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WO2019057852A1 - Utilisation de kap1 en tant que biomarqueur pour la détection ou la surveillance de l'inhibition d'atr chez un sujet - Google Patents

Utilisation de kap1 en tant que biomarqueur pour la détection ou la surveillance de l'inhibition d'atr chez un sujet Download PDF

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WO2019057852A1
WO2019057852A1 PCT/EP2018/075535 EP2018075535W WO2019057852A1 WO 2019057852 A1 WO2019057852 A1 WO 2019057852A1 EP 2018075535 W EP2018075535 W EP 2018075535W WO 2019057852 A1 WO2019057852 A1 WO 2019057852A1
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Prior art keywords
sample
subject
protein
pkap1
kinase inhibitor
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Sven Golfier
Antje Margret Wengner
Anna BEHNKE
Sabine SCHUBERT
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Bayer AG
Bayer Pharma AG
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Bayer AG
Bayer Pharma AG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation

Definitions

  • the present invention covers a method for detecting or monitoring ATR inhibition in a subject, the method comprising quantifying the amount of pKAP1 protein phosphorylated at serine 824 in a sample from said subject. It also covers the use of pKAP1 protein phosphorylated at serine 824 as a biomarker for detecting or monitoring ATR inhibition in a subject, the use comprising quantifying the amount of pKAP1 protein phosphorylated at serine 824 in a sample obtained from said subject.
  • ATM inhibitors For ATM inhibitors a number of potential biomarkers have been identified to serve this purpose, such as for example phosphorylation of the ATM downstream targets p53, KAP1 , SMC1 , CHK2 and H2AX (Weber and Ryan, ATM and ATR as therapeutic targets in cancer, Pharmacology & Therapeutics 149 (2015), 124-138).
  • these markers are thought to be not specific measures of ATM kinase activity as they may be also targets of other kinases, such as for example ATR kinase.
  • KAP1 as a downstream target of ATM kinase.
  • CHK1 phosphorylation is the most widely used preclinical biomarker.
  • ATM can also phosphorylate CHK1 albeit to a lesser extent and a recent study in ovarian cancer has suggested that CHK1 phosphorylation status may not be a reliable marker for inhibition of the ATR-CHK1 pathway.
  • ATR inhibitor activity could include increased DNA replication stress markers such as phosphorylation of Histone H2AX (gammaH2AX), but this marker is unlikely to be specific for ATR inhibition because it has been shown that H2AX is a common target for other kinases such as ATM kinase or DNA-PKcs (Weber and Ryan, ATM and ATR as therapeutic targets in cancer, Pharmacology & Therapeutics 149 (2015), 124-138).
  • DNA replication stress markers such as phosphorylation of Histone H2AX (gammaH2AX)
  • gammaH2AX gammaH2AX
  • Hall et al. show an increase in gammaH2AX and pKAP1 induction in combination treatment of cisplatin with increasing ATR kinase inhibitor VX-970 concentrations in H2009 lung cancer cells (see Figure 1 b of Hall et al.).
  • VX-970 alone shows only weak induction of gammaH2AX and, according to Hall et al., led to mild induction of P-H2AX and P-KAP 1 .
  • Figure 1 b of Hall et al. does not show an ATR kinase inhibitor concentration- dependent induction of pKAP1 in H2009 lung cancer cells ( Figure 1 b of Hall et al.).
  • At least 4 out of 5 ATR kinase inhibitor concentrations tested by Hall et al. in a VX-970 monotherapy treatment show no induction of pKAP1 (see Figure 1 b, lanes 2 to 5), This is in line with the fact that VX-970 has only weak anti-tumor activity in monotherapy treatment in contrast to cisplatin monotherapy and cisplatin/VX-970 combination treatment (see Figure 4 of Hall et al.). Based upon the data of Hall et al.
  • pKAP1 as a potential biomarker for ATR inhibition in tumors or surrogate tissue samples after ATR kinase inhibitor treatment, in particular in context with ATR kinase inhibitor monotherapy.
  • Pires et al. (British Journal of Cancer (2012) 107, 291 -299) describe the cellular effects of pharmacological inhibition of ATR kinase with VE-821 in severe hypoxic conditions and its potential as a radiosensitiser.
