WO2021039592A1 - Method for supporting drug discovery, device for supporting drug discovery and program - Google Patents

Abstract

A method for supporting drug discovery, said method comprising: an acquisition step for acquiring the expression data of a target living organism which is fluorescently labeled at non-normal cells (target parts) and normal cells (control parts), said normal cells being to be compared with the non-normal cells, in multiple organs (body regions); and a display step for displaying list data, in which the expression data of the individual parts acquired in the acquisition step is quantified and listed, and threshold data, in which preset thresholds are indicated, on a display unit.

Description

Drug discovery support methods, drug discovery support devices and programs

The present invention relates to a drug discovery support method, a drug discovery support device, and a program.

In recent years, the number of cancer patients has been increasing, and various studies are required at the drug discovery stage in order to realize personalized treatment for each patient.
As an auxiliary role in the study of the drug discovery stage, the development of a technology for quantitatively evaluating the expression level of target biological substances such as intracellular cancer proteins using phosphor integrated nanoparticles (PID) has been promoted. (See, for example, Patent Document 1).

International Publication No. 2012/029342

However, a method for guessing which biological part the drug is suitable for when administering the drug has not yet been established. That is, in order to estimate the sensitivity of the drug at the time of administration and the corresponding biological part, it is necessary to make a step-by-step judgment from various data, and it is difficult to make a quick and accurate judgment.

An object of the present invention is to provide a drug discovery support method, a drug discovery support device, and a program capable of quickly and accurately determining the sensitivity of a drug and a suitable biological part.

In order to solve the above problems, the drug discovery support method of the present invention is used.
An acquisition step of acquiring expression information of a target part in a plurality of biological parts and a fluorescently labeled target biological substance of the target part to be compared with the target part.
A display step of displaying the list information obtained by digitizing the expression information of each part acquired by the acquisition step and displaying the list information, and the threshold information indicating a predetermined threshold value on the display unit.
Have.

Further, the drug discovery support device of the present invention is
An acquisition means for acquiring expression information of a target part in a plurality of biological parts and a fluorescently labeled target biological substance of the target part to be compared with the target part.
A display control means for displaying the list information obtained by digitizing the expression information of each part acquired by the acquisition means and displaying the list information, and the threshold value information indicating a predetermined threshold value on the display unit.
To be equipped.

In addition, the program of the present invention
Computer,
An acquisition means for acquiring expression information of a target part in a plurality of biological parts and a fluorescently labeled target biological substance of the target part to be compared with the target part.
A display control means for displaying on the display unit the list information obtained by digitizing the expression information of each part acquired by the acquisition means and displaying the list information and the threshold value information indicating a predetermined threshold value.
It is a program that functions as.

According to the present invention, it is possible to quickly and accurately determine the sensitivity of a drug and a suitable biological part.

It is a figure which shows the schematic structure of the drug discovery support system which concerns on this invention. It is a block diagram which shows the functional structure of the information acquisition apparatus of FIG. It is a figure which shows an example of evaluation support information. It is a figure for demonstrating evaluation support information. It is a figure which shows an example of evaluation support information.

Hereinafter, embodiments for carrying out the present invention will be described, but the present invention is not limited thereto.

<Drug discovery support system>
FIG. 1 shows an overall configuration example of a drug discovery support system 100 that executes the drug discovery support method according to the present invention. The drug discovery support system 100 acquires a microscopic image of a tissue sample stained with a predetermined staining reagent, and analyzes the acquired microscopic image to quantitatively express the expression of a specific biological substance in the tissue sample to be observed. It is a system that can output the feature amount represented by.

As shown in FIG. 1, the drug discovery support system 100 is configured by connecting a microscope image acquisition device 1A and an information acquisition device 2A as a drug discovery support device so as to be able to transmit and receive data via an interface such as a cable 3A. Has been done. The connection method between the microscope image acquisition device 1A and the information acquisition device 2A is not particularly limited. For example, the microscope image acquisition device 1A and the information acquisition device 2A may be connected by a LAN (Local Area Network) or may be wirelessly connected.

The microscope image acquisition device 1A is a known optical microscope with a camera, which acquires a microscope image of a tissue sample on a slide placed on a slide fixing stage and transmits it to the information acquisition device 2A.
The microscope image acquisition device 1A is configured to include an irradiation means, an imaging means, an imaging means, a communication I / F, and the like. The irradiation means is composed of a light source, a filter, and the like, and irradiates a tissue sample on the slide placed on the slide fixing stage with light. The imaging means is composed of an eyepiece, an objective lens, or the like, and forms a transmitted light, a reflected light, or a fluorescence emitted from a tissue sample on a slide by the irradiated light. The imaging means is a microscope-installed camera equipped with a CCD (Charge Coupled Device) sensor or the like, and images an image formed on an imaging surface by the imaging means to generate digital image data of a microscope image. The communication I / F transmits the image data of the generated microscope image to the information acquisition device 2A. In the present embodiment, the microscope image acquisition device 1A includes a bright field unit in which an irradiation means and an imaging means suitable for bright field observation are combined, and a fluorescence unit in which an irradiation means and an imaging means suitable for fluorescence observation are combined. It is possible to switch between bright field and fluorescence by switching the unit.

