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WO2012052758A1 - Biomarqueurs de réponse pour des antagonistes de iap dans des cancers humains - Google Patents

Biomarqueurs de réponse pour des antagonistes de iap dans des cancers humains Download PDF

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Publication number
WO2012052758A1
WO2012052758A1 PCT/GB2011/052022 GB2011052022W WO2012052758A1 WO 2012052758 A1 WO2012052758 A1 WO 2012052758A1 GB 2011052022 W GB2011052022 W GB 2011052022W WO 2012052758 A1 WO2012052758 A1 WO 2012052758A1
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gene
resistant
mut
patient
mutant
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PCT/GB2011/052022
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Naomi Mae Laing
Steven Roels
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Astrazeneca Ab
Astrazeneca Uk Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention is based on identifying a link between gene status of tumor protein p53 (TP53) and susceptibility to treatment with an inhibitor of apoptosis protein (IAP) antagonist compound. This therefore provides opportunities, methods and tools for selecting patients for treatment with an IAP antagonist drug, particularly cancer patients, and/or avoiding treatment of patients less likely to respond therapeutically to the treatment thus avoiding unnecessary treatment and any side effects that may be associated with such ineffective treatment.
  • IAP apoptosis protein
  • the present invention relates to patient selection tools and methods (including personalised medicine).
  • the selection is based on whether the tumour cells to be treated possess wild-type or mutant TP53 genes.
  • the TP53 gene status can therefore be used as a biomarker of susceptibility to treatment with an IAP antagonist.
  • IAPs Inhibitor of apoptosis proteins
  • BIR baculo virus IAP repeat
  • IAPs A second motif found in the baculovirus IAP and some cellular IAPs is the really interesting new gene (RING) finger, a type of Zn-finger found in other proteins, which in the IAPs has E3-ubiquitin ligase activity.
  • the human genome encodes eight IAPs: cIAPl, cIAP2, XIAP, Ts-IAP, Livin, survivin, NAIP and Apollon or Bruce. (Hunter, A. M., E. C. LaCasse and R. G. Korneluk, 2007; The inhibitors of apoptosis (IAPs) as cancer targets, Apoptosis, 12:1543- 1568.)
  • XIAP has three BIR domains (BIR1, 2 and 3) and a RING finger. It can directly inhibit apoptosis through its ability to bind to the active form of several members of the caspase family of proapoptotic proteases.
  • the XIAP BIR3 domain binds to the N-terminus of activated caspase-9 preventing caspase-9 dimer formation, which is essential for activity.
  • Caspases-3 and -7 bind to the linker region between the BIR1 and 2 domains blocking the caspase active site.
  • cIAPl and 2 were initially identified by interaction with the type 2 tumor necrosis factor-a receptor complex [TNFR2] (Rothe, M. et al. 1995, The TNFR2-TRAF Signaling Complex Contains Two Novel Proteins Related to Baculoviral Inhibitor of Apoptosis
  • cIAPl Proteins, Cell, 83 : 1243-12512. Both cIAPl and 2 contain three BIR domains (BIR1, 2 and 3), a RING finger and a caspase recruitment domain (CARD). cIAPl binds to TRAF1/2 in the TNFR2 complex through its BIR1 domain (Samuel, T., K. Welsh, T. Lober, S. H. Togo, J. M. Zapata and J. C. Reed, 2006, Distinct BIR Domains of cIAPl Mediate Binding to and Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 2 and Second
  • c-IAPl and C-IAP2 are Critical Mediators of Tumor Necrosis Factor a (TNFa)-induced NF- ⁇ Activation, J. Biol. Chem., 283 : 24295-24299.).
  • cIAPl also acts to negatively regulate the non-canonical NF- ⁇ pathway by ubiquitination and subsequent proteosomal degradation of NIK.
