JP5900489B2 - Immunohistochemical staining method and method for determining the effectiveness of an antibody drug using the same - Google Patents

Description

  The present invention relates to a method for determining the effectiveness of an antibody drug using a highly accurate tissue staining method.

  Cancer is a disease that bisects the cause of death in adults together with vascular diseases represented by myocardial infarction and cerebral infarction. For example, the incidence of breast cancer in Japan is lower than in Western countries, but has been increasing in recent years. In 1998, it surpassed the incidence of gastric cancer and became the first female morbidity. According to a recent report by the Ministry of Health, Labor and Welfare in 2005, the annual incidence of breast cancer exceeds 50,000. Similarly, the number is increasing every year in the world, and according to a 2008 WHO report, breast cancer has the highest incidence rate for both men and women, and the annual incidence is over 1.38 million. It accounts for about 23% of women ’s cancer.

  In addition to diagnostic imaging such as X-ray CT and MRI, cancer diagnostics include methods that detect cancer markers that are specifically expressed in specific cancers, cancer markers that leak into blood and tissues, etc. Has been. General screening tests for breast cancer include interviews, palpation, soft x-ray mammography (mammography), ultrasonography, etc. If clinical suspicion arises, cytology and biopsy are performed and pathological diagnosis It is determined whether it is cancer. Pathological diagnosis is important for determining the course of cancer treatment and prognosis, and “HE (hematoxylin and eosin) staining for morphological observation” and “cancer markers” are the core of this diagnosis. An immunohistochemical method using an antibody against a factor ". In particular, with the advent of antibody drugs in recent years, the importance of immunohistochemistry has increased greatly.

  For example, it is marketed as Herceptin (registered trademark), an antibody drug targeting human epidermal growth factor receptor-2 (HER2), which is a factor involved in cancer growth. Trastuzumab is known to be a typical anticancer drug for breast cancer. Immunohistochemistry (IHC) method for analyzing the expression of HER2 protein and the like and FISH (Fluorescence in situ hybridization) method for analyzing the amplification of HER2 gene and the like are clinical methods for determining the effectiveness of this drug administration. Widely used in the field.

  Here, the IHC method is a method of staining and visualizing an anti-HER2 antibody bound to the HER2 antigen site using DAB (Diaminobenzidine), and the expression level of HER2 is detected through the visualized anti-HER2 antibody. can do. However, since the determination criterion is a rough determination criterion based on only four stages with a staining level of score 0 to 3, it lacks quantitativeness, and further, the determination criterion depends on the skill level of the pathologist, It is a clinical problem.

  On the other hand, the FISH method is a method for amplifying and analyzing the HER2 gene using a probe for detecting the HER2 gene and a probe for detecting the chromosome 17 centromere, and for each chromosome 17 analyzed by the FISH method. The presence or absence of amplification of the HER2 gene can be determined based on the gene copy number of HER2. Although the FISH method is a quantitative test method, it is not a method for directly evaluating the amount of HER2 protein or the intracellular localization of HER2.

  Also, for example, because the antigenic determinant (epitope) of a therapeutic agent (antibody) targeting HER2 protein is different from that used for detection of HER2 protein in the diagnosis of breast cancer, the IHC method using anti-HER2 antibody including the current Hercept test Therefore, it was considered to be inadequate as a method for selecting patients to be treated with trastuzumab. Under such circumstances, it is necessary to develop a method for determining the effectiveness of a medicine containing a highly accurate antibody as a component.

  In addition, in the conventional IHC method using an anti-HER2 antibody including Hercept test, an antibody different from trastuzumab is used as this anti-HER2 antibody, and knowledge about the staining by the IHC method using an antibody drug as an antibody is up to now. Has not been confirmed.

Nahta R et al, Breast Cancer Res. 2006; 8: 215-223 Scaltriti M et al, J. Natl. Cancer Inst. 2007; 99: 628-638

  The present invention has been made in view of the above problems, and immunohistological staining that enables high-precision staining by using an antibody drug containing an antibody such as trastuzumab as a component as a substance that recognizes a target biological substance. It is an object of the present invention to provide a method for determining the effectiveness of an antibody drug with high accuracy using such an immunohistochemical staining method.

  The present inventor has found a method capable of selecting a patient suitable for administration of an antibody drug equivalent to confirmation of gene amplification by the FISH method by performing a biological tissue staining method using an antibody drug containing an antibody such as trastuzumab as a component. Invented.

That is, the present invention provides an immunohistochemical staining method shown in the following [1] to [ 2 ] and a method for determining the effectiveness of an antibody drug.

[1] As a substance that recognizes an antigen targeted by an antibody drug, an antibody that is a component of the antibody drug is used,
The antibody is used in the form of a labeled antibody to which a labeling substance is bound;
The labeling substance is a nanoparticle having a diameter of 94 nm or more obtained by integrating quantum dots or organic fluorescent dyes ,
A distribution of the antigen present in the tissue is obtained in a visualized form depending on the amount of the antibody bound to the antigen; and
An immunohistochemical staining method wherein the antigen is any one selected from the group consisting of a cancer growth regulator, a metastasis regulator, a growth regulator receptor, and a metastasis regulator receptor.

