WO2026004792A1 - Procédé de mesure de l'activité de réponse immunitaire cellulaire et kit associé - Google Patents

Procédé de mesure de l'activité de réponse immunitaire cellulaire et kit associé

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Publication number
WO2026004792A1
WO2026004792A1 PCT/JP2025/022448 JP2025022448W WO2026004792A1 WO 2026004792 A1 WO2026004792 A1 WO 2026004792A1 JP 2025022448 W JP2025022448 W JP 2025022448W WO 2026004792 A1 WO2026004792 A1 WO 2026004792A1
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Prior art keywords
pbmcs
measuring
subject
plasma
cells
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English (en)
Japanese (ja)
Inventor
太郎 齊藤
裕太 東久世
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Canon Medical Diagnostics Corp
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Canon Medical Diagnostics Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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

Definitions

  • the present invention relates to a method for measuring cellular immune response activity and a kit for use therein.
  • Cellular immunity is one of the immune responses of the body that eliminates foreign substances that invade from the outside.
  • Cellular immunity is an immune response system that recognizes foreign substances, such as pathogens, that have invaded the body and activates CD4 + T cells and/or CD8 + T cells via antigen-presenting cells, thereby eliminating the foreign substances (or cells containing the foreign substances, such as infected cells) (Non-Patent Document 1). Measuring the activity of the cellular immune response allows us to determine the level of the body's immune response.
  • the activity of the cellular immune response can be measured by adding an antigen derived from a pathogen or other pathogen to a sample containing peripheral blood mononuclear cells (PBMCs) derived from a subject, stimulating white blood cells such as T cells contained in the PBMCs, and then detecting effector molecules such as cytokines (e.g., interferon- ⁇ ) produced by the stimulated white blood cells, detecting the gene expression of effector molecules in white blood cells, or detecting white blood cells expressing effector molecules.
  • PBMCs peripheral blood mononuclear cells
  • Non-Patent Document 2 As examples of methods for measuring the activity of a subject's cellular immune response to pathogens, methods for measuring the activity of a subject's cellular immune response to SARS-CoV-2 have been reported, including a method using ELISpot technology (Non-Patent Document 2), an ELISA method (Non-Patent Document 3), a reverse transcription polymerase chain reaction (RT-PCR) method (Patent Document 1), and a flow cytometry technology (Non-Patent Document 4).
  • ELISpot technology Non-Patent Document 2
  • ELISA method As examples of methods for measuring the activity of a subject's cellular immune response to SARS-CoV-2 have been reported, including a method using ELISpot technology (Non-Patent Document 2), an ELISA method (Non-Patent Document 3), a reverse transcription polymerase chain reaction (RT-PCR) method (Patent Document 1), and a flow cytometry technology (Non-Patent Document 4).
  • Samples containing white blood cells such as T cells used in methods for measuring cellular immune response activity include whole blood, which is collected as is, and PBMCs, which are separated from collected blood. From the perspective of long-term storage and transportation of samples, it is preferable that the samples can be frozen.
  • Cell cryopreservation solutions such as CELLBANKER (registered trademark) (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) are known as preservation solutions for stably cryopreserving cells.
  • PBMCs can be stored in a frozen state for long periods of time by using the above-mentioned cell cryopreservation solution, making them more suitable than whole blood for long-term storage.
  • PBMCs are often used as samples to measure cellular immune response activity.
  • the inventors compared the activity signals obtained when measuring cellular immune response activity using PBMCs as a subject's sample with the activity signals obtained when measuring cellular immune response activity using whole blood as a subject's sample, they found that the activity signals obtained when measuring cellular immune response activity using PBMCs as a subject's sample were lower.
  • the present invention aims to provide a method for measuring cellular immune response activity with sufficient sensitivity using PBMCs as a subject's sample, and a measurement kit for measuring cellular immune response activity with sufficient sensitivity using PBMCs as a subject's sample.
  • a method for measuring the cellular immune response activity of a subject comprising: (1) incubating an aqueous medium containing peripheral blood mononuclear cells (PBMCs) collected from the subject, an antigen, and human serum or plasma; and (2) measuring immune effector molecules in the PBMCs after step (1).
  • PBMCs peripheral blood mononuclear cells
  • the content of human serum or plasma in the aqueous medium in step (1) is 20% or more and less than 100% based on the total mass of the aqueous medium.
