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WO2022024881A1 - Procédé de génération d'informations - Google Patents

Procédé de génération d'informations Download PDF

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Publication number
WO2022024881A1
WO2022024881A1 PCT/JP2021/027167 JP2021027167W WO2022024881A1 WO 2022024881 A1 WO2022024881 A1 WO 2022024881A1 JP 2021027167 W JP2021027167 W JP 2021027167W WO 2022024881 A1 WO2022024881 A1 WO 2022024881A1
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WO
WIPO (PCT)
Prior art keywords
substance
image
target substance
binding
binding substance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2021/027167
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English (en)
Japanese (ja)
Inventor
哲 齊藤
優 高橋
洋希 藤原
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Konica Minolta Inc
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Konica Minolta Inc
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Filing date
Publication date
Application filed by Konica Minolta Inc filed Critical Konica Minolta Inc
Priority to JP2022540222A priority Critical patent/JPWO2022024881A1/ja
Publication of WO2022024881A1 publication Critical patent/WO2022024881A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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

Definitions

  • the present invention relates to an information generation method for generating information on a target substance and a binding substance that binds to the target substance in a sample.
  • Drug development involves the processes of drug discovery target identification, validation, candidate compound selection, screening, compound optimization, clinical trials, application, and approval. Moreover, in drug development, among many candidate compounds, only a few drugs are approved, and a long period of time and enormous cost are required. Therefore, improving productivity is an important issue in drug development. Each pharmaceutical company employs various criteria to identify projects to continue and projects to abandon in order to improve the productivity of medicines (see, for example, Non-Patent Document 1).
  • Non-Patent Document 1 describes, as an example of criteria for identifying projects to be continued and projects to be abandoned, exposure of the drug to the target tissue, binding to the target site of action, and target-mediated function expression of the drug. ..
  • the exposure of a drug to a target tissue includes not only the exposure of the target tissue but also the blood concentration of the drug and the concentration of the drug in the tissue.
  • Binding to a target point of action includes evaluation of drug occupancy with respect to receptors measured using PET (Positron emission tomography) and evaluation of drug distribution in tissues.
  • Function expression mediated by a drug includes a pharmacological mechanism of action.
  • An object of the present invention is to provide an information generation method capable of grasping information on a target substance and a binding substance and accurately evaluating the characteristics of the binding substance.
  • the information generation method as one means for solving the above-mentioned problems is a step of obtaining a first image in which the target substance in the sample is visualized, and a binding substance that can be bound to the target substance in the sample is visualized. It has a step of obtaining a second image and a step of generating information regarding the relationship between the target substance and the binding substance in the sample based on the first image and the second image.
  • the characteristics of the binding substance can be accurately evaluated.
  • FIG. 1 is a flowchart for explaining an information generation method according to an embodiment of the present invention.
  • 2A and 2B are conceptual diagrams for explaining an example of information obtained from the first image and the second image.
  • FIG. 1 is a flowchart for explaining an information generation method according to an embodiment of the present invention.
  • the information generation method includes a step of obtaining a first image in which a target substance is visualized (S100) and a step of obtaining a second image in which a binding substance is visualized (S100). S110) and a step (S120) of obtaining information on the relationship between the target substance and the binding substance from the first image and the second image.
  • the order in which the step of obtaining the first image (S100) and the step of obtaining the second image (S110) are performed is not particularly limited.
  • the step of obtaining the second image (S110) may be performed after performing the step of obtaining the first image (S100), or the step of obtaining the first image after performing the step of obtaining the second image (S110) (S100). ) May be performed. Further, since the subsequent image processing and the like can be simplified, it is preferable that the first image and the second image are images obtained for the same field of view in the same region.
  • the target substance is not particularly limited as long as the binding substance can be bound.
  • target substances include nucleic acids (single-stranded or double-stranded DNA, RNA, polynucleotides, oligonucleotides, PNAs (peptide nucleic acids), etc., or nucleosides, nucleotides and modified molecules thereof. ); Proteins (polypeptides, oligopeptides, receptors present on the cell membrane of cells, etc.); Amino acids (including modified amino acids); Sugars (oligosaccharides, polysaccharides, sugar chains, etc.); Lipids; Exosomes; or these Includes modified molecules and complexes of.
  • target substances include 5T4, AXL, BCMA, C4.4A, CA6, Cadherin3, Cadherin6, CEACAM5, CD16, CD19, CD22, CD37, CD56, CD71, CD138, CD142, CD352, DLL3, EphA2, EphrinA4. , ETBR, Fc ⁇ RIII, FOLR1, FGFR2, FGFR3, GCC, HER1 (EGFR), HER2, HER3, Integrin ⁇ V, LAMP1, LIV1, Mesothelin, MUC1, MUC16, NaPi2B, Nectin4, NOTCH3, PD Includes PTK7, SLAMF7, SLITRK6, STEAP1 and TROP2.
  • the binding substance is not particularly limited as long as it can bind to the target substance.
  • the binding substance is, for example, a substance administered to a sample and is a substance that binds to a target substance.
  • binding substances include antibodies, antibody drug conjugates (ADCs), drugs such as ordinary pharmaceutical compounds, and toxic substances.
  • Pharmaceuticals include substances that inhibit the replication of DNA in cells.
  • Examples of pharmaceuticals include therapeutic agents for various diseases, especially anti-cancer agents.
  • the target substance in the sample may be visualized to obtain the first image, or the target substance in the sample may already be visualized to obtain the first image. Further, in the step of obtaining the first image, images of the target substance visualized at different time points after the administration of the binding substance may be obtained as a plurality of first images.
  • the method of visualizing the target substance is not particularly limited.
  • methods for visualizing a target substance include immunostaining and staining using a molecular recognition group similar to an antibody.
  • a biological tissue section containing the target substance for example, a receptor present on the cell membrane of a cell
  • an immunostaining image in which the target substance is visualized by a fluorescent label or an enzyme label is obtained.
  • fluorescent label phosphor-accumulated particles (fluorescent dye-accumulated particles) can be used.