The microscope image acquisition device 1A is not limited to a microscope with a camera, and is, for example, a virtual microscope slide creation device (for example, a special microscope image acquisition device) that scans a slide on a slide fixing stage of a microscope to acquire a microscope image of the entire tissue specimen. (See Table 2002-514319) and the like may be used. According to the virtual microscope slide creating device, it is possible to acquire image data in which the entire image of the tissue sample on the slide can be viewed at once on the display unit.

The information acquisition device 2A analyzes the microscope image transmitted from the microscope image acquisition device 1A.
FIG. 2 shows an example of the functional configuration of the information acquisition device 2A. As shown in FIG. 2, the information acquisition device 2A is configured to include a control unit 21, an operation unit 22, a display unit 23, a communication I / F 24, a storage unit 25, and the like, and each unit is connected via a bus 26. There is.

The control unit 21 is configured to include a CPU (Central Processing Unit), a RAM (Random Access Memory), etc., executes various processes in cooperation with various programs stored in the storage unit 25, and executes various processes to obtain information acquisition device 2A. Controls the operation of.

The operation unit 22 is configured to include a keyboard equipped with character input keys, number input keys, various function keys, and a pointing device such as a mouse, and a key press signal operated by the keyboard and an operation signal by the mouse. Is output to the control unit 21 as an input signal.

The display unit 23 is configured to include, for example, a monitor such as a CRT (Cathode Ray Tube) or an LCD (Liquid Crystal Display), and displays various screens according to instructions of display signals input from the control unit 21.

The communication I / F 24 is an interface for transmitting and receiving data to and from an external device such as the microscope image acquisition device 1A, and inputs a fluorescent image captured by the microscope image acquisition device 1A to the information acquisition device 2A. Act as a means for.

The storage unit 25 is composed of, for example, an HDD (Hard Disk Drive), a semiconductor non-volatile memory, or the like. As described above, various programs, various data, and the like are stored in the storage unit 25.
In addition, the information acquisition device 2A may be provided with a LAN adapter, a router, or the like, and may be connected to an external device via a communication network such as a LAN.

<Drug discovery support method>
Hereinafter, the drug discovery support method according to the present invention will be described.
The drug discovery support methods according to this embodiment are mainly 1. 2. The process of staining sections of multiple organs (living parts) using a staining reagent. 2. The step of detecting the domain of the biological substance from the stained section and 3. It has a step of evaluating the expression level of the domain of the detected biological substance.

Above 1. In the above step, fluorescent nanoparticles can be used as a staining reagent.
Fluorescent nanoparticles are nano-sized particles that emit fluorescence when irradiated with excitation light, and are particles capable of emitting fluorescence having sufficient intensity to represent a target biological substance as a bright spot one by one.
As the fluorescent nanoparticles, preferably fluorescent substance integrated nanoparticles (PID) are used.

For example, when two types of nanoparticles are used, a biological substance recognition site that recognizes one domain is bound to one of the nanoparticles, and a predetermined fluorescent substance is contained therein. A fluorescent substance having a biological substance recognition site that recognizes a domain different from the biological substance recognition site of one nanoparticle is bound to the other nanoparticle, and has a fluorescence wavelength different from that of the fluorescent substance of one nanoparticle. It is included. That is, each nanoparticles contains biomaterial recognition sites that are different from each other and fluorescent substances having different fluorescence wavelengths. Therefore, different domains of the same protein can be clearly distinguished and stained due to the difference in fluorescence wavelength caused by the fluorescent substance.
If the biomaterial recognition site and the fluorescent substance (fluorescent wavelength) are different from each other, three or more types of nanoparticles may be used to detect three or more types of domains.
Details of the types and characteristics of fluorescent substances and methods for detecting biological substances are as follows.

(1) Target biomaterial The target biomaterial is a substance that is subject to immunohistochemical staining using a fluorescent label, mainly for detection or quantification from the viewpoint of pathological diagnosis, and is expressed in a tissue section. It is a biological substance.

Examples of the target biological substance applicable to the present embodiment include biological substances that can be used as biomarkers such as proteins (antigens) and mRNA. Specific examples include, but are not limited to, PD-L1, HER2, TIM-3, and the like.

(2) Fluorescent substance-accumulated nanoparticles Fluorescent substance-accumulated nanoparticles are based on particles made of organic substances or inorganic substances, and a plurality of fluorescent substances (for example, fluorescent organic dyes and quantum dots described later) are contained therein. Nano-sized particles having a structure that is and / or is adsorbed on the surface thereof.
As the fluorescent substance-accumulated nanoparticles, it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges and electrostatically interact with each other.
As the fluorescent substance-accumulated nanoparticles, fluorescent dye-accumulated nanoparticles, quantum dot-accumulated nanoparticles, and the like are used.

(2.1) Fluorescent substance Examples of the fluorescent substance used in the dyeing reagent for acquiring a fluorescent image include a fluorescent organic dye and quantum dots (semiconductor particles). When excited by ultraviolet-near-infrared light having a wavelength in the range of 200 to 700 nm, it is preferable to exhibit visible to near-infrared light having a wavelength in the range of 400 to 1000 nm.

Fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invigen) dye molecules, BODIPY (Invigen) dye molecules, cascade dye molecules, coumarin dye molecules, and eodin dyes Examples thereof include molecules, NBD-based dye molecules, pyrene-based dye molecules, Texas Red-based dye molecules, cyanine-based dye molecules, and the like.

Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2', 4,4', 5', 7,7'-hexachlorofluorescein, 6 -Carboxy-2', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy -Rhodamine, 5,6-dicarboxy-Rhodamine, Rhodamine 6G, Tetramethyl Rhodamine, X-Rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (manufactured by Invitrogen) ), methoxycumarin, eodin, NBD, pyrene, Cy5, Cy5.5, Cy7 and the like. It may be used alone or in a mixture of a plurality of types.

Quantum dots include group II-VI compounds, group III-V compounds, or group IV elements as components ("group II-VI quantum dots", "group III-V quantum dots", and "group III-V quantum dots, respectively". Either of "Group IV quantum dots") can be used. It may be used alone or in a mixture of a plurality of types.

Specific examples include, but are not limited to, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.

It is also possible to use a quantum dot having the above quantum dot as a core and a shell provided on the core. Hereinafter, as the notation of the quantum dot having a shell in the present specification, when the core is CdSe and the shell is ZnS, it is described as CdSe / ZnS. For example, CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO ₂ , Si / ZnS, Ge / GeO ₂ , Ge / ZnS and the like can be used, but are not limited thereto.
If necessary, the quantum dots may be surface-treated with an organic polymer or the like. For example, CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like can be mentioned.

As the fluorescent substance to be accumulated in the fluorescent substance-accumulated nanoparticles, in addition to the fluorescent organic dye and the quantum dot as described above, for example, Y ₂ O ₃ , Zn ₂ SiO _4, etc. are used as the parent body, and Mn 2+, Eu 3+, etc. are activated. Examples thereof include a "long afterglow phosphor" as an agent.

(2.2) Mother Body Among the mother bodies, the organic substances are resins generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin; styrene. Resins generally classified as thermoplastic resins such as resins, acrylic resins, acrylonitrile resins, AS resins (acrylonitrile-styrene copolymers), ASA resins (acrylonitrile-styrene-methyl acrylate copolymers); polylactic acid, etc. Other resins; polysaccharides can be exemplified.
Examples of the inorganic substance in the mother body include silica and glass.

(2.3) Quantum Dot Accumulated Nanoparticles Quantum dot integrated nanoparticles have a structure in which the quantum dots are contained in the mother body and / or are adsorbed on the surface thereof.
When the quantum dots are contained in the mother body, the quantum dots need only be dispersed inside the mother body, and may or may not be chemically bonded to the mother body itself.

(2.4) Fluorescent dye-accumulated nanoparticles The fluorescent dye-accumulated nanoparticles have a structure in which the fluorescent organic dye is contained in the mother body and / or is adsorbed on the surface thereof.
When the fluorescent organic dye is encapsulated in the mother body, the fluorescent organic dye may or may not be chemically bonded to the mother body itself as long as it is dispersed inside the mother body.

(2.5) Preparation of Fluorescent Material Accumulated Nanoparticles Fluorescent substance integrated nanoparticles can be produced according to a known method.
Specifically, for example, the fluorescent substance-encapsulating silica particles containing silica as a base and a fluorescent substance contained therein are fluorescent substances such as quantum dots and fluorescent organic dyes, and a silica precursor such as tetraethoxysilane. It can be prepared by dropping a solution in which and is dissolved in a solution in which ethanol and ammonia are dissolved and hydrolyzing the silica precursor.

On the other hand, for fluorescent substance-accumulated resin particles in which a resin is used as a base and a fluorescent substance is adsorbed on the surface of the resin particles or encapsulated in the resin particles, a solution of those resins or a dispersion of fine particles is prepared in advance. It can be produced by adding a fluorescent substance such as a quantum dot or a fluorescent organic dye to the mixture and stirring the mixture. Alternatively, the fluorescent substance-accumulated resin particles can be produced by advancing the polymerization reaction after adding the fluorescent substance to the solution of the resin raw material.
For example, when a thermosetting resin such as a melamine resin is used as a base resin, the raw material of the resin (monomer or oligomer or prepolymer, for example, methylol melamine which is a condensate of melamine and formaldehyde) and a fluorescent organic dye are used. Fluorescent dye-accumulated resin particles can be produced by heating a reaction mixture containing a surfactant and a polymerization reaction accelerator (acid or the like), and proceeding the polymerization reaction by an emulsion polymerization method. When a thermoplastic resin such as a styrene-based copolymer is used as the base resin, the raw material of the resin and the fluorescent organic dye (as the raw material monomer of the resin, the organic fluorescent dye is previously bonded by a covalent bond or the like). The reaction mixture containing the above-mentioned monomer (may be used) and a polymerization initiator (benzoyl peroxide, azobisisobutyronitrile, etc.) is heated, and the polymerization reaction is allowed to proceed by a radical polymerization method or an ionic polymerization method. Therefore, the fluorescent dye-accumulated resin particles can be produced.

