WO2022255401A1 - Ｅｒｋ－ｍａｐｋ経路の異常な活性化に伴い発現する疾患マーカー - Google Patents

Ｅｒｋ－ｍａｐｋ経路の異常な活性化に伴い発現する疾患マーカー Download PDF

Info

Publication number
WO2022255401A1
WO2022255401A1 PCT/JP2022/022276 JP2022022276W WO2022255401A1 WO 2022255401 A1 WO2022255401 A1 WO 2022255401A1 JP 2022022276 W JP2022022276 W JP 2022022276W WO 2022255401 A1 WO2022255401 A1 WO 2022255401A1
Authority
WO
WIPO (PCT)
Prior art keywords
mek1
cancer
mutant
ras
marker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/022276
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
睦寛 武川
裕二 久保田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Tokyo NUC
Original Assignee
University of Tokyo NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Tokyo NUC filed Critical University of Tokyo NUC
Priority to JP2023525886A priority Critical patent/JPWO2022255401A1/ja
Publication of WO2022255401A1 publication Critical patent/WO2022255401A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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 disease markers.
  • cancer marker molecules have been reported so far for the purpose of cancer diagnosis and post-treatment follow-up. Some of them have already been put to practical use, for example, blood PSA (prostate specific antigen) is used as a marker molecule for prostate cancer.
  • blood PSA prostate specific antigen
  • the expression levels of many other cancer marker molecules do not necessarily match the development of cancer.
  • there are no effective diagnostic markers yet for pancreatic cancer and the difficulty in early detection is a cause of poor prognosis.
  • many reports have been reported so far on activating mutations/gene amplifications of Ras, Raf, MEK, etc. that constitute the ERK-MAP kinase pathway (Raf-MEK-ERK) (pancreatic cancer).
  • KRas is mutated to an active form in more than 90% of pancreatic cancers
  • BRaf is mutated to an active form in about 50% of metastatic melanomas, constitutively activating the ERK pathway.
  • BRaf-activated malignant melanoma and ALK-activated lung cancer although molecular-targeted drugs temporarily disappear, the ERK pathway is eventually reactivated via collateral pathways. Acquisition of drug resistance has been shown to cause cancer recurrence.
  • Non-Patent Document 1 the MEK1 (K57N) mutation found in lung tumors has been shown to significantly promote cell proliferation through activation of the ERK pathway (Non-Patent Document 3).
  • Non-Patent Document 4 Non-Patent Document 4
  • a molecular marker that can sensitively detect abnormal activation of the ERK-MAP kinase pathway can be found, it can be used for early diagnosis of cancer and Ras/MAPK syndrome and prediction of cancer recurrence.
  • any abnormalities in the ERK-MAP kinase pathway are related to cancer or Ras/MAPK syndrome, and it is one of the important problems to be solved in the medical field. be.
  • the present invention clarifies what diseases are triggered by abnormal activation of the ERK-MAPK pathway and what gene expression is induced. It is an object of the present invention to provide markers for early detection of activation-related diseases, particularly cancer, and early detection of Ras/MAPK syndrome.
  • the inventors attempted to search for genes that are highly expressed only when the ERK pathway is constitutively and strongly activated, among genes whose expression is induced downstream of the ERK pathway. .
  • Human-derived epithelial cells were transfected with cancer-derived active MEK1 point mutants or Ras/MAPK syndrome-derived active MEK1 point mutants, and cDNA microarray analysis was performed to obtain genome-wide gene expression profiles in the cells. analyzed exhaustively. As a result, we found that the expression levels of various mRNAs fluctuate greatly.
  • cancer-derived MEK1 mutants we collected various cancer cells (malignant melanoma, lung cancer, pancreatic cancer, colorectal cancer, etc.) in which abnormal activation of the ERK pathway was observed, and examined these genes. When we monitored the expression level at the RNA and protein levels, we confirmed that it was actually strongly expressed in most cancer cell lines. In addition, for some molecules, analysis (immunostaining) using clinical cancer tissue is also performed, and at least in clinical samples of pancreatic cancer, lung cancer, and colon cancer, at high frequency (in 80% or more cases) High expression was confirmed. Interestingly, many of these molecules included secretory proteins and membrane proteins that could serve as novel cancer detection markers.
  • the present invention is the following (1) to (13).
  • a cancer detection marker comprising at least one molecule selected from a group of proteins whose expression is induced by ERK1 or ERK2, wherein said ERK1 and ERK2 are activated by autophosphorylation of the T-loop region.
  • a marker for cancer detection characterized by being activated by a MEK1 mutant or MEK2 mutant.
  • the marker for cancer detection according to (1) above, wherein the mutation in the MEK1 mutant is Q56P, K57N, C121S or E203K.
  • the marker for cancer detection according to (1) above, wherein the mutation in the MEK2 mutant is Q60P.
  • MMP10 EMP1, Rheb2, TM4SF1, TM4SF19, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR (CD87), PVR (CD155), IL7R (CD127), IL1R2 (CD121b), IL4R, TweakR (CD266), CD3D, CD44, SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1, BMP2, TFPI2,