[2] As a substance that recognizes an antigen targeted by an antibody drug, an antibody that is a component of the antibody drug is used,
The antibody is used in the form of a labeled antibody to which a labeling substance is bound;
The labeling substance is a nanoparticle having a diameter of 94 nm or more formed by integrating quantum dots or organic fluorescent dyes , and
The amount of the antibody bound to the amount of the antigen is measured using an immunohistochemical staining method in which the distribution of the antigen present in the tissue is visualized according to the amount of the antibody bound to the antigen. Thereby determining the effectiveness of the antibody drug.

  According to the present invention, the immunohistochemical staining method can be performed with high accuracy, whereby the patient to which the antibody drug is to be administered can be selected more accurately.

  Hereinafter, although the form for implementing this invention is demonstrated, this invention is not limited to these. Hereinafter, the present invention, its components, and the best mode for carrying out the invention will be described in detail.

[Labeling substance]
The labeling substance used in the present invention is not particularly limited as long as it does not interfere with the antigen-antibody reaction and does not interfere with the quantitativeness in the measurement. However, the antigen-antibody reaction is directly visualized, particularly in a visible form. From the viewpoint of being able to do so, a labeling substance that can detect the presence of the complex formed by the antigen-antibody reaction by color development is preferably used. Here, “direct visualization in a visible form” means that the location of the antigen-antibody reaction can be directly observed without secondary operations such as development. Examples of such a labeling substance include a substance that itself can develop color, or an enzyme that can generate a substance that develops color from a suitable substrate.

  Here, the “chromogenic substance itself” is not particularly limited as long as it is a substance that can easily identify the presence or absence of the complex produced by the antigen-antibody reaction, but it is easy to identify the color development. From the point of view, there are fluorescent labeling agents such as organic fluorescent dyes, quantum dots, and fluorescent aggregates obtained by integrating a plurality of fluorescent substances.

  Organic fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes. Examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, cyanine dye molecules, perylene dye molecules, and oxazine dye molecules.

  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, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 14, 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 635, Alexa Fluor 635, Alexa Fluor 633, Alexa Fluor 633, Alexa Fluor 633 Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, ODIPYB6 50/665 (all manufactured by Invitrogen), methoxy coumarin, eosin, NBD, pyrene, Cy5, Cy5.5, can be mentioned Cy7 like.

  These organic fluorescent dyes may be used alone or in combination of two or more.

  As quantum dots, II-VI group compounds, III-V group compounds, or quantum dots containing group IV elements as components ("II-VI group quantum dots", "III-V group quantum dots", " Or “Group IV quantum dots”). You may use individually or what mixed multiple 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 thereon. Hereinafter, as a notation of quantum dots having a shell in this specification, when the core is CdSe and the shell is ZnS, it is expressed 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.

  As the quantum dots, those subjected to surface treatment with an organic polymer or the like may be used as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.

  As rare earth phosphors, for example, neodymium oxide, neodymium chloride, neodymium nitrate, ytterbium oxide, ytterbium chloride, ytterbium nitrate, lanthanum oxide, lanthanum chloride, lanthanum nitrate, yttrium oxide, yttrium chloride, yttrium nitrate, pradiocem chloride, erbium chloride, Orthophosphoric acid, ammonium phosphate, ammonium dihydrogen phosphate and the like can be used.

  In addition, as a “fluorescent aggregate formed by accumulating a plurality of fluorescent substances”, a fluorescence having a core capable of immobilizing a large number of fluorescent substances and a shell made of a plurality of fluorescent substances immobilized on the core. In particular, fluorescent aggregate particles composed of a core made of nanoparticles of an appropriate material such as silica nanoparticles and a shell made of a plurality of the above organic fluorescent dyes are preferably used. When such a fluorescent aggregate is used as a labeling substance, it is preferable because tissue staining can be performed with a very high quantitativeness equivalent to that of the FISH method. In the present specification, the fluorescent aggregate particles may be referred to as “phosphor aggregate particles”. The term “nanoparticle” is used to mean a particle having a size on the order of nanometers (1 to several hundred nanometers).

  In the present invention, the material constituting the core is not particularly limited as long as it can fix the shell on the surface and does not quench the fluorescence from the fluorescent material constituting the shell. As a suitable material, silica and the like can be mentioned.

  Here, the fluorescent aggregate including a core made of silica nanoparticles and a shell made of a plurality of the organic fluorescent dyes is, for example, a functional group that can bind to a functional group of the organic fluorescent dye with respect to the organic fluorescent dye. It can be obtained as an organic fluorescent dye integrated / silica nanoparticle by reacting with a suitable silane coupling agent having the following formula and further reacting with a suitable tetraalkylsilane such as tetraethoxysilane.

  In addition, “an enzyme capable of generating a chromogenic substance from an appropriate substrate” includes an enzyme that changes a substrate to generate a fluorescent species, such as horseradish peroxidase (HRP). Examples include peroxidase, alkaline phosphatase (ALP), and glucosidase enzymes.

