WO2022244807A1 - Gène de fusion de la ltk - Google Patents

Gène de fusion de la ltk Download PDF

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WO2022244807A1
WO2022244807A1 PCT/JP2022/020673 JP2022020673W WO2022244807A1 WO 2022244807 A1 WO2022244807 A1 WO 2022244807A1 JP 2022020673 W JP2022020673 W JP 2022020673W WO 2022244807 A1 WO2022244807 A1 WO 2022244807A1
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polynucleotide
fusion
ltk
gene
polypeptide
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Japanese (ja)
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功一 後藤
慎吾 松本
大樹 泉
進 小林
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国立研究開発法人国立がん研究センター
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    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a fusion gene involved in canceration of cells. More specifically, the present invention relates to a novel LTK fusion gene involved in canceration of cells.
  • Non-Patent Document 1 Non-Patent Document 2, Patent Documents 1-3.
  • These driver mutations have been found in non-small cell lung cancer among lung cancers, especially non-squamous non-small cell lung cancer (mainly adenocarcinoma).
  • the purpose of the present invention is to provide a novel oncogenic driver gene that serves as a new therapeutic target for cancer.
  • the present invention further provides a means for identifying cancer patients or subjects at risk of cancer for whom a drug targeting a gene having the mutation or the protein encoded by the gene has a therapeutic effect, based on a novel oncogenic driver gene. for the purpose of providing
  • the present inventors discovered a novel LTK fusion gene in the genetic screening platform (LC-SCRUM-Asia) constructed by the present inventors. Furthermore, in in vitro studies, we performed a functional analysis of the novel LTK fusion gene and clarified that it is involved in canceration of cells, and that its activity is suppressed by tyrosine kinase inhibitors. The present invention was completed by discovering that treatment with a tyrosine kinase inhibitor is effective for positive patients. That is, the present invention includes, but is not limited to, the following aspects.
  • a method for detecting a gene fusion that is an oncogenic driver mutation comprising: in an isolated sample from a subject with cancer, a fusion polynucleotide represented by formula (I) below or a polypeptide encoded thereby: A method comprising detecting: 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position)
  • the fusion polynucleotide encodes a polypeptide comprising the kinase domain of LTK and having kinase activity.
  • a method according to aspect 4, wherein the fusion polynucleotide is a CLIP1-LTK fusion polynucleotide encoding a polypeptide selected from the group consisting of (a) to (c) below: (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and having kinase activity; and (c) a sequence A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by number 2 and having kinase activity.
  • fusion polynucleotide is any of (a') to (d') below: (a') a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1; (b') a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 and that encodes a polypeptide having kinase activity , (c') a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, and encoding a polypeptide having kinase activity; or (d') a polynucleotide consisting of a nucleotide sequence
  • Aspect 8 Aspect 7, wherein the mutation in the LTK gene portion is a mutation resulting in an LTK mutation selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof. the method of.
  • a method comprising the steps of: (1) a method comprising detecting, in an isolated sample from a subject, a fusion polynucleotide represented by formula (I) below or a polypeptide encoded thereby: 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position) wherein said fusion polynucleotide encodes a polypeptide comprising the kinase domain of LTK and having kinase activity; and (2) when said fusion polynucleotide or a polypeptide encoded
  • fusion polynucleotide is a CLIP1-LTK fusion polynucleotide encoding a polypeptide selected from the group consisting of (a) to (c): (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and having kinase activity; and (c) a sequence A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by number 2 and having kinase activity.
  • fusion polynucleotide is any of (a') to (d'): (a') a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1; (b') a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 and that encodes a polypeptide having kinase activity , (c') a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, and encoding a polypeptide having kinase activity; or (d') a polynucle
  • the mutation in the LTK gene portion is a mutation resulting in an LTK mutation selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof. the method of.
  • a kit for detecting gene fusions that are oncogenic driver mutations comprising any of (A) to (C) or a combination thereof:
  • the fusion polynucleotide is a CLIP1-LTK fusion polynucleotide encoding a polypeptide selected from the group consisting of (a) to (c): (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and having kinase activity; and (c) a sequence A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by number 2 and having kinase activity.
  • a kit according to aspects 24 or 25, wherein the fusion polynucleotide is any of (a') to (d'): (a') a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1; (b') a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 and that encodes a polypeptide having kinase activity , (c') a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, and encoding a polypeptide having kinase activity; or (d') a polynucle
  • the mutation in the LTK gene portion is a mutation resulting in an LTK mutation selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof. kit.
  • FIG. 30 An isolated fusion polynucleotide of formula (I): 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position)
  • the fusion polynucleotide encodes a polypeptide comprising the kinase domain of LTK and having kinase activity.
  • the fusion polynucleotide according to aspect 33 which is a CLIP1-LTK fusion polynucleotide encoding a polypeptide selected from the group consisting of (a) to (c): (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and having kinase activity; and (c) a sequence A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by number 2 and having kinase activity.
  • a fusion polynucleotide according to aspects 33 or 34 which is any of (a') to (d'): (a') a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1; (b') a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 and that encodes a polypeptide having kinase activity , (c') a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, and encoding a polypeptide having kinase activity; or (d') a polynucleot
  • the mutation in the LTK gene portion is a mutation resulting in an LTK mutation selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof.
  • Aspect 42 Determined by the method according to any one of aspects 11 to 20 that a substance that suppresses the expression and/or activity of a polypeptide encoded by a fusion polynucleotide generated by gene fusion that is an oncogenic driver mutation has a therapeutic effect
  • a method for screening an active ingredient of a cancer therapeutic agent comprising the steps of: (1) contacting a cell expressing the fusion polypeptide according to any one of aspects 38 to 41 with a test substance; (2) determining whether the expression and/or activity of said fusion polypeptide is inhibited; Selecting as an active ingredient of a drug.
  • a cancer therapeutic agent comprising, as an active ingredient, a substance selected by the screening method according to aspect 47.
  • a method for treating cancer wherein the method according to any one of aspects 11 to 20 suppresses expression and/or activity of a polypeptide encoded by a fusion polynucleotide generated by gene fusion that is an oncogenic driver mutation. administering a substance that suppresses the expression and/or activity of the polypeptide encoded by the fusion polynucleotide to a cancer patient or a subject determined to be at risk of cancer, for whom the substance that performs including, method.
  • the present invention makes it possible to provide novel oncogenic driver genes that serve as new therapeutic targets for cancer. Furthermore, according to the present invention, a cancer patient or a subject at risk of cancer for whom a drug targeting a gene having the mutation or a protein encoded by the mutation produces a therapeutic effect is identified based on the novel oncogenic driver gene. It becomes possible to provide a method, a method of treating a patient with cancer, a cancer therapeutic agent, a screening method for a cancer therapeutic agent, and the like.
  • FIG. 1 shows the CLIP1-LTK fusion gene identified by total RNA sequencing analysis of non-small cell lung cancer patients. Spanning reads (top) and junction reads (bottom) of the detected CLIP1-LTK fusion gene transcript are shown.
  • FIG. 2 shows schematic representations of wild-type CLIP1 protein (top), wild-type LTK protein (middle), and CLIP1-LTK fusion protein (bottom). Straight lines indicate breakpoints. CC, coiled-coil domain; TM, transmembrane domain.
  • FIG. 3 shows RT-PCR detection of CLIP1-LTK gene transcripts in patient #1. Primer positions are indicated at the top.
  • FIG. 4 shows an electropherogram of Sanger sequencing analysis of the CLIP1-LTK fusion gene transcript.
  • cDNA was purified from patient-derived RNA and subjected to RT-PCR using CLIP1-LTK-F3 and CLIP1-LTK-R3 primers. PCR products were directly sequenced using each primer.
  • FIG. 4 shows an electropherogram of Sanger sequencing analysis of the CLIP1-LTK fusion gene transcript.
  • FIG. 5 shows screening of lung cancer specimens for CLIP1-LTK gene transcripts using the primer set of primer F5 and primer R3.
  • a representative RT-PCR screen panel including two positive samples (#2 and #3) and GAPDH is shown.
  • Figure 6 shows the constitutive tyrosine kinase activity of the CLIP1-LTK fusion protein.
  • NIH3T3 cells were transiently transfected with empty vector, wild-type LTK expression plasmid, or CLIP1-LTK expression plasmid. Cell extracts were subjected to Western blotting analysis.
  • FIG. 7 shows light microscope images of NIH3T3 stably expressing cells.
  • Figure 9 shows LTK phosphorylation by CLIP1-LTK (a, Ba/F3 cells; b, NIH3T3 cells).