  • cIAPl and 2 can bind to caspases in vitro, however, the affinity by which they bind does not appear to be physiologically relevant (Eckelman, B. P. and G. S. Salvesen, 2006, The Human Anti-apoptotic Proteins cIAPl and cIAP2 Bind but Do Not Inhibit Caspases, J. Biol. Chem. 281 : 3254-3260.).
  • SMAC second mitochondrial activator of caspases
  • SMAC binding to XIAP prevents XIAP from inhibiting caspases-3, -7 and -9 and thus can be proapoptotic.
  • SMAC binding to cIAPl and 2 leads to
  • cIAPl and 2 inhibits signaling downstream of the TNFR through the canonical NF-KB pathway.
  • caspase-8 activation through the formation of a complex between TRAD, RIPK1 and procaspase-8.
  • XIAP BIR2 and BIR3 domain regions binds to the N-terminal 4 amino acids of each of the two identical subunits of SMAC, Ala-Val-Pro-Ile (AVPI).
  • Biochemical studies have shown that AVPI and related peptides also bind to cIAPl, cIAP2 and Livin BIR domains.
  • Cell permeable, small molecule mimetics of AVPI (SMAC mimetics) that bind to XIAP, ML-IAP, cIAPl and cIAP2 have been made.
  • SMAC mimetics can be described as monomers, which mimic one AVPI protein motif, or dimers which mimic two adjacent AVPI protein motifs present in the mature SMAC homodimer.
  • SMAC mimetics are IAP inhibitors.
  • Monomeric SMAC mimetics are generally less potent at inducing apoptosis in cancer cells than are dimeric SMAC mimetics, since they bind to IAP proteins with reduced potency.
  • TNFa tumor necrosis factor alpha
  • Determination of the basal level of TNFa mRNA or protein in a tumor is a potential biomarker to identify some of the tumors that may respond to SMAC mimetics because they have high basal level of TNFa.
  • WO 2008/137930 (Tetralogic Pharmaceuticals Corp.) teaches the use of TNFalpha gene expression as a biomarker of sensitivity to IAP antagonists.
  • p53 (TP53) wild-type can be used as a biomarker for determining sensitivity to treatment with mTOR inhibitors in combination with a cytotoxic agent.
  • a biomarker can be described as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or
  • a biomarker is any identifiable and measurable indicator associated with a particular condition or disease where there is a correlation between the presence or level of the biomarker and some aspect of the condition or disease (including the presence of, the level or changing level of, the type of, the stage of, the susceptibility to the condition or disease, or the responsiveness to a drug used for treating the condition or disease).
  • the correlation may be qualitative, quantitative, or both qualitative and quantitative.
  • a biomarker is a compound, compound fragment or group of compounds. Such compounds may be any compounds found in or produced by an organism, including proteins (and peptides), nucleic acids and other compounds.
  • Biomarkers may have a predictive power, and as such may be used to predict or detect the presence, level, type or stage of particular conditions or diseases (including the presence or level of particular microorganisms or toxins), the susceptibility (including genetic susceptibility) to particular conditions or diseases, or the response to particular treatments (including drug treatments). It is thought that biomarkers will play an increasingly important role in the future of drug discovery and development, by improving the efficiency of research and development programs. Biomarkers can be used as diagnostic agents, monitors of disease progression, monitors of treatment and predictors of clinical outcome. For example, various biomarker research projects are attempting to identify markers of specific cancers and of specific cardiovascular and immunological diseases. It is believed that the development of new validated biomarkers will lead both to significant reductions in healthcare and drug development costs and to significant improvements in treatment for a wide variety of diseases and conditions.
  • a biomarker may be required.
  • the marker may be measurable in surrogate and tumour tissues. Ideally these markers will also correlate with efficacy and thus could ultimately be used for patient selection.
  • the technical problem underlying the present invention is the identification of means for stratification of patients for treatment with an IAP antagonist.
  • the technical problem is solved by provision of the embodiments characterized in the claims.