  • the cancer detection marker according to (4) above comprising at least one protein selected from the group consisting of EMP1, TM4SF1, TM4SF19, c11orf96, PHLDA1, PHLDA2, TFPI2, Rheb2 and GDF15.
  • MMP10 EMP1, Rheb2, TM4SF1, TM4SF19, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR (CD87), PVR (CD155), IL7R (CD127), IL1R2 (CD121b), IL4R, TweakR (CD266), CD3D, CD44, SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1, BMP2, TFPI2, A cancer detection kit comprising antibodies or aptamers against GDF15, PAEP, CCL7, IL11 or CRLF.
  • a method for diagnosing cancer or diagnosing cancer comprising the step of measuring the expression level of the cancer detection marker according to any one of (1) to (6) above present in a sample derived from a subject. Auxiliary method.
  • a marker for cancer detection comprising the step of searching for a protein that induces the expression of ERK1 or ERK2 that is activated by a MEK1 mutant or MEK2 mutant that is activated by autophosphorylation of the T-loop region. screening method.
  • a marker for detecting Ras/MAPK syndrome containing at least one molecule selected from the group of proteins whose expression is induced by ERK1 or ERK2, wherein the ERK1 and ERK2 are not phosphorylated in the T-loop region A marker for detecting Ras/MAPK syndrome, characterized by being activated by a MEK1 mutant or MEK2 mutant having activity even in the case of Ras/MAPK syndrome.
  • the mutation in the MEK2 mutant is F53C, F53V, F57L, K61E, A62P, P128R, G132V, T134C, or Y134H.
  • (12) comprising the step of measuring the expression level of the Ras/MAPK syndrome detection marker according to any one of (9) to (11) present in a subject-derived sample, a method for diagnosing Ras/MAPK syndrome or A diagnostic aid for Ras/MAPK syndrome.
  • (123) Ras/MAPK, including the step of searching for proteins that induce the expression of ERK1 or ERK2 activated by MEK1 or MEK2 mutants that are active even if the T-loop region is not phosphorylated Screening method for markers for syndrome detection.
  • the sign "-" indicates a numerical range including the values on the left and right of it.
  • cancers caused by abnormal activation of the ERK pathway can be detected with high sensitivity and accuracy.
  • Ras/MAPK syndrome detection marker according to the present invention, early diagnosis and detection of Ras/MAPK syndrome, which is difficult to diagnose, becomes possible.
  • (A) shows the results of measuring the kinase activity (using ERK2 as a substrate) of cancer-derived MEK1 mutants and other MEK1 mutants.
  • Kinase-deficient Myc-ERK2(K/N) and HA-MEK1 were co-transfected into HEK293 cells.
  • Phosphorylated ERK2 was detected by immunoblotting and its band intensity was quantified (P-ERK). The same filter was blotted again with an anti-Myc antibody to confirm the amount of ERK present.
  • Mutants with Q56P and K57N mutations are cancer-derived MEK1 mutants. Other variants are those derived from the Ras/MAPK syndrome.
  • K/M K97M mutation that lacks kinase activity
  • AA non-phosphorylated mutant in which Ser218 and Ser222 of the T-loop are replaced with alanine.
  • GST-MEK1 or cancer-derived MEK1 mutants Q56P or K57N were purified from E. coli and immunoblotted with an anti-phospho-MEK antibody. K/M; indicates the kinase activity-deficient K97M mutation.
  • B HA-MEK1 or its mutants (Q56P and K57N) were transiently expressed in HEK293 cells, and T-loop phosphorylation was detected in the same manner as in (A).
  • C Purified MEK1 protein was incubated with ERK2 (K/N) lacking kinase activity, and ERK phosphorylation was detected by blotting with anti-P-ERK antibody. The band intensity of P-ERK was quantified.
  • D GST-MEK1(Q56P) mutants were incubated with kinase activity-deficient MEK1(K/M) or MEK2(K/M), phosphorylation of MEK (GST-MEK1(Q56P)) was inhibited by anti-P-MEK detected with an antibody.
  • AA represents non-phosphorylated mutations.
  • A62P is a RAS/MAPK syndrome-derived variant.
  • B HEK293 cells were transfected with either HA-MEK2 or its mutants, and T-loop phosphorylation was detected with a P-MEK antibody. K/M represents mutations that lack kinase activity. Effects of MEK mutations on ERK pathways.
  • A HEK293 cells expressing HA-MEK1(WT), HA-MEK1(F53S) (derived from Ras/MAPK syndrome) or HA-MEK1(K57N) (derived from cancer) were stimulated with EGF (5 ng/ml). did.
  • Phosphorylation of ERK (“P-ERK”), MEK (“P-MEK”), Raf-1 (“P-Raf1(S338)”) and S6 ribosomal protein (“P-S6K”) after stimulation with EGF was detected with a phosphorylated antibody. Egr1 expression levels were also monitored (“Egr1”).
  • Egr1 Egr1 expression levels were also monitored (“Egr1”).
  • B HEK293 cell lines stably co-expressing ERK1-GFP and various MEK1 proteins were stimulated with EGF (5 ng/ml), and ERK localization was continuously monitored using a time-lapse imaging system. . The amount of ERK1-GFP fluorescence was quantified, and the percentage of ERK1 translocated to the nucleus was graphed.
  • HEK293 (with MEK1(WT) or MEK1(K57N)), H1299 (NRas Q61K ) (from lung cancer), A375 (BRaf V600E ) (from malignant melanoma), sk-Mel28 (BRaf V600E ) and WiDr (BRaf V600E ) (Colon cancer-derived) cells were treated in the presence or absence of U0126 (20 ⁇ M, 24 h), and the results of blotting of cell lysates or cell culture supernatants with antibodies against each protein are shown. Expression levels of C11orf96 protein in human malignancies.
