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WO2017213117A1 - Procédé de coloration multiple et trousse de coloration - Google Patents

Procédé de coloration multiple et trousse de coloration Download PDF

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Publication number
WO2017213117A1
WO2017213117A1 PCT/JP2017/020927 JP2017020927W WO2017213117A1 WO 2017213117 A1 WO2017213117 A1 WO 2017213117A1 JP 2017020927 W JP2017020927 W JP 2017020927W WO 2017213117 A1 WO2017213117 A1 WO 2017213117A1
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Prior art keywords
antibody
molecule
protein
detecting
staining
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Ceased
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PCT/JP2017/020927
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English (en)
Japanese (ja)
Inventor
賢吾 竹内
誠司 小川
圭亮 片岡
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Japanese Foundation for Cancer Research
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Japanese Foundation for Cancer Research
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Priority to JP2018522496A priority Critical patent/JP6781252B2/ja
Publication of WO2017213117A1 publication Critical patent/WO2017213117A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a multiple staining method in immunostaining.
  • the present invention relates to a staining method that can be easily diagnosed during pathological diagnosis.
  • Pathological diagnosis is used for various examinations such as tumor classification, malignancy estimation, and search for specific viruses and bacteria using various samples such as surgical tissues, biopsy specimens, and body cavity fluids. Along with clinical symptoms, it is an essential diagnostic technique that determines the severity and malignancy of a lesion site and is responsible for selecting a treatment policy and method. In recent years, it has come to play an extremely important role as a diagnostic technique for determining the indication of molecular target therapy.
  • hematoxylin and eosin staining in which two types of dyes, hematoxylin and eosin, are used to dye the cell nucleus and the part other than the nucleus is used.
  • immunostaining methods for detecting disease-related antigen molecules using specific antibodies that bind to target molecules have been widely used.
  • Immunostaining using specific antibodies is an important analysis method for pathological diagnosis and molecular studies of lesion tissue.
  • different antigens can be stained with different labeled antibodies, and the molecular localization can be analyzed on the same specimen, so that a great deal of information can be obtained.
  • different cells such as B cells and T cells can be dyed on the same pathological tissue section, normal cells and cancer cells expressing cancer antigens can be dyed, or multiple antigens can be localized in the same cell. By detecting at the same time, the correlation between antigens can be observed and analyzed.
  • the expression of the target antigen molecule is not always the same.
  • small molecule drugs and antibody drugs against the molecule can be expected to have therapeutic effects, but therapeutic effects are expected for patients with cancer cells that do not express the molecules.
  • therapeutic effects are expected for patients with cancer cells that do not express the molecules.
  • Patent Documents 1 to 3 there is a method for diagnosing whether the therapeutic drug acts effectively prior to the administration of the antibody drug due to the presence of a patient having resistance as seen in rituximab and the low response rate as seen in trastuzumab.
  • the multiple immunostaining used in the conventional pathological examination is a method of identifying and staining different molecules, and not a method of simultaneously detecting changes in specific molecules.
  • the target tissue often expresses a mixture of mutant and wild type. Therefore, the expression and mutation of the causative gene must be performed by serially sectioning and staining the expression of a specific gene on one side, while separately expressing the expression of the mutant and comparing both. This takes a very long time for the examination itself and requires skill in diagnosis. Therefore, despite the fact that testing for the expression of proteins targeted by small molecule drugs and antibody drugs that are molecular target drugs and mutations in those proteins are necessary tests to confirm the effectiveness of the drug, The current situation is not usually done.
  • the present invention relates to the expression of mutant proteins due to gene mutations such as deletions and point mutations, protein changes such as modifications such as phosphorylation and three-dimensional structure changes on the same section of the pathological tissue and the original protein. It is an object of the present invention to provide a method for staining in a distinguishable manner and a staining kit.
  • the present invention relates to a method and kit for multiple staining of a specimen containing cells on the same specimen.
  • a multiple staining method that uses a plurality of antibodies to detect in a specimen in which a specimen containing cells is fixed, and detects an antibody for detecting the presence of a specific molecule and a change in the specific molecule
  • a multiple staining method characterized by using an antibody for (2)
  • a kit for detecting a change in a molecule in a sample in which a specimen containing cells is immobilized, and an antibody for detecting the presence of a specific molecule and at least one for detecting a change in the specific molecule Comprising a label for distinguishably labeling at least one antibody, an antibody for detecting the presence of the specific molecule, and at least one antibody for detecting a change in the specific molecule, respectively.