  • measuring the immune effector molecule comprises measuring the expression level of mRNA of the immune effector molecule.
  • PBMCs peripheral blood mononuclear cells
  • kits according to any one of [9] to [13], further comprising a blood collection tube.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs which can be stored frozen for long periods, as a subject's sample.
  • 1 is a graph showing the results of measuring IFN- ⁇ (IFNG) gene expression using fresh blood.
  • 1 is a graph showing the results of measuring IFN- ⁇ (IFNG) gene expression using PBMC.
  • 10 is a graph showing the results of measuring IFN- ⁇ (IFNG) gene expression after antigen stimulation of PBMC in plasma from the same subject. This graph shows the results of a Student's t-test on the cellular immune activity when PBMCs were stimulated with antigen in the plasma of the same subject, and the cellular immune activity when PBMCs were stimulated with antigen in RPMI medium that did not contain human serum or human plasma.
  • This graph shows the average values for six cases of the relative values (relative values when the measurement value of fresh blood is set to 100) of the cellular immune activity when PBMCs were stimulated with antigen in plasma from the same subject and when PBMCs were stimulated with antigen in RPMI medium not containing human serum or human plasma, to the measurement value of fresh blood.
  • the method for measuring the cellular immune response activity of a subject of this embodiment includes: The method includes (1) incubating an aqueous medium containing peripheral blood mononuclear cells (PBMCs) collected from a subject, an antigen, and human serum or plasma, and (2) measuring immune effector molecules in the PBMCs after step (1).
  • PBMCs peripheral blood mononuclear cells
  • the incubation step (step (1)) is a step in which PBMCs are stimulated by adding an antigen. This causes the stimulated PBMCs to produce immune effector molecules.
  • PBMCs are stimulated by adding an antigen.
  • antigen-presenting cells By incubating PBMCs with antigen, antigen-presenting cells take up the antigen, process it as needed, and present it on the cell surface together with major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • the subject may be a human or a non-human animal.
  • the non-human animal is preferably a mammal.
  • the subject may be an individual infected with a specific pathogen, an individual suspected of being infected, an uninfected individual, or an individual vaccinated against a specific pathogen.
  • the specific pathogen is SARS-CoV-2
  • the subject may be an individual infected with SARS-CoV-2, an individual suspected of being infected with SARS-CoV-2, an individual uninfected with SARS-CoV-2, or an individual vaccinated against SARS-CoV-2.
  • Whole blood is usually collected from a vein and stored in a blood collection tube containing an anticoagulant.
  • blood collection tubes include EDTA blood collection tubes and sodium heparin blood collection tubes.
  • EDTA may affect lymphocyte activity, so sodium heparin blood collection tubes are more preferred.
  • Peripheral blood mononuclear cells are obtained by removing and separating plasma components, red blood cells, platelets, and granulocytes from whole blood. They include monocytes and lymphocytes such as T cells (CD4 + cells/CD8 + cells), B cells, NK cells, and dendritic cells.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells collected from a subject
  • PBMCs peripheral blood mononuclear cells collected from a subject
  • PBMCs peripheral blood mononuclear cells collected from a subject
  • antigen and human serum or plasma
  • the PBMCs and human serum or plasma can be simultaneously separated from whole blood and used.
  • the method of this embodiment may include, prior to step (1), a step of preparing PBMCs derived from a subject.
  • the step of preparing PBMCs derived from a subject may be or may include a step of separating and/or isolating PBMCs from whole blood (specimen) collected from the subject. Separation of PBMCs from whole blood may be performed by density gradient centrifugation, which may be performed using a density gradient medium such as Ficoll or LymphoPrep.
  • whole blood may be stratified in a centrifuge tube containing a density gradient medium, and by centrifuging, PBMCs will gather at the interface between the density gradient medium and plasma, and PBMCs can be obtained by collecting the white layer (PBMC layer) formed at this interface.
  • PBMC layer white layer
  • PBMCs can be cryopreserved, and cell function can be maintained for long periods of time. They can be cryopreserved using a preservation solution that stably freezes and preserves cells. Examples of such preservation solutions include cell cryopreservation solutions such as CELLBANKER (registered trademark) (manufactured by Nippon Zenyaku Kogyo Co., Ltd.).
  • CELLBANKER registered trademark
  • the PBMCs used in the method of this embodiment may also be frozen and thawed after collection from a subject. Even when frozen and thawed PBMCs are used, the method of this embodiment can still measure cellular immune response activity with sufficient sensitivity.
