WO2022059601A1 - Procédé de visualisation et procédé d'acquisition d'informations - Google Patents

Procédé de visualisation et procédé d'acquisition d'informations Download PDF

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Publication number
WO2022059601A1
WO2022059601A1 PCT/JP2021/033230 JP2021033230W WO2022059601A1 WO 2022059601 A1 WO2022059601 A1 WO 2022059601A1 JP 2021033230 W JP2021033230 W JP 2021033230W WO 2022059601 A1 WO2022059601 A1 WO 2022059601A1
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substance
image
drug
visualizing
target cell
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PCT/JP2021/033230
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English (en)
Japanese (ja)
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大輔 富岡
優 高橋
洋 根岸
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コニカミノルタ株式会社
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Priority to JP2022550516A priority Critical patent/JPWO2022059601A1/ja
Publication of WO2022059601A1 publication Critical patent/WO2022059601A1/fr

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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
    • 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
    • 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
    • G01N33/483—Physical analysis of biological material
    • 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

Definitions

  • the present invention relates to a visualization method for visualizing a first substance and a second substance in a sample, and an information acquisition method for acquiring information on the first substance and the second substance in a sample.
  • the candidate compounds that can be used as pharmaceuticals only the compounds evaluated to be effective are selected by screening.
  • Drugs often bind to target substances such as receptors, enzymes, ion channels, and transporters in the body. Then, the drug reaches the cell, binds to the target substance, and then acts on the target substance to show its medicinal effect. Therefore, the judgment as to whether the drug has reached the target substance or whether the drug that has reached the target substance has a medicinal effect is an important criterion in screening.
  • Non-Patent Documents 1 and 2 As a method for detecting a target substance or the like, an immunostaining method using an antigen-antibody reaction or the like on a prepared tissue specimen is known (see, for example, Non-Patent Documents 1 and 2).
  • Non-Patent Document 1 describes a phase 1 study in which ABT-888, which is a PARP inhibitor, is used in combination with topotecan.
  • ABT-888 is orally administered
  • topotecan is intravenously administered
  • PAR poly (ADP-ribose)
  • ⁇ H2AX DNA damage marker
  • the presence or absence of ⁇ H2AX has been confirmed by an immunostaining method using a fluorescent labeling substance.
  • Non-Patent Document 2 describes the relationship between DNA double-strand breaks caused by cytotoxic drugs and DNA double-strand breaks related to apoptosis.
  • Non-Patent Document 2 by observing the co-expression of a plurality of markers in cells, whether the expression of ⁇ H2AX in a specific cell is DNA double-strand break by a cell-damaging drug or DNA 2 related to apoptosis. We are analyzing whether it is a main-strand break. The presence or absence of ⁇ H2AX has been confirmed by an immunostaining method using a fluorescent labeling substance.
  • Non-Patent Documents 1 and 2 since only the result of administration of a drug or the like is evaluated, it can be determined whether or not the drug was effective, but the cause cannot be grasped. Therefore, the methods described in Non-Patent Documents 1 and 2 are insufficient in evaluating the characteristics of a drug. For example, when a drug is metabolized, the existence of the drug cannot be confirmed, and even if the drug is successful as a result, it may be determined that the drug is not successful. In addition, because the amount of drug delivered to individual cells is unclear, the effect may not have been exhibited because the drug did not actually arrive, or the drug arrived but the effect was exhibited due to another factor. It remained unclear if it was not done.
  • An object of the present invention is to provide a visualization method and an information acquisition method that can accurately evaluate the characteristics of an agent.
  • the visualization method as one means for solving the above-mentioned problems is caused by the step of visualizing the first substance expressed in the target cell of the target on which the agent acts in the sample, and the action of the agent in the sample. It has a step of visualizing a second substance.
  • the information acquisition method as one means for solving the above-mentioned problems includes a step of obtaining a first image in which the first substance expressed in the target cell of the target on which the acting substance acts in the sample is visualized, and a step in the sample. A step of obtaining a second image in which the second substance generated by the action of the working substance is visualized, and a step of obtaining information on the first substance and the second substance from the first image and the second image.
  • the characteristics of the acting substance can be accurately evaluated.
  • FIGS. 1A to 1C are schematic views for explaining information regarding the first substance and the second substance.