  • FIG. 10 shows IL-3 independent proliferation (Ba/F3 cells) by CLIP1-LTK.
  • FIG. 11 shows the screening of chemical inhibitors of CLIP1-LTK.
  • NIH3T3 cells were transiently transfected with pCMV3-CLIP1-LTK, cultured for 24 hours, and then treated with the indicated drugs for 6 hours. Cell extracts were subjected to Western blotting analysis.
  • FIG. 12 shows inhibition of anchorage-independent colony formation in NIH3T3-CLIP1-LTK cells.
  • Figure 13 shows a cell viability assay of Ba/F3-CLIP-LTK cells. Ba/F3-CLIP-LTK cells were treated with increasing concentrations of the indicated drugs for 48 hours.
  • Figure 14 shows inhibition of CLIP-LTK kinase activity and its downstream signaling by lorlatinib.
  • FIG. 1 shows the time course of CT scan images of the right lung and liver of patient 1 who received 100 mg of lorlatinib orally per day.
  • FIG. 16 shows evaluation of transforming activity by CLIP1-LTK in vivo.
  • Figure 17 shows the effect of lorlatinib on CLIP1-LTK expressing tumors in vivo.
  • FIG. 18 shows the time course of lung and liver CT scan images (baseline, 2 months, 5 months) of patient 1 who received lorlatinib 100 mg orally per day.
  • FIG. 1 shows the time course of CT scan images of the right lung and liver of patient 1 who received 100 mg of lorlatinib orally per day.
  • FIG. 16 shows evaluation of transforming activity by CLIP1-LTK in vivo.
  • Figure 17 shows the effect of lorlatinib on CLIP1-LTK expressing tumors in vivo.
  • FIG. 18 shows the time course of lung and liver CT scan images (baseline, 2 months, 5 months) of patient 1 who received lorlatini
  • FIG. 19 shows the time series of whole-body PET images of patient 1 who was orally administered 100 mg of lorlatinib per day.
  • FIG. 20 shows detection of LTK gene rearrangements in human tumor and non-tumor cells by break-apart fluorescence in situ hybridization (FISH) assay.
  • Figures 21A-C show the effect of LTK gene mutations in LTK fusion genes on lorlatinib sensitivity.
  • Figures 21A-C show the effect of LTK gene mutations in LTK fusion genes on lorlatinib sensitivity.
  • Figures 21A-C show the effect of LTK gene mutations in LTK fusion genes on lorlatinib sensitivity.
  • Figures 22A to 22C show evaluation of sensitivity to various drugs having ALK/LTK inhibitory activity in cells expressing CLIP1-LTK fusion protein with LTK mutation.
  • Figures 22A to 22C show evaluation of sensitivity to various drugs having ALK/LTK inhibitory activity in cells expressing CLIP1-LTK fusion protein with LTK mutation.
  • Figures 22A to 22C show evaluation of sensitivity to various drugs having ALK/LTK inhibitory activity in cells expressing CLIP1-LTK fusion protein with LTK mutation.
  • the present inventors were the first to discover an LTK gene fusion as an oncogenic driver mutation. Based on such findings, the present invention provides a method for detecting the gene fusion, a method for identifying a cancer patient or a subject at risk of cancer for whom cancer treatment based on the presence of the oncogenic driver mutation is effective, The present invention provides therapeutic methods, cancer therapeutic agents, screening methods for cancer therapeutic agents, and the like.
  • a gene that directly causes carcinogenesis is called an oncogenic driver gene (herein, sometimes simply referred to as a driver gene).
  • a mutation that occurs in an oncogenic driver gene is referred to as an oncogenic driver mutation (herein, sometimes simply referred to as a driver mutation), which is a term used interchangeably with cancer-responsible mutations.
  • driver mutations driver mutations listed in the National Comprehensive Cancer Network (NCCN) guidelines
  • NCCN National Comprehensive Cancer Network
  • EGFR / ALK / ROS1 / BRAF / MET / NTRK / HER2 / RET / KRAS gene mutations are not present (i.e., if the mutation is mutually exclusive with the known driver mutation), the mutation is It can be said to be an oncogenic driver mutation.
  • fusions which are specific oncogenic driver mutations in the present invention, are described below.
  • a gene resulting from gene fusion is called a fusion gene.
  • the "fusion point" in the fusion polynucleotide means the boundary where the 5'-side gene portion and the 3'-side gene portion are connected, which is the boundary between the two nucleotide residues. Boundary.
  • the "fusion point” in the fusion polypeptide means the boundary where the N-terminal side polypeptide and the C-terminal side polypeptide are connected, and this is the boundary between two amino acid residues, or, if the gene fusion occurred within a single codon, the single amino acid residue encoded by that codon itself.
  • a gene fusion that is an oncogenic driver mutation in the present invention is a mutation that results in the expression of a polypeptide encoded by a fusion polynucleotide represented by formula (I) below: 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position).
  • formula (I) represented by formula (I) below: 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position).
  • the fusion polynucleotide encodes a polypeptide containing the kinase domain of LTK and having kinase activity.
  • LTK is a member of the insulin receptor superfamily that forms the ALK/LTK subfamily together with ALK, is encoded by a gene located at 15q15 in humans, and is typically represented by SEQ ID NO: 4. It is a protein consisting of an amino acid sequence (NCBI Accession No. NP_002335.2). The LTK protein is characterized by having a kinase domain, which corresponds to the amino acid sequence of positions 510-786 of the amino acid sequence represented by SEQ ID NO: 4 in humans. The LTK gene has never been reported as a driver gene either alone or as a fusion gene.
  • the present invention is based on the discovery for the first time that the LTK gene functions as a driver when fused with other genes. Therefore, the partner gene that serves as a fusion partner for the LTK gene is not limited as long as it can be fused with the LTK gene and as a result, the gene exhibits a function as a driver.
  • a partner gene in the present invention is, for example, the CLIP1 gene.
  • CLIP1 protein is a member of the microtubule plus-end accumulation protein family, is encoded by a gene present at 12q24 in humans, and typically has an amino acid sequence represented by SEQ ID NO: 6 (NCBI Accession No. NP_001234926.1 ) is a protein consisting of The CLIP1 protein is characterized by having a coiled-coil domain, which corresponds to the amino acid sequence of positions 350-1353 of the amino acid sequence represented by SEQ ID NO: 6 in humans.
  • the fusion polypeptide of the present invention is a polypeptide that contains the kinase domain of the LTK protein and has kinase activity.
  • the fusion polypeptide of the present invention may contain the entire kinase domain of the LTK protein, or may contain a portion of the kinase domain as long as the fusion polypeptide of the present invention has kinase activity.
  • the fusion polypeptide of the present invention "has kinase activity” means that it has activity as an enzyme that phosphorylates tyrosine due to the kinase domain derived from the LTK protein.
  • Kinase activity of LTK fusion polypeptides is measured by standard methods, usually by detecting phosphorylated tyrosine of the substrate after incubation with a substrate (such as a synthetic peptide substrate) and ATP under appropriate conditions. It can also be measured using a commercially available measurement kit.
  • the fusion polypeptide of the present invention may contain the dimerization domain of the partner gene.
  • a fusion polypeptide of the invention may contain all of the dimerization domain or, as long as the fusion polypeptide of the invention is capable of dimerizing, the dimer is It may also contain part of a somatogenic domain.
  • Whether or not the fusion polypeptide of the present invention forms dimers can be confirmed by known methods such as gel filtration chromatography, combination of cross-linking agent treatment and SDS-polyacrylamide gel electrophoresis, and the like.
  • the fusion polypeptides of the present invention dimerize via a coiled-coil domain present on the N-terminal side, autophosphorylate, and constitutively activate. It is thought that it contributes to the
  • kinase domain As used by protein domains, “kinase domain”, “dimerization domain”, and “coiled-coil domain” are also used for the corresponding gene domains.
  • the polynucleotide encoding the fusion polypeptide of the present invention is a fusion polynucleotide represented by the following formula (I): 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position).
  • the fusion polynucleotide of the present invention may be mRNA, cDNA or genomic DNA.
  • the partner gene polynucleotide may contain a dimerization domain such as a coiled-coil domain.
  • the partner gene polynucleotide may be the CLIP1 gene polynucleotide.
  • a fusion polynucleotide of the invention can be, for example, a CLIP1-LTK fusion polynucleotide encoding a polypeptide selected from the group consisting of (a) to (c) below: (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and having kinase activity, or (c) a sequence A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by number 2 and having kinase activity.
