltage -40.0V, and ion implantation time of 22 ms.

[0062] 1-8. Protein Identification and Label-Free Quantification Protein identification and label-free quantification were performed using Proteome Discoverer 2.5 software (Thermo Fisher Scientific). For protein identification, the proteome dataset was searched in the UniprotKB Human Database using the Sequest HT search engine (Thermo Fisher Scientific). The false detection rate (FDR) threshold for peptide identification was set to <1%. Minora Feature Detector was used for label-free quantification. The search criteria were set as follows: trypsin specificity, dynamic modification of oxidation, static modification of carbamide methyl, and tolerance for up to two cleavage errors. A peptide length of at least 6 amino acids was required. The mass tolerance for precursor mass was 10 ppm, and the tolerance for fragment ions was 0.6 Da.

[0063] 1-9. Multiple Reaction Monitoring (MRM) For MRM (Multiple reaction monitoring) analysis, a Shimadzu LCMS-8060 triple quadrupole mass spectrometer and a Thermo Fisher Scientific UltiMate 3000 RSLC nanosystem were used. A two-step linear gradient was used, increasing acetonitrile containing 0.1% formic acid from 2% to 40% over 10 minutes, and then from 40% to 98% over 2 minutes, at a flow rate of 300 nL / min for a total of 25 minutes. The following parameters were used: nebulizer gas 0.5 L / min, dry gas 10.0 L / min, heated gas 10.0 L / min, interface ESI, interface voltage 2.0 kV, interface temperature 300 °C, DL temperature 100 °C, heat block temperature 200 °C, conversion di 10.0 kV, CID gas pressure 300 kPa. Details of the optimized MRM parameters are shown in Table 1. The absolute quantification was performed using the peak area ratio of endogenous peptides and their corresponding stable isotope-labeled internal standards (Scrum, Inc., Tokyo, Japan), and the data was analyzed using LabSolutions (ver. 5.97).

[0064]

[0065] 1-10. RNA Extraction and Real-Time Quantitative PCR Assay: RNA was extracted from bone marrow CD138-positive cells using the RNeasy Mini Kit (Qiagen) or RNeasy Micro Kit (Qiagen) according to the manufacturer's protocol. cDNA was synthesized using SuperScript III Reverse Transcriptase (#18090050, Invitrogen). A cDNA array of 48 normal human tissues was purchased from OriGene (HMRT104). RNA levels were measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific). RT-qPCR was performed using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific) with TaqMan FastAdvanced Master Mix for qPCR (#4444556, Thermo Fisher Scientific). 100 pg of cDNA was used for BCMA, and 2-5 ng of cDNA was used for IL5RA. The TaqMan primers used were as follows: BCMA (Assay ID: Hs00171292_m1), GAPDH (Assay ID: Hs02786624_g1), IL5RA (Assay ID: Hs01064360_m1). The relative expression levels of each target were 2 -ΔCT The formula was used to normalize for GAPDH.

[0066] 1-11. Serum-soluble BCMA ELISA assay method. Frozen serum samples were thawed and diluted to 1:100. A human BCMA / TNFRSF17 DuoSet ELISA Kit (#DY193E, R&D Systems) was used according to the manufacturer's protocol. ELISA plates were read at 450 nm using a SUNRISE absorbance microplate reader (TECAN) with XFLUOR4 software version 4.51.

[0067] 1-12. Statistical Analysis In bone marrow EV proteome analysis, the proteomic composition of MM patients and controls was compared using a two-sided Welch's t-test. Bone marrow EV proteins that met the following criteria were selected as biomarker candidates: 1) For proteins detected only in MM patients, unique peptides ≥ 2 and valid value ≥ 50%. 2) For proteins with elevated expression in MM patients, p < 0.05, FC ≥ 10, unique peptides ≥ 2, and valid value ≥ 50%. In serum EV proteome analysis, the concentrations of each peptide were compared between MM patients and healthy controls using the Mann-Whitne U test. Progression-free survival (PFS) was estimated using the Kaplan-Meier method and compared between groups using the log-rank test. In multivariate analysis, Cox proportional hazards regression was used to identify factors associated with PFS. PFS was defined as the time from diagnosis to disease progression or death from any cause. All statistical analyses were performed using R version 4.4.0 with the EZR package (Jichi Medical University Saitama Medical Center, Japan). A p<0.05 value was considered statistically significant.