  Here, a substrate generally used as a fluorogenic substrate in a conventionally known assay method based on a fluorogenic substrate conversion method can be used as a substrate that is converted into a chromogenic substance by these enzymes. Examples of such substrates include oxidoreductase substrates such as horseradish peroxidase (HRP) substrates, phosphatase substrates such as alkaline phosphatase (ALP) substrates, and glycosidase substrates such as β-galactosidase substrates. Although it is mentioned, it is not limited to these. Specific examples of the substrate used for the enzymatic reaction by HRP include 3,3′-diaminobenzidine (DAB), 3-p-hydroxyphenylpropionic acid (HPPA), ECL plus (trademark), 4-chloro-1-naphthol, 4-chloronaphthalen-1-ol and the like. Moreover, as a substrate used for the enzyme reaction by alkaline phosphatase (ALP), 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium salt (BCIP / NBT), 4-methylumbelliferyl phosphate (MUP) ), 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), AttoPhos®, 9H- (1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) Examples include phosphate (DDAOP). As a substrate used for the enzyme reaction by β-galactosidase, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), 9H- (1,3-dichloro-9,9 -Dimethylacridin-2-one-7-yl) β-D-galactopyranoside (DDAOG), 4-methylumbelliferyl-β-D-galactoside (MUG) and the like.

  Among these labeling substances shown above, a fluorescent labeling agent is preferably used because color development according to the amount of the complex produced by the antigen-antibody reaction can be obtained more reliably. Among fluorescent labeling agents, a fluorescent aggregate obtained by accumulating a plurality of fluorescent substances is particularly preferably used because high emission intensity can be easily obtained and excellent quantitativeness can be obtained.

[Buffer]
The buffer solution according to the present invention is a solvent for stably maintaining an environment suitable for an antigen-antibody reaction. For example, phosphate buffer physiological saline (PBS), phosphate buffer, Tris buffer, MES buffer, citrate-phosphate buffer, and the like.

[Dyeing method]
Hereinafter, the staining method of the present invention will be described.

  The immunohistochemical staining method visualizes the antigen-antibody reaction between a biological material present on the surface of a pathological slice tissue or cell and a substance recognizing the biological material, so that the biological material on the surface of the pathological slice tissue or cell is visualized. It is a method of judging whereabouts. This immunohistochemical staining method is a technique generally called an immunohistochemical method. Here, since visualization is performed through a change in color or brightness due to emission of fluorescence or the like, a change in color absorption, and the like, this visualization is sometimes referred to as “staining” in this specification.

  Here, in the staining method of the present invention, in this immunohistological staining method, the antigen targeted by the antibody drug is positioned as a target biological substance to be stained, and the antibody drug is used as a substance that recognizes the biological substance. It is characterized by using the antibody itself which is a constituent of In other words, the staining method of the present invention is an immunohistochemical staining method using an antibody that is a component of the antibody drug as a substance that recognizes the antigen targeted by the antibody drug. The staining method of the present invention is not limited to a pathological section tissue and can also be applied to cell staining.

  A method for preparing a pathological section to which the staining method of the present invention can be applied (hereinafter sometimes referred to as “section”) is not particularly limited, and a section prepared by a known method can be used. As a method for preparing such a section, there is a paraffin method in which the collected tissue is embedded in paraffin and then sliced to obtain a section, or a method in which the collected tissue is frozen and sliced to obtain a section. Examples include frozen section method.

1) Deparaffinization step When the staining method of the present invention is applied to a pathological section tissue, when the pathological section including the pathological section tissue is prepared by the paraffin method, the paraffin that hinders staining is removed from the pathological section. Need to be removed from. As the paraffin removal method at this time, a conventionally known method can be used in the present invention.

  Paraffin removal can be performed, for example, in the following manner.

  The pathological section is immersed in a container containing xylene and the paraffin is removed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.

  Next, the pathological section is immersed in a container containing ethanol to remove xylene. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, ethanol may be exchanged during the immersion.

  Thereafter, the pathological section is immersed in a container containing water and ethanol is removed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, water may be exchanged during the immersion.

2) Activation treatment step In the staining method of the present invention, as in the case of a conventionally known immunohistochemical staining method, in many cases, a purpose included in a pathological section or cell in advance so that good immunostaining is performed. The biological material activation process is performed. Here, the activation process of the target biological substance can be performed by a conventionally known method.

  The activation conditions are not particularly defined, but as the activation liquid, use 0.01M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1M Tris-HCl buffer, etc. Can do. 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 can be performed at room temperature. The temperature can be 50-130 ° C. and the time can be 5-30 minutes.

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

3) Staining Step In the staining method of the present invention, the staining step detects an antigen targeted by the antibody drug present on the surface of a pathological section tissue or other cellular tissue (hereinafter sometimes referred to as “tissue”). In order to do so, in the step of staining the pathological section tissue or cells using an antibody that is a component of the antibody drug as a substance that recognizes the antigen, and using an antigen-antibody reaction between the antigen and the antibody is there.

Antibody, antibody drug In the staining method of the present invention, when a tissue is stained, an antibody that is a component of the antibody drug is used as an antibody that recognizes an antigen targeted by the antibody drug present on the surface of the tissue. To do.

Here, in the present invention, the term “antibody” is used to include any antibody fragment or derivative. For example, Fab, Fab ′ ₂ , CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv ) Etc. The term “antibody drug” refers to a pharmaceutical product containing an antibody as a constituent component. In addition to the antibody, if necessary, it includes other components such as an adjuvant such as a stabilizer and a pharmaceutically acceptable filler as a constituent component. It is a waste. However, in the present specification, the term “antibody drug” may be used for an antibody that is a constituent component as long as the presence or absence of the other components is not a problem.

  In the following description of the present specification, an antibody that is a component of an antibody drug may be referred to as an “antibody drug component antibody”.