  • Fluorescent integrated particles are based on particles made of organic or inorganic substances, and have a structure in which multiple phosphors (for example, fluorescent dyes and semiconductor nanoparticles) are contained therein and / or adsorbed on the surface thereof. It is a nano-sized particle.
  • fluorescent dyes constituting the phosphor-accumulated nanoparticles include rhodamine-based dyes, Cy-based dyes, AlexaFluor® dyes, BODIPY-based dyes, squarylium-based dyes, cyanine-based dyes, aromatic ring-based dyes, and oxazines.
  • the fluorescent substance integrated particles can be produced according to a known method (see, for example, Japanese Patent Application Laid-Open No. 2013-57937).
  • a recognition substance for specifically binding to the target substance is added to the phosphor-accumulated particles.
  • the recognition material is selected so that the fluorophore-accumulated particles and the target material bind directly or indirectly.
  • recognition substances include proteins such as nucleotide chains, avidin, streptavidin, antibodies, and low molecular weight compounds such as biotin.
  • an antibody primary antibody
  • the recognition substance is an antibody (secondary antibody) that specifically binds to an antibody (primary antibody) that specifically binds to the target substance.
  • a substance that specifically binds to the secondary antibody for example, avidin, streptavidin, or biotin
  • an aptamer or SNAP-tag is used as a molecular recognition group to obtain a first image.
  • the binding substance in the sample may be visualized to obtain the second image, or the binding substance in the sample may be visualized to obtain the second image. Further, in the step of obtaining the second image, the visualized images of the binding substance may be obtained as a plurality of second images at different time points after the administration of the binding substance.
  • the binding substance in the step of obtaining the second image may be one bound to the target substance or may be one before binding to the target substance.
  • the method of visualizing the binding substance is not particularly limited.
  • methods for visualizing binding substances include immunostaining and staining with molecular recognition groups similar to antibodies.
  • a biological tissue section containing the binding substance is immunostained, and the binding substance is labeled with a fluorescent label or an enzyme label.
  • a visualized immunostaining image (second image) is obtained.
  • the fluorescent label or enzyme label that can be used for the immunostaining method is the same as when the first image was obtained.
  • the recognition substance added to the phosphor-accumulated particles is the same as that at the time of acquiring the first image, except that the phosphor-accumulated particles and the binding substance are selected to be directly or indirectly bonded. ..
  • an aptamer or SNAP-tag is used as a molecular recognition group to obtain a second image.
  • step (S120) of obtaining information on the relationship between the target substance and the binding substance information on the relationship between the target substance and the binding substance in the sample is obtained from the first image and the second image.
  • the type of information regarding the relationship between the target substance and the binding substance is not particularly limited as long as it is obtained from the first image and the second image.
  • Examples of information on the relationship between the target substance and the binding substance include information on the positional relationship between the target substance and the binding substance, information on the relationship between the amount of the target substance and the binding substance, and changes in the positional relationship between the target substance and the binding substance. Includes information and information on changes in the relationship between the amount of target and binding substances.
  • the information on the positional relationship between the target substance and the binding substance is the information on the positional relationship of the binding substance with respect to the target substance.
  • the position of the target substance is specified from the first image
  • the position of the binding substance is specified from the second image, and based on this information, whether or not the binding substance reaches within a predetermined range from the target substance. Can be obtained as information regarding the positions of the target substance and the binding substance. This makes it possible to determine how close the binding substance is to the target substance and whether the binding substance can exert its action.
  • the information regarding the change in the position of the target substance and the binding substance is the information on the change in the positional relationship of the binding substance with respect to the target substance.
  • the position of the target substance at a certain first time point after administration of the binding substance is specified from the first image
  • the position of the target substance at the second time point after a predetermined time elapses from the first time point is specified from the other first image.
  • the position of the binding substance at the first time point is specified from the second image, and the position of the binding substance at the second time point is specified from the other second image.
  • the information regarding the relationship between the amount of the target substance and the amount of the binding substance is the information on the relationship between the amount of the binding substance and the amount of the target substance. For example, by specifying the amount of the target substance from the first image, specifying the amount of the binding substance from the second image, and calculating the ratio of the amount of the binding substance to the amount of the target substance, how much with respect to the target substance. It is possible to evaluate whether or not the binding substance of the above is bound, that is, the amount (occupancy rate) of the binding substance bound to the target substance.
  • the amount of the target substance or the binding substance is the amount of fluorescence or light emitted from the fluorescent label or the enzyme label bound to the target substance or the binding substance. It can be identified by measuring the number of points or the number of fluorophore-accumulated particles bound to the target substance or binding substance.
  • the information regarding the amount of the target substance and the change in the amount of the binding substance is the information on the relationship between the change in the amount of the binding substance and the change in the amount of the target substance.
  • the amount of the target substance at a certain first time point after administration of the binding substance is specified from the first image
  • the amount of the target substance at the second time point after a predetermined time elapses from the first time point is specified from the other first image.
  • the amount of the binding substance at the first time point is specified from the second image, and the amount of the binding substance at the second time point is specified from the other second image.