  • VE-821 inhibits ATR-mediated signalling in response to replication arrest induced by severe hypoxia. Under these severe hypoxia conditions ( ⁇ 0.02% O2) , VE-821 induced DNA damage and increased ATM-mediated phosphorylation of H2AX and KAP1 .
  • Pires et al. (Figure 3A) show no induction of pKAP1 by VE-821 alone under non-hypoxic conditions.
  • KAP1 phosphorylation of KAP1 is considered to be ATM-dependent.
  • KAP1 phosphorylation is functionally associated with ATM kinase and no upregulated phosphorylation of Ser824 KAP1 is shown after treatment with ATR kinase inhibitor VE-821.
  • phosphorylation of Ser824 KAP1 can be used as a biomarker for detecting or monitoring the activity of a ATR kinase inhibitor on the ATR kinase target in ATR kinase monotherapy and/or in ATR kinase combination therapy.
  • the present invention covers a method of detecting or monitoring ATR inhibition in a subject, the method comprising
  • pKAP1 protein as used herein means human KAP1 -protein, which is phosphorylated at serine 824.
  • the present invention also covers a method of detecting or monitoring ATR inhibition in a subject, the method comprising
  • the method(s) of the present invention are performed outside the human body, they are in vitro method(s).
  • an elevation of the amount of pKAP1 protein measured in the second sample compared to the amount of pKAP1 protein measured in the first sample indicates that the ATR kinase inhibitor has bound to the ATR kinase target and/or that the ATR kinase target has been inhibited by the ATR kinase inhibitor or by the ATR kinase inhibitor treatment regimen in the subject.
  • the target engagement of an ATR kinase inhibitor to the ATR kinase target is detected or monitored.
  • the administration of the ATR kinase inhibitor to the subject is a ATR kinase inhibitor monotherapy treatment.
  • the second sample is from the same subject after single dose treatment with the ATR kinase inhibitor.
  • the second sample is from the same subject after or during multiple dose treatment with the ATR kinase inhibitor.
  • the subject is characterized by one or more deleterious mutation(s) of the ATM gene/protein.
  • the first and the second sample are surrogate tissue samples from the same surrogate tissue.
  • the surrogate tissue sample is a blood sample, a skin sample or a hair follicle sample, particulartly a hair follicle or skin sample.
  • the first and the second sample are blood samples from the subject enriched in circulating tumor cells.
  • the first and the second sample each is a blood sample, in which circulating tumor cells are enriched in the first and in the second blood sample, and the quantification of the amount of pKAP1 protein is performed in said circulating tumor cell enriched blood samples.
  • the first and the second sample are circulating tumor cells, particularly circulating tumor cells isolated from a blood sample, from said subject.
  • the first and the second sample each is a blood sample, from which circulating tumor cells are isolated in the first and in the second sample, and the quantification of the amount of pKAP1 protein is performed in said isolated circulating tumor cells.
  • the first and the second sample are tumor tissue samples from the same tumor tissue.
  • the first and the second sample is a tumor tissue sample and the first and the second sample are obtained from the same tumor tissue.
  • the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue.
  • the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue and/or each does not comprise moderate hypoxic tumor tissue.
  • the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each consists of non- hypoxic tumor tissue.
  • the first and the second sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.
  • the first and the second sample is a surrogate tissue sample from the same surrogate tissue and the second surrogate tissue sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.
  • the first and the second sample is a blood sample enriched in circulating tumor cells and the second blood sample enriched in circulating tumor cells was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.
  • the first and the second sample is a circulating tumor cell sample and the second circulating tumor cell sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.
  • the first and the second sample is a tumor tissue sample from the same tumor tissue and the second tumor tissue sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.
  • the amount of the pKAP1 protein is determined by using an antibody specific for pKAP1 protein, preferably an antibody specific for pKAP1 , which is phosphorylated at serine 824.
  • the antibody specific for pKAP1 protein is selected from Phospho-TIF1 ⁇ (Ser824) Antibody and Phospho-TRIM28 (Ser824) Antibody.
  • Phospho-TIF1 ⁇ (Ser824) Antibody refers to a polyclonal antibody specific for the detection of Ser824 phosphorylation of KAP1 protein, which can be purchased from Cell Signaling Technology, USA, Product No. 4127.
  • Phospho-TRIM28 (Ser824) Antibody refers to a recombinant rabbit monoclonal antibody specific for the detection of Ser824 phosphorylation of KAP1 protein, which can be purchased for example from ThermoFisher Scientific, USA, Product No. 702084.