  • Human serum or plasma is obtained by centrifuging collected blood and recovering the liquid portion. Serum is obtained by centrifuging the collected blood after allowing it to clot naturally. On the other hand, plasma is obtained by centrifuging in the presence of an anticoagulant. Serum does not contain clotting factors such as fibrinogen.
  • Human serum or plasma may be derived from a single donor, for example, from the subject in the method of this embodiment. When human serum or plasma is derived from a subject, whole blood collected from the subject can be separated and used separately as PBMCs and human serum or plasma.
  • the antigens used in the method of this embodiment are not particularly limited, but include, for example, proteins derived from pathogens such as viruses, bacteria, and parasites; proteins derived from cells recognized as non-self in the body, such as tumor cells, infected cells, and inflammatory cells; and peptides containing the amino acid sequences that make up the above proteins. Two or more types of antigens may be combined.
  • Viruses from which antigens are derived include, for example, SARS-CoV-2, human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus, measles virus, rubella virus, poliovirus, and human papillomavirus (HPV).
  • Virus-derived proteins include, for example, the spike protein (S protein) of SARS-CoV-2, HIV Gag protein, HIV envelope protein (gp120), influenza virus M1 protein, influenza virus hemagglutinin (HA), CMV pp65 protein, hepatitis B virus surface antigen (HBsAg), and HPV E7 protein.
  • Bacteria from which antigens can be derived include, for example, Mycobacterium tuberculosis, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, and Escherichia coli. Proteins derived from bacteria include, for example, ESAT-6 from Mycobacterium tuberculosis and listeriolysin O (LLO) from Listeria monocytogenes.
  • ESAT-6 from Mycobacterium tuberculosis
  • LLO listeriolysin O
  • Tumor cells from which antigens are derived include, for example, cells expressing neoantigens, gp100, MART-1, PSA, CEA, AFP, MUC-1, CA125, CA19-9, HER2, Survivin, and WT-1 positive cells.
  • Proteins derived from tumor cells include, for example, tumor-specific proteins expressed on the cell surface of tumor cells.
  • Infected cells from which antigens are derived include cells infected with the viruses listed above.
  • Proteins derived from infected cells include, for example, viral proteins displayed on the cell surface of infected cells.
  • Inflammatory cells from which antigens are derived include cells involved in the autoimmune response that occurs in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, and type 1 diabetes.
  • Proteins derived from inflammatory cells include, for example, proteins expressed on the cell surface of inflammatory cells.
  • antigen presentation and recognition can be carried out in an aqueous medium in vitro or ex vivo, and those skilled in the art can select or prepare an aqueous medium suitable for that purpose based on their general knowledge.
  • the aqueous medium contains PBMCs, an antigen, and human serum or plasma.
  • the content of human serum or plasma in the aqueous medium during the incubation process may be 20% or more and less than 100% based on the total mass of the aqueous medium.
  • the medium other than the PBMCs and antigens e.g., storage buffer
  • the PBMCs and antigens prepared in this manner are added to human serum or plasma, the aqueous medium excluding the PBMCs and antigens becomes approximately 100% human serum or plasma.
  • the content of human serum or plasma in the aqueous medium in the incubation step may be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more, based on the total mass of the aqueous medium.
  • the aqueous medium may contain, for example, water and/or a liquid medium for cell culture in addition to PBMCs, antigen, and human serum or plasma, or may not contain a liquid medium.
  • the mass of human serum or plasma in the aqueous medium before incubation is not particularly limited as long as the effects of the present invention are achieved, but may be, for example, 0.05 mL to 1.0 mL, 0.05 mL to 0.5 mL, 0.1 mL to 0.3 mL, or 0.1 mL to 0.2 mL for 0.3 x 10 6 cells of PBMCs.
  • the conditions for incubating an aqueous medium containing PBMCs, an antigen, and human serum or plasma are not particularly limited and can be set appropriately by one skilled in the art.
  • the incubation can be carried out at a temperature ranging from 30°C to 40°C, preferably from 36.5°C to 37.5°C.
  • the incubation can be carried out for, for example, 1 hour or more, 1 hour to 72 hours, 1 hour to 48 hours, 1 hour to 24 hours, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 7 hours, 6 hours to 8 hours, 8 hours to 24 hours, 1 hour to 4 hours, or 2 hours to 4 hours.