  • the specific molecule is PD-L1 or PD-L2, and an antibody for detecting the presence of PD-L1 or PD-L2, and changes in the C-terminal region of PD-L1 or PD-L2
  • ⁇ Multiple staining can be performed on the same specimen to identify molecular changes, so that not only can diagnostic results be obtained quickly, but also accurate diagnosis can be performed.
  • staining method of this invention The figure which shows typically the identification method using 3 or more types of antibodies.
  • the top photo is a magnified image.
  • the present invention recognizes and detects the presence of an antigen protein molecule in a clinical specimen immobilized on a preparation, as well as modifications such as deletion mutation, protein change by point mutation, phosphorylation, glycosylation, etc. It is a method of simultaneously detecting molecular changes such as interactions between them and changes in steric structure. Accordingly, it is possible to detect the presence or absence of a structural change in a protein such as a wild-type protein and a mutated protein, or an activated protein and a non-activated protein in the same specimen.
  • a plurality of site-specific antibodies against the protein to be examined are used, and the antigen protein is directly used. Changes in the structure of the molecule can be detected. Therefore, all proteins expressed on the cell surface and in the cell can be targeted, and on the same specimen, wild-type protein and mutated protein, or activated protein and non-activated protein, etc. It is possible to detect the presence and extent of changes in the structure of the protein.
  • immunostaining is performed with a site-specific antibody against the same protein, it is possible to simultaneously detect the presence site in the cell, such as the cell membrane, cytoplasm, nucleus, and intracellular structure of the target protein. Furthermore, it is possible to simultaneously stain and analyze different parameters such as intracellular localization sites, degree of accumulation, presence / absence of higher-order structure changes, and higher-order structure change sites.
  • This method is very useful for examining “cancer cell heterogeneity” that the antigen expression of each cancer cell is different and diverse, even in a lump of cancer.
  • a molecular pathological diagnosis of what therapeutic antibody can be applied as an anticancer drug can be performed.
  • the time and equipment requirements of the detection method are also at a level applicable to normal pathological examination work of a medical institution and have a great practical advantage.
  • any antibody may be used as long as it can detect a specific epitope in the target molecule and has binding properties.
  • it is preferably a monoclonal antibody, even a polyclonal antibody can be suitably used as long as it can detect only a specific region in the molecule, such as an anti-peptide antibody.
  • antibodies that recognize a portion where a mutation occurs and antibodies that recognize a wild-type antibody are used so that they are separated from each other to the extent that steric hindrance does not occur at the time of binding.
  • protein expression and mutation can be detected simultaneously.
  • cells expressing the wild-type and mutant-type can be detected simultaneously.
  • the label used in the present invention may be any label as long as it is two or more distinguishable labels.
  • “identifiable” includes not only those that can be identified by an observer through normal microscopic observation, but also those that can be identified as mechanical signals even if they cannot be identified with the naked eye.
  • the label may be selected from known labels, and is selected from those usually used as labels, such as dyes, quantum dots, and colloids. Moreover, specifically as a pigment
  • fluorescent dyes examples include rhodamine dye molecules, Alexa Fluor (made by Invitrogen) dye molecules, cyanine dye molecules, Texas Red dye molecules, squarylium dye molecules, oxazine dye molecules, and aromatic ring dye molecules. Carbopyronine dye molecules can be used.
  • any label may be used as long as a plurality of molecular species can be finally identified.
  • a labeled antibody obtained by directly binding a label to an antibody used for detection may be used, or the detection may be finally performed using a secondary antibody or a tertiary antibody.
  • a label usually used in immunostaining methods such as avidin and biotin may be used.
  • the reagent which colors in a different color should just be used.
  • the detection kit of the present invention includes an antibody for detecting the presence of a molecule, an antibody for detecting a molecular change such as deletion mutation, point mutation, modification, interaction, conformational change of the molecule, and each antibody. It includes a sign that can identify As each antibody, a commercially available antibody may be used, or an antibody that detects a molecular change may be prepared and used. Further, reagents necessary for the enzyme antibody method and reagents necessary for a normal immunostaining method such as a washing solution can be included.