  • a first embodiment is a cancer detection marker comprising at least one molecule selected from a group of proteins whose expression is induced by ERK1 or ERK2, wherein the ERK1 and ERK2 autophosphorylate the T-loop region.
  • a marker for cancer detection characterized by being activated by a MEK1 mutant or a MEK2 mutant that is activated by .
  • ERK1 and ERK2 are members of the MAPK (Mitogen-activated protein kinase) family, have molecular weights of 44 kDa and 42 kDa, respectively, and have approximately 85% homology in their amino acid sequences.
  • Ras activated by extracellular growth factor stimulation binds to Raf and promotes its activation, and Raf phosphorylates and activates MEK, which then phosphorylates ERK. oxidize and activate.
  • Activated ERK translocates from the cytoplasm to the nucleus and activates transcription factors such as ELK, CREB, c-Myc, c-Fos and Sp-1 to induce gene expression.
  • MEK1 and MEK2 mutants have their T-loop regions phosphorylated by intramolecular autophosphorylation and exhibit constitutive and strong kinase activity (see below).
  • MEK1 mutants and/or MEK2 mutants having such characteristics are also described as "MEK1/2 mutants according to the first embodiment") for the first time.
  • ERK1 and ERK2 activated by this MEK1 mutant or MEK2 mutant induce the expression of various cancer cell-specific proteins through the activation of various transcription factors.
  • cancer-derived MEK1/2 mutants which are characterized by their activation by intramolecular autophosphorylation of the T-loop region, indirectly induce the expression of cancer cell-specific proteins.
  • the protein whose expression is induced can be used as an effective marker for detecting the presence of cancer cells (hereinafter also referred to as a "cancer detection marker according to the first embodiment").
  • MEK1/2 and ERK1/2 when MEK1/2 and ERK1/2 are described, they mainly refer to MEK1 protein and/or MEK2 protein, ERK1 protein and/or ERK2 protein. Make a note to that effect.
  • the MEK1/2 T-loop region exists, for example, in the 208th to 233rd amino acid region on the MEK1 amino acid sequence and in the 212th to 237th amino acid region on the MEK2 amino acid sequence.
  • amino acid residues present in the T-loop region are intramolecularly autophosphorylated. It is characterized by being activated, showing constant and strong kinase activity.
  • MEK1/2 mutants according to the first embodiment include, but are not limited to, MEK1 mutants with Q56P, K57N, C121S or E203K mutations, and MEK2 mutants with Q60P mutations. can.
  • the MEK1/2 mutant according to the first embodiment may be naturally occurring or newly produced by genetic engineering or the like. Whether or not the MEK1/2 mutant has the ability to intramolecularly autophosphorylate can be determined, for example, by performing a kinase assay in which the MEK1/2 mutant and the MEK1/2 mutant that has lost the kinase activity are allowed to coexist. In this case, if the MEK1/2 mutant that has lost the kinase activity is not phosphorylated, it can be determined that the MEK1/2 mutant has intramolecular autophosphorylation ability (for details, see Examples checking).
  • a cancer detection marker according to the first embodiment or a cancer detection marker candidate molecule can be searched for, for example, as follows.
  • the MEK1/2 mutant according to the first embodiment is introduced into normal cells that are not cancerous, and the expression levels of various mRNAs of genes in the cells are comprehensively examined using, for example, microarrays and RNAseq. Analyzes are performed, and genes whose expression levels are statistically significantly increased are selected as compared with the expression levels in stably expressing cells transfected with wild-type MEK1/2. If the gene product (protein) of the selected gene is expressed in cancer cells or if the gene product expressed in cancer cells is present in serum-secreted exosomes, the molecule is It can be used as a marker for cancer detection.
  • the cancer detection marker according to the first embodiment or the candidate molecule for the cancer detection marker is a protein
  • a protein is selected, and if the protein is expressed in cancer cells, that molecule can be used as a marker for cancer detection.
  • a second embodiment includes the step of searching for a protein that induces the expression of ERK1 or ERK2 that is activated by a MEK1 mutant or MEK2 mutant that is activated by autophosphorylation of the T-loop region. It is a screening method for cancer detection markers.
  • the search for molecules induced by MEK1 mutants activated by autophosphorylation of the T-loop region or ERK1 or ERK2 activated by MEK2 mutants can be performed by microarray analysis or proteome analysis, as described above.
  • the expression levels of proteins expressed in cells introduced with the MEK1/2 mutant according to the first embodiment and cells introduced with the wild-type MEK1/2 are compared, and MEK1/2 according to the first embodiment It can be carried out by selecting a protein whose expression is significantly increased in cells into which two mutants have been introduced. Here, among the selected proteins, those that have been confirmed to be specifically expressed in cancer cells can be used as cancer detection markers.