  • the information acquisition method includes a step of obtaining a first image in which the first substance expressed in the target cell on which the agent acts in the sample is visualized, and a step of obtaining the first image of the agent in the sample. It has a step of obtaining a second image in which the second substance produced by the action is visualized, and a step of obtaining information on the first substance and the second substance from the first image and the second image.
  • the order in which the steps for obtaining the first image and the steps for obtaining the second image are performed is not particularly limited.
  • the order of performing the steps of obtaining the first image and the step of obtaining the second image may be the step of obtaining the first image first, the step of obtaining the second image first, or simultaneously. good.
  • the first image in which the first substance expressed on the target cell on which the active substance acts in the sample is visualized is obtained.
  • the first image may be an image in which the first substance before the agent is administered is visualized, or an image in which the first substance after the agent is administered may be visualized.
  • sample is not particularly limited.
  • specimens include blood (serum, plasma), urine, nasal fluid, saliva, stool, body cavity fluid (medullary fluid, ascites, pleural effusion, etc.), biological tissues, and cells or tissues produced by culture.
  • the acting substance is a substance that acts on the target cell.
  • agents include antibodies, antibody drug conjugates (ADCs), drugs such as ordinary pharmaceutical compounds, and toxic substances.
  • Pharmaceutical compounds include substances that inhibit DNA replication in cells.
  • Specific examples of pharmaceuticals include therapeutic agents for various diseases, especially anti-cancer agents.
  • "acting on a target cell” includes a case of directly acting on a target cell and a case of binding to a receptor or the like in the target cell.
  • target cells include effector T cells, effector memory T cells, helper CD8 T cells including central memory T cells, B cells, NK cells, macrophages, and dendritic cells.
  • helper CD8 T cells including central memory T cells, B cells, NK cells, macrophages, and dendritic cells.
  • cells are helper CD8 T cells from the perspective of developing cancer immunotherapeutic agents.
  • the first substance is a cell marker that is specifically expressed on the target cell on which the acting substance acts, and may be a substance that the acting substance acts directly on, or may be another substance that does not directly act on the acting substance. good.
  • cell markers include CD3, CD4, CD11c, CD20, CD21, CD56, CD68, CD163.
  • “specifically expressed” does not necessarily mean that it is expressed only in the target cell, but in the target cell to the extent that the target cell and other cells existing around the target cell can be distinguished. It suffices if the expression level is high and the expression level in other cells is low.
  • the method of visualizing the first substance is not particularly limited.
  • Examples of the method for visualizing the first substance include an immunostaining method and a staining method using a molecular recognition group similar to an antibody.
  • a biological tissue section containing the first substance specifically expressed in the target cell on which the agonist acts after a certain period of time has passed after the administration of the agonist is immunostained to obtain an immunostained image (first image) in which the first substance is visualized by a fluorescent label or an enzyme label.
  • fluorescent substance-accumulated particles can be used as the fluorescent label in the immunostaining method.
  • 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.
  • an aptamer or SNAP-tag is used as a molecular recognition group to obtain a first image.
  • the second substance produced by the action of the working substance is visualized to obtain the second image.
  • the first image and the second image are obtained at the same site (for example, the same tissue section) or the corresponding site (for example, adjacent tissue sections) in the sample.
  • the second substance is a substance produced by the action of the acting substance.
  • the second substance may be a biomarker produced by the inhibition of replication.
  • the target on which the agent acts is not particularly limited, and is, for example, an intracellular organelle or a substance present in a cell (for example, a protein such as an enzyme or a receptor).
  • the second substance include a DNA damage response marker, a microtubule polymerization marker, a microtubule depolymerization marker, an apoptosis marker, a surrogate marker, and a stress marker.
  • DNA damage response markers include 53BP1, Ku80, MDC1, Nbs1, pATM, pATR, pChk1, pDNA-PK, Rad51, ⁇ H2AX.
  • microtubule polymerization markers and depolymerization markers include APC, CLASP, CLIP170, EB1, EB3, MACF, MCAK, STIM1, XMAP215, ⁇ -Tubulin, ⁇ -Tubulin.
  • apoptosis markers include the active form of Caspase-3.
  • the method of visualizing the second substance is not particularly limited.
  • methods for visualizing the second substance include an immunostaining method and a staining method using a molecular recognition group similar to an antibody.
  • the second substance for example, DNA
  • the biological tissue section (specimen) containing the fragment) is immunostained to obtain an immunostaining image (second image) in which the second substance produced by the action of the first substance is visualized by a fluorescent label or an enzyme label.