  • the amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence encoded by a CLIP1-LTK fusion polynucleotide found in a sample derived from human cancer tissue, as shown in the Examples below.
  • the fusion point is located between positions 1083 and 1084 in the amino acid sequence represented by SEQ ID NO:2. That is, CLIP1 is up to 1083rd place, and LTK is after 1084th place.
  • one or more amino acids usually means 1 to 50, preferably 1 to 30, more preferably 1 to 10, still more preferably 1 to several (e.g., 1 ⁇ 5, 1-4, 1-3, 1 or 2, or 1) amino acids.
  • sequence identity is preferably 85% or more, more preferably 90% or more, 95% or more, even more preferably 97% or more, 98% or more, 99% or more sequence identity.
  • Amino acid sequence identity is called BLASTX or BLASTP based on the algorithm BLAST by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) It can be determined using a program (Altschul SF, et al: J Mol Biol 215: 403, 1990).
  • BLAST and Gapped BLAST programs use the default parameters for each program. Specific methods of these analysis methods are well known to those skilled in the art (eg, http://www.ncbi.nlm.nih.gov/).
  • the fusion polynucleotide of the present invention can be, for example, any of the following (a') to (d') polynucleotides: (a') a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1; (b') a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 and that encodes a polypeptide having kinase activity , (c') a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, and encoding a polypeptide having kinase activity; or (d')
  • the nucleotide sequence represented by SEQ ID NO: 1 is the nucleotide sequence of a CLIP1-LTK fusion polynucleotide found in a sample derived from human cancer tissue, as shown in Examples below.
  • the fusion point is located between positions 4383 and 4384 in the nucleotide sequence represented by SEQ ID NO:1. In other words, up to 4383th place is CLIP1, and 4384th place and later is LTK.
  • under stringent conditions refers to moderately or highly stringent conditions, unless otherwise specified.
  • Moderately stringent conditions can be easily designed by those skilled in the art, for example, based on the length of the target polynucleotide. Basic conditions are presented in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Typically, moderately stringent conditions are prewash conditions of 5 ⁇ SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) for nitrocellulose filters; % formamide, 2-6 ⁇ SSC (or other similar hybridization solution such as Stark's solution in about 50% formamide at about 42° C.); Includes wash conditions of 6 ⁇ SSC, 0.1% SDS. Moderately stringent conditions preferably include hybridization conditions at about 50° C., 6 ⁇ SSC, and may include washing conditions and/or washing conditions as described above.
  • Highly stringent conditions can also be easily designed by those skilled in the art, for example, based on the length of the target polynucleotide.
  • Highly stringent conditions include higher temperatures and/or lower salt concentrations than moderately stringent conditions.
  • it includes hybridization conditions of about 65° C., 0.2-6 ⁇ SSC, preferably 6 ⁇ SSC, more preferably 2 ⁇ SSC, even more preferably 0.2 ⁇ SSC.
  • wash conditions of about 65-68°C, 0.2 x SSC, 0.1% SDS.
  • SSPE (1 ⁇ SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4.
  • washing can be performed for about 15 minutes after hybridization is complete.
  • nucleotides is usually 1 to 50, preferably 1 to 30, more preferably 1 to 10, still more preferably 1 to several (e.g., 1-5, 1-4, 1-3, 1 or 2, or 1) nucleotides.
  • sequence identity is preferably 85% or more, more preferably 90% or more, 95% or more, even more preferably 97% or more, 98% or more, 99% sequence identity above.
  • the fusion polynucleotide of the present invention may have mutations in its LTK gene portion.
  • the LTK gene portion refers to the portion after position 4384 in the nucleotide sequence represented by SEQ ID NO: 1 in the case of humans, and refers to the orthologue corresponding to the human LTK gene portion in the case of organisms other than humans.
  • Mutations in the LTK gene portion are preferably mutations leading to LTK mutations selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof, more preferably , mutations that lead to LTK mutations, including L650F.
  • the present invention provides a method for detecting gene fusion (hereinafter also referred to as the detection method of the present invention).
  • the detection methods of the present invention comprise detecting any of the fusion polynucleotides or polypeptides encoded thereby in an isolated sample from a subject with cancer.
  • subjects are not particularly limited as long as they are mammals.
  • mammals include rodents such as mice, rats, hamsters, chipmunks and guinea pigs, rabbits, pigs, cows, goats, horses, sheep, minks, dogs, cats, humans, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, Examples include primates such as orangutans and chimpanzees, but humans are preferred.
  • a subject with cancer may be not only a subject with cancer, but also a subject suspected of having cancer or a subject at risk of developing cancer in the future.
  • the “cancer” to which the detection method of the present invention is applied is not particularly limited as long as the gene fusion can be detected, but lung cancer is preferred, and non-small cell lung cancer is more preferred.
  • isolated samples derived from subjects include not only biological samples (e.g., cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, sputum, alveolar/bronchial lavage, urine, stool), Nucleic acid extracts (genomic DNA extracts, mRNA extracts, cDNA preparations and cRNA preparations prepared from mRNA extracts, etc.) and protein extracts obtained from these biological samples are also included. Genomic DNA, mRNA, cDNA or protein can be prepared by a person skilled in the art by selecting a suitable known technique in consideration of the type and condition of the sample. Moreover, the sample may be formalin-fixed, alcohol-fixed, frozen, or paraffin-embedded.
  • biological samples e.g., cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, sputum, alveolar/bronchial lavage, urine, stool
  • Nucleic acid extracts genomic DNA extracts, mRNA extracts,
  • the "isolated sample” is preferably derived from an organ where the cancer exists or is suspected to exist, for example, small intestine, spleen, kidney, liver, stomach, lung, adrenal gland, heart, brain, Examples include those derived from the pancreas, aorta, etc., and more preferably those derived from the lung.
  • the fusion polynucleotide or the polypeptide encoded by it can be detected using a technique known per se.
  • RNA or cDNA prepared from mRNA When targeting transcripts from genomic DNA (mRNA or cDNA prepared from mRNA), RT-PCR, sequencing, TaqMan probe method, northern blotting, dot blotting, cDNA microarray analysis, etc. Fusion polynucleotides that are mRNA or cDNA can be detected.
  • ISH in situ hybridization
  • genomic PCR method sequencing
  • TaqMan probe method Southern blotting
  • genomic microarray analysis etc. are used to detect fusion polynucleotides that are genomic DNA. can do.
  • each of these methods can be used independently, but they can also be used in combination.
  • the gene fusion is thought to contribute to canceration by expressing the fusion polypeptide
  • when detecting a fusion polynucleotide that is genomic DNA for example, by in situ hybridization, etc.
  • transcription it is also preferable to further confirm that the product or protein is produced (eg, by RT-PCR, immunostaining, etc.).
  • the fusion polynucleotide is detected by hybridization technology (e.g., TaqMan probe method, Northern blotting, Southern blotting, dot blotting, microarray analysis, in situ hybridization (ISH), etc.), the fusion polynucleotide is Polynucleotides can be used that are probes designed to recognize
  • “specifically recognizing a fusion polynucleotide” includes wild-type genes from which the 5'-end and 3'-end portions of the fusion point of the fusion polynucleotide are derived under stringent conditions. It refers to distinguishing and recognizing the fusion polynucleotide from polynucleotides other than the fusion polynucleotide.
  • genomic DNA to be detected is stable even under formalin-fixation. It is preferable to use in situ hybridization from the viewpoint of high detection sensitivity.
  • the following ( ⁇ ) or ( ⁇ ) having a chain length of at least 15 bases as a polynucleotide that is a probe designed to specifically recognize the fusion polynucleotide in the biological sample
  • the genomic DNA encoding the fusion polypeptide (fusion polynucleotide) can be detected by hybridizing the polynucleotides of .
  • ( ⁇ ) For each fusion gene at least one selected from the group consisting of a probe that hybridizes to the nucleotide sequence of the 5' fusion partner gene (e.g. CLIP1 gene) and a probe that hybridizes to the nucleotide sequence of the 3' LTK gene.
  • the 5'-side fusion partner gene of the present invention is typically a gene specified by NCBI Entrez Gene: 6249 if it is human-derived.
  • the LTK gene according to the present invention is typically a gene specified by NCBI Entrez Gene: 4058 if it is human-derived.
  • the DNA sequence of a gene can change naturally (that is, non-artificially) due to its mutation. Therefore, such naturally occurring mutants can also be subject of the present invention (the same shall apply hereinafter).
  • the present inventors have found that the fusion gene of the present invention containing a mutated LTK gene segment can also serve as a therapeutic target.