  • a washing solution can be included.
  • Detection of a molecule having a deletion mutation A method of simultaneously staining a wild-type molecule A and a molecule A 'having a deletion mutation on the same section will be described (Fig. 1, deletion mutant).
  • Fig. 1, deletion mutant When distinguishing cells expressing wild-type molecule A and deletion mutant A ′, antibody a and deletion capable of reliably recognizing both wild-type molecule A and mutant molecule A ′
  • An antibody b that recognizes a region where a mutation often occurs may be used for detection.
  • Antibody a recognizes both wild type and deletion mutant molecules.
  • antibody b cannot bind when deletion occurs. Therefore, only the wild-type molecule A is recognized because it does not bind to the deletion mutant A '. That is, antibody a and antibody b bind to wild-type molecule A, and antibody a only binds to the deletion mutant.
  • the a antibody is labeled so as to be colored in brown and the b antibody is colored in black so that the brown is masked, the cells expressing the wild type molecule A are black.
  • Cells expressing mutant A ′ can be dyed brown.
  • a label that can be easily distinguished from multiple staining may be used.
  • deletion mutants can be detected by this method in cancers that highly express PD-L1 (Programmed cell death 1) lacking the C-terminal region.
  • PD-L1 Programmed cell death 1
  • PD-L1 is highly expressed (Non-patent Document 1), but when an antibody that recognizes the deficient portion is used, its expression cannot be detected.
  • a patient who highly expresses such a C-terminal region-deficient PD-L1 may be a patient (super responder) that is highly effective against an anti-PD-L1 antibody drug.
  • Non-patent Document 2 Non-patent Document 2
  • Embodiment 2 Detection of a molecule having a chromosomal translocation A case will be described in which a translocation occurs in a chromosome and a fusion protein B 'is expressed by transposition of a part of the gene by translocation (FIG. 1 , Chromosome translocation).
  • Antibody c represents an antibody that recognizes proteins B and B ′ regardless of the presence or absence of translocation.
  • Antibody d is an antibody that detects only wild-type protein B and does not recognize when translocation occurs.
  • Mutant C ′ can be distinguished and stained. Even when different point mutations are introduced at the same location, it can be confirmed that the point mutation has been introduced at the relevant location depending on the presence or absence of binding of antibody f that detects only wild-type C.
  • arginine (R172) at position 172 of the gene IDH2 encoding isocitrate dehydrogenase (IDH) is substituted with multiple amino acids by point mutation, but the mutation is a predictor of brain tumors and the like are known. Conventionally, each specific antibody was prepared for each mutation and the mutation was confirmed. If the method of this embodiment is used, an antibody that specifically recognizes wild-type R172 is used. Good. In addition, amino acid at position 132 of IDH1, position 12, position 13 and position 61 of KRAS are known as such hot spots for gene mutation.
  • an antibody that does not recognize the point mutation C ′ is taken as an example, but an antibody that recognizes the point mutation and does not recognize the wild type may be used.
  • FIG. 1 presence / absence of modification (phosphorylation, addition of sugar, etc.)
  • FIG. 1 presence / absence of modification (phosphorylation, addition of sugar, etc.)
  • Some molecules may undergo modifications such as phosphorylation upon activation.
  • the presence or absence of sugar chains plays an important role in biological reactions.
  • numerator which received the phosphorylation is demonstrated, the modification by a sugar_chain
  • Antibody g represents an antibody that is recognized regardless of the presence or absence of activation.
  • the antibody h is an antibody that recognizes only the protein D that is not phosphorylated and does not recognize the phosphorylated epitope.
  • An antibody h that recognizes a protein that has not undergone phosphorylation can be distinguished from an antibody g that is recognized regardless of activation (D) and an antibody g that is bound only (D ′). By selecting the label so that it becomes, it is possible to perform staining by distinguishing the presence or absence of phosphorylation.
  • staining may be performed in the same manner as described above using an antibody that recognizes only the phosphorylation state of the phosphorylation site, that is, an antibody that recognizes an activated form and an antibody that recognizes whether or not activated.