  • the fluorescent label in the immunostaining method the above-mentioned fluorescent substance-accumulated particles can be used.
  • information on the first substance and the second substance is obtained from the first image and the second image.
  • FIGS. 1A to 1C are schematic views for explaining information regarding the first substance and the second substance.
  • FIG. 1A shows an example when the drug effect (antitumor effect) is high
  • FIGS. 1B and 1C show an example when the drug effect (antitumor effect) is low.
  • the small black circles in FIGS. 1A to 1C indicate CD8, and the small white circles indicate Granzyme B.
  • the schematic views of FIGS. 1A to 1C show the degree to which CD8 is expressed in the immune cells reached by the drug, and the action of the drug produces Granzyme B, which causes apoptosis of Granzyme B cancer cells.
  • FIG. 1A shows an example when the drug effect (antitumor effect) is high
  • FIGS. 1B and 1C show an example when the drug effect (antitumor effect) is low.
  • the small black circles in FIGS. 1A to 1C indicate CD8, and the small white circles indicate Granzyme B.
  • the schematic views of FIGS. 1A to 1C show
  • FIG. 1A it is shown that the medicinal effect is large because many drugs (acting substances) reach the target cells and many Granzyme B cause apoptosis.
  • FIG. 1B many drugs (acting substances) have reached the target cells, but since there are not many Granzyme B, the degree of causing apoptosis is low, and the drug efficacy is shown to be small.
  • FIG. 1C it is shown that the medicinal effect is extremely small because the amount of the drug (acting substance) reached is small and the amount of Granzyme B is also small.
  • the type of information regarding the first substance and the second substance is not particularly limited as long as it is obtained from the first image and the second image.
  • the information regarding the first substance and the second substance includes information regarding the position or amount of the first substance and information regarding the position or amount of the second substance.
  • the step of obtaining information it is possible to obtain the amount of the first substance specifically expressed in the target cell to which the agent is bound and the amount of the second substance produced by the action of the agent. Further, it is possible to further obtain information on the action or effect of the agent based on the obtained information on the first substance and the second substance.
  • further information on the relationship between the position or amount of the first substance specific to the target cell and the position or amount of the second substance generated by the action of the agent May be generated. Specifically, with respect to information on how much the second substance was produced with respect to the amount of the first substance or the agent expressed from the target cell, or with respect to the amount of the first substance or the agent expressed from the target cell. Contains information on how many target cells produced the second substance.
  • information on the first substance and the second substance is obtained from the first image in which the first substance is visualized and the second image in which the second substance is visualized.
  • both the information on the first substance and the information on the second substance can be grasped, and the relationship between the first substance and the second substance can be accurately specified.
  • the drug is a drug in which the active substance is metabolized or cannot be labeled
  • the relationship between the active substance and the action of the active substance can be accurately identified, and the characteristics of the active substance can be accurately identified. Can be evaluated.
  • a step of visualizing a first substance expressed on a target cell on which an agent acts in a sample and a second substance generated by the action of the agent in the sample are used. It has a step of visualization.
  • the order in which the step of visualizing the first substance and the step of visualizing the second substance are performed is not particularly limited.
  • the step of visualizing the first substance may be performed first, the step of visualizing the second substance may be performed first, or the process may be performed at the same time.
  • the first substance expressed on the target cell on which the acting substance acts in the sample is visualized.
  • the step of visualizing the first substance can be performed by the same method as the "step of obtaining the first image" in the first embodiment.
  • the agent, target cell and first substance are the same as in the first embodiment.
  • the second substance produced by the action of the active substance in the sample is visualized.
  • the step of visualizing the second substance can be performed by the same method as the "step of obtaining the second image" in the first embodiment.
  • the second substance is the same as that of the first embodiment.
  • a lung cancer patient who received a drug for example, Opdivo
  • a drug for example, Opdivo
  • a lung cancer patient who did not receive the drug and received anti-PD-1 treatment We obtained tissue slides of lung cancer patients who did not perform the procedure.
  • the Texas red dye-encapsulating melamine resin particles thus produced were surface-modified by the following procedure.
  • the particles subjected to the surface amination treatment were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid) so that the final concentration of the solution was 10 mM.