This “antibody drug-constituting antibody” recognizes an antigen targeted by the antibody drug and binds to the antigen through an antigen-antibody reaction to thereby exert a predetermined medicinal effect as a drug. As an aspect of "antibody drug constituent antibody",
(i) an antibody having both a function of recognizing an antigen targeted by an antibody drug and a function of exerting a predetermined drug effect after binding to the antigen; and
(ii) An antibody drug has only a function of recognizing a target antigen, but exhibits a predetermined drug effect by transporting the drug active substance to a cell or tissue having the antigen as a complex with the drug active substance. And antibodies that contribute to.

  In the present invention, the means for labeling the complex produced by the antigen-antibody reaction between the antigen targeted by the antibody drug and the antibody drug-constituting antibody is not particularly limited, but the form of the labeled antibody to which the labeling substance is bound. It is preferable to use an antibody pharmaceutical composition antibody.

  As the “antibody drug” used in the present invention, an antibody drug generally used for the treatment of autoimmune diseases such as rheumatoid arthritis, malignant tumors such as cancer, viral infections and the like can be used.

Among these, as an antibody drug administered to a cancer patient, any one of a cancer growth regulator, a metastasis regulator, a growth regulator receptor, a metastasis regulator receptor, etc. is used as a target antigen, and the target antigen And those containing an antibody that binds to as an active ingredient. As an antibody (that is, antibody drug component antibody) contained as a component of such an antibody drug,
(i ') an antibody that suppresses the growth of cancer cells by itself binding to cancer cells, or kills cancer cells;
(ii ′) An antibody that functions as a means for transporting antitumor active substances such as anticancer agents, antiviral agents, antibiotics, and radiation-emitting substances to cancer cells.

  Table 1 below shows typical antibody drugs used clinically. Here, for reference, antibody drugs used for the treatment of autoimmune diseases and infectious diseases are also listed in Table 1.

  In Table 1, for example, “Herceptin” is an antibody drug, and “Trastuzumab” is an antibody that is included as a component (that is, an antibody drug constituent antibody).

  Among the antibody drugs shown in Table 1 above, gemtuzumab is used in the form of Gemutuzumab-Ozogamicin formed by binding to an antitumor active substance, calicheamicin.

  Of the antibody drugs shown in Table 1 above, an antibody drug containing trastuzumab as a constituent component (that is, Herceptin (registered trademark)) is preferably used.

  Examples of cancers to which the staining method of the present invention is applied include colorectal cancer, rectal cancer, kidney cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, endometrial cancer, and esophagus. Cancer, blood cancer, liver cancer, pancreatic cancer, skin cancer, lung cancer, breast cancer and the like.

Antigen The antigen to be subjected to the staining method of the present invention is not particularly limited as long as it is a biological substance that functions as an antigen to be a target molecule of the above-described antibody drug.

  In the present invention, the term “antigen” refers to a biological substance, in particular, a molecule or molecular fragment. Examples of such “molecule” or “molecular fragment” include nucleic acids (single-stranded, DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid) etc., or nucleoside, nucleotide and their modified molecules), protein (polypeptide, oligopeptide etc.), amino acid (modified) Amino acids are also included), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules or complexes thereof, such as tumor markers, signaling substances, hormones, etc. There may be, and it is not specifically limited. However, in the staining method of the present invention, an antibody that is a constituent component of an antibody drug is used as the antibody, and therefore, among these “antigens”, those targeted by the antibody drug are to be detected. In the following description of the present specification, an antigen targeted by an antibody drug may be referred to as an “antibody drug target antigen”.

  However, considering that many antibody drugs are used as anticancer agents, cancer growth regulators, metastasis regulators, growth regulator receptors and metastasis regulator receptors are suitable target antigens. As mentioned.

  Among such growth control factors, metastasis control factors, growth control factor receptors and metastasis control factor receptors, examples of cancer growth control factors and receptors include epidermal growth factor (EGF), The EGF receptor (EGFR), platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR), insulin-like growth factor (IGF), the IGF receptor (IGFR) ), Fibroblast growth factor (FGF), the FGF receptor (FGFR), vascular endothelial growth factor (VEGF), the VEGF receptor (VEGFR), hepatocyte growth factor (HGF). Hepatocyte Growth Factor), the HGF receptor (HGFR), neurotrophic factor (NT: Neurotropin), transforming growth factor-β (TGFβ) family, Cell growth factor and its receptor, such as ER2, and cyclin, cyclin-dependent kinase (CDK), cyclin A, cyclin B, cyclin D, cyclin E, CDK1, CDK2, CDK4, Examples include factors that regulate the cell cycle, such as CDK6, p16INK, p15, p21, p27, and RB (Retinoblastoma). Examples of cancer metastasis regulators and receptors thereof include matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), PAR1 (Protease Activated Receptor 1), and CXCR4 (Chemokine [CX-C motif]. ] receptor 4), CCR7 (Chemokine [C-C motif] receptor 7), etc., and among these, trastuzumab targeting HER2 is widely used, so HER2 can be preferably exemplified .

  In addition to cancer-related antigens, inflammatory cytokines such as TNF-α (Tumor Necrosis Factor α) and IL-6 (Interleukin-6) receptor, virus-related molecules such as RSV F protein, and the like are also included in the present invention. Can be detected by the staining method.