  • Method of generating information on the amount of target substance and the amount of binding substance Here, an example of a method for generating information regarding the amount of target substance and the amount of binding substance will be described in detail. Here, a method of estimating the amount of the target substance and the amount of the binding substance in the sample using the calibration curve will be described.
  • a tissue section containing a target substance and a binding substance is reacted with a biotinylated antibody against the target substance and a biotinylated antibody against the binding substance, and then the spray avidin-modified phosphor-accumulated particles are reacted to cause an immune tissue.
  • Perform staining For staining of the target substance and the binding substance, two types of the target substance and the binding substance may be stained with dyes having different emission wavelengths for one tissue section. In addition, each of the target substance and the binding substance may be stained with individual tissue sections. When the target substance and the binding substance are stained with individual tissue sections, the tissue section for staining the target substance and the tissue section for staining the binding substance are preferably adjacent sections.
  • the first image and the second image are obtained from the stained tissue section using a DSU confocal microscope (Olympus Corporation) or the like. Specifically, a digital image of a first image in which the target substance is visualized and a second image in which the binding substance is visualized is obtained. The emission intensity of the target substance stained in the obtained first image and the emission intensity of the binding substance stained in the obtained second image are quantified, respectively.
  • the method of quantifying is not particularly limited. Examples of the method for digitizing include, for example, digitization using image processing software (Imaris; Bitplane). In this case, the total value of the color development intensities of the target substance and the binding substance is used as a numerical value representing each of the amount of the target substance and the amount of the binding substance.
  • the total value of the color development intensities of the target substance and the binding substance is included in the fluorescent substance-accumulated particles labeled with each. It is measured at the emission wavelength of the dye to be used, and is a numerical value representing each of the amount of the target substance and the amount of the binding substance.
  • the target substances and the bonds that are close to each other are replaced.
  • a substance that is, a target substance having a binding substance nearby and a binding substance having a target substance nearby
  • the total value of the luminescence intensity of the extracted target substance and the binding substance may be quantified. ..
  • the measured luminescence intensity value of the target substance can be converted into the amount of the target substance using the calibration curve.
  • a standard sample in which a recombinant protein expressing a known concentration of a target substance is immobilized on a slide glass at a constant concentration or a constant density can be used to prepare a calibration curve for converting to the amount of the target substance. .. Then, the sample is stained in the same manner as described above, and the emission intensity of the target substance is quantified based on the image in which the stained tissue section is visualized.
  • the relationship between the amount of the target substance and the numerical value of the emission intensity of the target substance (calibration curve of the target substance) can be obtained.
  • a calibration curve can also be obtained by using a homogenate of the whole cell expressing the target substance or the cell membrane fraction instead of the recombinant protein expressing the target substance.
  • the measured luminescence intensity value of the binding substance can be converted into the amount of the binding substance using the calibration curve.
  • a standard binding substance in which a known concentration of a binding substance is fixed to a slide glass at a constant concentration or a constant density can be used to prepare a calibration curve for converting to the amount of the binding substance.
  • the sample is stained in the same manner as described above, and the emission intensity of the bound substance is quantified based on the image in which the stained bound substance is visualized.
  • the relationship between the numerical value of the emission intensity of the binding substance with respect to the amount of the binding substance by using multiple standard binding substances including the binding substances fixed at different concentrations or densities (calibration curve of the binding substance). Is obtained.
  • the amount of the target substance is obtained from the total value of the color development intensity of the target substance and the calibration curve of the target substance.
  • the amount of the binding substance is obtained from the total value of the color development intensity of the binding substance and the calibration curve of the binding substance.
  • the amount of the target substance and the amount of the binding substance are obtained from the estimated amount of the target substance and the estimated amount of the binding substance.
  • a binding substance eg, a drug
  • the binding substance that is, near
  • the amount of the binding substance in which the target substance is present as shown in FIG. 2A, the dose of the binding substance and the occupancy rate of the binding substance with respect to the target substance (how much the binding substance is bound to the target substance). Is it possible to find the relationship with?). Further, even if they are not at the same position, it is possible to estimate whether or not the binding substance can exert an effect (for example, medicinal effect) by obtaining the amount of the binding substance within a predetermined range from the target substance.
  • a first image stained with the target substance and a second image stained with the binding substance are obtained.
  • the first image and the second image are a first time point, which is a predetermined time point after the binding substance is administered to the sample, and a second time point, which is a time point after a predetermined time has elapsed from the first time point. Get about each.
  • the amount of the target substance and the amount of the binding substance for each of the first image and the second image by the same method as the above-mentioned method, the amount of the target substance and the amount of the target substance after a certain period of time have passed.
  • the change with the amount of binding substance can be obtained.
  • a binding substance for example, a drug
  • Occupancy rate with respect to can be known over time. For example, by finding the maximum value of the relational curve between the elapsed time and the occupancy rate, the dynamics of the binding substance (for example, pharmacokinetics) such as how long the binding substance should be bound to the target substance. ) Can be known.
  • the deviation rate of the binding substance from the target substance can be obtained from the first image and the second image at a time after the time showing the maximum value.
  • the present invention it is useful for, for example, screening of a drug, proof of the mechanism of action of a drug, evaluation of toxicity of a drug, and the like.