(2.6) Average particle size The average particle size of the fluorescent substance-accumulated nanoparticles used in the present embodiment is not particularly limited, but those having a large particle size have difficulty in accessing the antigen, and the particle size is small and the brightness value is low. Since the signal of the nanoparticles accumulating fluorescent substances is buried in background noise (camera noise and autofluorescence of cells), those having a wavelength of about 20 to 500 nm are preferable.
The coefficient of variation (= (standard deviation / average value) × 100%) indicating the variation in particle size is not particularly limited, but 20% or less can be used, preferably 5 to 15%.
The average particle size is determined by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of a sufficient number of particles, and using each measured value as the area of a circle. Asked as. In the present application, the arithmetic mean of the particle sizes of 1000 particles is taken as the average particle size. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.

(3) Antibody As the primary antibody, an antibody (IgG) that specifically recognizes and binds to a protein as a target biological substance as an antigen can be used.
In the present embodiment, as the primary antibody, an antibody that recognizes and binds to a specific domain of the same target biological substance (protein) is used.

For example, when PD-L1 is the target biological substance, "SP263", "SP142" (all manufactured by Ventana), and "E1L3N" (Cell Signaling Technology) are used as anti-PD-L1 antibodies that can recognize the intracellular domain. As an anti-PD-L1 antibody that can recognize the extracellular domain, "22c3" (manufactured by Dako) and "28-8" (manufactured by Abcam) can be used.
When HER2 is used as the target biological substance, "4B5" (manufactured by Ventana), "CB11" (manufactured by BioGenex), and extracellular domain can be recognized as anti-HER2 antibodies capable of recognizing the intracellular domain. As an anti-HER2 antibody, "SV2-61γ" (manufactured by Nichirei Bioscience) can be used.
When TIM-3 is used as the target biological substance, "F38-2E2", "RMT3-23" (all manufactured by BioLegend), and "MM0936" are used as anti-TIM-3 antibodies that can recognize the intracellular domain. "-14S23", "RM0135-6F46" (both manufactured by Abcam), and "344823" (manufactured by R & D Systems) can be used as an anti-TIM-3 antibody capable of recognizing the extracellular domain.

The primary antibody may be an antibody fragment or derivative instead of a natural full-length antibody as long as it has the ability to specifically recognize and bind to a specific biological substance (antigen). That is, the term "antibody" as used herein refers to not only full-length antibody, but also antibody fragments such as Fab, F (ab) '2, Fv, scFv, chimeric antibody (humanized antibody, etc.), and multifunctional antibody. Derivatives such as are included.

As the secondary antibody, an antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used.

Both the primary antibody and the secondary antibody may be polyclonal antibodies, but monoclonal antibodies are preferable from the viewpoint of quantitative stability. The type of animal (immune animal) that produces the antibody is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like as in the conventional case.

(4) Immunostaining agent An immunostaining agent is obtained by dispersing a labeled antibody in which an antibody capable of directly or indirectly binding to a target biological substance and a labeling substance are directly or indirectly bound in an appropriate medium. Will be generated.
In order to improve the efficiency of the fluorescent label and suppress the passage of time leading to the deterioration of fluorescence as much as possible, the primary antibody and the fluorescent substance-accumulated nanoparticles indirectly, that is, the antigen-antibody reaction or the avidin-biotin reaction was used. , It is preferable to use a complex linked by a bond other than a covalent bond, but the present invention is not limited to this.

As an example of an immunostaining agent in which an antibody and fluorescent nanoparticles are indirectly linked, [primary antibody against the target biological substance] ... [antibody against the primary antibody (secondary antibody)] to [fluorescent nanoparticles (fluorescent nanoparticle) )]. Here, “…” indicates that the bond is formed by an antigen-antibody reaction, and the mode of binding indicated by “～” is not particularly limited, and is, for example, covalent bond, ionic bond, hydrogen bond, coordination bond, or physical bond. Examples thereof include adsorption or chemisorption, and may be mediated by a linker molecule if necessary.

The secondary antibody-fluorescent substance-accumulated nanoparticle conjugate can be prepared, for example, by using a silane coupling agent which is a compound widely used for binding an inorganic substance and an organic substance. This silane coupling agent is a compound having an alkoxysilyl group that gives a silanol group by hydrolysis at one end of the molecule and a functional group such as a carboxyl group, an amino group, an epoxy group, or an aldehyde group at the other end. It binds to an inorganic substance via the oxygen atom of the silanol group. Specifically, mercaptopropyltriethoxysilane, glycidoxypropyltriethoxysilane, aminopropyltriethoxysilane, a silane coupling agent having a polyethylene glycol chain (for example, PEG-silaneno. SIM6492.7 manufactured by Gelest) and the like can be used. Can be mentioned. When a silane coupling agent is used, two or more kinds may be used in combination.

A known method can be used for the reaction procedure between the fluorescent substance-accumulated nanoparticles and the silane coupling agent. For example, the obtained silica nanoparticles containing a fluorescent substance are dispersed in pure water, aminolopyrtriethoxysilane is added, and the mixture is reacted at room temperature for 12 hours. After completion of the reaction, silica nanoparticles having a surface modified with an aminopropyl group can be obtained by centrifugation or filtration. Subsequently, by reacting the amino group with the carboxyl group in the antibody, the antibody can be bound to silica nanoparticles containing a fluorescent substance via an amide bond. If necessary, a condensing agent such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbodiimide Hydrochloride: manufactured by Pierce) can also be used.