Staining In the present invention, the tissue staining means is not particularly limited as long as it can visualize the antigen-antibody reaction between the antibody drug target antigen and the antibody drug constituent antibody. However, from the viewpoint of ease of visualization, it is preferable to perform staining by using an antibody drug-constituting antibody in the form of a labeled antibody by binding a labeling substance as the antibody. In particular, when a tissue is stained using a labeled antibody obtained by binding the above-described “chromogenic substance itself” as a labeling substance to an antibody drug constituent antibody, the antigen-antibody reaction is caused by the labeling. Since it can be expressed as a color change, the distribution of the antigen present in the tissue can be visualized according to the amount of the antibody bound to the antigen.

<Labeled antibody>
In the present invention, the labeled antibody contains the above-mentioned antibody drug-constituting antibody and the above-mentioned labeling substance, and the above-mentioned antibody drug-constituting antibody and the above-mentioned labeling substance comprise an amide bond, an ester bond, an imide bond, It has a structure bonded via an appropriate chemical bond such as a bond utilizing thiol addition to a maleimide group, or via a biotin-avidin bond or a biotin-streptavidin bond.

  Such a labeled antibody can be obtained by binding the above-described labeling substance to the above-described antibody drug constituent antibody according to a conventional method. Specific labeling methods include a method using an antibody (secondary antibody) having specific affinity for the above-mentioned antibody-drug-constituting antibody, a biotin-avidin method, a thiol group-maleimide group coupling reaction method , A method using an existing chemical linker, a cross-linking reaction method using a cross-linking agent (EDC, etc.), an ion binding method, etc. can be mentioned, and when the antibody is a humanized antibody or a human antibody, Preferred examples include a thiol group-maleimide group coupling reaction method with an antibody or avidin.

  A specific forming procedure is, for example, as follows.

  First, a first binding group is introduced into an antibody drug constituent antibody, and a second binding group capable of binding to the first binding group is introduced into a labeling substance. At this time, linkers having appropriate chain lengths may be interposed between the first binding group and the antibody drug constituent antibody and between the second binding group and the labeling substance. Here, the first and second linking groups may be chemical functional groups such as a carboxyl group, an amino group, an aldehyde group, a thiol group, and a maleimide group, or molecules such as biotin, avidin, and streptavidin. It may be. When a secondary antibody is used as the second binding group, the first binding group may be a site other than the site that recognizes the target antigen that constitutes the antibody drug constituent antibody.

  Thereafter, a labeled antibody is obtained by reacting the antibody-drug-constituting antibody introduced with the first binding group with the labeled substance introduced with the second binding group.

  This labeled antibody is prepared in advance in the absence of the tissue to be stained by reacting the antibody-drug-constituting antibody introduced with the first binding group with the labeled substance introduced with the second binding group. Alternatively, in the staining step, after reacting the unlabeled antibody drug-constituting antibody introduced with the first binding group with the tissue, the antibody incorporated in the tissue It may be formed by reacting a labeling substance introduced with a second binding group.

<< Staining with labeled antibody >>
The staining using the labeled antibody can be performed by bringing the labeled antibody obtained by the labeling method shown in the section “<< Labeled antibody >>” into contact with the tissue to be stained. Alternatively, an unlabeled antibody drug constituent antibody into which the first binding group has been introduced (hereinafter referred to as “an unlabeled antibody drug constituent antibody into which the first binding group has been introduced”) is brought into contact with the tissue to be stained. It can also be carried out by forming an antigen-antibody complex and then binding the antigen-antibody complex with a labeling substance into which the second binding group has been introduced. By this operation, when the antibody drug target antigen is present on the surface of the slice tissue or cell, a complex composed of the labeled antibody and the antibody drug target antigen is formed. As a result, the labeled antigen present on the surface of the slice or cell is specifically labeled.

  Here, it is preferable that the tissue to be stained is preliminarily subjected to an activation treatment by the above-described activation treatment step so that good staining is performed, and nonspecific adsorption of a labeled antibody or the like is suppressed. Therefore, it is preferable to perform a blocking treatment in advance on the tissue surface to be stained using a known blocking agent such as a BSA-containing buffer solution.

Staining with this labeled antibody typically involves
(i) using a known blocking agent, a step of performing a blocking treatment on the tissue surface to be stained;
(ii) contacting the labeled antibody with the tissue surface that has undergone the step (i) to obtain a stained tissue;
(iii) It can be carried out through a series of steps including the step of washing away the unreacted labeled antibody remaining on the stained tissue obtained in the step (ii).

However, instead of the above steps (ii) to (iii),
(ii′-1) contacting the above-mentioned “unlabeled antibody pharmaceutical constituent antibody into which the first binding group has been introduced” with the tissue surface that has undergone the step (i);
(ii'-2) The above-mentioned "labeling substance having the second binding group introduced" is reacted with the tissue surface on which the antigen-antibody complex has been formed by the step (ii'-1), and the antigen-antibody complex Obtaining a stained tissue by binding a label to the antibody part of the body;
(iii ′) In order to remove unreacted “unlabeled antibody drug-constituting antibody introduced with the first binding group” and “labeled substance introduced with the second binding group”, an appropriate buffer solution is used to You may perform through the process including the process of wash | cleaning the dyeing | staining structure | tissue obtained by (ii'-2). Here, in order to remove the unreacted “unlabeled antibody drug-constituting antibody into which the first binding group has been introduced”, the above step is performed between the step (ii′-1) and the step (ii′-2). A cleaning step similar to (iii ′) may be performed.

  Here, an example of staining in the case where the above-described phosphor-aggregated particles are used as a labeling substance is shown.