  • the antigen content in the aqueous medium before incubation in the incubation step is also not particularly limited and can be set appropriately by those skilled in the art, but may be, for example, 0.1 to 20 ⁇ g/mL, 0.2 to 10 ⁇ g/mL, or 0.5 to 5 ⁇ g/mL.
  • the number of PBMC cells in the aqueous medium before incubation in the incubation step is not particularly limited and can be appropriately determined by those skilled in the art, but may be, for example, 0.2 ⁇ 10 6 cells to 0.3 ⁇ 10 6 cells/mL.
  • the measurement step (step (2)) in this embodiment is a step of measuring immune effector molecules in PBMCs after stimulating the PBMCs with an antigen in the incubation step.
  • the term "measuring" used in the context of cellular immune response activity may include not only quantifying cellular immune response activity, but also determining or testing for the presence or absence of significant cellular immune response activity based on the quantification results (e.g., in comparison with a negative control and/or a positive control).
  • White blood cells include lymphocytes, neutrophils, eosinophils, basophils, monocytes, etc. Monocytes differentiate into macrophages and dendritic cells, so they are also considered to be part of white blood cells. Examples of lymphocytes include T cells, B cells, and natural killer cells. Furthermore, T cells can include CD4 + T cells and CD8 + T cells. PBMCs are white blood cells excluding granulocytes (neutrophils, eosinophils, basophils, and mast cells).
  • the activity of the cellular immune response can be measured by measuring immune effector molecules in PBMCs after stimulating the PBMCs with antigen during the incubation process.
  • Immune effector molecules can be measured by detecting the immune effector molecules, detecting gene expression of immune effector molecules in PBMCs, detecting PBMCs expressing immune effector molecules, etc. Measuring immune effector molecules may involve measuring the expression level of mRNA of the immune effector molecule, or detecting and/or quantifying the protein that is the immune effector molecule.
  • Methods for measuring the mRNA expression levels of immune effector molecules include, for example, hybridization-based methods such as Northern blots and microarrays, RNase protection, and RNA-Seq (sequencing).
  • hybridization-based methods such as Northern blots and microarrays, RNase protection, and RNA-Seq (sequencing).
  • RT-PCR reverse transcription polymerase chain reaction
  • real-time RT-PCR include the SYBR® Green method and the TaqMan® method.
  • LAMP loop mediated isothermal amplification
  • Other methods with higher quantitative capabilities include, for example, the digital droplet PCR method and the digital droplet LAMP method.
  • RNA When measuring the expression level of mRNA of an immune effector molecule, RNA may be extracted from PBMCs, or specific cell subsets (e.g., CD4 + T cells or CD8 + T cells) may be isolated from PBMCs using flow cytometry or magnetic beads, and then RNA may be extracted. RNA extraction can be performed by a total RNA extraction method using commonly available commercially available magnetic beads, centrifugal columns, etc. Examples of RNA include mRNA, RNA fractions containing mRNA, and total RNA.
  • the mRNA to be measured may be mature mRNA that has undergone various processes such as splicing, or RNA (pre-mRNA) that has not yet been processed to become mature mRNA.
  • RT-PCR reverse transcription polymerase chain reaction
  • qPCR quantitative PCR
  • the qPCR master mix used for quantitative PCR can be the aforementioned SYBR® Green or TaqMan®.
  • the conditions for the qPCR reaction can be set appropriately by those skilled in the art.
  • the mRNA expression level of the immune effector molecule can be measured by normalizing the obtained relative mRNA expression level to an internal control gene. In the case of real-time RT-PCR, fluorescent signals are collected for each cycle of the qPCR reaction, allowing amplification of the PCR product to be monitored in real time.
  • the step of measuring immune effector molecules in PBMCs may be, for example, an RT-PCR method comprising the following steps (a) to (b) or (a) to (c): (a) lysing PBMCs to obtain a lysate containing mRNA; (b) capturing mRNA from the lysate, and (c) measuring mRNA levels.
  • Step (a) includes, for example, lysing cells using a cell lysis buffer.
  • Cell lysis elutes mRNA contained in PBMCs, yielding a lysate containing mRNA.
  • Any known cell lysis buffer can be used as the cell lysis buffer.
  • Step (b) may involve, for example, transferring the lysate obtained in step (a) to an mRNA capture substrate (e.g., an oligo(dT)-immobilized substrate) and incubating it at 4°C for 1 to 24 hours to capture the mRNA on the substrate.