  • a third embodiment provides a method for diagnosing cancer or diagnosing cancer, comprising the step of measuring the expression level of the cancer detection marker according to the first embodiment of the present invention present in a sample derived from a subject. It is an auxiliary method.
  • the cancer detection marker according to the first embodiment is not limited to specific cancers, and can be used for early detection of various cancers. For example, malignant melanoma, colon cancer, thyroid cancer, lung cancer, ovarian cancer, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, bladder cancer, It can be used for early detection of blood cancer, sarcoma, and the like.
  • the abundance of the cancer detection marker according to the first embodiment in a sample derived from a subject is When the amount is statistically significantly higher than the abundance of the subject, it can be determined that the subject has or is likely to have some cancer.
  • samples derived from a subject include tissues suspected of developing cancer, blood (including exosomes), and body fluids such as pancreatic fluid and urine.
  • body fluids such as blood, pancreatic fluid and urine derived from healthy subjects.
  • the fourth embodiment is a kit for cancer detection or cancer diagnosis.
  • the kit of the present invention contains at least an antibody or aptamer for detecting the cancer detection marker according to the first embodiment. More specifically, for example, MMP10, EMP1, Rheb2, TM4SF19, TM4SF1, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR(CD87), PVR(CD155), IL7R(CD127), IL1R2(CD121b), IL4R, TweakR (CD266), CD3D, CD44, SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1 , BMP2, TFPI2, GDF15
  • a fifth embodiment is a marker for detecting Ras/MAPK syndrome containing at least one molecule selected from the group consisting of proteins whose expression is induced by ERK1 or ERK2, wherein the ERK1 and ERK2 are T-loop regions
  • ERK1 and ERK2 are T-loop regions
  • a marker for detecting Ras/MAPK syndrome characterized by being activated by a MEK1 mutant or MEK2 mutant that has activity even if is not phosphorylated.
  • MEK1 and MEK2 mutants from Ras/MAPK syndrome constitutively have kinase activity even though their T-loop regions are not phosphorylated.
  • MEK1 and/or MEK2 mutants having such characteristics are also described as “MEK1/2 mutants according to the fifth embodiment”). Furthermore, the inventors found that ERK1/2 activated by this MEK1/2 mutant induces the expression of proteins specific to Ras/MAPK syndrome through the activation of various transcription factors. rice field.
  • MEK1/2 mutants which are characterized by having constitutive kinase activity even when the T-loop region is not phosphorylated, indirectly induce the expression of proteins specific to Ras/MAPK syndrome, The protein whose expression is induced is a marker effective for detecting the presence or absence or possibility of the onset of Ras/MAPK syndrome (hereinafter referred to as "marker for detecting Ras/MAPK syndrome according to the fifth embodiment of the present invention").
  • MEK1/2 mutants include, but are not limited to, MEK1 mutations having F53S, T55P, D67N, P124L, P124Q, G128V, G128N, Y130C, Y130N, Y130H, or E203Q mutations.
  • MEK2 mutants with F53C, F53V, F57L, K61E, A62P, P128R, G132V, T134C, or Y134H mutations.
  • the MEK1/2 mutant according to the fifth embodiment may be naturally occurring or newly produced by genetic manipulation or the like.
  • As a marker for detecting Ras / MAPK syndrome according to the fifth embodiment in cells expressing Ras / MAPK syndrome-derived MEK1 mutant (F53S) (one of the MEK1 mutants according to the fifth embodiment) , the Col14A1 protein whose expression level was significantly increased can be exemplified.
  • a sixth embodiment includes the step of searching for a protein that induces the expression of ERK1 or ERK2 activated by a MEK1 mutant or MEK2 mutant that has activity even if the T-loop region is not phosphorylated. , a screening method for markers for detecting Ras/MAPK syndrome. Molecules induced by ERK1 or ERK2, which is activated by MEK1 or MEK2 mutants that are active even if the T-loop region is not phosphorylated, can be explored by methods such as microarray analysis or proteome analysis.
  • the expression levels of proteins expressed in cells introduced with the MEK1/2 mutant according to the fifth embodiment and in cells introduced with the wild-type MEK1/2 are compared, and the MEK1/2 mutant according to the fifth embodiment can be carried out by selecting a protein whose expression is significantly increased in cells into which is introduced.
  • a seventh embodiment comprises the step of measuring the expression level of a Ras/MAPK syndrome detection marker according to the fifth embodiment of the present invention present in a sample derived from a subject, a method for diagnosing Ras/MAPK syndrome or It is a diagnostic aid for Ras/MAPK syndrome.
  • Ras/MAPK syndrome is characterized by specific facial features (interocular dissociation, oblique palpebral fissure, temporal stenosis, etc.), cardiac hypertrophy, mental retardation, and cutaneous symptoms. It is a chromosomal dominant genetic disease (generic term for Costello/Noonan/CFC syndrome, etc.) (Hum Mutat. 2008 Aug;29(8):992-1006).