  • the polynucleotide that is the probe according to ( ⁇ ) of the present invention is the base sequence of the 5'-side fusion partner gene (for example, CLIP1 gene) and/or the base of the 3'-side LTK gene, which is the target base sequence of the polynucleotide.
  • Any polynucleotide that can detect the presence of genomic DNA encoding the fusion polypeptide in the biological sample by hybridizing to the sequence but is preferably a polynucleotide described in ( ⁇ 1) to ( ⁇ 3) below.
  • ( ⁇ 1) A polynucleotide that hybridizes to the nucleotide sequence of the upstream region on the 5′ side of the breakpoint of the 5′ fusion partner gene (hereinafter also referred to as “5′ fusion partner gene probe 1”), and the 3′ fusion Combination with a polynucleotide that hybridizes to the nucleotide sequence of the downstream region on the 3' side of the breakpoint of the partner gene (hereinafter also referred to as “3' fusion partner gene probe 1"); ( ⁇ 2) A polynucleotide hybridizing to the nucleotide sequence of the upstream region on the 5′ side of the breakpoint of the 5′ fusion partner gene (hereinafter also referred to as “5′ fusion partner gene probe 1”), and the 5′ fusion combination with a polynucleotide that hybridizes to the nucleotide sequence of the downstream region on the 3' side of the breakpoint of the partner gene (hereinafter also referred to as "5'
  • the nucleotide sequence of the upstream region on the 5′ side of the breakpoint of the gene includes the entire coiled-coil domain of the CLIP1 protein or Some coding regions are included.
  • the nucleotide sequence of the downstream region on the 3' side of the breakpoint of the 3' LTK gene includes the coding region of all or part of the kinase domain of the LTK protein.
  • polynucleotides described in ( ⁇ 1) above include combinations of the following polynucleotides ( ⁇ 1-1) to ( ⁇ 1-3): ( ⁇ 1-1) A polynucleotide that hybridizes to the coding region of all or part of the dimerization domain of the 5′-side fusion partner protein and a polynucleotide that hybridizes to the coding region of all or part of the kinase domain of LTK a combination of nucleotides; ( ⁇ 1-2) A combination of a polynucleotide that hybridizes to all or part of the coding region of the coiled-coil domain of the 5′-side fusion partner protein and a polynucleotide that hybridizes to all or part of the coding region of the kinase domain of LTK ;or ( ⁇ 1-3) A combination of a polynucleotide that hybridizes to all or part of the coding region of the coiled-coil domain of CLIP1 and a
  • the region (target base sequence) to which the polynucleotide described in ( ⁇ ) used for in situ hybridization hybridizes from the viewpoint of specificity to the target base sequence and sensitivity of detection, the 5′ side A region within 1,000,000 bases from the fusion point between the fusion partner gene (eg CLIP1 gene) and the 3'-side LTK gene is preferred.
  • the polynucleotide described in ( ⁇ ) used for in situ hybridization is a fusion point between the 5' fusion partner gene and the 3' LTK gene, which is the target nucleotide sequence of the polynucleotide.
  • the polynucleotide according to ( ⁇ ) or ( ⁇ ) used for in situ hybridization covers the entire target nucleotide sequence from the viewpoint of specificity and detection sensitivity for the target nucleotide sequence.
  • a population consisting of multiple types of polynucleotides is preferred.
  • the length of the polynucleotides constituting the population is at least 15 bases, preferably 100-1000 bases.
  • the polynucleotide described in ( ⁇ ) or ( ⁇ ) used for in situ hybridization is preferably labeled with a fluorescent dye or the like for detection.
  • fluorescent dyes include, but are not limited to, DEAC, FITC, R6G, TexRed, Cy5.
  • radioactive isotopes eg 125 I, 131 I, 3 H, 14 C, 33 P, 32 P, etc.
  • enzymes eg ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, Malate dehydrogenase, etc.
  • luminescent substances eg, luminol, luminol derivatives, luciferin, lucigenin, 3,3′-diaminobenzidine (DAB), etc.
  • DAB 3,3′-diaminobenzidine
  • labeling of polynucleotides can be performed by a known method.
  • a substrate base labeled with a fluorescent dye or the like can be incorporated into a polynucleotide to label the polynucleotide.
  • the conditions for hybridizing the polynucleotide described in ( ⁇ ) or ( ⁇ ) with the biological sample may vary depending on various factors such as the length of the polynucleotide.
  • Stringent hybridization conditions include, for example, 0.2 x SSC and 65°C conditions
  • low stringency hybridization conditions include, for example, 2.0 x SSC and 50°C conditions.
  • a surfactant By appropriately selecting various conditions such as the concentration of NP-40, etc., the concentration of formamide, and pH, stringent hybridization conditions similar to those described above can be achieved.
  • Examples of methods for detecting genomic DNA encoding a fusion polypeptide using the polynucleotide described in ( ⁇ ) or ( ⁇ ) include Southern blotting, Northern blotting and dot blotting, in addition to the in situ hybridization. be done.
  • the fusion gene is detected by hybridizing the polynucleotide described in ( ⁇ ) or ( ⁇ ) to a membrane onto which a nucleic acid extract obtained from the biological sample is transcribed.
  • polynucleotide ( ⁇ ) When the polynucleotide ( ⁇ ) was used, a polynucleotide hybridizing to the nucleotide sequence of the 5′-side fusion partner gene and a polynucleotide hybridizing to the nucleotide sequence of the 3′-side LTK gene were developed on the membrane. When the same band is recognized, it can be judged that the genomic DNA encoding the fusion polypeptide has been detected.
  • Methods for detecting the genomic DNA encoding the fusion polypeptide using the ( ⁇ ) polynucleotide further include genome microarray analysis and DNA microarray analysis.
  • an array is prepared by immobilizing the polynucleotide ( ⁇ ) on a substrate, and the genomic DNA is detected by contacting the biological sample with the polynucleotide on the array.
  • the substrate is not particularly limited as long as the oligo or polynucleotide can be immobilized thereon, and examples thereof include glass plates, nylon membranes, microbeads, silicon chips and capillaries.
  • the detection method of the present invention it is also preferable to detect the fusion polynucleotide using PCR.
  • PCR it is possible to use a polynucleotide that is a pair of primers designed to specifically amplify a fusion polynucleotide using DNA (genomic DNA, cDNA) or RNA prepared from the biological sample as a template.
  • DNA genomic DNA, cDNA
  • RNA RNA prepared from the biological sample as a template.
  • “capable of specifically amplifying the fusion polynucleotide” means that the fusion polynucleotide can be It means that only nucleotides can be amplified, and the whole of the fusion polynucleotide may be amplified, or a part of the fusion polynucleotide containing the fusion point may be amplified.
  • the "polynucleotide that is a pair of primers" used in PCR etc. consists of a sense primer (forward primer) and an antisense primer (reverse primer) that specifically amplify the target fusion polynucleotide. It is designed from the nucleotide sequence on the 5'-terminal side from the fusion point of the fusion polynucleotide, and the antisense primer is designed from the nucleotide sequence on the 3'-terminal side from the fusion point of the fusion polynucleotide. These primers are usually designed so that the PCR product is 5 kb or less from the viewpoint of detection accuracy and sensitivity by PCR.
  • Primers can be appropriately designed by a known technique, for example, Primer Express (registered trademark) software (Applied Biosystems) can be used.
  • the length of these polynucleotides is usually 15 bases or more (preferably 16, 17, 18, 19 or 20 bases or more, more preferably 21 bases or more) and 100 bases or less (preferably 90, 80, 70, 60, 50 or 40 bases or less, more preferably 30 bases or less).
  • Preferred examples of the "polynucleotide that is a pair of primers" include a polynucleotide consisting of the base sequence represented by SEQ ID NO: 7 and/or a polynucleotide represented by SEQ ID NO: 8 as a forward primer for the CLIP1-LTK fusion polynucleotide. and a primer set consisting of a polynucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 9 as a reverse primer (see Table 1 below).
  • direct sequencing is performed on the PCR product, and by determining the base sequence including the fusion point, the 5' side gene part and the 3' side gene part are identified. are linked in-frame and/or the inclusion of a given domain in the fusion polynucleotide. Sequencing can be performed by a known technique, and can be easily performed by using a sequencer (e.g., ABI-PRISM 310 Genetic Analyzer (Applied Biosystems Inc.), etc.) according to the instruction manual.
  • a sequencer e.g., ABI-PRISM 310 Genetic Analyzer (Applied Biosystems Inc.), etc.
  • the TaqMan probe method confirms that the 5'-side gene portion and the 3'-side gene portion are linked in-frame and/or in the fusion polynucleotide. You can check that the given domain is included.