  • Collaborative Enzyme Enhanced Reactive-immunoassay is a method known to the person skilled in the art, which is for example described in Kim et al., Proteome Sci. 201 1 ; 9: 75.
  • PDA Proximity extension assay
  • PKA proximity ligation assay
  • the ATR kinase inhibitor is selected from VX-803, VX-970, AZD-6738 and 2-[(3R)-3-methylmorpholin-4-yl]-4-(1 -methyl- 1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine, preferably the ATR kinase inhibitor is 2- [(3R)-3-methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7- naphthyridine.
  • the second sample is from the same subject after the administration of the ATR kinase inhibitor to the subject and after the administration to the subject of one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper- proliferative disease.
  • the second sample is treated in vitro with a DNA damaging agent.
  • the DNA damaging agent is selected from ionizing radiation, UV radiation, 4-nitroquinoline, a platinating agent, an inhibitor of topoisomerase I, an inhibitor of topoisomerase II, an antimetabolite, an alkylating agent and a cytotoxic antibiotic.
  • the present invention covers the use of pKAP1 protein for detecting or monitoring ATR inhibition in a subject, the use comprising quantifying the amount of pKAP1 protein in a sample obtained from said subject.
  • the use comprises: a) quantifying the amount of pKAP1 protein in a first sample obtained from the subject prior to the administration of the ATR kinase inhibitor to the subject;
  • pKAP1 protein as used herein means human KAP1 -protein, which is phosphorylated at serine 824.
  • an elevation of the amount of pKAP1 protein measured in the second sample compared to the amount of pKAP1 protein measured in the first sample indicates that the ATR kinase inhibitor has bound to the ATR kinase target and/or that the ATR kinase target has been inhibited by the ATR kinase inhibitor or by the ATR kinase inhibitor treatment regimen in the subject.
  • the target engagement of an ATR kinase inhibitor to the ATR kinase target is detected or monitored.
  • the second sample was obtained from the same subject after single dose treatment with the ATR kinase inhibitor.
  • the second sample is obtained from the same subject after or during multiple dose treatment with the ATR kinase inhibitor.
  • the subject is characterized by one or more deleterious mutation(s) of the ATM gene/protein.
  • the second sample is obtained from the same subject after the administration of the ATR kinase inhibitor to the subject and after the administration of one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper-proliferative disease to the subject.
  • the second sample is obtained from a subject, which has received a combination therapy, which comprises a treatment of the subject with the ATR kinase inhibitor in combination with one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances.
  • the second sample is treated in vitro with a DNA damaging agent.
  • the DNA damaging agent is selected from ionizing radiation, UV radiation, 4-nitroquinoline, a platinating agent, an inhibitor of topoisomerase I, an inhibitor of topoisomerase II, an antimetabolite, an alkylating agent and a cytotoxic antibiotic.
  • the first and the second sample is a surrogate tissue sample and the first and the second sample are obtained from the same surrogate tissue.
  • the surrogate tissue sample is a blood sample, a skin sample or a hair follicle sample, particularly a skin sample or hair follicle sample.
  • the first and the second sample each is a blood sample, in which circulating tumor cells are enriched in the first and in the second blood sample, and the quantification of the amount of pKAP1 protein is performed in said circulating tumor cell enriched blood samples.
  • the first and the second sample each is a blood sample, from which circulating tumor cells are isolated in the first and in the second sample, and the quantification of the amount of pKAP1 protein is performed in said isolated circulating tumor cells.
  • the first and the second sample is a tumor tissue sample and the first and the second sample are obtained from the same tumor tissue.
  • the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue.
  • the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue and/or each does not comprise moderate hypoxic tumor tissue.
  • the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each consists of non-hypoxic tumor tissue.
  • the amount of the pKAP1 protein is determined by using an antibody specific for pKAP1 protein, preferably an antibody specific for pKAP1 , which is phosphorylated at serine 824.
  • the antibody specific for pKAP1 protein is selected from Phospho-TIF1 ⁇ (Ser824) Antibody and Phospho-TRIM28 (Ser824) Antibody.
  • the ATR kinase inhibitor is selected from VX-803, VX-970, AZD-6738 and 2-[(3R)-3-methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol- 5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine, preferably the ATR kinase inhibitor is 2-[(3R)-3- methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine.