  • SM (PEG) 12 succinimidyl- [(N-maleimideropionamide) -dodecaethylneglycol] ester, manufactured by Thermoscientific Co., Ltd. was mixed and reacted for 1 hour.
  • streptavidin (Wako Pure Chemical Industries, Ltd.) can be bound to Texas red accumulated melamine particles by performing thiol group addition treatment using N-succinimidyl S-aceticylthioacetylate (SATA) and then filtering with a gel filtration column. A streptavidin solution was obtained.
  • SATA N-succinimidyl S-aceticylthioacetylate
  • the above Texas red accumulated melamine particles and streptavidin were mixed in PBS containing 2 mM of EDTA and reacted at room temperature for 1 hour. 10 mM mercaptoethanol was added to terminate the reaction. After concentrating the obtained solution with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification, and the obtained streptavidin-bound Texas red accumulated melamine particles were used as a red PID stain.
  • Streptavidin 32243-24; Nacalai Tesque, Inc. was bound to a red PID stain to prepare a streptavidin-bound red PID.
  • EZ-Link maleimide-PEG11-biotin (21911; Thermo Fisher Scientific Co., Ltd.) was bound to the anti-mouse IgG1 antibody SB77e (ab99601; Abcam Co., Ltd.) to obtain a biotin-bound anti-mouse IgG1 antibody.
  • the red PID stain stains CD8.
  • An anti-FITC antibody (A150-112A; Fluor Laboratories, Inc.) was bound to a green PID stain to prepare an anti-FITC antibody-bound green PID.
  • anti-mouse IgG2a antibody LO-MG2a-7 (MCA421; Bio-Rad Laboratories Co., Ltd.) and fluorescein-5-maleimide (containing up to 2% of N, N-dimethylformamide) (F0810; Tokyo Chemical Industry) Co., Ltd.) was combined to prepare a FITC-bound anti-mouse IgG2a antibody.
  • Granzyme B is stained with the green PID stain.
  • DAB-stained tissue slides are deparaffinized, activated, and blocked, and a primary antibody mixed solution (anti-CD8 antibody C8 / 144B (mouse IgG1, M7103; Agilent Technologies, Inc.)) is added at room temperature. Reacted for 1 hour. Then, after washing the tissue slide with PBS, Fistfine Simple Stain MAX-PO (MULTI) (049-22831; Nichirei Bioscience Co., Ltd.) was added and reacted at room temperature for 30 minutes. The tissue slides were then washed with PBS, DAB with 30% hydrogen peroxide was added, reacted at room temperature for 3 minutes and then immersed in pure water for 5 minutes. Finally, the emission intensity of the DAB-stained area was measured with an optical microscope. In DAB staining, Granzyme B is stained.
  • MULTI Simple Stain MAX-PO
  • tissue slides were then washed with PBS, HistoGreen (E109; AbCys) was added, reacted at room temperature for 3 minutes and then immersed in pure water for 5 minutes. Finally, the emission intensity of the HistoGreen-stained area was measured with an optical microscope. With HistoGreen staining, CD8 is stained.
  • Table 1 shows the results of each measurement. "A” and “B” shown below are the results of tissue slides collected from lung cancer patients who received anti-PD-1 treatment, and “C” are lung cancer patients who did not receive anti-PD-1 treatment. Results for tissue slides taken from.
  • the presence or absence of the effect of anti-PD-1 treatment can be evaluated as the ratio of the number of bright spots of CD8 to the number of bright spots of Granzyme B.
  • the ratio of the number of bright spots of CD8 to the number of bright spots of Granzyme B is close to 1, and it can be seen that an equivalent number of Granzyme B is produced with respect to the number of expressed CD8. It can be evaluated that the effect is extremely high.
  • Example B it can be seen that the ratio of the number of bright spots of CD8 to the number of bright spots of Granzyme B is large, and the number of Granzyme B produced is small with respect to the number of expressed CD8. It can be evaluated as lower than A.
  • Example C the ratio of the number of bright spots of CD8 to the number of bright spots of Granzyme B is even larger, and the number of Granzyme B produced is less than one tenth of the number of expressed CD8. As it is known, it can be evaluated that the drug has no effect or is extremely low.
  • Comparative Examples A, B, and C the same value is shown regardless of the presence or absence of administration of the drug and the superiority or inferiority of the drug efficacy. From this, it can be seen that the effect of the drug cannot be accurately evaluated because color separation and the like are difficult in the stained image by DAB staining or histogreen staining.
  • Table 2 shows the result of dividing the number of bright spots of Granzyme B by the number of cells expressing CD8, not by the number of expressed CD8. Since CD8 is a protein and cannot directly evaluate the activity of the cells themselves, it is more accurate to evaluate the effect of the drug by the number of cells expressing the protein rather than the number of expressed proteins. Can be evaluated.
  • the number of bright spots of Granzyme B extracellularly is defined as the amount of Granzyme B released, regardless of the presence or absence of anti-PD-1 treatment, and is intracellular.
  • the number of bright spots of Granzyme B in the above can be evaluated as the production amount (expression level) of Granzyme B. From the examples of A, B and C in Table 1, the same number of T cells were generated, but when A, B and C in Table 1 were compared, the expression level of Granzyme B was different. From this, it can be seen that apoptosis does not occur in C (the drug efficacy is small).
  • the present invention it is useful for, for example, screening of an active substance and evaluation of the medicinal effect of the active substance.