Further, if necessary, a linker compound having a site capable of directly binding to silica nanoparticles containing an organic molecule-modified fluorescent substance and a site capable of binding to a molecular target substance can be used. As a specific example, sulfo-SMCC (Sulfosuccinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate: manufactured by Pierce) having both a site that selectively reacts with an amino group and a site that selectively reacts with a mercapto group. ) Is used to bind the amino group of silica nanoparticles containing a fluorescent substance modified with aminopropyltriethoxysilane to the mercapto group in the antibody to obtain silica nanoparticles containing the fluorescent substance to which the antibody is bound. Be done.

When a biological substance recognition site (a site that can specifically recognize a biological substance, for example, biotin, avidin, an antibody, etc.) is bound to polystyrene particles containing a fluorescent substance, the fluorescent substance is either a fluorescent organic dye or a quantum dot. In some cases, similar procedures can be applied. That is, by impregnating polystyrene nanoparticles having a functional group such as an amino group with quantum dots or a fluorescent organic dye, fluorescent substance-accumulated polystyrene particles having a functional group can be obtained, and thereafter, by using EDC or sulfo-SMCC. , Fluorescent substance-accumulated polystyrene particles to which the antibody is bound are formed.

As another example of an immunostaining agent in which an antibody and fluorescent nanoparticles are indirectly linked, [primary antibody against a target biological substance] ... [antibody against a primary antibody (secondary antibody)]-[biotin] / [avidin]- [Fluorescent substance (fluorescent substance-accumulated nanoparticles)] (Here, "..." indicates that the bond is bound by an antigen-antibody reaction, and "-" is a covalent bond that may be mediated by a linker molecule, if necessary. It indicates that they are bound, and "/" indicates that they are bound by the avidin-biotin reaction).) Examples thereof include a complex consisting of three molecules linked in a manner.

For the secondary antibody-biotin conjugate (biotin-modified secondary antibody), for example, a commercially available biotin labeling reagent (kit) is used based on a known method capable of binding biotin to a desired antibody (protein). Can be produced. Further, if a biotin-modified secondary antibody itself in which biotin is bound to a desired antibody in advance is commercially available, it may be used.

Fluorescent material-accumulated nanoparticles-avidin conjugate (avidin-modified fluorophore) are also produced based on a known method capable of binding avidin to a phosphor, for example, using a commercially available avidin labeling reagent (kit). can do. The avidin in this case may be an improved form such as streptavidin or neutravidin, which has a higher binding force with biotin than avidin.

Specific examples of the method for producing the fluorescent substance-accumulated nanoparticles-avidin conjugate are as follows.
When the fluorescent substance-accumulated nanoparticles are based on a resin, the functional groups of the resin and the functional groups of avidin (protein) are, if necessary, a linker molecule such as PEG having functional groups at both ends of the molecule. It can be combined by means of. For example, in the case of a melamine resin, a functional group such as an amino group can be used, and in the case of an acrylic resin, a styrene resin, etc., a monomer having a functional group (for example, an epoxy group) is copolymerized in the side chain. , The functional group itself or a functional group converted from the functional group (for example, an amino group produced by reacting aqueous ammonia) can be utilized, and further, another functional group can be utilized by utilizing those functional groups. A group can also be introduced.

When the phosphor-accumulated nanoparticles are based on silica, a desired functional group can be introduced by surface modification with a silane coupling agent. For example, an amino group is introduced by using aminopropyltrimethoxysilane. be able to.
On the other hand, for avidin, a thiol group can be introduced by reacting, for example, N-succinimidyl S-acetylthioacetate (SATA) with the amino group of avidin. Then, by using a crosslinker reagent having an N-hydroxysuccinimide (NHS) ester having a reactivity with an amino group and a maleimide group having a reactivity with a thiol group at both ends of a polyethylene glycol (PEG) chain, the amino is used. Fluorescent substance-accumulated nanoparticles having a group and avidin into which a thiol group has been introduced can be linked.

(5) Tissue Specimen A tissue specimen is a tissue section of an organ collected from a subject (such as a cancer patient) or a cell obtained by culturing cells contained in the tissue collected from the subject. In the present embodiment, three or more organs of a subject are targeted, and lesion cells such as tumor cells (target portion) from each organ and normal cells (target portion) to be compared with the lesion cells are selected. It is collected as a tissue sample. The source of the lesion cells and the normal cells is not limited to the same subject. That is, lesion cells and normal cells may be collected from different subjects.
Tissue specimens generally take the form of specimen slides on which tissue sections or cells are placed, as is commonly used when evaluating the expression level of a target biological substance by immunohistochemical staining.
The method for preparing the tissue specimen is not particularly limited, and generally, for example, a tissue section collected from a subject is fixed with formalin or the like, dehydrated with alcohol, treated with xylene, and subjected to high-temperature paraffin. A tissue sample prepared by embedding paraffin in it can be obtained by making a section of 3 to 4 μm, and the sample slide can be obtained by placing the tissue section on a slide glass and drying it. Can be made.

(6) Staining method The staining method for tissue specimens will be described below. The staining method described below is not limited to tissue sections and can also be applied to cell staining.

(6.1) Specimen preparation step (6.1.1) Deparaffin treatment The section is immersed in a container containing xylene to remove paraffin. The temperature is not particularly limited, but it can be carried out at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, xylene may be replaced during immersion.