  • an mRNA capture substrate e.g., an oligo(dT)-immobilized substrate
  • Step (c) includes, for example, adding a reverse transcription buffer containing a primer and reverse transcriptase to the mRNA isolated in step (b) to produce complementary DNA (cDNA), and contacting the cDNA with sense and antisense primers specific to the nucleic acid sequence of the immune effector molecule gene and DNA polymerase to obtain a DNA amplification product. This allows the expression level of the immune effector molecule mRNA to be measured.
  • the expression level of the immune effector molecule mRNA is quantified. Quantification may be calculated by comparing the amount of mRNA encoding one or more markers with a reference value.
  • reference values include the expression level of a gene whose expression is neither induced nor suppressed by a stimulant (e.g., an antigen peptide), and a specific example is the expression level of a known housekeeping gene (e.g., ⁇ -actin).
  • the mRNA expression level can be evaluated, for example, based on the cycle threshold value (Ct value) obtained using real-time RT-PCR.
  • Normalizing the Ct value of the immune effector molecule with the Ct value of the housekeeping gene allows for easy comparison of the expression levels of immune effector molecules between samples.
  • the detected amounts of one or more immune effector molecules and housekeeping genes are measured, the immune effector molecule is normalized using the Ct value of the housekeeping gene, and the normalized Ct value is converted to gene expression level, allowing for an approximate calculation of the amount of mRNA whose expression has changed (e.g., increased) due to antigen stimulation.
  • the expression level of one or more immune effector molecules induced by stimulation with an antigen with the expression level of the same immune effector molecules from a non-induction treated (solvent only control) sample it is possible to evaluate changes in the expression level of immune effector molecules due to stimulation.
  • the degree of induction can be roughly calculated from the fold increase (F.I.) value, which is the comparison of the gene expression level of the induced immune effector molecule with the gene expression level of the immune effector molecule that has not been subjected to induction treatment.
  • Methods for detecting and/or quantifying immune effector molecule proteins include, for example, methods using ELISpot technology, ELISA, and flow cytometry.
  • ELISpot technology is a method for detecting and/or quantifying molecules secreted by individual cells. By capturing the secreted immune effector molecules with antibodies and visualizing them as spots, the number of individual cells secreting immune effector molecules can be quantified.
  • ELISA is a method for detecting and/or quantifying the concentration of a specific protein. It uses specific antibodies to capture immune effector molecules secreted by stimulating PBMCs, and can quantify their concentration using chemiluminescence or fluorescence.
  • Flow cytometry technology uses fluorescently labeled antibodies to detect immune effector molecules on the surface or inside individual cells, and by measuring the fluorescence intensity of each cell, it is possible to analyze cellular characteristics and molecular expression.
  • the method of this embodiment allows the activity of cellular immune responses to be measured ex vivo.
  • the kit of this embodiment is for measuring the cellular immune response activity of a subject, and the measurement of the cellular immune response activity of a subject may be performed by the method described above.
  • the kit of this embodiment may be for measuring the cellular immune response activity of a subject using peripheral blood mononuclear cells (PBMCs) incubated with an antigen in human serum or plasma.
  • PBMCs peripheral blood mononuclear cells
  • the kit of this embodiment may include reagents for measuring antigens and/or immune effector molecules.
  • the antigens, immune effector molecules, and methods for measuring them are as described above.
  • the reagent for measuring immune effector molecules may be a reagent for measuring the expression level of mRNA of an immune effector molecule.
  • reagents for measuring the expression level of mRNA of an immune effector molecule include a total RNA extraction reagent, reverse transcriptase, random primers or oligo-dT primers, a qPCR master mix (typically containing DNA polymerase, dNTPs, MgCl 2 , a buffer, a fluorescent dye, etc., such as SsoAdvanced Universal SYBR Green Supermix (Bio-Rad)), primers specific to the gene of the target immune effector molecule, primers for an internal control gene (e.g., a housekeeping gene), and combinations thereof.
  • a RNA extraction reagent typically containing DNA polymerase, dNTPs, MgCl 2 , a buffer, a fluorescent dye, etc., such as SsoAdvanced Universal SYBR Green Supermix (Bio-Rad)
  • the reagent for measuring the expression level of mRNA of an immune effector molecule may include a reagent for purifying RNA from PBMCs and/or a reagent for synthesizing cDNA from mRNA.