[0068] 2. Results 2-1. A schematic diagram of the workflow for proteomic analysis of EVs isolated from bone marrow and serum is shown in Figure 1. EVs were isolated using the Tim4 affinity method. The isolated EV samples showed enrichment of the EV marker proteins CD9 and CD63 compared to whole bone marrow aspirate and serum (Figure 2). Furthermore, nanoparticle tracking analysis (NTA) revealed that the median size distribution of EVs isolated from bone marrow and serum was 150–200 nm (Figure 3). Overall, these data confirmed the successful isolation of EVs from both bone marrow aspirate and serum, and validated the quality for further analysis.

[0069] 2-2. Selection of Biomarker Candidates by Comprehensive Proteomic Analysis of Bone Marrow EV Proteins Bone marrow aspiration samples were collected from nine MM patients for diagnostic purposes. The characteristics of the MM patients are shown in Table 2. Bone marrow samples from 10 lymphoma patients without bone marrow infiltration were used as controls (Table 3). Label-free quantification of EVs obtained from bone marrow aspiration fluids from MM patients and controls identified 8839 proteins, of which 463 were detected only in MM patients' EVs and 138 were detected only in control EVs (Figure 4). Furthermore, in MM patients' EVs, 126 proteins were significantly elevated in expression compared to controls (fold change [FC] ≥ 2, p < 0.05), while 144 proteins were decreased in expression (FC ≤ 0.5, p < 0.05) (Figure 5).

[0070]

[0071]

[0072] From 463 proteins detected only in EVs of MM patients, eight proteins meeting the criteria of having ≥2 unique peptides and an effective value ≥50% were selected as biomarker candidates (Table 4). Furthermore, from 126 proteins that showed significantly increased expression in EVs of MM patients, six proteins meeting the criteria of FC ≥10, ≥2 unique peptides, and an effective value ≥50% were also selected as biomarker candidates (Table 5). Thus, a total of 14 proteins were identified as biomarker candidates through comprehensive proteomic analysis of bone marrow EVs (Figure 6). These 14 proteins were subjected to a secondary screening process.

[0073]

[0074]

[0075] 2-3. Identification of Serum EV Biomarkers by Targeted Proteome Analysis Because bone marrow examination is invasive and sometimes insufficient material is obtained, the feasibility of detecting these biomarkers in serum using less invasive routine blood tests was evaluated. Thirteen MM patients and 30 healthy individuals were included in this secondary screening process (Tables 2 and 6). Absolute quantification of 14 previously selected biomarker candidate proteins was performed by targeted proteome analysis using multiple reaction monitoring (MRM) with corresponding stable isotope-labeled peptides. The expression levels of each protein were compared between MM patients and healthy individuals. As a result, BCMA (FC=3.64, p<0.001), IL5RA (FC=1.33, p=0.048), and LSR (FC=1.63, p=0.017) were significantly elevated in serum-derived EVs from MM patients compared to those in healthy individuals (Figure 7).

[0076]

[0077] To clarify the relationship between these EV proteins and prognosis, receiver operating characteristic (ROC) curves were plotted and cutoff values ​​were set for each EV protein (Figure 8abc). At a median follow-up period of 21 months, patients with elevated serum EV BCMA levels at diagnosis (≥1.95 fmol / mL) had a significantly shorter progression-free survival (PFS) compared to patients without elevated serum EV BCMA levels (15-month PFS; 60% [95% confidence interval (95% CI), 12.6-88.2] vs 87.5% [95% CI, 38.7-98.1], p = 0.018; Figure 9a). Furthermore, patients with elevated serum EV IL5RA levels at diagnosis (≥2.85 fmol / mL) had a significantly shorter PFS compared to patients without elevated serum EV IL5RA levels (15-month PFS; 33.3% [95% CI, 0.9-77.4] vs. 90.0% [95% CI, 47.3-98.5], p < 0.001; Figure 9b). Notably, all three patients with elevated serum IL5RA experienced disease progression during follow-up. No correlation was found between serum EV LSR and PFS (15-month PFS, 77.8% [95% CI, 36.5-93.9] vs. 75.0% [12.8-96.1], p = 0.083; Figure 9c).

[0078] These data suggest that serum EV BCMA, serum EV IL5RA, and serum EV LSR can serve as diagnostic biomarkers for newly diagnosed MM patients (NDMM patients), and furthermore, that serum EV BCMA and serum EV IL5RA can function as prognostic biomarkers for this patient group.