  • the use is an in vitro use.
  • detecting means a singular measurement of a relevant parameter
  • monitoring refers to multiple measurements of a relevant parameter within a certain time period.
  • pKAP1 protein means human KAP1 -protein, which is phosphorylated at serine 824.
  • KAP1 protein which is also called “KRAB-associated protein 1 ", “E3 SUMO-protein ligase TRIM28” or "Transcription intermediary factor 1 -beta (TIF1 ⁇ )" means a human protein which amino acid sequence is described under SEQ ID NO: 1 .
  • KAP1 protein was initially identified as a universal transcriptional co-repressor because it interacts with a large KRAB-containing zinc finger protein (KRAB-ZFP) transcription factor family.
  • KRAB-ZFP zinc finger protein
  • KAP1 structure, post-translational modifications and interacting proteins are described in Figure 2 of Chun-Ting Cheng, Ching-Ying Kuo, David K Ann, KAPtain in charge of multiple missions: Emerging roles of KAP1 , World J Biol Chem. Aug 26, 2014; 5(3): 308-320.
  • KAP1 affects gene expression by regulating the transcription of KRAB-ZFP-specific loci, trans-repressing as a transcriptional co-repressor or epigenetically modulating chromatin structure.
  • KAP1 also functions independent of gene regulation by serving as a SUMO/ubiquitin E3 ligase or signaling scaffold protein to mediate signal transduction.
  • KAP1 is subjected to multiple post-translational modifications (PTMs), including serine/tyrosine phosphorylation, SUMOylation, and acetylation, which coordinately regulate KAP1 function and its protein abundance.
  • PTMs post-translational modifications
  • KAP1 is involved in multiple aspects of cellular activities, including DNA damage response, virus replication, cytokine production and stem cell pluripotency.
  • knockout of KAP1 results in embryonic lethality, indicating that KAP1 is crucial for embryonic development and possibly impacts a wide-range of (patho)physiological manifestations.
  • KAP1 -deficiency significantly impairs vital physiological processes, such as immune maturation, stress vulnerability, hepatic metabolism, gamete development and erythropoiesis (Cheng et al, World J Biol Chem. Aug 26, 2014; 5(3): 308-320).
  • treatment regimen means a treatment plan that specifies the dosage and the schedule of treatment.
  • ATR kinase inhibitor treatment regimen means a treatment plan that specifies the dosage and the schedule of treatment with an ATR kinase inhibitor, particularly in ATR kinase inhibitor monotherapy or in combination therapy with one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper- proliferative disease.
  • ATR kinase inhibitor monotherapy treatment means a treatment of the hyper- proliferative disease solely with an ATR kinase inhibitor but without the additional administration of one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper-proliferative disease to the subject.
  • ATR kinase inhibitor refers to any inhibitor of ATR kinase, particularly an inhibitor of ATR kinase selected from VX-803, VX-970, AZD-6738 and 2-[(3R)-3-methylmorpholin-4-yl]-4- (1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine (Compound A) as described infra.
  • VX-803 is understood as meaning 2-amino-6- fluoro-N-[5-fluoro-4-(4- ⁇ [4-(oxetan-3-yl)piperazin-1 -yl]carbonyl ⁇ piperidin-1 -yl)pyridin-3- yl]pyrazolo[1 ,5-a]pyrimidine-3-carboxamide.
  • VX-803 is of structure
  • VX-970 is understood as meaning 3-(3- ⁇ 4- [(methylamino)methyl]phenyl ⁇ -1 ,2-oxazol-5-yl)-5-[4-(propan-2-ylsulfonyl)phenyl]pyrazin-2- amine.
  • VX-970 is of structure
  • AZD-6738 is understood as meaning 4- ⁇ 4- [(3R)-3-methylmorpholin-4-yl]-6-[1 -(S-methylsulfonimidoyl)cyclopropyl]pyrimidin-2-yl ⁇ -1 H- pyrrolo[2,3-b]pyridine.
  • AZD-6738 is of structure
  • the ATR kinase inhibitor is 2-[(3R)-3- methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.
  • target engagement refers to the binding of a ligand, e.g. the ATR kinase inhibitor, to the target of interest, e.g. the ATR kinase, thereby inhibiting the target ' s activitiy, e.g. ATR kinase activity.
  • treating or “treatment” as used herein is used conventionally, for example the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of a disease or disorder, such as a carcinoma.