  • the kit of this embodiment may optionally include an RNase inhibitor, and/or a PCR reaction plate, and/or reagents for isolating mRNA from total RNA.
  • the reagent for measuring immune effector molecules may be a reagent for detecting and/or quantifying proteins that are immune effector molecules.
  • Reagents for detecting and/or quantifying proteins that are immune effector molecules may include, for example, reagents selected from a capture antibody specific to an immune effector molecule, a secondary antibody that binds to the captured immune effector molecule, a substrate solution for an enzyme reaction, a stop solution for an enzyme reaction, a cell culture buffer, and combinations thereof; fluorescently labeled antibodies that detect immune effector molecules or cell surface markers, cell fixation/permeabilization reagents, cell staining buffers, dead cell staining reagents, and combinations thereof.
  • the kit of this embodiment may further include one or more selected from the following: a blood collection tube (e.g., a heparin sodium blood collection tube); a PBMC separation tube (e.g., SepMate-50 (manufactured by STEMCELL TECHNOLOGIES)) or reagent (e.g., density gradient media such as Ficoll (registered trademark) or Lymhoprep (registered trademark)) for separating and/or isolating PBMCs from whole blood; a cell preservation solution (e.g., CultureSure DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), CELLBANKER (registered trademark) (manufactured by Nippon Zenyaku Kogyo Co., Ltd.)); a wash buffer; a detergent; a surfactant; a cell culture vessel (e.g., a cell culture plate); and a cell culture medium (e.g., RPMI medium).
  • the blood collection tube may
  • the above-mentioned components that may be contained in a reagent for measuring immune effector molecules can be combined in any manner and used in the above-mentioned method for measuring cellular immune response activity.
  • the components may be combined to prepare one or more mixed solutions, which can then be used in the above-mentioned method for measuring cellular immune response activity.
  • the kit of this embodiment may include one or more containers containing the reagents, etc., as described above.
  • the reagents, etc. may be contained in separate containers for each type, or may be contained in any combination in a container.
  • the kit of this embodiment may include instructions for using the kit and/or a label indicating how to use the kit.
  • a label includes any written or recorded material provided on or with the kit, or otherwise associated with the kit.
  • the instructions for use and the label may be written directly on the instructions and label, or may be written as a link (e.g., a URL, QR code (registered trademark)) to a site that describes the instructions for use.
  • kits of this embodiment include the aspects described above in relation to the method for measuring cellular immune response activity, and can be applied without limitation.
  • Sample Preparation refers to samples collected in heparin sodium collection tubes, mixed by inversion, and then used for antigen stimulation within 24 hours. After collection, the number of cells in fresh blood was counted using Turk's solution.
  • Samples described as "frozen PBMC” refer to samples in which PBMCs were separated from blood collected in heparin sodium collection tubes and stored frozen. The PBMC separation process is shown below. Blood collected in heparin sodium collection tubes was dispensed into 50 mL Falcon tubes and centrifuged at 300 g and 25°C for 10 minutes to separate it into plasma and blood cell components.
  • the plasma was dispensed into another 50 mL Falcon tube, frozen in a -80°C freezer, and stored.
  • the blood cells remaining in the 50 mL Falcon tube were mixed with an equal volume of "PBS buffer containing 2% FBS (referred to as PBS-2%FBS)" as the plasma dispensed into another 50 mL Falcon tube.
  • PBS-2%FBS "PBS buffer containing 2% FBS
  • An equal volume of PBS-2% FBS as the blood volume collected was then added and mixed.
  • the blood cell suspension prepared in PBS-2% FBS as described above was dispensed into a SepMate-50 tube containing Lymphoprep and centrifuged at 1200 g and 25°C for 10 minutes to separate PBMCs.
  • the PBMCs were transferred to another 50 mL Falcon tube, and 10 mL of PBS-2% FBS was added to the suspension and the suspension was suspended. The suspension was then centrifuged at 300 g and 25°C for 8 minutes, after which the supernatant was removed. The same procedure was repeated to wash the PBMCs. The PBMCs were then suspended in 1 mL of Cell Banker and frozen in a -80°C freezer for storage.
  • PepGen Sf an overlapping peptide pool product based on the amino acid sequence of the spike protein constituting the wild-type Wuhan-Hu-1 strain of SARS-CoV-2, was prepared. These peptide pools consisted of multiple peptides corresponding to 15-amino acid fragments of the spike protein amino acid sequence, with adjacent peptides overlapping by 11 amino acids.