[0079] 2-4. Serum soluble BCMA is elevated in MM patients. We identified BCMA in serum-derived EV as a diagnostic and prognostic biomarker for NDMM patients. BCMA is mainly expressed in MM cells. It is also known that BCMA undergoes proteolysis by γ-secretase, resulting in its release into the bloodstream as a soluble form. Several reports indicate that this soluble BCMA is elevated in the serum of MM patients, and that elevated serum concentrations may be associated with poor outcomes. We confirmed these findings in this cohort. Serum soluble BCMA concentrations at diagnosis were significantly higher in MM patients (n=20) compared to healthy individuals (n=30) (Figure 10). We also evaluated the correlation between serum soluble BCMA at diagnosis and prognosis. This analysis included 13 patients from the same cohort as those whose serum EV proteins were analyzed, with a follow-up period of 12 months or more. Thresholds were determined by plotting ROC curves (Figure 8d). Patients with elevated serum soluble BCMA levels at diagnosis tended to have shorter progression-free survival (PFS) compared to patients without elevated levels, but the difference was not statistically significant (Figure 11).

[0080] 2-5. To further clarify the significance of serum EV IL5RA as an independent prognostic biomarker for NDMM, along with serum EV BCMA, a multivariate analysis was performed incorporating established prognostic factors (age, bone marrow plasma cell percentage, revised International Staging Classification, and serum soluble BCMA). Among these established prognostic factors, serum EV IL5RA was the only significant factor associated with PFS after multivariate analysis (hazard ratio 33.11 [95% CI, 2.34-468.00], p = 0.009; Table 7). These results indicate that serum EV IL5RA is an independent and potentially more potent prognostic biomarker than other known prognostic factors.

[0081]

[0082] 2-6. BCMA and IL5RA Expression is Specifically Increased in MM Cells To evaluate the expression of BCMA and IL5RA in MM cells and compare their expression with that of normal human tissue, reverse transcription quantitative polymerase chain reaction (RT-qPCR) of BCMA and IL5RA was performed using RNA extracted from bone marrow CD138-positive cells, and their expression levels were compared with those of normal tissue. As a result, the expression of both TNFRSF17, the RNA encoding BCMA, and IL5RA was significantly elevated in CD138-positive cells from MM patients compared with that of normal human tissue (Figure 12). cDNA of TNFRSF17 and IL5RA was detected in bone marrow, lymph nodes, lymphocytes, and several lymphoid tissues such as the tonsils. Furthermore, small amounts were also present in organs rich in plasma cells and lymphocytes, such as the respiratory tract, gastrointestinal tract, and thymus. However, the expression levels were significantly higher in CD138-positive cells compared to lymphoid tissues and lymphocyte-rich tissues.

[0083] 3. Discussion During the exploratory phase of this study, 8,839 proteins were identified from bone marrow-derived extracellular proteins (EVs) from MM patients and controls. To our knowledge, this is the largest proteomic dataset of bone marrow-derived EVs reported to date. While there have been several reports on proteomic data of EVs specifically derived from bone marrow mesenchymal stem cells, this study is unique in that it analyzed EVs obtained from the entire bone marrow supernatant, including those secreted from plasma cells and other cells within the bone marrow microenvironment.

[0084] In the validation phase of this study, we identified serum EV BCMA, serum EV IL5RA, and serum EV LSR as diagnostic biomarkers for MM patients from our comprehensive proteomic dataset. Furthermore, serum EV BCMA and serum EV IL5RA also function as prognostic biomarkers for NDMM patients. The current gold standard for MM diagnosis requires bone marrow examination, but such tests are invasive, sometimes do not yield sufficient material, and make evaluation difficult. In addition, cytogenetic abnormalities such as del(17p), t(4;14), t(14;16), and gain / amp(1q) are established prognostic factors in MM patients, but the detection of these abnormalities also requires bone marrow examination, presenting similar challenges. Moreover, these abnormalities do not necessarily reflect the spatial heterogeneity of the tumor. Our biomarkers overcome these limitations by being available through routine blood tests, being less invasive, more accessible, and potentially more accurately reflecting the heterogeneous tumor ecology.