  • Probes used in the TaqMan probe method include, for example, the polynucleotides described in ( ⁇ ) or ( ⁇ ) above. Probes are labeled with a reporter dye (eg, FAM, FITC, VIC, etc.) and a quencher (eg, TAMRA, Eclipse, DABCYL, MGB, etc.).
  • the above primers and probes may be DNA, RNA, or DNA/RNA chimeras, but are preferably DNA.
  • PNA polyamide nucleic acid, peptide nucleic acid
  • LNA registered trademark, locked nucleic acid, Bridged Nucleic Acid, crosslinked nucleic acid
  • ENA registered trademark, 2'-O, 4'-C) -Ethylene-bridged nucleic acid
  • GNA Glycerol nucleic acid, glycerol nucleic acid
  • TNA threose nucleic acid, threose nucleic acid
  • the primers and probes may be double-stranded or single-stranded, but are preferably single-stranded.
  • primers and probes may contain one or more nucleotide mismatches as long as they can specifically hybridize to the target sequence. 91, 92, 93, 94% or greater, more preferably 95, 96, 97, 98, 99% or greater identity, most preferably 100% identity.
  • primers and probes can be synthesized according to conventional methods using a DNA/RNA automatic synthesizer, for example, based on the nucleotide sequence information described in this specification.
  • the fusion polynucleotide may be detected by whole transcriptome sequencing (RNA sequencing) or genome sequencing.
  • RNA sequencing whole transcriptome sequencing
  • genome sequencing e.g., Genome Analyzer IIx (Illumina), Hi-Sec Sequencer (HiSeq2000, Illumina), Genome Sequencer FLX System (Roche), etc.
  • a next-generation sequencer e.g., Genome Analyzer IIx (Illumina), Hi-Sec Sequencer (HiSeq2000, Illumina), Genome Sequencer FLX System (Roche), etc.
  • a commercially available kit e.g., mRNA-Seq sample preparation kit (Illumina), etc.
  • Illumina mRNA-Seq sample preparation kit
  • fusion polynucleotides examples include immunostaining, Western blotting, RIA, ELISA, flow cytometry, and immunoprecipitation. , antibody array analysis, etc. can be used to detect the translation product.
  • Antibodies that specifically recognize the fusion polypeptide are used in these methods.
  • the term "specifically recognizes the fusion polypeptide” refers to proteins other than the fusion polypeptide, including wild-type proteins derived from the N-terminal and C-terminal portions of the fusion point of the fusion polypeptide. It means recognizing only the fusion polypeptide without recognizing it.
  • An antibody that "specifically recognizes a fusion polypeptide" used in the detection method of the present invention may be a single antibody or a combination of two or more antibodies.
  • Antibody that specifically recognizes a fusion polypeptide includes an antibody specific to a polypeptide containing a fusion point of the fusion polypeptide (hereinafter also referred to as a "fusion point-specific antibody”).
  • fusion point-specific antibody means an antibody that specifically binds to a polypeptide containing the fusion point, but does not bind to the wild-type protein from which the N-terminal and C-terminal portions are derived. do.
  • the "antibody that specifically recognizes the fusion polypeptide” includes an antibody that binds to the polypeptide consisting of the region on the N-terminal side from the fusion point of the fusion polypeptide and the region on the C-terminal side from the fusion point of the fusion polypeptide. Also included are combinations of antibodies that bind to different polypeptides.
  • the fusion polypeptide can be detected by sandwich ELISA, immunostaining, immunoprecipitation, western blotting, or the like using these two antibodies.
  • antibodies include polyclonal antibodies, natural antibodies such as monoclonal antibodies (mAb), chimeric antibodies, humanized antibodies, single-chain antibodies that can be produced using genetic recombination technology, and binding fragments thereof. including but not limited to.
  • a binding fragment means a partial region of the antibody having specific binding activity, and specifically includes, for example, Fab, Fab', F(ab')2, Fv, and single-chain antibodies.
  • the class of the antibody is not particularly limited, and the antibody may have any isotype such as IgG, IgM, IgA, IgD, or IgE, but IgG is more preferable in consideration of ease of purification and the like.
  • an "antibody that specifically recognizes a fusion polypeptide” can be prepared by a person skilled in the art by appropriately selecting a known technique.
  • known techniques include a polypeptide containing the fusion point of the fusion polypeptide, a polypeptide consisting of a region on the N-terminal side from the fusion point of the fusion polypeptide, or a region on the C-terminal side from the fusion point of the fusion polypeptide.
  • a method for producing a monoclonal antibody Commercially available antibodies may be used. Moreover, by using an antibody to which a labeling substance is bound, it is possible to directly detect the target protein by detecting the label.
  • the labeling substance is not particularly limited as long as it can bind to an antibody and is detectable.
  • thiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, and radioactive substances a method of indirectly detecting the target protein using a secondary antibody conjugated with a labeling substance, protein G or protein A, etc. can also be used.
  • the gene fusion is an oncogenic driver mutation, and is thought to contribute to malignant transformation of cancer and the like by constitutively activating LTK kinase activity. Therefore, in cancer patients in whom such gene fusion is detected, there is a high probability that treatment with a substance that suppresses the expression and/or activity of the polypeptide encoded by the fusion polynucleotide generated by the gene fusion will be effective. .
  • the present invention provides cancer patients or cancer risks for whom a substance that suppresses the expression and/or activity of a polypeptide encoded by a fusion polynucleotide generated by a gene fusion that is an oncogenic driver mutation has a therapeutic effect.
  • a method for identifying a subject with the disease (hereinafter also referred to as the identification method of the present invention) is provided.
  • the identification method of the invention includes the following steps: (1) a method comprising detecting, in an isolated sample from a subject, a fusion polynucleotide represented by formula (I) below or a polypeptide encoded thereby: 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position) wherein said fusion polynucleotide encodes a polypeptide comprising the kinase domain of LTK and having kinase activity; and (2) when said fusion polynucleotide or a polypeptide encoded thereby is detected, said Determining that a substance that inhibits the expression and/or activity of a polypeptide has a therapeutic effect in said subject.
  • formula (I) a fusion polynucleotide represented by formula (I) below or a polypeptide encoded thereby
  • the present inventors have found that even the fusion gene of the present invention having a mutation in the LTK gene portion can serve as a therapeutic target. Therefore, the fusion polynucleotide used in the identification method of the present invention may have a mutation in its LTK gene portion.
  • the LTK gene portion refers to the portion after position 4384 in the nucleotide sequence represented by SEQ ID NO: 1 in the case of humans, and refers to the orthologue corresponding to the human LTK gene portion in the case of organisms other than humans.
  • Mutations in the LTK gene portion are preferably mutations leading to LTK mutations selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof, more preferably , mutations that lead to LTK mutations, including L650F.
  • the fusion polypeptide used in the identification method of the present invention may have mutations in the LTK portion.
  • a "cancer patient or a subject at risk of cancer” is a mammal, preferably a human, suffering from cancer or suspected of suffering from cancer.
  • the "cancer” to which the identification method of the present invention is applied is not particularly limited as long as it is a cancer in which any of the gene fusions can be detected, preferably lung cancer, and more preferably non-small cell cancer. have lung cancer.
  • the "therapeutic effect” is the effect of cancer treatment, and is not particularly limited as long as it is a beneficial effect on the patient, for example, tumor reduction effect, progression-free survival effect, prolongation of life effects, etc.
  • expression of a polypeptide encoded by a fusion polynucleotide generated by a gene fusion that is an oncogenic driver mutation which is an object for evaluating the efficacy of cancer treatment with respect to gene fusion that is an oncogenic driver mutation and/or activity
  • a target substance in the identification method of the present invention is a substance that directly or indirectly inhibits the expression and/or function of the fusion polypeptide of the present invention. is not particularly limited.
  • Substances that inhibit the expression of the fusion polypeptide of the present invention include, for example, siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), and antisense that suppress the expression of the fusion polypeptide of the present invention.
  • Nucleic acids, expression vectors capable of expressing these polynucleotides, low-molecular-weight compounds, and the like are included.
  • Substances that inhibit the function of the fusion polypeptide of the present invention include, for example, substances that inhibit LTK kinase activity (eg, low-molecular-weight compounds), antibodies that bind to the fusion polypeptide of the present invention, and the like.
  • substances that inhibit LTK kinase activity eg, low-molecular-weight compounds
  • antibodies that bind to the fusion polypeptide of the present invention and the like.