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Abstract

L'invention concerne un procédé de génération d'informations qui comprend : une étape consistant à obtenir une première image dans laquelle une substance cible dans un échantillon est visualisée ; une étape consistant à obtenir une seconde image dans laquelle un liant capable de se lier à la substance cible dans l'échantillon est visualisé ; et une étape consistant à générer des informations sur une relation entre la substance cible dans l'échantillon et le liant sur la base de la première image et de la seconde image.
PCT/JP2021/027167 2020-07-31 2021-07-20 Procédé de génération d'informations Ceased WO2022024881A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022540222A JPWO2022024881A1 (fr) 2020-07-31 2021-07-20

Applications Claiming Priority (2)

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JP2020-130490 2020-07-31
JP2020130490 2020-07-31

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WO2022024881A1 true WO2022024881A1 (fr) 2022-02-03

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015045962A1 (fr) * 2013-09-26 2015-04-02 コニカミノルタ株式会社 Procédé pour déterminer la quantité de matière biologique dans une section de tissu
WO2018159212A1 (fr) * 2017-02-28 2018-09-07 コニカミノルタ株式会社 Procédé de détection d'un composant constitutif d'un conjugué anticorps-médicament

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015045962A1 (fr) * 2013-09-26 2015-04-02 コニカミノルタ株式会社 Procédé pour déterminer la quantité de matière biologique dans une section de tissu
WO2018159212A1 (fr) * 2017-02-28 2018-09-07 コニカミノルタ株式会社 Procédé de détection d'un composant constitutif d'un conjugué anticorps-médicament

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