  • the amount of the marker for detecting Ras/MAPK syndrome according to the fifth embodiment in a sample derived from a subject is A determination is made that the subject has or is at risk of having some form of Ras/MAPK syndrome if it is statistically significantly greater than the abundance in the control sample. be able to.
  • a subject-derived sample for example, by targeting amniotic fluid collected from a pregnant subject, there is a possibility that a newborn can be diagnosed congenitally with Ras/MAPK syndrome before birth. .
  • the cancer detection marker according to the first embodiment and the Ras/MAPK syndrome detection marker according to the fifth embodiment are proteins
  • their expression levels can be detected and measured by known methods.
  • methods include methods using antibodies or aptamers (such as nucleic acid aptamers or peptide aptamers) that specifically bind to the cancer detection marker protein or Ras/MAPK syndrome detection marker protein used.
  • the detection and quantification of marker proteins can be performed by immunological techniques or methods utilizing the binding properties of antibodies or aptamers to marker proteins, such as immunochromatography and Western blotting. , ELISA (e.g.
  • ERK1-GFP fluorescence from cells in randomly selected wells was monitored every 90 seconds. Specifically, EGF was added to the medium to a final concentration of 5 ng/ml, and fluorescence from living cells was observed continuously for 60 minutes using a Nikon Eclipse Ti fluorescence microscope (Rolera EM- C2) equipped with a CCD camera. , QImaging).
  • the ratio of ERK-GFP translocated to the nucleus in each cell was calculated by dividing the fluorescence intensity of the whole cell by the fluorescence intensity of the nuclear region of the same cell. Quantitative analysis of fluorescence intensity was performed using MetaFluor software (Molecular Devices).
  • the three cancer tissue microarrays used in this example were purchased from Biomax US. Tissue slides were deparaffinized and antigen-retrieved by incubation in citrate buffer (pH 6) at 95°C for 40 minutes. Endogenous peroxidase activity in each tissue sample was blocked by treatment with 0.3% hydrogen peroxide. Rehydrated tissue slides were incubated with BlockAce (Yukijirushi) for 10 minutes at room temperature, followed by anti-C11orf96 antibody (1:250 dilution) overnight at 4°C.
  • BlockAce Yukijirushi
  • An anti-rabbit-HRP antibody (Dako Envision systems) was used as a secondary antibody, and DAB (3,3'-Diaminobenzidine) was used to visualize the binding between the primary antibody and the antigen. Immunohistological evaluation of C11orf96 was performed by a pathologist.
  • Plasmid pcDNA3HA, pcDNA3Flag and pcDNA4Myc vectors were used to express MEK1, MEK2, ERK2, BRaf, Raf-1, KSR1 and their mutants.
  • MEK1/2 mutants were generated by site-directed mutagenesis by PCR.
  • pCold (Takara Bio, Japan)
  • pRSF-Duet Merck-Millipore
  • pGEX-6P vectors GE healthcare
  • ERK2 The catalytically inactive form of ERK2 (K/N) replaces the codon of Lys52 with that of asparagine, and the MEK1 (K/M) and MEK2 (K/M) inactive mutants replace codons of Lys97 and Lys101, respectively. was prepared by substituting the codon for methionine.
  • Raf-1 ⁇ N was produced as previously reported (Takekawa et al., Mol Cell 18, 295-306 2005).
  • the DD sequence destabilization region, Clontech
  • Retrovirus infection HEK293 cell lines stably expressing MEK mutants were generated by retrovirus infection. Retroviruses were generated in GP2-293 packaging cells by transient transfection with pVSV and pQCXIP or pQCXIH plasmids. Culture supernatants were harvested 48 hours after transfection, filtered and added with 8 ⁇ g/ml polybrene. Cells were retrovirally infected and cells expressing the protein of interest were selected with puromycin or hygromycin. Retroviral infection of MEFs was performed as described above, except Plat-E packaging cells were used.
  • GST-MEK1, GST-MEK2, GST-ERK2(K/N) and their mutants were expressed in E. coli DH5 ⁇ by adding IPTG (0.5 ⁇ M) and glutathione- It was purified using Sepharose beads (GE healthcare).
  • Phosphorylated MEK1 was purified from E. coli co-expressing GST-MEK1 and 6xHis-BRaf(V600E). His-MEK1 and MEK2 were purified on a His-Trap HP column using FPLC AKTA system (GE healthcare).
  • HEK293 cells were cultured on coverslips and transiently transfected with HA-MEK1 or its mutants. Eighteen hours after transfection, cells were fixed in PBS (pH 7.4) containing 3% paraformaldehyde for 10 minutes. The cells were washed with PBS, incubated with 0.1% Triton X-100 for 5 minutes for membrane permeabilization, washed again, and treated with BlockAce (Yukijirushi) for 1 hour at room temperature. Cells were then incubated with 1 ⁇ g/ml anti-HA mAb 16B12 (Covance) in PBS containing 2% BSA for 50 minutes at room temperature.
  • PBS pH 7.4
  • BlockAce Yukijirushi
  • Co-immunoprecipitation assay HEK293 cells were transiently transfected with the plasmid and lysed with buffer C after 48 hours. The cell lysate was pretreated with protein G at 4°C for 1 hour and then incubated with an anti-Flag antibody at 4°C for 3 hours. Immunoprecipitates were collected with protein G sepharose and washed with cold buffer C several times. Proteins were spun on SDS-PAGE and subjected to immunoblotting analysis with anti-Myc antibody.