  • an antibody that recognizes only the phosphorylation state of the phosphorylation site that is, an antibody that recognizes an activated form and an antibody that recognizes whether or not activated.
  • detecting the presence or absence of a sugar chain it may be detected by using an antibody that recognizes an amino acid sequence to which no sugar chain is added.
  • post-translational modifications such as acetylation and ubiquitination can be detected by the same method.
  • Antibody i represents an antibody that is recognized regardless of the presence or absence of complex formation.
  • the antibody j is an antibody that cannot recognize the protein E when a complex with the protein X is formed.
  • E complex
  • a label that makes it possible to distinguish between them it is possible to distinguish and stain the presence or absence of complex formation.
  • an example is given in which a protein X different from protein E forms a complex, but dimers and trimers of the same protein can also be distinguished and stained by the same technique.
  • Detection of three-dimensional structure change A three-dimensional structure itself of a protein may be greatly changed by modification or complex formation. It is also possible to capture the change in the three-dimensional structure by the staining method of the present embodiment (FIG. 1, change in the three-dimensional structure).
  • Antibody k represents an antibody that is recognized regardless of changes in the three-dimensional structure.
  • the antibody 1 is an antibody that does not bind to the protein F ′ having a changed three-dimensional structure.
  • an example is given in which an antibody that is not recognized due to a change in the three-dimensional structure is used, but it is also possible to identify a change in the three-dimensional structure using an antibody that recognizes the change in the three-dimensional structure.
  • FIG. 2 shows an example in which point mutation occurs in two places, but a plurality of changes occurring on the same protein may be any combination of changes.
  • Antibody m represents an antibody that recognizes regardless of changes in protein G.
  • Antibody n represents an antibody that cannot be recognized due to the occurrence of point mutation y. The antibody n cannot recognize the protein G "" in which the point mutation y occurs alone and the protein G "" in which the double mutation with the point mutation z occurs.
  • Antibody o indicates an antibody that cannot be recognized when a point mutation z occurs. That is, the antibody o cannot recognize the protein G ′ in which the point mutation z occurs alone and the protein G ′ ′′ in which the mutation is doubled.
  • a label capable of distinguishing the molecules (G) recognized by the three antibodies from the antibodies m, n, and o may be used.
  • a fluorescent label or a label with a quantum dot or the like it is possible to distinguish the three types of antibodies in a distinguishable manner, so that the modes of all molecules on the same section can be distinguished.
  • Isocitrate dehydrogenase is an enzyme known to have three subtypes of IDH1, IDH2, and IDH3 in humans.
  • IDH1 / 2 Several point mutations have been reported in IDH1 / 2 in several cancers including glioma and acute myeloid leukemia. Since the point mutation found in IDH1 / 2 is known as a prognostic marker, prognosis can be easily performed by distinguishing and distinguishing these molecular mutations. Examples in which the multiple staining method of the present invention is applied are shown below as examples.
  • PD-1 (Programmed cell death 1) is a T cell regulatory molecule expressed on the surface of T cells and functions as an immune checkpoint protein together with ligands PD-L1 and PD-L2. It is known that cancer cells often escape immune cell attacks. This is thought to be because PD-L1 and PD-L2 antigens expressed in cancer cells bind to PD-1 to suppress cytotoxic T cells and act on immune checkpoint mechanisms.
  • PD-L1 is overexpressed in human cancer cells isolated from ovarian cancer, breast cancer, melanoma, lung cancer, colon cancer, bladder cancer, cervical cancer, etc. It is considered that the immune response is suppressed by the interaction with No. 1 and is beneficial for cancer cell survival. Cancers with high PD-L1 expression are highly reactive to anti-PD-L1 antibody drugs, so clinically confirm the expression of PD-L1 with a companion diagnostic agent. Anti-PD-L1 antibody drugs are used.
  • Non-patent Document 1 Abnormalities in the 3 'region are found in multiple cancers, including adult T-cell leukemia / lymphoma, diffuse large B-cell lymphoma, and gastric adenocarcinoma, and are clearly responsible for the high expression of PD-L1 It was made.
  • Abnormalities in the 3 ′ region include not only abnormalities in UTR but also mutations in which the C-terminus is deleted.