  • substances may be substances that specifically suppress the expression and/or activity of the fusion polypeptide of the present invention, or substances that also suppress the expression and/or activity of the wild-type LTK protein. good.
  • substances include, for example, substances having ALK inhibitory activity, but are not limited to crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, entrectinib ( entrectinib), repotrectinib, and giltertinib.
  • These substances can be prepared by methods known per se, for example, based on the sequence information of the CLIP1-LTK fusion polynucleotide and/or CLIP1-LTK fusion polypeptide disclosed herein.
  • a substance selected by a screening method, which will be described later, may be used, or a commercially available substance may be used.
  • These substances are cancer therapeutic agents administered to a subject with cancer when a CLIP1-LTK fusion polynucleotide or a polypeptide encoded by it is detected in an isolated sample derived from a subject with cancer. can be used as an active ingredient in
  • Step (1) in the identification method of the present invention can be performed in the same manner as the steps included in the detection method of the present invention.
  • step (2) in the identification method of the present invention the fusion polynucleotide or its When the polypeptide encoded by is detected, the substance of interest in the identification method of the present invention is determined to have a therapeutic effect in the subject, while the fusion polynucleotide or the polypeptide encoded by it is not detected If so, it is determined that the target substance in the identification method of the present invention is unlikely to have a therapeutic effect in the subject.
  • the identification method of the present invention positive examples of gene fusion newly found as an oncogenic driver mutation are detected from cancer patients or subjects at risk of cancer, and fusion polynucleotides generated by the gene fusion are detected. It is possible to identify cancer patients or subjects at risk of cancer for whom a substance that suppresses the expression and/or activity of a polypeptide encoded by It is useful in that it enables appropriate treatment.
  • a fusion polynucleotide resulting from a gene fusion that is an oncogenic driver mutation or a polypeptide encoded thereby can be detected using the primers, probes, antibodies, or combinations thereof, and can detect the gene fusion.
  • kits for the detection of gene fusions that are oncogenic driver mutations comprising any or a combination of the following:
  • B a polynucleotide that is a pair of primers designed to specifically amplify the fusion polynucleotide represented by the formula (I); or
  • C the fusion polynucleotide represented by the formula
  • the present inventors have found that even the fusion gene of the present invention having a mutation in the LTK gene portion can serve as a therapeutic target. Therefore, when the fusion gene is an LTK fusion gene, the fusion polynucleotide used in the kit of the present invention may have a mutation in the LTK gene portion.
  • the LTK gene portion refers to the portion after position 4384 in the nucleotide sequence represented by SEQ ID NO: 1 in the case of humans, and refers to the orthologue corresponding to the human LTK gene portion in the case of organisms other than humans.
  • Mutations in the LTK gene portion are preferably mutations leading to LTK mutations selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof, more preferably , mutations that lead to LTK mutations, including L650F.
  • kits of the present invention include substrates necessary for detecting labels attached to polynucleotides and antibodies, positive controls (for example, fusion polynucleotides of the present invention, or cells harboring these). and negative controls, PCR reagents, counterstaining reagents (DAPI, etc.) used in in situ hybridization, molecules necessary for antibody detection (e.g., secondary antibodies, protein G, protein A), sample dilution and A buffer or the like used for washing can be included in an appropriate combination. Kits of the invention can also include instructions for use. By using the kit of the present invention, the detection method of the present invention can be easily carried out.
  • the kit of the present invention is used to determine whether a subject is a cancer patient for whom a substance that suppresses the expression and/or activity of a polypeptide encoded by a fusion polynucleotide generated by gene fusion that is an oncogenic driver mutation produces a therapeutic effect. It can also be used to diagnose whether or not you are at risk for cancer.
  • the detection method and detection kit of the present invention make it possible to detect gene fusions newly found as carcinogenic driver mutations, and as described later, identify positive cases of the gene fusions and apply personalized medicine. It is extremely useful for
  • the identification method of the present invention identifies cancer patients in whom a substance that suppresses the expression and/or activity of the polypeptide encoded by the fusion polynucleotide resulting from gene fusion produces therapeutic effects. Therefore, cancer can be treated efficiently by selectively administering the substance to patients carrying the fusion gene among cancer patients. Therefore, the present invention provides a method for treating cancer, comprising the step of administering the substance to a subject determined by the identification method of the present invention that the substance has a therapeutic effect (hereinafter referred to as the method of the present invention). (also referred to as methods of treatment of
  • the present invention further provides expression of a polypeptide encoded by a fusion polynucleotide generated by the gene fusion and/or A therapeutic agent for cancer (hereinafter also referred to as a therapeutic agent for cancer of the present invention) containing, as an active ingredient, a substance that suppresses its activity is provided.
  • a therapeutic agent for cancer hereinafter also referred to as a therapeutic agent for cancer of the present invention
  • the active ingredient of the cancer therapeutic agent of the present invention is described as a substance that suppresses the expression and/or activity of the polypeptide encoded by the fusion polynucleotide generated by the gene fusion. substances that have
  • the cancer therapeutic agent of the present invention can be prepared as a pharmaceutical composition using pharmacologically acceptable carriers, excipients, and/or other additives that are commonly used for their formulation.
  • the administration method of the cancer therapeutic agent of the present invention is appropriately selected according to the type of the inhibitor and the type of cancer. (aerosol), rectal, intravaginal, etc. modes of administration can be employed.
  • the dosage of the therapeutic agent for cancer of the present invention depends on the activity and type of the active ingredient, mode of administration (e.g., oral, parenteral), severity of disease, animal species to be administered, drug acceptability of the subject to be administered, It can be determined as appropriate in consideration of body weight, age, and the like.
  • the therapeutic method and cancer therapeutic agent of the present invention are useful because they enable the treatment of patients with specific oncogenic driver mutations that were previously unknown and clarified by the present invention.
  • the present invention provides a method of screening for an active ingredient of a cancer therapeutic agent that exerts a therapeutic effect on cancer patients having the gene fusion (hereinafter also referred to as the screening method of the present invention).
  • a substance that suppresses the expression and/or activity of any of the fusion polypeptides of the present invention eg, CLIP1-LTK
  • CLIP1-LTK fusion polypeptides of the present invention
  • a test substance to be subjected to the screening method of the present invention may be any compound or composition, such as nucleic acids (eg, nucleosides, oligonucleotides, polynucleotides), carbohydrates (eg, monosaccharides, oligosaccharides, polysaccharides), lipids (e.g., saturated or unsaturated linear, branched and/or cyclic fatty acids), amino acids, proteins (e.g., oligopeptides, polypeptides), low-molecular-weight compounds, compound libraries, Examples include random peptide libraries, natural ingredients (eg, ingredients derived from microorganisms, animals, plants, marine organisms, etc.), foods, and the like.
  • nucleic acids eg, nucleosides, oligonucleotides, polynucleotides
  • carbohydrates eg, monosaccharides, oligosaccharides, polysaccharides
  • lipids e.g.,
  • the screening method of the present invention may take any form as long as it can evaluate whether or not a test substance suppresses the expression and/or activity of the fusion polypeptide of the present invention.
  • the screening methods of the invention comprise the following steps: (1) contacting cells expressing the fusion polypeptide of the present invention with a test substance; (2) determining whether the expression and/or activity of said fusion polypeptide is inhibited; Selecting as an active ingredient of a drug.
  • step (1) cells expressing the fusion polypeptide of the present invention are brought into contact with the test substance.
  • a solvent eg, DMSO, etc.
  • the contacting can take place in a medium.
  • the medium is appropriately selected depending on the type of cells used. 199 medium, etc.
  • the culture conditions are also appropriately determined depending on the type of cells used. 12 to about 72 hours.
  • Cells expressing the fusion polypeptide of the present invention include, for example, cancer tissue-derived cells endogenously expressing the fusion polypeptide, cell lines derived from the cell, and genetically engineered cells. cell lines, etc., which have been identified, but are not limited to these. Whether or not a certain cell expresses the fusion polypeptide of the present invention can also be confirmed using the detection method of the present invention. Also, the cells are usually mammalian cells, preferably human cells.
  • step (2) it is determined whether the expression and/or activity of the fusion polypeptide is suppressed.
  • Fusion polypeptide expression can be measured by determining mRNA levels or protein levels in cells using known analytical methods such as Northern blotting, quantitative PCR, immunoblotting, ELISA, and the like. .
  • the activity of the fusion polypeptide can also be measured by known analysis methods (eg, kinase activity measurement, etc.).
  • the measured values obtained are compared with the measured values in control cells which have not been contacted with the test substance. Comparison of measured values is preferably performed based on the presence or absence of a significant difference. If the measured value in cells contacted with the test substance is significantly lower compared to controls, it can be determined that the test substance inhibits the expression and/or activity of the fusion polypeptide.