[0085] BCMA is expressed in mature B cells and plasma cells, and its expression is elevated in myeloma cells (MMs). BCMA is a target for myeloma treatments such as antibody-drug conjugates, chimeric antigen receptor T-cell therapy, and bispecific antibodies. Furthermore, BCMA is cleaved by γ-secretase and released into the bloodstream as a soluble form. This soluble BCMA is elevated in the serum of MM patients, and elevated serum concentrations are thought to be related to poor progression-free survival (PFS). However, this study is the first to report elevated BCMA expression in extracellular viable cells (EVs) of MM patients.

[0086] The role of IL5RA in the pathogenesis of multicellular matrix (MM) is currently unknown. IL5RA is a subunit that, along with IL5βc, constitutes the interleukin-5 (IL-5) receptor. The intracellular domain of IL5RA is associated with JAK2, and the intracellular domain of IL5βc is associated with JAK1. When IL-5 and IL5RA bind, IL5βc is recruited, and a heterodimer is formed. Heterodimerization leads to tyrosine phosphorylation and subsequent activation of JAK2. This triggers activation and dimerization of STAT-5, which then translocates to the nucleus and binds to the promoters of target genes, promoting the transcription of genes such as anti-apoptotic genes (BCL2L1, BCL-2), genes involved in cell proliferation (MYC, PIM1), and cell cycle regulatory genes (CCND1). We hypothesize that IL5RA promotes the survival and proliferation of MM cells by enhancing the transcription of these genes, resulting in more active and rapid MM cell proliferation and consequently a poor prognosis.

[0087] The current standard of care for MM patients eligible for transplantation is multi-drug induction therapy, high-dose chemotherapy / autologous peripheral blood stem cell transplantation, and subsequent maintenance therapy. For patients not eligible for transplantation, combination therapy including daratumumab is the standard of care. However, the optimal duration of these treatments remains unclear. The EV biomarkers identified in this study may provide guidance for determining the duration of treatment when monitored during the treatment period. Furthermore, while the prognosis for MM patients has improved significantly in recent years with the introduction of new drugs, the prognosis for patients who no longer respond to such drugs remains extremely poor. IL5RA expression was elevated in MM cells but only slightly in normal tissues. This suggests that IL5RA may be a promising new therapeutic target.

[0088] Recent studies have reported a poor prognosis for patients who relapse within 18 months of treatment initiation or within 12 months of autologous peripheral blood stem cell transplantation, and these patients are often referred to as "functionally high-risk" patients. It is noteworthy that all three patients in this study with elevated serum EV-IL5RA levels experienced disease progression, and that this progression occurred within 18 months of treatment initiation. In other words, the biomarkers discovered in this study allowed us to identify clinically important high-risk patients who need better treatment options.

[0089] Several clinical trials are underway to evaluate treatment intensification, such as early introduction of CAR-T cell therapy, in high-risk MM patients. Identifying patients at high risk of disease progression before treatment initiation will allow for better selection of suitable candidates for such future clinical trials. This study provides valuable insights into the ecology of MM and helps identify patients at high risk of disease progression. In conclusion, we were able to identify a novel diagnostic and prognostic EV biomarker in NDMM patients.

Claims

1. A method for predicting the prognosis of multiple myeloma, comprising the step of detecting IL5RA in extracellular vesicles derived from a subject's biological sample.

2. A composition for predicting the prognosis of multiple myeloma, comprising a reagent for detecting IL5RA in extracellular vesicles derived from a subject's biological sample.

3. A kit for predicting the prognosis of multiple myeloma, including reagents for detecting IL5RA in extracellular vesicles derived from biological samples of subjects.

4. A method for diagnosing multiple myeloma, comprising the step of detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject.

5. A diagnostic composition for multiple myeloma, comprising a reagent for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a biological sample of a subject.

6. A diagnostic kit for multiple myeloma comprising reagents for detecting one or more selected from IL5RA, LSR, and BCMA in extracellular vesicles derived from a subject's biological sample.

7. The method according to claim 1 or 4, wherein the biological sample is serum.

8. A method for screening for a preventive or therapeutic agent for multiple myeloma, comprising the steps of: administering a candidate substance to a subject; detecting one or more extracellular vesicles derived from the subject, selected from IL5RA, LSR, and BCMA; and selecting a target preventive or therapeutic agent for multiple myeloma using the obtained results as an indicator.

9. A prophylactic or therapeutic agent for multiple myeloma comprising an active ingredient that targets one or two selected from IL5RA and LSR.

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