  • peptide plates were dissolved in antigen diluent (PBS buffer containing 20% DMSO) to a concentration of 20 ⁇ g/mL. 5 ⁇ L of each peptide pool solution was dispensed into each well of a cell culture plate. In addition to each peptide pool solution, 5 ⁇ L of the antigen diluent was also dispensed into other wells of the cell culture plate as a negative control.
  • the cell culture plates into which each solution was dispensed (hereinafter referred to as peptide plates) were frozen and stored in a -80°C freezer.
  • the RPMI medium was then transferred to the 50 mL Falcon tube (at this point, 10 mL of RPMI medium containing PBMCs was present in the 50 mL Falcon tube).
  • the RPMI medium containing the PBMCs was centrifuged at 300 g for 10 minutes at 25°C, and the supernatant was removed.
  • the PBMCs were washed again with human serum-free RPMI medium in the same manner as above, and then suspended in human serum-free RPMI medium. After that, the cells were counted and adjusted to a cell concentration similar to that of fresh blood.
  • the peptide plate which had been frozen, was kept at 25°C and the peptide pool solution was thawed.
  • Fresh blood or PBMCs with an adjusted cell concentration were added to each well of the peptide plate in an amount of 120 ⁇ L and mixed by stirring. After stirring, the mixture was incubated at 37°C for 4 hours to stimulate the leukocytes contained in the fresh blood or PBMCs (frozen PBMCs). After incubation, the peptide plate containing the peptide-stimulated leukocytes or PBMCs was frozen and stored at -80°C.
  • PBMCs cryopreserved in (4) were also subjected to the same procedure as for fresh blood, and the PBMCs were captured on the filter.
  • centrifugation refers to rotating the plate in a centrifuge and applying centrifugal force toward the bottom of the plate.
  • mRNA capture plate Another multiwell plate (hereinafter referred to as the mRNA capture plate) with oligo(dT) immobilized in each well was placed below the filter plate, with the wells of both plates stacked vertically. The plate was then centrifuged at 2500 g and 4°C for 5 minutes. The filter plate was removed, and the mRNA capture plate containing the cell lysate in its wells was incubated at 4°C for 1 to 24 hours. After incubation, each well was washed with wash buffer to remove any non-oligo(dT)-binding substances contained in the cell lysate.
  • log 2 F.I. A larger value indicates an increased level of mRNA expression induced by peptide stimulation.
  • Test Example 2 [When PBMCs were stimulated with antigen in human serum-free RPMI medium] Blood was collected in sodium heparin blood collection tubes from six subjects who had received the COVID-19 mRNA vaccine, and fresh blood and frozen PBMCs from the same subjects were used to compare the cellular immune activity when fresh blood was used with that when frozen PBMCs were used, according to the method described in Test Example 1. In the measurement of cellular immune activity using PBMCs, the step of antigen stimulation of PBMCs was carried out in RPMI medium that did not contain human serum or human plasma.
  • Example 1 [When PBMCs were stimulated with antigen in human plasma] Blood was collected in heparin sodium blood collection tubes from six subjects who had received the COVID-19 mRNA vaccine. Fresh blood and frozen PBMCs from the same subjects were used to compare the cellular immune activity when using fresh blood with that when using frozen PBMCs, according to the method described in Test Example 1. In the cellular immune activity measurement using PBMCs in this Example 1, the antigen stimulation step was performed in the plasma of the same subject, rather than in RPMI medium containing no human serum or plasma as in Test Example 2. For subjects 1, 2, and 5, 0.1 mL of plasma was added per 0.3 x 10 PBMC cells, and for subjects 3, 4 , and 6 , 0.1 mL of plasma was added per 0.2 x 10 PBMC cells.
  • PBMCs were stimulated with an antigen in the plasma of the same subject, and the IFN- ⁇ (IFNG) gene was measured as an indicator of cellular immune activity.
  • the results are shown in Table 2 and Figure 3.
  • PBMCs were stimulated with an antigen in the plasma of the same subject, expression increased beyond the margin of error for gene expression fluctuations (-1 ⁇ LOG 2 F.I. ⁇ 1).
  • the average values of the results for six cases were calculated and are shown in Figure 4.