  • proliferation of the cells can be used as an index for determination in this step.
  • the proliferation of the cells contacted with the test substance is measured. Measurement of cell proliferation can be performed by a method known per se such as cell counting, 3H thymidine incorporation, BRDU method and the like.
  • the growth of the cells contacted with the test substance is then compared to the growth of control cells not contacted with the test substance. Comparison of proliferation levels is preferably performed on the basis of the presence or absence of significant differences.
  • the proliferation of the control cells not contacted with the test substance may be a value measured beforehand or at the same time as the measurement of the proliferation of the cells contacted with the test substance, but the accuracy of the experiment, From the viewpoint of reproducibility, values measured simultaneously are preferred. As a result of the comparison, it can be determined that the test substance inhibits the expression and/or activity of the fusion polypeptide if the proliferation of the cells contacted with the test substance is inhibited.
  • step (3) the test substance determined to suppress the expression and/or activity of the fusion polypeptide in step (2) is selected as the active ingredient of the cancer therapeutic agent.
  • the present inventors have found that even the fusion gene of the present invention having a mutation in the LTK gene portion can serve as a therapeutic target. Therefore, when the fusion gene is an LTK fusion gene, the fusion polypeptide used in the screening method of the present invention may have mutations in the LTK portion.
  • the mutation in the LTK portion is preferably a mutation that, in humans, results in a LTK mutation selected from the group consisting of I565N, F568C, L590M, L592F, G596R, D597N, L650F, G663A, and combinations of two or more thereof. , more preferably mutations that lead to LTK mutations, including L650F.
  • mutations in the LTK portion are preferably mutations that result in LTK mutations at positions corresponding to the human mutations described above.
  • the screening method of the present invention it is possible to obtain cancer therapeutic agents applicable to the treatment of patients with previously unknown carcinogenic driver mutations. Furthermore, it is also possible to obtain effective cancer therapeutic agents for patients with carcinogenic driver mutations in which the LTK gene portion is further mutated. For example, by the screening method of the present invention, it is possible to obtain a cancer therapeutic agent that targets an oncogenic driver mutation that does not have a mutation in the LTK gene portion, and furthermore, it is possible to develop resistance to such a cancer therapeutic agent. It is also possible to obtain alternative cancer therapeutic agents that target oncogenic driver mutations with mutations in the LTK gene portion as shown.
  • the present invention provides an isolated fusion polynucleotide of formula (I): 5'-A-B-3' (Formula I) (Wherein, 5' represents the 5' terminal position, A represents the partner gene polynucleotide, B represents the LTK gene polynucleotide, and 3' represents the 3' terminal position)
  • the fusion polynucleotide encodes a polypeptide comprising the kinase domain of LTK and having kinase activity.
  • an "isolated" substance means a substance in the environment in which it naturally exists (e.g., within the cells of an organism) other substances (preferably biological agents) (e.g., nucleic acids). in some cases, substantially separated or purified from non-nucleic acid factors and nucleic acids containing nucleic acid sequences other than the nucleic acid of interest; if proteins, non-protein factors and proteins containing amino acid sequences other than the protein of interest, etc.). It means what was done.
  • isolated preferably means 75 wt% or more, more preferably 85 wt% or more, even more preferably 95 wt% or more, and most preferably 96 wt% or more, 97 wt% or more. , having a purity of 98% by weight or more, 99% by weight or more, or 100%.
  • isolated polynucleotides and polypeptides include polynucleotides and polypeptides purified by standard purification methods, and also include chemically synthesized polynucleotides and polypeptides.
  • the polynucleotide of the present invention may be mRNA, cDNA or genomic DNA. Moreover, it may be double-stranded or single-stranded.
  • a typical example of cDNA encoding the fusion polypeptide of the present invention is a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO:1.
  • the polynucleotide of the present invention can be produced by a method known per se. For example, it can be extracted using a known hybridization technique from a cDNA library or a genomic library prepared from cancer tissue or the like that retains the fusion polynucleotide of the present invention. It can also be prepared by amplifying mRNA, cDNA or genomic DNA prepared from the cancer tissue or the like as a template using a known gene amplification technique (PCR). Furthermore, using the cDNA of the wild-type gene from which the 5'-terminal side and 3'-terminal side of each fusion polynucleotide are derived, as a material, PCR, restriction enzyme treatment, site-directed mutagenesis method (Kramer) , W. & Fritz, HJ., Methods Enzymol, 1987, 154, 350.) and other known gene amplification techniques or recombinant techniques.
  • PCR gene amplification technique
  • the present invention also provides an isolated fusion polypeptide encoded by the fusion polynucleotide of the present invention, comprising the kinase domain of an LTK protein and having kinase activity (hereinafter also referred to as the fusion polypeptide of the present invention), or Also provide fragments.
  • “Fragment” refers to a fragment of the fusion polypeptide of the present invention, consisting of a continuous partial sequence including sequences upstream and downstream of the fusion point.
  • the sequence upstream of the fusion point contained in the partial sequence is 1 or more amino acid residues from the fusion point (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 , 100 amino acid residues or more), and may include up to the N-terminus of the fusion polypeptide of the present invention.
  • the sequence downstream of the fusion point contained in the partial sequence is 1 or more amino acid residues from the fusion point (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 , 100 amino acid residues or more), and may include up to the C-terminus of the fusion polypeptide of the present invention.
  • the length of the fragment is not particularly limited, but is usually 8 amino acid residues or more (eg, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 100 amino acid residues or more).
  • a fusion polypeptide of the invention can be, for example, an isolated fusion polypeptide of: An isolated CLIP1-LTK fusion polypeptide comprising all or part of the coiled-coil domain of the CLIP1 protein and the kinase domain of the LTK protein and having kinase activity.
  • a fusion polypeptide of the invention can be, for example, an isolated fusion polypeptide of: (i) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (ii) a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and having kinase activity, or (iii) a sequence A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by number 2 and having kinase activity.
  • the fusion polypeptide of the present invention or a fragment thereof can also be produced by a method known per se. For example, by inserting the polynucleotide prepared as described above into an appropriate expression vector, introducing the vector into a cell-free protein synthesis system (e.g., reticulocyte extract, wheat germ extract) and incubating, Alternatively, the fusion polypeptide of the present invention can be prepared by introducing the vector into suitable cells (eg, E. coli, yeast, insect cells, animal cells) and culturing the resulting transformants.
  • suitable cells eg, E. coli, yeast, insect cells, animal cells
  • the fusion polypeptide of the present invention or a fragment thereof can be used as a marker in the detection method and the like of the present invention, and can also be used to prepare antibodies against the fusion polypeptide of the present invention.
  • the isolated fusion polynucleotides and fusion polypeptides of the present invention may have mutations in the LTK gene portion or LTK portion.
  • NIH3T3 cells and Phoenix-AMPHO cells were purchased from the American Type Culture Collection (CRL-1658 and CRL-3213, respectively).
  • Ba/F3, WEHI, and BOSC23 cells were provided by Dr. Daniel G. Tennen (Harvard Medical School).
  • PC9 cells were provided by Dr. Pasi Jannet (Dana-Farber Cancer Institute).
  • Crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, entrectinib, repotrectinib, and giltertinib were purchased from CEREC.
  • LC-SCRUM-Asia National Industry-Academia Collaborative Cancer Genome Screening Project
  • LC-SCRUM-Asia Multicenter Lung Cancer Genomic Screening Project Platform
  • DNA/RNA was extracted from fresh and frozen tissues and/or formalin-fixed paraffin-embedded (FFPE) samples using the AllPrep DNA/RNA Mini Kit (Qiagen).
  • ALK, ROS1, and RET fusion genes were analyzed by RT-PCR and positive by FISH.
  • Molecular screening was also performed using targeted next-generation sequencing (NGS) systems (Oncomine Comprehensive Assay (version 1 or 3), Oncomine Precision Assay, and multigene quantitative PCR (qPCR) assays.
  • NGS next-generation sequencing
  • qPCR multigene quantitative PCR
  • RNA samples (20-100 ng) using TruSeq RNA Exome (Illumina, San Diego, CA, USA). The library was subjected to paired-end sequencing of 75 bp reads using the NextSeq 500/550 High Output Kit version 2.5 on the NextSeq 550 (Illumina).
  • a kinase-dead (KD) CLIP1-LTK-K1440M (corresponding to LTK K544M) expression plasmid was generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (#210518, Agilent).