  First, by performing biotinylation or the like on an antibody drug, an antibody drug constituent antibody into which the first binding group is introduced is obtained in the form of a biotinylated antibody drug constituent antibody, for example. On the other hand, for phosphor-aggregated particles, for example, streptavidin-modified phosphors are aggregated by binding streptavidin or the like via an appropriate linker in an appropriate buffer such as PBS (phosphate buffered saline). A phosphor-aggregated particle having the second bonding group introduced therein is obtained in the form of a particle or the like.

  Here, when performing staining using different antibody pharmaceutical constituent antibodies, the phosphor integrated particles obtained from the antibody pharmaceutical constituent antibodies introduced with the first binding group and the phosphor integrated particles introduced with the second binding group Each labeled antibody dispersion in PBS is prepared, placed on a pathological section, and reacted with the target biological substance. In the present invention, the “target biological substance” is a target antigen targeted by the antibody drug constituent antibody. The PBS dispersion of the phosphor-aggregated particle-labeled antibody may contain a known blocking agent such as BSA-containing PBS and a surfactant such as Tween20. The temperature is not particularly limited, but can be performed at room temperature.

  The reaction time with the phosphor-aggregated particle-labeled antibody is preferably 30 minutes or more and 24 hours or less.

  It should be noted that a known blocking agent such as BSA-containing PBS is preferably added dropwise before staining with the phosphor-aggregated particle-labeled antibody.

  Next, the stained section is immersed in a container containing PBS to remove the unreacted phosphor-aggregated particle-labeled antibody. A PBS solution may contain a surfactant such as Tween20. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, PBS may be replaced during the immersion.

  In the above description, an example in which PBS is used as a buffer solution is given, but other buffer solutions described above may be used instead of PBS.

  Hematoxylin-eosin staining may be performed for tissue morphology observation.

  After performing the above operation, the pre-operation required for performing the observation mentioned later about the dyed structure | tissue can be performed as needed. For example, when a pathological section is used as the tissue to be stained, a cover glass is placed on the section and sealed. At this time, you may use a commercially available mounting agent as needed.

4) Observation under a fluorescence microscope In the staining method of the present invention, the tissue stained by the steps 1) to 3) is measured for changes in color and brightness due to changes in light emission and color absorption. By doing so, it is possible to evaluate the amount of the target biological material existing on the stained tissue.

  Here, when a fluorescent labeling agent or an enzyme that generates a fluorescent luminescent chemical species is used as the labeling substance, the target biological substance existing on the stained tissue is measured by measuring the intensity of the fluorescent emission. Can evaluate the number. In particular, the observation of the tissue stained in the form of a color pattern in the visible region can be performed by visual inspection without performing a secondary operation such as development.

  Specifically, the expression level of the target biological substance can be measured based on the number of bright spots or light emission luminance using a fluorescent microscope on a stained tissue such as a stained pathological section. Here, by using a confocal microscope as a microscope, it is also possible to obtain three-dimensional information about the stained tissue, and from the three-dimensional image obtained through fluorescence, such as cancer cells contained in the stained tissue A distribution can also be obtained. As the excitation light source for fluorescence excitation, a fluorescent material corresponding to the absorption maximum wavelength and fluorescence wavelength of the fluorescent substance used can be used as appropriate, and in order to separate excitation light from emitted fluorescence, fluorescence can be used as necessary. An optical filter for detection can be used in combination.

  Measurement of the number of bright spots or emission luminance can be performed using image analysis software, for example, public analysis software ImageJ, or G-Count, a total bright spot automatic measurement software manufactured by Zeonstrom.

[Method for determining the effectiveness of an antibody drug]
The staining method of the present invention described above can be applied to a method for determining the effectiveness of an antibody drug. Here, the present invention relates to the effectiveness of the antibody drug by measuring the amount of binding of the antibody that is a constituent of the antibody drug with respect to the amount of the antigen targeted by the antibody drug using the staining method described above. A method for determining gender is also provided. That is, the determination method of the present invention intends to determine the effectiveness of an antibody drug by evaluating the amount of binding of the constituent antibody to the target antigen amount by the staining method described above. It is.

  Specifically, in the determination method of the present invention, an antigen-antibody complex is produced by reacting and staining an antibody drug as a subject to a tissue in which a certain amount of effective target antigen is present. It is intended to determine the effectiveness of an antibody drug by visualizing it and evaluating the amount of antigen-antibody complex produced based on the color change at that time.

  However, in determining the effectiveness of a medicine containing an antibody as a component, the amount of an effective target antigen in a tissue may vary depending on the condition of the acquired tissue, the activation process, and the like. In order to correct the influence of the tissue condition, activation treatment, and the like, for example, a genetic diagnosis method, a conventionally known immunohistochemical method using an antibody drug as an antibody using a coloring reaction between an enzyme and a substrate, etc. It is preferable to make a comparison with the conventional determination method. Examples of gene diagnosis methods include PCR method, Southern hybridization method, FISH method and the like. Examples of immunohistochemical methods using coloring reaction of enzyme and substrate include horseradish peroxidase (HRP) and the HRP. Examples include a method using DAB as a substrate, alkaline phosphatase (ALP) and BCIP / NBT as a substrate for the ALP, β-galactosidase and X-gal as a substrate for the β-galactosidase, among these. When determining the efficacy of trastuzumab in breast cancer, the FISH method and / or the immunohistochemistry (IHC-DAB) method using HRP and DAB can be preferably exemplified.