  • the invention provides a method of determining sensitivity of cells to an IAP antagonist.
  • the method is applicable to dimeric SMAC mimetics.
  • the method comprises determining the status of TP53 genes in said cells.
  • the cells are identified as likely to be sensitive to an IAP antagonist if the cells possess a mutated TP53 gene.
  • a cell is sensitive to an IAP antagonist if it undergoes apoptosis in response to the IAP antagonist.
  • Methods of the invention are useful for predicting which cells are more likely to respond to an IAP antagonist by undergoing apoptosis.
  • the present invention is further based, in part, on methods that can be used to determine a patient's responsiveness to an IAP antagonist including determining whether to administer an IAP antagonist.
  • the methods of the present invention include the determination of the gene status of TP53. The presence of a mutated TP53 gene indicating that the tumor cells are more likely to respond to cell killing when contacted with an IAP antagonist. The TP53 gene status can therefore be used to select patients for treatment with an IAP antagonist.
  • an in vitro method for the identification of a responder for or a patient sensitive to an IAP antagonist is disclosed. Also disclosed are uses of an oligo- or
  • polynucleotide primers or probes capable of detecting the mutation status of the TP53 gene is provided.
  • the invention pertains to an in vitro method for determining whether a patient suffering from cancer is likely to be a responder to a pharmaceutical treatment with an IAP antagonist, said method comprising the steps of: (i) obtaining a sample representative of the tumor that was previously collected from said patient; and, (ii) determining whether the TP53 genes contain a mutation in said sample.
  • a mutation in TP53 is indicative of increased likelihood of a response to the IAP antagonist.
  • identification of tumors that contain a TP53 mutation will enrich for response to an IAP antagonist.
  • a sample "representative of the tumor” can be the actual tumour sample isolated, or may be a sample that has been further processed, e.g. a sample of PCR amplified nucleic acid from the tumor sample.
  • Allele refers to a particular form of a genetic locus, distinguished from other forms by its particular nucleotide or amino acid sequence.
  • Amplification reactions are nucleic acid reactions which result in specific amplification of target nucleic acids over non-target nucleic acids.
  • the polymerase chain reaction (PCR) is a well known amplification reaction.
  • Cancer is used herein to refer to neoplastic growth arising from cellular
  • Gene is a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including a promoter, exons, introns, and other sequence elements which may be located within 5 ' or 3 ' flanking regions (not within the transcribed portions of the gene) that control expression.
  • Gene status refers to whether the gene is wild type or not (i.e. mutant).
  • Label refers to a composition capable of producing a detectable signal indicative of the presence of the target polynucleotide in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Non-synonymous variation refers to a variation (variance) in or overlapping the coding sequence of a gene that results in the production of a distinct (altered) polypeptide sequence. These variations may or may not affect protein function and include missense variants (resulting in substitution of one amino acid for another), nonsense variants (resulting in a truncated polypeptide due to generation of a premature stop codon) and insertion/deletion variants.
  • “Synonymous variation” refers to a variation (variance) in the coding sequence of a gene that does not affect sequence of the encoded polypeptide. These variations may affect protein function indirectly (for example by altering expression of the gene), but, in the absence of evidence to the contrary, are generally assumed to be innocuous.
  • Nucleic acid refers to single stranded or double stranded DNA and RNA molecules including natural nucleic acids found in nature and/or modified, artificial nucleic acids having modified backbones or bases, as are known in the art.
  • Primer refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied.
  • the length and sequence of the primer must be such that they are able to prime the synthesis of extension products.
  • a typical primer contains at least about 7 nucleotides in length of a sequence substantially complementary to the target sequence, but somewhat longer primers are preferred. Usually primers contain about 15-26 nucleotides, but longer or shorter primers may also be employed.
  • Polymorphic site is a position within a locus at which at least two alternative sequences are found in a population.