  • antibodies that are candidates for companion diagnostic agents antibodies that bind to this region Is also included.
  • mutant PD-L1 lacking 3 ′ cannot be recognized even though PD-L1 is highly expressed. Therefore, when this companion diagnostic agent is used, a super responder in which an anti-PD-L1 antibody drug works very well is overlooked.
  • Example 1 by using the double staining method shown in Example 1, not only can the information obtained by antibodies recognizing other than the C-terminal among the conventional methods, that is, the expression level of PD-L1, be confirmed, but Among them, a super responder having a mutation in which the C-terminal is deleted can be simultaneously identified.
  • FIG. 3 shows a case of Hodgkin's lymphoma, and shows the result of staining cells expressing PD-L1 molecules in which wild type and defects have occurred.
  • Antibody that recognizes PD-L1 molecule is brown when PD-L1 is expressed regardless of wild type or deletion mutant, and antibody that recognizes a region present only in wild type PD-L1 The sign is selected so that it is colored black.
  • clone E1J2J manufactured by CST
  • clone SP142 manufactured by SPRING
  • anti-CD68 antibody manufactured by DAKO
  • the colors are brown, black and red, respectively. Immunostaining is performed according to a standard method.
  • BCL present in the germinal center of lymphoid follicles does not show BCL2 expression, but in follicular lymphoma, BCL2 and immunoglobulin heavy chain genes are translocated, and BCL2 expression is constantly observed. Therefore, expression of BCL2 is observed at the neoplastic germinal center. However, it is known that mutations frequently occur in BCL2 in follicular lymphoma, and the expression may not be confirmed depending on the antibody used.
  • FIG. 4 shows double follicular lymphoma tissue stained with two anti-BCL2 antibodies, clone 124 (manufactured by Dako) and clone C-2 (manufactured by Santacruz), and colored with blue and brown, respectively. is there.
  • clone 124 manufactured by Dako
  • clone C-2 manufactured by Santacruz

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Abstract

La présente invention concerne l'identification simultanée, dans le même échantillon, de cellules exprimant une molécule particulière et de cellules exprimant la molécule lorsque la molécule a été modifiée, cette identification étant réalisée par coloration à l'aide d'un anticorps destiné à détecter la présence d'une molécule spécifique marquée de manière identifiable, et d'un anticorps destiné à détecter toute modification apportée à la molécule.
PCT/JP2017/020927 2016-06-10 2017-06-06 Procédé de coloration multiple et trousse de coloration Ceased WO2017213117A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020050373A1 (fr) * 2018-09-06 2020-03-12 コニカミノルタ株式会社 Procédé d'acquisition d'informations, dispositif d'acquisition d'informations et programme

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014151006A2 (fr) * 2013-03-15 2014-09-25 Genentech, Inc. Biomarqueurs et méthodes de traitement d'états associés à pd-1 et pd-l1
WO2016175275A1 (fr) * 2015-04-30 2016-11-03 国立大学法人京都大学 Procédé pour prévoir l'effet thérapeutique d'un inhibiteur de pd-1/pd-l1 en utilisant une anomalie dans pd-l1(cd274) comme indice

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014151006A2 (fr) * 2013-03-15 2014-09-25 Genentech, Inc. Biomarqueurs et méthodes de traitement d'états associés à pd-1 et pd-l1
WO2016175275A1 (fr) * 2015-04-30 2016-11-03 国立大学法人京都大学 Procédé pour prévoir l'effet thérapeutique d'un inhibiteur de pd-1/pd-l1 en utilisant une anomalie dans pd-l1(cd274) comme indice

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG W ET AL.: "A Frequent Somatic Mutation in CD 274 3' -UTR Leads to Protein Over-Expression in Gastric Cancer by Disrupting miR-570 Binding", HUMAN MUTATION, vol. 33, December 2011 (2011-12-01), pages 480 - 484, XP055327183, ISSN: 1059-7794, DOI: doi:10.1002/humu.22014 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020050373A1 (fr) * 2018-09-06 2020-03-12 コニカミノルタ株式会社 Procédé d'acquisition d'informations, dispositif d'acquisition d'informations et programme

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