  • hyper-proliferative disease includes but is not limited to, for example: psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), as well as malignant neoplasia.
  • BPH benign prostate hyperplasia
  • malignant neoplasia include solid and hematological tumors. Solid tumors can be exemplified by tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, anum, endocrine glands (e.g.
  • malignant neoplasias include inherited cancers exemplified by Retinoblastoma and Wilms tumor. In addition, malignant neoplasias include primary tumors in said organs and corresponding secondary tumors in distant organs ("tumor metastases").
  • Hematological tumors can be exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non- Hodgkins disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, and cancers of unknown primary site as well as AIDS related malignancies.
  • breast cancer examples include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ, particularly with bone metastases.
  • cancers of the respiratory tract include, but are not limited to small-cell and non- small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.
  • brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor.
  • Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer.
  • Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.
  • Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
  • Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, urethral and human papillary renal cancers.
  • Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
  • liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
  • Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • Head-and-neck cancers include, but are not limited to laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oral cavity cancer and squamous cell.
  • Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.
  • Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
  • Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
  • subject means a human or an animal, such as for example mice, rat, rabbit, dog and monkey.
  • subject is a human, particularly a human having a hyper-proliferative disease.
  • sample refers to biological material obtained from the subject.
  • the sample assayed by the method/use of the present invention is not limited to any particular type.
  • Samples include, as non-limiting examples, single cells, circulating tumor cells, multiple cells, tissues, tumor tissue, biological fluids, biological molecules, or supernatants or extracts of any of the foregoing. Examples include tissue obtained for biopsy, tissue obtained during resection, blood, urine, skin tissue, hair follicles, lymph tissue, lymph fluid, cerebrospinal fluid, mucous, and stool samples.
  • the sample used will vary based on the assay format, the detection method and the nature of the tumors, tissues, cells or extracts to be assayed. Methods for preparing samples are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method/use of the present invention.
  • surrogate tissue sample means a sample obtained from a surrogate tissue.
  • surrogate tissue means any tissue, cell or body fluid of the subject having the hyper-proliferative disease, excluding the primary tumor tissue.
  • a surrogate tissue responds to the pharmacological intervention of the ATR kinase inhibitor treatment and reflects its impact on the subject ' s organism, especially on the primary site of the disease. Measurable effects in surrogate tissue can therefore indicate ATR inhibition, particularly they can indicate target engagement of the ATR kinase inhibitor to the ATR kinase target.
  • Surrogate tissue includes for example hair follicle tissue/cells, skin tissue/cells, peripheral blood mononuclear cells (PBMCs), blood, plasma, serum, lymph, urine, tears, synovial fluid, wound fluid and/or cerebrospinal fluid.
  • PBMCs peripheral blood mononuclear cells
  • Surrogate tissue samples may be gathered using a variety of methods known in the art, e.g., via a skin swab, hair pluck, skin puncture or intravenous bleed.
  • severe hypoxic tumor tissue means a tumor tissue which shows an oxygen concentration of equal or less than 0.02% 0 2 .
  • tumor tissue means a tumor tissue which shows an oxygen concentration in the range of 0.02% to 2,00% 0 2 .
  • non-hypoxic tumor tissue means a tumor tissue which shows an oxygen concentration of more than 2,00%, particularly an oxygen concentration in the range of 2% to 8%, preferably an oxygen concentration in the range of 2.5% to 5%.
  • the oxygen concentration of the first and the second tumor tissue sample can be determined by methods known to the person skilled in the art. Preferably, it is determined by using a two-channel fiberoptic oxygen- sensing device, such as for example Oxylite 2000 (Oxford Optronix, Oxford, UK), particularly it is determined by using the method described by Brurberg et al. (Int. J. Radiation Oncology Biol. Phys., Vol. 58, No. 2, (2004), pp. 403-409).
  • the oxygen concentration of the tumor tissue is determined in vivo to identify tumor tissue of the patient, which does not comprise severe hypoxic tumor tissue and/or which does not comprise moderate hypoxic tumor tissue.
  • the first and the second tumor tissue sample which each does not comprise severe hypoxic tumor tissue and/or which each does not comprise moderate hypoxic tumor tissue, is then isolated from the subject and is then further tested according to the method(s) of the present invention.
  • in vitro means that said method/use is performed in a controlled environment (e.g. test tube, reaction vessel) outside of a living subject (e.g. human, animal).