  • a Student's t-test was performed on the cellular immune activity of PBMCs stimulated with an antigen in the plasma of the same subject compared to the cellular immune activity of PBMCs stimulated with an antigen in RPMI medium containing no human serum or human plasma.
  • the cellular immune activity of PBMCs stimulated with an antigen in the plasma of the same subject was significantly higher (p ⁇ 0.0001). Furthermore, the relative values (relative values when the measured value of fresh blood is set to 100) of the cellular immune activity when PBMCs were stimulated with antigen in plasma from the same subject and when PBMCs were stimulated with antigen in RPMI medium containing no human serum or human plasma, for six cases, are shown in Figure 5. The cellular immune activity when PBMCs were stimulated with antigen in plasma from the same subject showed a value close to 100%.

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Abstract

L'invention concerne un procédé de mesure de l'activité de réponse immunitaire cellulaire d'un sujet, le procédé consistant à : (1) incuber un milieu aqueux comprenant des cellules mononucléaires du sang périphérique (PBMC) collectées auprès d'un sujet, un antigène et du sérum ou du plasma humain ; et (2) après (1), mesurer des molécules effectrices immunitaires à l'intérieur des PBMC.
PCT/JP2025/022448 2024-06-24 2025-06-23 Procédé de mesure de l'activité de réponse immunitaire cellulaire et kit associé Pending WO2026004792A1 (fr)

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US20090081649A1 (en) * 2005-04-15 2009-03-26 Therim Diagnostica Ab Diagnostic method for detecting cancer by measuring amount of cytokine like il -6
JP2010527917A (ja) * 2007-05-03 2010-08-19 メディミューン，エルエルシー インターフェロンα誘導性薬力学的マーカー
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JP2017012010A (ja) * 2015-06-26 2017-01-19 チャ バイオテック カンパニー リミテッド 自然殺害細胞増殖方法、及び自然殺害細胞増殖用の組成物
CN108651439A (zh) * 2018-04-13 2018-10-16 上海韵飞生物科技有限公司 一种外周血单核细胞完全无血清冻存液及其方法
JP2019537712A (ja) * 2016-10-25 2019-12-26 バイオンテック エールエヌアー ファーマシューティカルズ ゲーエムベーハー 免疫療法剤のための用量決定
JP2021500912A (ja) * 2017-10-31 2021-01-14 チェム−フォルシュングスツェントルン フュル モレクラーレ メディツィン ゲゼルシャフト ミット ベシュレンクテル ハフツング 試験化合物の選択性を決定する方法
JP2023541366A (ja) * 2020-09-09 2023-10-02 キュー バイオファーマ， インコーポレイテッド １型真性糖尿病（ｔ１ｄ）を治療するためのｍｈｃクラスｉｉ ｔ細胞調節多量体ポリペプチド及びその使用方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090081649A1 (en) * 2005-04-15 2009-03-26 Therim Diagnostica Ab Diagnostic method for detecting cancer by measuring amount of cytokine like il -6
JP2010527917A (ja) * 2007-05-03 2010-08-19 メディミューン，エルエルシー インターフェロンα誘導性薬力学的マーカー
JP2016518828A (ja) * 2013-03-22 2016-06-30 インペリアル・イノベ−ションズ・リミテッド サイトカインストーム応答を査定するためのｉｎｖｉｔｒｏ方法
JP2017012010A (ja) * 2015-06-26 2017-01-19 チャ バイオテック カンパニー リミテッド 自然殺害細胞増殖方法、及び自然殺害細胞増殖用の組成物
JP2019537712A (ja) * 2016-10-25 2019-12-26 バイオンテック エールエヌアー ファーマシューティカルズ ゲーエムベーハー 免疫療法剤のための用量決定
JP2021500912A (ja) * 2017-10-31 2021-01-14 チェム−フォルシュングスツェントルン フュル モレクラーレ メディツィン ゲゼルシャフト ミット ベシュレンクテル ハフツング 試験化合物の選択性を決定する方法
CN108651439A (zh) * 2018-04-13 2018-10-16 上海韵飞生物科技有限公司 一种外周血单核细胞完全无血清冻存液及其方法
JP2023541366A (ja) * 2020-09-09 2023-10-02 キュー バイオファーマ， インコーポレイテッド １型真性糖尿病（ｔ１ｄ）を治療するためのｍｈｃクラスｉｉ ｔ細胞調節多量体ポリペプチド及びその使用方法

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