  • MEF proliferation assay cells were plated in 3 wells at 1 x 10 3 cells/well in DMEM containing 10% FBS, and the cell count was determined using the Cell Counting Kit. Counted at -8 (Dojindo).
  • the anchorage-independent growth assay in soft agar the dishes were previously covered with SeaPlaque Agarose (DMEM containing 0.5% agar and 10% FBS) and then covered with 0.35% MEF cells. Agar (containing 10% FBS) was added. Medium was added on top to prevent the agar from drying out.
  • MEF stably expressing cells 3 x 104 cells in agar Colonies (>0.1 mm) were counted after inoculation and 4 weeks of culture. Mean colony numbers were calculated based on values obtained from triplicate assays.
  • genes with expression levels >2 or 0.5 ⁇ compared to the gene expression levels in MEK1(WT)-expressing cells were identified in the DAVID database (http://www.daviddatabase.org/). (http://david.abcc.ncifcrf.gov) and subjected to functional annotation analysis.
  • q-PCR Quantitative analysis of mRNA expression levels
  • cDNA samples derived from the HEK293 stable cell line were subjected to q-PCR analysis using the Takara Thermal Cycler Dice real time system (Takara Bio, Japan) and Thunderbird SYBR qPCR mix (Toyobo, Japan). Relative gene expression levels were normalized to GAPDH. All q-PCR experiments were performed in triplicate and represent the mean ⁇ SEM. Primers used in q-PCR analysis are shown in Tables 1 and 2.
  • the MEK variants used here are those from cancer or RAS/MAPK syndrome. All tested MEK mutants showed higher kinase activity than wild-type MEK (Fig. 1A), but the strength of the activity differed depending on the position of the MEK mutation. That is, cancer-derived MEK mutants (e.g., Q56P and K57N) are "highly active” with strong kinase activity, and Ras/MAPK syndrome-derived MEK mutants (e.g., F53S, T55P, D67N and Y130C) , was a "moderately active" type with weaker kinase activity than cancer-derived MEK mutants.
  • cancer-derived MEK mutants e.g., Q56P and K57N
  • Ras/MAPK syndrome-derived MEK mutants e.g., F53S, T55P, D67N and Y130C
  • non-phosphorylated wild-type MEK1(AA) mutant is able to phosphorylate ERK in vivo, its kinase activity is lower than that of non-phosphorylated MEK mutants with the Q56P or K57N mutation (MEK1(Q56P+)). AA) and MEK1 (K57N+AA)), it decreased to about 50% (Fig. 2C).
  • Gln56 of MEK1 is conserved as Gln60 in MEK2, it is possible that these conserved amino acid substitutions in MEK2, namely cancer-derived MEK2(Q60P) mutants, induce T-loop autophosphorylation. As expected, the MEK2(Q60P) mutant showed autophosphorylation activity as well as the MEK1(Q56P) mutant (FIGS. 3A and B).
  • HEK293 cells stably expressing ERK-GFP and HA-MEK1 or its mutants, and observed nuclear translocation of ERK in response to EGF stimulation using a time-lapse microscopy system.
  • Expression of Ras/MAPK syndrome-derived MEK1(F53S) resulted in EGF-dependent ERK translocation to the nucleus and sustained nuclear localization.
  • MEK1(K57N)-expressing cells accumulation of ERK in the nucleus was confirmed independently of EGF treatment (Fig. 4B).
  • Egr1 one of the IEGs
  • MEK1(WT)-expressing cells depended on the time of EGF treatment, peaking at 5 hours after treatment and then decreasing, whereas MEK1(F53S) expression Its expression was prolonged in cells, and MEK1(K57N)-expressing cells exhibited constitutive expression independent of EGF stimulation (Fig. 4C).
  • Col14A1 (collagen type 14A1) is a Ras/MAPK syndrome-derived MEK1 with moderate constitutive activity. We found that it was specifically upregulated only when (F53S) was expressed (Fig. 5). Indeed, no upregulation of Col14A1 was observed when the highly active cancer-derived MEK1(K57N) mutant was expressed. Col14A1 is thought to act as a bridging molecule between extracellular fibers and matrix components (Eyre et al., Biochem Soc Trans 30, 844-848 2002; Schuppan et al., J Biol Chem 265, 8823 -8832 1990).
  • Ras/MAPK syndrome it has been reported that collagen fiber deposition is observed in various organs (Hinek et al. Am J Med Genet A 133A, 1-12. 2005; Mori et al., Am J Med Genet 61, 304-309 1996 ), suggesting that expression of genes regulated by Ras/MAPK syndrome-derived MEK1 mutants, mutants that induce constitutive activation of MEK with or without T-loop phosphorylation, It is suggested that it is associated with the onset of Ras/MAPK syndrome.
  • c11orf96 protein one of the gene products induced by ERK signaling activation by MEK1(K57N), was significantly expressed in colon, lung and pancreatic tumors compared to normal tissues. , was confirmed by immunohistochemical analysis using TMA (tissue microarrays) (Fig. 8).
  • the present invention enables early detection of diseases such as cancer and Ras/MAPK syndrome, and is expected to be used in the medical field.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Plant Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
PCT/JP2022/022276 2021-06-03 2022-06-01 Ｅｒｋ－ｍａｐｋ経路の異常な活性化に伴い発現する疾患マーカー Ceased WO2022255401A1 (ja)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023525886A JPWO2022255401A1 (https=) 2021-06-03 2022-06-01