  • MIGR1 retroviral vector which encodes the EGFR-L858R fusion gene, has been previously reported (Yasuda, H. et al. Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer. Sci Transl Med 5, 216ra177, doi:10.1126/scitranslmed.3007205 (2013)).
  • the oligonucleotides shown in Table 1 were used for cloning. The integrity of all vector constructs was confirmed by Sanger sequencing analysis.
  • NIH3T3 cells and PC9 cells were transfected with 8 ⁇ g/mL polybrene (#12996-81, Nacalai Tesque). Transfected cells were selected by GFP sorting.
  • Ba/F3 stably expressing cells were maintained in RPMI medium supplemented with 10% FBS, 5% WEHI conditioned medium (as IL-3 source), 100 units/mL penicillin, and 100 ⁇ g/mL streptomycin.
  • NIH3T3 and PC9 stably expressing cells were maintained in DMEM medium supplemented with 10% FBS, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin.
  • Phospho-ALK/LTK (pTyr1278/672: #6941), Phospho-Akt (pS473: #4060), Phospho-ERK 1/2 (pT202/pY204: #9106), total Akt (#4685), total Erk 1/ 2 (#9102), BIM (#2819), CLIP1 (#8977), PARP (#9532), and cleaved PARP (#5625) antibodies were purchased from Cell Signaling Technologies, Inc. (Beverly, MA, USA). Images were acquired using an ImageQuant LAS 4000 (GE Healthcare).
  • ⁇ Cell viability assay> Bs/F3 cells (10,000 cells per well) were seeded in a 96-well plate, treated with the drug described in the ⁇ Reagent> section for 48 hours, and treated using Cell Counting Kit-8 (CK04, Fujifilm). and incubated for 2 hours at 37°C. Absorbance was read at 450 nm.
  • a PCR product of the expected size was specifically detected from two metastatic sites (supraclavicular lymph node and liver) (Fig. 3).
  • the CLIP1-LTK fusion protein is constitutively activated and exhibits oncogenic potential>
  • CLIP1-LTK or LTK expression constructs were transiently introduced into NIH3T3 cells, strong phosphorylation of CLIP1-LTK protein was detected, but phosphorylation of wild-type LTK was much weaker (Fig. 6).
  • Antibodies against phosphorylated LTK detected strong phosphorylated bands in CLIP1-LTK-expressing NIH3T3 cells or Ba/F3 cells (NIH3T3-CLIP1-LTK or Ba/F3-CLIP1-LTK), but LTK or No phosphorylated bands were detected in CLIP1-LTK-K1140M-expressing NIH3T3 cells or Ba/F3 cells despite the expression of these foreign proteins. These results confirmed that the CLIP1-LTK fusion protein was constitutively activated, ie phosphorylated, in the stably expressed cell lines.
  • the NIH3T3 cell line a mouse fibroblast cell line, is widely used as a model for oncogenic activity.
  • NIH3T3 cells expressing CLIP1 (NIH3T3-CLIP1) or NIH3T3 cells expressing LTK (NIH3T3-LTK) were contact inhibited and morphologically indistinguishable from NIH3T3 mock cells (cobblestone fibroblast).
  • NIH3T3 cells expressing CLIP1-LTK (NIH3T3-CLIP1-LTK) displayed a round morphology and lost contact inhibition (Fig. 7).
  • NIH3T3 cells expressing CLIP1-LTK-K1140M lost their ability to change morphology and proliferated in a density-dependent manner.
  • soft agar colony formation assays revealed that NIH3T3-CLIP1-LTK colonies were significantly larger after 2 weeks of growth compared to mock cell colonies, NIH3T3-CLIP1 colonies, or NIH3T3-LTK colonies.
  • NIH3T3-CLIP1-LTK colonies were also larger than NIH3T3 cells expressing EGFR-L858R, which is known to have oncogenic potential and served as a positive control.
  • NIH3T3 cells expressing CLIP1-LTK-K1140M lost the ability to induce anchorage-independent growth.
  • the present inventors further evaluated the oncogenic potential of the CLIP1-LTK fusion protein.
  • Ba/F3 are murine B progenitor cells that require IL-3 for proliferation and expression of oncogenic kinases renders them IL-3 independent.
  • Ba/F3-CLIP1-LTK a Ba/F3 cell transfected with the CLIP1-LTK fusion gene, the LTK protein was constitutively activated (phosphorylated) (Fig. 9), and even in the absence of IL-3. Survival and proliferation were allowed (Fig. 10).
  • Lorlatinib also inhibited anchorage-independent growth in soft agar colony formation assays ( Figure 12). Furthermore, in a cell viability assay using Ba/F3-CLIP1-LTK cells, lorlatinib exhibited a high 50% cytostatic concentration ( IC50 ) of 4.2 nM, and other tyrosine kinase inhibitors also had an IC50 of 20 nM. It showed a cell growth inhibitory effect as high as below (Fig. 13). In contrast, the EGFR tyrosine kinase inhibitor osimertinib was not as effective as the ALK inhibitor, with an IC 50 greater than 100 nM.
  • ALK inhibitor is effective as a substance that suppresses the activity of the LTK fusion protein of the present invention.
  • LTK fusion gene as a therapeutic target>
  • Patient 1 an NSCLC patient, is a 44-year-old Asian female with a history of light smoking and stage IV lung adenocarcinoma.
  • the patient received first-line therapy with 4 cycles of carboplatin, pemetrexed, and pembrolizumab, resulting in tumor regression.
  • CT computed tomography
  • PET positron emission tomography
  • FIG. 15 A tumor was shown (Fig. 15).
  • WTS and Sanger sequencing analysis of the patient's tumor sample detected the CLIP1-LTK fusion gene.
  • lorlatinib After consenting to treatment with LTK-targeted therapy, lorlatinib at the usual dose of 100 mg per day was started in the patient. Two-week and four-week follow-up imaging in this patient showed a dramatic reduction in tumor size at the primary site and all metastatic sites ( Figure 15). These results demonstrate hypersensitivity to lorlatinib in LTK kinase-dependent tumors. And it is suggested that LTK kinase inhibitors such as lorlatinib show high therapeutic efficacy in NSCLC patients with LTK fusion gene such as CLIP1-LTK fusion gene.
  • LTK fusion gene as a therapeutic target>
  • NSCLC patient 2 months and 5 months after initiation of the usual dose of lorlatinib at 100 mg per day, follow-up CT images showed rapid and dramatic tumor progression in the primary tumor and multiple metastatic tumors. It showed a reduction in size (Fig. 18).
  • all primary and metastatic tumors at baseline responded dramatically to lorlatinib treatment, as shown by whole-body positron emission tomography (PET) imaging (FIG. 19).
  • PET whole-body positron emission tomography
  • FISH fluorescence in situ hybridization
  • the probe showed an orange (5′)-green (3′) fusion signal (FIG. 20, left panel), whereas tumor cells had only one fusion signal and a kinase domain.
  • a single green (3') signal was shown (Fig. 20, right panel). This result indicates the presence of LTK gene rearrangement in the tumor. Loss of the orange signal (5') in tumor cells suggests deletion of this chromosomal region.
  • the present inventors focused on secondary ALK gene mutations that occur during lorlatinib treatment for the ALK fusion gene, and LTK mutations homologous to them was tested for its effect on lorlatinib susceptibility.
  • LTK gene mutations especially the L650F mutation, could contribute to resistance to lorlatinib therapy targeting the LTK fusion gene.
  • Gilteritinib is a molecular targeted drug approved for FLT3 gene mutation-positive acute myeloid leukemia and has LTK inhibitory activity.
  • NIH3T3 cells transfected with CLIP1-LTK were subcutaneously injected into nude mice, and lorlatinib (10 mg/kg, once daily), gilteritinib (30 mg/kg , once daily) or vehicle control for 2 weeks. Similar to the cell viability assay results, gilteritinib treatment significantly inhibited tumor growth, whereas lorlatinib failed to reduce tumor size (FIG. 22C).

Abstract

La présente invention concerne un nouveau gène pilote de cancer servant de nouvelle cible thérapeutique pour le cancer. La présente invention concerne en outre : une méthode permettant d'identifier, sur la base du nouveau gène pilote de cancer, soit des patients cancéreux soit des sujets présentant un risque de cancer, pour lesquels un effet thérapeutique sera produit grâce à médicament qui cible soit des gènes ayant une mutation soit une protéine codée par ceux-ci; une méthode de traitement et un médicament de traitement du cancer qui sont appropriés pour de tels patients cancéreux; une méthode de criblage pour des médicaments de traitement du cancer, et analogues.
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