Next, the section is immersed in a container containing ethanol to remove xylene. The temperature is not particularly limited, but it can be carried out at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, ethanol may be replaced during immersion.

Immerse the section in a container filled with water and remove ethanol. The temperature is not particularly limited, but it can be carried out at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, the water may be replaced during immersion.

(6.1.2) Activation treatment The activation treatment of the target biological substance is carried out according to a known method. The activation conditions are not particularly specified, but the activation solution is 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, and 0.1 M Tris-hydrochloric acid buffer. A liquid or the like can be used.
The pH condition is such that a signal is output from the range of pH 2.0 to 13.0 depending on the tissue section to be used and the tissue roughness is such that the signal can be evaluated. Normally, the pH is 6.0 to 8.0, but for special tissue sections, for example, pH 3.0 is also used.
As the heating device, an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used. The temperature is not particularly limited, but it can be carried out at room temperature. The temperature can be 50 to 130 ° C. and the time can be 5 to 30 minutes.

Next, the section after activation treatment is immersed in a container containing PBS and washed. The temperature is not particularly limited, but it can be carried out at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, PBS may be replaced during immersion.

(6.2) Immunohistochemical Staining Step In the immunohistochemical staining step, in order to stain the target biological substance, a solution of the immunostaining agent is placed on a section and reacted with the target biological substance.
The solution of the immunostaining agent used in the immunohistochemical staining step may be prepared in advance before this step.

The conditions for performing the immunohistochemical staining step, that is, the temperature and the immersion time when immersing the tissue specimen in the solution of the immunostaining agent, are based on the conventional immunohistochemical staining method so that an appropriate signal can be obtained. It can be adjusted as appropriate.
The temperature is not particularly limited, but it can be carried out at room temperature. The reaction time is preferably 30 minutes or more and 24 hours or less.
It is preferable to drop a known blocking agent such as PBS containing BSA or a surfactant such as Tween 20 before performing the treatment as described above.

For example, when the immunostaining agent is a complex of [primary antibody (probe)] ... [secondary antibody]-[biotin] / [avidin]-[fluorescent nanoparticles (fluorescent substance-accumulated nanoparticles, etc.)], the first Immersing the tissue sample in the solution of the primary antibody (primary reaction process), then immersing the tissue sample in the solution of the secondary antibody-biotin conjugate (secondary reaction process), and finally according to the present invention. A treatment (fluorescence labeling treatment) may be performed in which a tissue section as a tissue sample is immersed in a solution of avidin-fluorescent nanoparticles dispersed in a diluted solution for fluorescent nanoparticles.

(6.3) Specimen Post-treatment Step It is preferable that the tissue specimen after the immunohistochemical staining step is subjected to treatments such as immobilization / dehydration, permeation, and encapsulation so as to be suitable for observation.

For the immobilization / dehydration treatment, the tissue specimen may be immersed in a fixation treatment solution (crosslinking agent such as formalin, paraformaldehyde, glutaaldehyde, acetone, ethanol, methanol). The permeation treatment may be performed by immersing the tissue specimen after the immobilization / dehydration treatment in a permeation solution (xylene or the like). The encapsulation treatment may be performed by immersing the tissue specimen that has undergone the permeation treatment in the encapsulation liquid.
The conditions for performing these treatments, for example, the temperature and the immersion time when immersing the tissue specimen in a predetermined treatment solution, may be appropriately adjusted so as to obtain an appropriate signal according to the conventional immunostaining method. it can.

(6.4) Morphological Observation Staining Step Apart from the immunohistochemical staining step, morphological observation staining may be performed so that the morphology of cells, tissues, organs, etc. can be observed in a bright field.
The morphological observation dyeing step can be performed according to a conventional method.
For morphological observation of tissue specimens, staining with eosin, in which cytoplasm, interstitium, various fibers, erythrocytes, and keratinocytes are stained in red to deep red, is standardly used. Staining with hematoxylin, in which the cell nucleus, lime, cartilage tissue, bacteria, and mucus are stained in blue to pale blue, is also standardly used (the method of performing these two stainings at the same time is hematoxylin / eosin staining). Known as (HE staining)). In addition, staining with a fluorescent dye such as DAPI (4', 6-diamidino-2-phenylindole) that specifically stains the cell nucleus may be performed.
When the morphological observation staining step is included, it may be performed after the immunohistochemical staining step or before the immunohistochemical staining step.

(7) Evaluation method (7.1) Observation / imaging step In the observation / imaging step, the microscope image acquisition device 1A is set to a desired magnification, and the target biological substance used in the immunohistochemical staining step is displayed in the same field of view. The tissue specimen is irradiated with excitation light corresponding to each fluorescent substance to be fluorescently labeled, and a fluorescent image due to fluorescence emitted from those fluorescent substances is observed and photographed.