In the method for judging the effectiveness of a pharmaceutical comprising the antibody of the present invention as a component, and in the system for judging the effectiveness of a pharmaceutical comprising the above-mentioned antibody as a component, the pharmaceutical for which the effectiveness is judged includes an antibody as an active ingredient The present invention is not limited to pharmaceuticals, and when a signal transduction pathway exists in a protein targeted by an antibody, a pharmaceutical that targets a factor (protein) in the signal transduction pathway is also included. When the antibody in the pharmaceutical comprising the above antibody as a component is HER2, for example, Ras, Raf, etc. as factors involved in cell proliferation downstream of HER2 signaling pathway, PI3K, AKT, etc. as factors involved in anti-apoptosis Can be mentioned. As an antibody in a pharmaceutical comprising an antibody as a component, an antibody that specifically recognizes a cancer growth regulator or a metastasis regulator is preferable, and examples of the type of antibody include a monoclonal antibody and a polyclonal antibody. The class and subclass of the antibody are not particularly limited. Examples of the class include IgA, IgG, IgE, IgD, IgM, and the like. Examples of the subclass include IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and the like. Can be mentioned. The term “antibody” used herein is used to include any antibody fragment or derivative. For example, Fab, Fab ′ ₂ , CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv), etc. including. Such antibodies can be produced by known methods (see, for example, Harlow E. & Lane D., Antibody, Cold Spring Harbor Laboratory Press (1988)).

[Example 1: Preparation of labeled body]
《Biotinylated trastuzumab》
As trastuzumab, by using Herceptin (registered trademark) in powder form manufactured by Roche in the form of pharmaceuticals, biotinylated using Biotin Labeling kit-SH (Dojindo), Biotinylated trastuzumab was obtained.

<Streptavidin-modified HRP>
High Sensitivity Streptavidin-HRP (Thermo Scientific) was used as streptavidin-modified HRP.

<Streptavidin-modified phosphor aggregated particles>
Synthesis of fluorescent particles (organic fluorescent dye)
Tetramethylrhodamine (TAMRA-SE manufactured by Invitrogen) (excitation wavelength: 550 nm, emission wavelength: 570 nm) 6.6 mg and 3-aminopropyltrimethoxysilane (3-aminopropyltrimethoxysilane, manufactured by Shin-Etsu Silicone, KBM903) 3 μL were mixed in DMF. An organoalkoxysilane compound was obtained. 0.6 ml of the obtained organoalkoxysilane compound was mixed with 48 ml of ethanol, 0.6 ml of TEOS (tetraethoxysilane), 2 ml of water, 2 ml of 28% ammonia water for 3 hours.

  The mixed solution prepared in the above step was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed twice. As a result, 10 mg of tetramethylrhodamine-aggregated / silica nanoparticles were obtained as phosphor-aggregated particles.

  When the obtained tetramethylrhodamine-aggregated / silica nanoparticles were observed by SEM, the average particle size was 104 nm and the coefficient of variation was 12%.

Synthesis of fluorescent particles (quantum dots)
Dissolve In (acac) ₃ and tris (trimethylsilyl) phosphine in 6 ml of octadecene in a three-necked flask in 0.1 ml each in 1 ml of octadecene so that the ratio of In to P is In / P = 1/1. The resulting solution was added and reacted in an argon atmosphere at 300 ° C. for 1 h to obtain an InP core particle dispersion.

  Next, using this InP core particle dispersion, InP / ZnS core / shell particles were synthesized. After allowing the InP core particle dispersion to cool to 80 ° C., a solution in which zinc stearate and sulfur are dissolved in 1 ml of octadecene at a ratio of Zn / S = 1/1 to the dispersion, They were added so that the molar ratio of In, P, Zn, and S was In / P / Zn / S = 1/1/1/1. The mixture was heated from 80 ° C. to 230 ° C., reacted for 30 minutes, and acetone, which is a poor solvent, was added to the resulting solution. As a result, InP / ZnS core / shell particles were obtained as a precipitate. The thus obtained InP / ZnS core / shell semiconductor nanoparticles were particles having a maximum emission wavelength at 630 nm.

  A water-soluble semiconductor nanoparticle solution can be obtained by adding 0.1 mg of dodecylamine and 1 mL of ultrapure water to 0.1 mg of the InP / ZnS core / shell particles and stirring strongly for 1 h. The water-soluble semiconductor nanoparticle solution was subjected to hydrolysis by adding 0.1 mg of TEOS, 0.01 mL of ethanol, and 0.03 mL of concentrated ammonia water to obtain an InP, CdSe, CdTe semiconductor nanoparticle aggregate.

  When the obtained InP / ZnS core / shell particle aggregated silica nanoparticles were observed with an SEM, the average particle size was 94 nm and the coefficient of variation was 14%.

Binding of Streptavidin to Phosphor Accumulated Particles The binding of streptavidin to the above phosphor aggregated particles (that is, tetramethylrhodamine integrated silica nanoparticles and InP / ZnS core / shell particle integrated silica nanoparticles) The following procedure was followed.

  Phosphor-aggregated particles are dissolved in PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid) and adjusted to 3 nM, and SM (PEG) 12 ( Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) was mixed and reacted for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The silica nanoparticle for antibody binding was obtained by performing washing | cleaning by the same procedure 3 times.