  • a controlled environment e.g. test tube, reaction vessel
  • a living subject e.g. human, animal
  • the term "deleterious mutation of the ATM gene/protein” as used herein means a mutation of the ATM gene which has a deleterious effect on the function of said gene or on the function of its corresponding RNA or its corresponding protein.
  • the deleterious mutation of the ATM gene may result in a reduced gene expression level of said gene, a reduced amount or a reduced activity of the ATM protein, or it may result in a nonfunctional ATM gene/protein ("loss-of-function") compared to the respective wildtype ATM gene/protein.
  • a deleterious mutation include but are not limited to the following:
  • the deleterious mutation can be a nonsense mutation, which is a point mutation in the ATM gene, resulting in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and nonfunctional ATM protein.
  • the deleterious mutation can be a missense mutation, which is a point mutation in the ATM gene, resulting in the production either of a nonfunctional ATM protein (complete loss of function) or in a ATM protein with partial loss of function compared to the respective wildtype ATM protein.
  • the deleterious mutation can also result in a frameshift mutation, which is a genetic mutation in the ATM gene caused by insertions or deletions of one or more nucleotides in such gene, wherein the number of nucleotides is not divisible by three, and resulting in a (sometimes truncated) nonfunctional ATM protein.
  • a frameshift mutation is a genetic mutation in the ATM gene caused by insertions or deletions of one or more nucleotides in such gene, wherein the number of nucleotides is not divisible by three, and resulting in a (sometimes truncated) nonfunctional ATM protein.
  • the deleterious mutation can also be a large rearrangement mutation, for example a deletion of one or more exons disrupting the reading frame or a critical functional domain of the ATM protein.
  • Another example for a large rearrangement mutation is a duplication of one or more nonterminal exons disrupting the reading frame or a critical functional domain of the ATM protein.
  • the deleterious mutation can also be a splice site mutation, which is a genetic mutation that inserts, deletes or changes a number of nucleotides in the specific site at which splicing takes place during the processing of precursor messenger RNA into mature messenger RNA.
  • Splice site consensus sequences that drive exon recognition are located at the very termini of introns. The deletion of the splicing site results in one or more introns remaining in mature mRNA thereby resulting in the production of a nonfunctional ATM protein.
  • the deleterious mutation can also be a copy number variant (CNV), particularly a decrease of the ATM gene copy number (e.g. a homozygous or heterozygous deletion) compared to the normal gene copy number of the ATM gene.
  • CNV copy number variant
  • the deleterious mutation(s) of the ATM gene/protein result(s) in the loss of the ATM protein in the sample.
  • IHC immunohistochemistry
  • Western Blot preferably by Western Blot as described in the Experimental Section, Example 4.
  • the term "antihyperproliferative, cytostatic or cytotoxic substances for treatment of cancers" as used herein includes, for example, the following active ingredients:
  • DNA damaging agent means any agent which can cause damage either directly or indirectly to the nucleotides in the genome, particularly the DNA damaging agent is selected from ionizing radiation, UV radiation, 4-nitroquinoline, a platinating agent, an inhibitor of topoisomerase I, an inhibitor of topoisomerase II, an antimetabolite, an alkylating agent and a cytotoxic antibiotic.
  • platinum-based antineoplastic drug which is used to treat hyper-proliferative a hyper-proliferative disease. It includes, for example, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, lobaplatin, triplatin tetranitrate and picoplatin.
  • the DNA damaging agent is a platinating agent, particularly cisplatin or carboplatin.
  • inhibitor of topoisomerase I covers any drug that inhibits topoisomerase I. It includes, for example, agents such as camptothecin, topotecan, irinotecan, SN38, rubitecan and belotecan.
  • inhibitor of topoisomerase II covers any drug that inhibits topoisomerase II. It includes, for example, agents such as etoposide, daunorubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin and teniposide.
  • antimetabolite covers any drug that inhibits the use of a metabolite and that can be used in the treatment of a hyper-proliferative disease. It includes, for example, purine antagonists or pyrimidine antagonists, such as methotrexate, pemetrexed, thioguanine, fludarabine, cladribine, 6-mercaptopurine, cytarabine, gemcitabine, 5-fluorouracil (5FU), aminopterin, raltitrexed, pentostatin, clofarabine, capecitabine, tegafur, carmofur, floxuridine, azacitidine and hydroxyurea.