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Abstract

Ce procédé de visualisation comprend les étapes consistant à : visualiser une première substance qui est exprimée sur une cellule cible sur laquelle agit une substance active dans un analyte; et visualiser une seconde substance qui est générée par l'action de la substance active dans l'analyte.
PCT/JP2021/033230 2020-09-16 2021-09-10 Procédé de visualisation et procédé d'acquisition d'informations WO2022059601A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012029342A1 (fr) * 2010-08-30 2012-03-08 コニカミノルタエムジー株式会社 Procédé de marquage tissulaire, procédé d'évaluation tissulaire et procédé de détection de biosubstance
JP2015502742A (ja) * 2011-11-10 2015-01-29 エネジェシス ファーマシューティカルズ，インク． ヒト骨格筋中の褐色脂肪細胞前駆細胞
US20160123964A1 (en) * 2014-11-05 2016-05-05 The Regents Of The University Of California Methods for stratifying non-responders to therapies that block pd1/pdl1 axis
JP2018536163A (ja) * 2015-11-22 2018-12-06 ヴェンタナ メディカル システムズ， インク． Ｐｄ−ｌ１陽性腫瘍組織における免疫細胞を同定する方法
WO2019131727A1 (fr) * 2017-12-27 2019-07-04 コニカミノルタ株式会社 Procédé pour évaluer un médicament
WO2019221062A1 (fr) * 2018-05-17 2019-11-21 コニカミノルタ株式会社 Procédé d'évaluation d'un médicament
WO2021106840A1 (fr) * 2019-11-27 2021-06-03 コニカミノルタ株式会社 Procédé de visualisation et procédé d'acquisition d'informations

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012029342A1 (fr) * 2010-08-30 2012-03-08 コニカミノルタエムジー株式会社 Procédé de marquage tissulaire, procédé d'évaluation tissulaire et procédé de détection de biosubstance
JP2015502742A (ja) * 2011-11-10 2015-01-29 エネジェシス ファーマシューティカルズ，インク． ヒト骨格筋中の褐色脂肪細胞前駆細胞
US20160123964A1 (en) * 2014-11-05 2016-05-05 The Regents Of The University Of California Methods for stratifying non-responders to therapies that block pd1/pdl1 axis
JP2018536163A (ja) * 2015-11-22 2018-12-06 ヴェンタナ メディカル システムズ， インク． Ｐｄ−ｌ１陽性腫瘍組織における免疫細胞を同定する方法
WO2019131727A1 (fr) * 2017-12-27 2019-07-04 コニカミノルタ株式会社 Procédé pour évaluer un médicament
WO2019221062A1 (fr) * 2018-05-17 2019-11-21 コニカミノルタ株式会社 Procédé d'évaluation d'un médicament
WO2021106840A1 (fr) * 2019-11-27 2021-06-03 コニカミノルタ株式会社 Procédé de visualisation et procédé d'acquisition d'informations

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