  On the other hand, streptavidin is reduced with 1M dithiothreitol (DTT) or thiol group addition treatment such as SATA, and a reduced antibody solution capable of binding to silica particles is removed by removing excess reaction reagent with a gel filtration column. Obtained.

  The antibody-binding silica nanoparticles obtained above and the reduced antibody were mixed in PBS containing 2 mM of EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. The resulting solution was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. By performing washing by the same procedure three times, streptavidin-bonded / phosphor-integrated silica nanoparticles were obtained.

  In Example 3 described later, the streptavidin-binding / phosphor-integrated silica nanoparticles were used as streptavidin-modified phosphor-integrated particles.

[Example 2: tissue staining (HRP)]
Human breast tissue was immunostained using biotinylated trastuzumab and streptavidin-modified HRP prepared in Example 1. The staining method of the present invention using this biotinylated trastuzumab and streptavidin-modified HRP may be hereinafter referred to as “trastuzumab-HRP”.

  As a stained section, a tissue array slide (CB-A712) manufactured by Cosmo Bio, whose FISH score was calculated in advance using a Pathvision HER-2 DNA probe kit (Abbott) was used. The tissue array slide was deparaffinized, washed with water, and autoclaved in 10 mM citrate buffer (pH 6.0) for 15 minutes to activate the antigen. The tissue array slide after the antigen activation treatment was washed with PBS buffer, and then subjected to blocking treatment with a 1% BSA-containing PBS buffer in a wet box for 1 hour. After blocking treatment, biotinylated trastuzumab diluted to 0.05 nM with PBS buffer containing 1% BSA was reacted with the tissue section for 2 hours and then washed. Furthermore, when it was reacted with streptavidin-modified HRP for 0.5 hours and washed, trastuzumab and a tissue modified with HRP (hereinafter referred to as “trastuzumab-HRP-modified tissue”) were obtained. This Herceptin-HRP modified tissue was stained by a conventional method using a DAB (diaminobenzidine tetrahydrochloride) solution as a substrate solution.

  Further, as examples of tissue staining without using antibody drugs, Histofine HER2 kit (Nichirei: hereinafter referred to as “N company staining kit”), Dako Hercept Test II (Dako: hereinafter referred to as “D company staining kit”), Ventana I-VIEW pathway Staining was performed using HER2 (4B5) (Roche: “R company staining kit”).

  For the stained tissue, the score was calculated based on the criteria of the HER2 inspection guide-revised third edition according to the staining concentration.

  Table 2 shows a comparison of the staining score and FISH score using each company's kit and trastuzumab-HRP. Here, a staining score of “trastuzumab-HRP” corresponds to a staining score for a tissue stained by the staining method of the present invention.

  Table 3 shows the correlation between the staining score using each company's kit and trastuzumab-HRP and the FISH score. It can be seen that the staining score using trastuzumab-HRP shows a high correlation with the FISH score as compared with the staining score of each company's kit not using the antibody drug.

[Example 3: Tissue staining (phosphor-aggregated particles)]
Using the same method as in Example 2, human breast tissue was immunostained using streptavidin-modified phosphor-aggregated particles as a label. That is, except that the tissue section that had been reacted with biotinylated trastuzumab was reacted with each of the streptavidin-modified phosphor-aggregated particles obtained in Example 1 instead of streptavidin-modified HRP. The immunostaining was performed according to the same procedure as in No. 2 (the staining method of the present invention using biotinylated trastuzumab and streptavidin-modified phosphor-aggregated particles may be hereinafter referred to as “trastuzumab-phosphor-aggregated particles”). Stained tissue sections were imaged using an Olympus DSU confocal microscope, the cancer cell area was identified, binary processing using image J was performed, and bright spot measurement was performed after noise removal processing. The number of bright spots was measured.

  Table 4 shows a comparison of the FISH score with the staining score using trastuzumab-HRP, the number of bright spots per 10 cells in the tissue stained with each trastuzumab-phosphor-aggregated particle.

  Table 5 shows a correlation comparison with the FISH score for the staining score using trastuzumab-HRP and each trastuzumab-phosphor-aggregated particle. It can be seen that the data on the number of bright spots measured using each trastuzumab-phosphor aggregated particle shows a higher correlation with the FISH score than the staining score using trastuzumab-HRP.

Claims (2)

As a substance that recognizes an antigen targeted by an antibody drug, an antibody that is a component of the antibody drug is used,
The antibody is used in the form of a labeled antibody to which a labeling substance is bound;
The labeling substance is a nanoparticle having a diameter of 94 nm or more obtained by integrating quantum dots or organic fluorescent dyes ,
A distribution of the antigen present in the tissue is obtained in a visualized form depending on the amount of the antibody bound to the antigen; and
An immunohistochemical staining method wherein the antigen is any one selected from the group consisting of a cancer growth regulator, a metastasis regulator, a growth regulator receptor, and a metastasis regulator receptor. As a substance that recognizes an antigen targeted by an antibody drug, an antibody that is a component of the antibody drug is used,
The antibody is used in the form of a labeled antibody to which a labeling substance is bound;
The labeling substance is a nanoparticle having a diameter of 94 nm or more formed by integrating quantum dots or organic fluorescent dyes , and
The amount of the antibody bound to the amount of the antigen is measured using an immunohistochemical staining method in which the distribution of the antigen present in the tissue is visualized according to the amount of the antibody bound to the antigen. Thereby determining the effectiveness of the antibody drug.