(7.2) Quantification step In the quantification step, image processing of a fluorescent image and quantification of the expression level are performed. The quantification step according to the present embodiment corresponds to the acquisition step according to the present invention, and is executed by the control unit 21.
The control unit 21 acquires expression information of fluorescently labeled target biological substances of normal cells and abnormal cells in three or more organs. In this embodiment, about 3 to 100 samples of normal cells and about 3 to 100 samples of abnormal cells are used to obtain the above expression information.
Specifically, the control unit 21 measures the fluorescence labeling signal such as the number of bright spots of fluorescence or the emission brightness corresponding to the target biological substance based on the image processing in the information acquisition device 2A for the fluorescent image taken with respect to the target biological substance. Then, the value obtained by quantifying the expression level of each target biological substance in the cell region is acquired as expression information. For example, the number of PID particles corresponding to the number of bright spots is measured, and the value obtained by calculating the number of particles per cell can be used as expression information. Further, the expression information may be a value obtained by calculating the number of particles per unit area of the tissue.
Examples of software that can be used for image processing and quantification of expression level include "ImageJ" (open source). By using such image processing software, processing such as extracting bright spots of a predetermined wavelength (color) from a fluorescent image and measuring the number of bright spots having a predetermined brightness or higher is semi-automatically and quickly. Can be done.
Moreover, since the bright spot is derived from one fluorescent nanoparticle, the size is constant and can be recognized by microscopic observation. A signal whose size is larger than a constant value (for example, the average value of observed fluorescent nanoparticles) is judged to be an aggregated bright spot. The bright spots and aggregated bright spots can be semi-automatically and quickly distinguished using software.

(7.3) Display step In the display step, the display unit 23 displays the list information obtained by digitizing the expression information acquired in the quantification step and displaying the list information, and the evaluation support information including the threshold value indicating a predetermined threshold value. Let me.

FIG. 3 is an example showing the evaluation support information displayed on the display unit 23.
As shown in FIG. 3, the list information is a bar-shaped display of the distribution of expression information (number of particles per cell) of normal cells and abnormal cells for each organ.
Further, the threshold value information is a linear display of a predetermined threshold value (indicated by a broken line in FIG. 3).

The threshold (cutoff value) is taken from multiple finalally effective tissue specimens taken before or during preoperative chemotherapy. Numerical value representing the expression level of a specific target biological substance contained in each sample in the tissue sample (pCR group) and the tissue sample (non-pCR group) collected from a plurality of subjects who did not finally obtain the effect. Can be derived by obtaining and comparing the results of the pCR group with the results of the non-pCR group.
Specifically, an ROC curve (receiver operating characteristic curve) for the PID score (mean value of the number of PID particles per cell) of HER2 in each of the pCR group and the non-pCR group should be created and set statistically. Can be done.

In addition, the threshold is a background (negative control: PID at the time of reaction without a primary antibody, as used in ELISA (Enzyme-Linked ImmunoSorbent Assay), which is a general biological measurement. It is also possible to add 3 times the standard deviation σ to the mean value of the score) (mean value of negative control + 3σ).

Further, the method of setting the threshold value is not limited to this, and other than this, the threshold value setting method may be set based on the result of comparing the expression information of normal cells and abnormal cells. Further, using AI (Artificial Intelligence), a threshold value may be set by iteratively analyzing a large amount of information and finding a pattern from the information.
As described above, the drug discovery support method used in the present invention is used for adjusting the sensitivity of the drug after setting the threshold value from the information obtained from the actual tissue sample, or the threshold value is determined from the list information obtained by quantifying the expression information. However, it can be widely used for drug discovery creation, such as by using it as a method for determining the sensitivity of a drug.

In such evaluation support information, whether or not the drug is effective for the subject who collected the tissue sample because the value of the expression information of each cell is larger than the threshold value (the effect is obtained by performing preoperative chemotherapy). Whether or not there is) can be predicted.
That is, it is shown that the lower the threshold value, the more effective it is for a sample in which the expression level of the target biological substance is small, and the higher the sensitivity. In addition, by comparing the values of normal cells and abnormal cells for each organ, it is predicted whether or not it is suitable for that organ.

The relationship between the value of the expression information of normal cells and abnormal cells and the threshold value will be described with reference to FIG.
Pattern (1) is an example showing the case where the maximum value of normal cells <threshold value and the minimum value of abnormal cells> threshold value. In the case of pattern (1), it can be inferred that the sensitivity to the drug is good and the drug is suitable for this organ. That is, when this happens, it can be determined as a target candidate.
Pattern (2) is an example showing the case where the minimum value of normal cells> the threshold value and the minimum value of abnormal cells> the threshold value. In the case of pattern (2), it is necessary to adjust the sensitivity to the drug, and it can be inferred that the drug is suitable for this organ. That is, when this happens, it can be determined that side effects are a concern.
Pattern (3) is an example showing the case where the minimum value of normal cells> the threshold value and the maximum value of abnormal cells <threshold value. In the case of pattern (3), it can be inferred that it is inappropriate for this organ. That is, when this happens, it can be determined that it is bad for the organ.
Pattern (4) is an example showing the case where the maximum value of normal cells <threshold value and the maximum value of abnormal cells <threshold value. In the case of pattern (4), it can be inferred that the sensitivity needs to be adjusted.

Since the evaluation support information of the present embodiment lists the expression information of a plurality of organs and displays them together with the threshold value, it enables the user to visually grasp the sensitivity and the suitable organ at a glance. Is. Drug discovery is performed according to the threshold value.
Further, on the display unit 23, the position of the threshold information can be adjusted by an instruction operation via the operation unit 22 of the user. This makes it possible to quickly and easily infer the ideal position for a plurality of organs.