  • purine antagonists or pyrimidine antagonists such as methotrexate, pemetrexed, thioguanine, fludarabine, cladribine, 6-mercaptopurine, cytarabine, gemcitabine, 5-fluorouracil (5FU), aminopterin, raltitrexed
  • alkylating agent covers any drug used in in the treatment of a hyper- proliferative disease that attaches an alkyl group to DNA. It includes, for example, agents such as nitrogen mustards, triazenes, alkyl sulphonates, procarbazines and aziridines, in particular cyclophosphamide, melphalan, chlorambucil, carmustine, dacarbazine, temozolomide, busulfan, mechlorethamine, ifosfamide, trofosfamide, prednimustine, bendamustine, uramustine, estramustine, carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, mannosulfan, treosulfan, triaziquone, triethylenemelamine, altretamine, mitobronitol.
  • agents such as nitrogen mustards, triazenes, alkyl sulphonates, proc
  • cytotoxic antibiotic covers any drug used in in the treatment of a hyper-proliferative disease that interrupts cell division.
  • the most important subgroup is the anthracyclines and the bleomycins; other examples include mitomycin C, plicamycin, mitoxantrone, and actinomycin.
  • the term also includes, for example, agents such as doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone.
  • the oral application volume was 10 ml/kg.
  • the time interval between two applications per day was 6-7h.
  • blood and tumors were sampled at 0.5h, 1 h, 3h, 8h, 24h, 48h and 72h post treatment. Matched samples were frozen and formalin fixed respectively and used for further analysis.
  • pKAP1 Frozen tumors were subjected to protein extraction and 12.5 ⁇ g protein were separated by capillar electrophoresis. Subsequently pKAP1 was detected by Phospho-TIF13 (Ser824) Antibody (Cell Signaling Technology, USA, Product No. 4127) and immunoprobed using an HRP-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is quantitated.
  • FFPE Formalin fixed and Paraffin embedded
  • Figure 4 shows an exemplary staining of untreated and treated tumors [24 hours after the last dose application (2QDx2 + QDx1 ) of 30 mg/kg Compound A] in mice.
  • Immunohistochemical analysis of tumor tissue is suitable for monitoring ATR activity after ATR inhibitor treatment.
  • ATR inhibitor/cisplatin combination treatment induces a strong phosphorylation of KAP1 at Ser824 and to a less extend at Ser473, as well of H2AX at Ser139.
  • ATM protein could be detected in NCI-H460 postive control cells (pos. Ctr.) as well in DLD1 parental cells. In contrast the lack of protein signal in DLD1 ATM -/- cells confirmed the loss of ATM protein. The detection of control protein GAPDH confirmed the protein loading of all samples ( Figure 6).
  • Example 5
  • FFPE Formalin fixed and Paraffin embedded 5 ⁇ tissue section of tumor samples were subjected to immunohistochemical analysis of Ser824 KAP1 phosphorylaton and compared to respective H2Ax Ser139 phosphorylation. Therefore sections were incubated for 1 hour at room temperature with Phospho-TIF1 ⁇ (Ser824) Antibody (Cell Signaling Technology, USA, Product No. 4127) or Phospho Histone H2AX (Millipore, USA, Product No. 05-636) at 1 ⁇ / ⁇ final concentration in TBS-T. Bound antibody was detected by rabbit EnVision System (Agilent K4010) and quantitated by automated image analysis (3DHistech; DensitoQuant).

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Abstract

La présente invention concerne un procédé de détection ou de surveillance de l'inhibition d'ATR chez un sujet, le procédé comprenant la quantification de la quantité de protéine pKAP1 phosphorylée à la sérine 824 dans un échantillon provenant dudit sujet. L'invention concerne en outre l'utilisation de la protéine pKAP1 phosphorylée à la sérine 824 en tant que biomarqueur pour détecter ou surveiller l'inhibition d'ATR chez un sujet, l'utilisation comprenant la quantification de la quantité de protéine pKAP1 phosphorylée à la sérine 824 dans un échantillon obtenu à partir dudit sujet.
PCT/EP2018/075535 2017-09-22 2018-09-20 Utilisation de kap1 en tant que biomarqueur pour la détection ou la surveillance de l'inhibition d'atr chez un sujet Ceased WO2019057852A1 (fr)

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