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021093552 2021-06-03
JP2021-093552 2021-06-03

Publications (1)

Publication Number Publication Date
WO2022255401A1 true WO2022255401A1 (ja) 2022-12-08

Family

ID=84323437

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/022276 Ceased WO2022255401A1 (ja) 2021-06-03 2022-06-01 Ｅｒｋ－ｍａｐｋ経路の異常な活性化に伴い発現する疾患マーカー

Country Status (2)

Country Link
JP (1) JPWO2022255401A1 (https=)
WO (1) WO2022255401A1 (https=)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12228574B2 (en) 2020-12-21 2025-02-18 Freenome Holdings, Inc. Markers for the early detection of colon cell proliferative disorders
CN120971731A (zh) * 2025-06-20 2025-11-18 上海市第十人民医院 一种标志物组合及其在诊断解痉多肽表达化生中的应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007506442A (ja) * 2003-05-30 2007-03-22 ゲノミック ヘルス， インコーポレイテッド Ｅｇｆｒ阻害薬への応答に関する遺伝子発現マーカー
WO2016115376A1 (en) * 2015-01-14 2016-07-21 The Regents Of The University Of California Detection and treatment of double drug resistant melanomas
JP2016140262A (ja) * 2015-01-30 2016-08-08 国立大学法人山口大学 肝細胞がんの転移性再発リスクの予測方法
JP2016176942A (ja) * 2010-06-09 2016-10-06 デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド Ｒａｆ及びｍｅｋ阻害剤に対する耐性を与えるｍｅｋ１変異
WO2018212192A1 (ja) * 2017-05-18 2018-11-22 京都府公立大学法人 癌の予後及び／又は転移可能性を評価する方法及びマーカー
JP2019518426A (ja) * 2016-04-15 2019-07-04 ジェネンテック， インコーポレイテッド がんの診断及び治療方法
WO2019142636A1 (ja) * 2018-01-16 2019-07-25 公立大学法人横浜市立大学 腎癌の検出方法及び検査薬
WO2020039321A2 (en) * 2018-08-20 2020-02-27 Pfizer Inc. Anti-gdf15 antibodies, compositions and methods of use
JP2020532745A (ja) * 2017-09-05 2020-11-12 アイジェノミクス エセ．エレ． 子癇前症と関連したバイオマーカーを検出するための方法及び装置
JP2020534351A (ja) * 2017-08-28 2020-11-26 アンジーエックス・インコーポレーテッド 抗ｔｍ４ｓｆ１抗体およびそれを使用する方法

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007506442A (ja) * 2003-05-30 2007-03-22 ゲノミック ヘルス， インコーポレイテッド Ｅｇｆｒ阻害薬への応答に関する遺伝子発現マーカー
JP2016176942A (ja) * 2010-06-09 2016-10-06 デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド Ｒａｆ及びｍｅｋ阻害剤に対する耐性を与えるｍｅｋ１変異
WO2016115376A1 (en) * 2015-01-14 2016-07-21 The Regents Of The University Of California Detection and treatment of double drug resistant melanomas
JP2016140262A (ja) * 2015-01-30 2016-08-08 国立大学法人山口大学 肝細胞がんの転移性再発リスクの予測方法
JP2019518426A (ja) * 2016-04-15 2019-07-04 ジェネンテック， インコーポレイテッド がんの診断及び治療方法
WO2018212192A1 (ja) * 2017-05-18 2018-11-22 京都府公立大学法人 癌の予後及び／又は転移可能性を評価する方法及びマーカー
JP2020534351A (ja) * 2017-08-28 2020-11-26 アンジーエックス・インコーポレーテッド 抗ｔｍ４ｓｆ１抗体およびそれを使用する方法
JP2020532745A (ja) * 2017-09-05 2020-11-12 アイジェノミクス エセ．エレ． 子癇前症と関連したバイオマーカーを検出するための方法及び装置
WO2019142636A1 (ja) * 2018-01-16 2019-07-25 公立大学法人横浜市立大学 腎癌の検出方法及び検査薬
WO2020039321A2 (en) * 2018-08-20 2020-02-27 Pfizer Inc. Anti-gdf15 antibodies, compositions and methods of use