Here, in the example of FIG. 3, the expression information values of normal cells and abnormal cells for the liver, kidney, large intestine, and small intestine are displayed as list information.
For example, in FIG. 3, when the threshold information L1 is shown, it can be inferred that the liver, kidney, and large intestine are target candidates and are inappropriate for the small intestine.
Further, in FIG. 3, when the threshold value information L2 is shown, it is considered to be effective for the liver and the large intestine, but there is a concern about side effects on the kidney and the small intestine.
Further, when the threshold information L3 is shown in FIG. 3, the effect can be expected in any of the liver, kidney, large intestine, and small intestine, but side effects are a concern.
In this way, for example, the threshold value and the expression information value of the cell are compared, and when a certain ratio of the expression information value displayed in a rod shape is larger than the threshold value, it can be judged to be effective. Since a certain percentage that can be judged to be effective differs depending on the antigen, it is judged for each antigen.

In addition, as shown in FIG. 5, for example, expression information may be acquired from a more localized structure (Golgi apparatus, endoplasmic reticulum, mitochondria, cytoplasm, cell membrane, cell nucleus, etc.). In the case of FIG. 5, it is an example in which the Golgi apparatus, endoplasmic reticulum, and mitochondria are extracted from the fluorescent image of the abnormal cell, and the expression information of the target biological substance (HER2 protein or HER2 mRNA) is acquired from the extracted image. ..
By doing so, more localized analysis becomes possible. In addition, since protein and mRNA can be detected on the same tissue section, it is possible to identify a subject or an organ in which the target mRNA is expressed but the protein is not expressed.

[Effect of embodiment]
As described above, the drug discovery support method of the present embodiment is fluorescently labeled with abnormal cells (target part) in a plurality of organs (biological sites) and normal cells (target part) to be compared with the abnormal cells. The acquisition process for acquiring the expression information of the target biological substance, the list information obtained by digitizing the expression information of each part acquired in the acquisition process, and the threshold information indicating a predetermined threshold are displayed on the display unit. It has a process.
In addition, the display step displays list information in which the expression information of each part is arranged in a bar shape and threshold information in which the threshold value is displayed in a straight line.
Therefore, the value indicating the expression information of the abnormal cells and the normal cells of a plurality of organs and the threshold value can be recognized at a glance, and the sensitivity of the drug and the suitable organ can be quickly and accurately determined.

Further, according to the present embodiment, there is a threshold value setting step of setting a threshold value based on background data representing a background component included in the expression information of each part.
The threshold value can be set based on the background data.

Further, according to the present embodiment, the acquisition step acquires the expression information of the fluorescently labeled target biological substance of the abnormal cells and the prenormal cells in the three or more organs. Therefore, the list information for the three or more organs is obtained. And by comparing the threshold information, it is more useful in improving the efficiency of the drug discovery stage.

Although the preferred embodiment to which the present invention is applied has been described above, the description content in the above embodiment is a preferable example of the present invention, and is not limited thereto.

For example, in the above embodiment, the expression information is acquired from three or more organs of the subject, but the expression information may be acquired from at least two organs.

The present invention can be used to quickly and accurately determine the sensitivity of a drug and a suitable biological site.

100 Pathological diagnosis support system 1A Microscopic image acquisition device 2A Information acquisition device 21 Control unit (acquisition means, display control means)
22 Operation unit 23 Display unit 24 Communication I / F
25 Memory

Claims (9)

1. An acquisition step of acquiring expression information of a target part in a plurality of biological parts and a fluorescently labeled target biological substance of the target part to be compared with the target part.
   A display step of displaying the list information obtained by digitizing the expression information of each part acquired by the acquisition step and displaying the list information, and the threshold information indicating a predetermined threshold value on the display unit.
   Drug discovery support method with.
2. The drug discovery support method according to claim 1, wherein the biological part is an organ.
3. The drug discovery support method according to claim 1 or 2, wherein the target part is an abnormal cell and the target part is a normal cell.
4. The drug discovery support method according to claim 3, wherein the target unit is a predetermined structure in the abnormal cell.
5. The wound according to any one of claims 1 to 4, wherein the display step displays the list information in which the expression information of each part is displayed side by side in a bar shape and the threshold information in which the threshold value is linearly displayed. Drug support method.
6. The drug discovery support method according to any one of claims 1 to 5, wherein the acquisition step acquires expression information of the target portion and the fluorescently labeled target biological substance in the target portion in three or more biological parts. ..
7. The drug discovery support method according to any one of claims 1 to 6, further comprising a threshold setting step of setting the threshold value based on background data representing a background component included in the expression information of each part.
8. An acquisition means for acquiring expression information of a target part in a plurality of biological parts and a fluorescently labeled target biological substance of the target part to be compared with the target part.
   A display control means for displaying the list information obtained by digitizing the expression information of each part acquired by the acquisition means and displaying the list information, and the threshold value information indicating a predetermined threshold value on the display unit.
   Drug discovery support device equipped with.
9. Computer,
   An acquisition means for acquiring expression information of a target part in a plurality of biological parts and a fluorescently labeled target biological substance of the target part to be compared with the target part.
   A display control means for displaying on the display unit the list information obtained by digitizing the expression information of each part acquired by the acquisition means and displaying the list information and the threshold value information indicating a predetermined threshold value.
   A program that functions as.

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