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU QING-HUA, SHI MEI-LIN, SUN CHAO, BAI JIN, ZHENG JUN-NIAN: "Role of the ERK1/2 pathway in tumor chemoresistance and tumor therapy", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 25, no. 2, 1 January 2015 (2015-01-01), Amsterdam NL , pages 192 - 197, XP093009381, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2014.11.076 *
NAVA CAROLINE; HANNA NADINE; MICHOT CAROLINE; PEREIRA SABRINA; POUVREAU NATHALIE; NIIHORI TETSUYA; AOKI YOKO; MATSUBARA YOICHI; AR: "Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype-phenotype relationships and overlap with Costello syndrome", JOURNAL OF MEDICAL GENETICS, BMJ PUBLISHING GROUP, LONDON, GB, vol. 44, no. 12, 1 December 2007 (2007-12-01), GB , pages 763 - 771, XP009129671, ISSN: 0022-2593 *
YOKO AOKI; TETSUYA NIIHORI; YOKO NARUMI; SHIGEO KURE; YOICHI MATSUBARA: "The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders", HUMAN MUTATION, JOHN WILEY & SONS, INC., US, vol. 29, no. 8, 9 May 2008 (2008-05-09), US , pages 992 - 1006, XP071974666, ISSN: 1059-7794, DOI: 10.1002/humu.20748 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12228574B2 (en) 2020-12-21 2025-02-18 Freenome Holdings, Inc. Markers for the early detection of colon cell proliferative disorders
CN120971731A (zh) * 2025-06-20 2025-11-18 上海市第十人民医院 一种标志物组合及其在诊断解痉多肽表达化生中的应用
CN120971731B (zh) * 2025-06-20 2026-03-27 上海市第十人民医院 一种标志物组合及其在诊断解痉多肽表达化生中的应用

Also Published As

Publication number Publication date
JPWO2022255401A1 (https=) 2022-12-08

Similar Documents

Publication Publication Date Title
Ni et al. Regulation of PERK signaling and leukemic cell survival by a novel cytosolic isoform of the UPR regulator GRP78/BiP
Rauch et al. Heterogeneous nuclear ribonucleoprotein H blocks MST2-mediated apoptosis in cancer cells by regulating A-Raf transcription
Kulkarni et al. RUNX1 and RUNX3 protect against YAP-mediated EMT, stem-ness and shorter survival outcomes in breast cancer
Hong et al. AKR1C1 activates STAT3 to promote the metastasis of non-small cell lung cancer
Pérez et al. Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways
Barber et al. Characterization of desmoglein expression in the normal prostatic gland. Desmoglein 2 is an independent prognostic factor for aggressive prostate cancer
Nouri et al. A kinome-wide screen using a NanoLuc LATS luminescent biosensor identifies ALK as a novel regulator of the Hippo pathway in tumorigenesis and immune evasion
He et al. α-Mannosidase 2C1 attenuates PTEN function in prostate cancer cells
Hung et al. The motor protein KIF14 inhibits tumor growth and cancer metastasis in lung adenocarcinoma
Guo et al. The E3 ligase Smurf1 regulates Wolfram syndrome protein stability at the endoplasmic reticulum
Xiao et al. S100A11 promotes human pancreatic cancer PANC-1 cell proliferation and is involved in the PI3K/AKT signaling pathway
Mikawa et al. Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2
Yang et al. DRAK2 participates in a negative feedback loop to control TGF-β/Smads signaling by binding to type I TGF-β receptor
Liu et al. Upregulation of KLHL17 promotes the proliferation and migration of non-small cell lung cancer by activating the Ras/MAPK signaling pathway
Rines et al. Snf1-related kinase inhibits colon cancer cell proliferation through calcyclin-binding protein-dependent reduction of β-catenin
Rasool et al. Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance
Fang et al. Somatic mutational analysis of FAK in breast cancer: a novel gain-of-function mutation due to deletion of exon 33
Liu et al. FBXW10-S6K1 promotes ANXA2 polyubiquitination and KRAS activation to drive hepatocellular carcinoma development in males
Bhakat et al. Regulation of limited N-terminal proteolysis of APE1 in tumor via acetylation and its role in cell proliferation
Zhang et al. Deubiquitinase USP28 inhibits ubiquitin ligase KLHL2-mediated uridine-cytidine kinase 1 degradation and confers sensitivity to 5'-azacytidine-resistant human leukemia cells
WO2022255401A1 (ja) Ｅｒｋ－ｍａｐｋ経路の異常な活性化に伴い発現する疾患マーカー
Guo et al. Linear ubiquitination of PTEN impairs its function to promote prostate cancer progression
US10828377B2 (en) Method for determining presence or absence of suffering from malignant lymphoma or leukemia, and agent for treatment and/or prevention of leukemia
Han et al. SMURF2 facilitates ubiquitin-mediated degradation of ID2 to attenuate lung cancer cell proliferation
Xiong et al. PPARG activation of fatty acid metabolism drives resistance to Anti-HER2 therapies in HER2-Positive breast cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22816147

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023525886

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22816147

Country of ref document: EP

Kind code of ref document: A1