  • Polymorphism refers to the sequence variation observed in an individual at a polymorphic site. Polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function. In the absence of evidence of an effect on expression or protein function, common polymorphisms, including non-synonomous variants, are generally considered to be included in the definition of wild-type gene sequence.
  • NCBI NCBI (dbSNP: htt ://www.ncbi ,nlm.nih. gov/projects/SNP ).
  • Probe refers to single stranded sequence-specific oligonucleotides which have a sequence that is exactly complementary to the target sequence of the allele to be detected.
  • Response is defined by measurements taken according to Response Evaluation Criteria in Solid Tumours (RECIST) involving the classification of patients into two main groups: those that show a partial response or stable disease and those that show signs of progressive disease.
  • “Stringent hybridisation conditions” refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10%> dextran sulphate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
  • “Survival” encompasses a patients' overall survival and progression-free survival.
  • OS Overall survival
  • progression-free survival is defined as the time from the initiation of drug administration to first appearance of progressive disease or death from any cause.
  • a method for selecting a patient for treatment with an IAP antagonist comprising providing a tumour cell containing sample from a patient; determining whether the TP53 gene in the patient's tumour cell containing sample are wild type or mutant; and selecting a patient for treatment with an IAP antagonist based thereon.
  • the patient is selected for treatment with an IAP antagonist.
  • the TP53 gene in the patient's tumour cell containing sample is mutant the patient is selected for monotherapy treatment with an IAP antagonist.
  • the TP53 gene in the patient's tumour cell containing sample is mutant the patient is selected for combination treatment with an IAP antagonist and another therapeutic agent.
  • the other therapeutic agent is a small molecule compound.
  • the method may include or exclude the actual patient sample isolation step.
  • a method for selecting a patient for treatment with an IAP antagonist comprising determining whether the TP53 gene in a tumour cell containing sample previously isolated from the patient are wild type or mutant; and selecting a patient for treatment with an IAP antagonist based thereon.
  • the patient is selected for treatment with the IAP antagonist if the tumour cell has a mutant TP53 gene.
  • An IAP antagonist for use in the invention is any molecule which binds to and inhibits the activity of one or more IAPs, such as cellular IAP (cIAP, e.g., cIAP-1 or cIAP-2) or X- linked IAP (XIAP).
  • IAP antagonist binds to cIAP-1 and cIAP-2 with greater affinity than it binds to XIAP.
  • the IAP antagonist binds cIAP-1 with at least 3-fold greater affinity than to XIAP, and in others, the IAP antagonist binds cIAP-1 with at least 100-fold greater affinity than XIAP.
  • the IAP antagonist binds XIAP with greater affinity than cIAP-1 or cIAP-2.
  • the IAP antagonist can include, for example, a peptide, an antibody, an antisense molecule or a small molecule. Flygare and Fairbrother (Expert Opinion Therapeutic Patents. 20(2):251-267, 2010), incorporated herin by reference, provide a patent review of small-molecule pan-IAP antagonists compounds, and any of the molecules mentioned therein may be used in the present invention. Small molecule compounds are particularly appropriate for use in this invention and the invention can be applied to select patients for treatment with small molecule IAP antagonists for monotherapy treatment or in combination, such as with another small molecule agent.
  • Other IAP antagonists useful in the present invention include but are not limited to, those described or claimed in the following publications the entire disclosures of which are incorporated by reference herein
  • IAP antagonist compound for use in this invention is (2S,2'S)-N,N'- ⁇ hexa-2,4-diyne- 1 ,6-diylbis[oxy(l S,2R)-2,3-dihydro- lH-indene-2, 1 -diyl] ⁇ bis ⁇ 1 - [(2S)-2- cyclohexyl-2- ⁇ [(2S)-2-(methylamino)propanoyl]amino ⁇ acetyl]pyrrolidine-2-carboxamide ⁇ , which is disclosed in Example 1 of WO 2010/142994 and USSN 12/796089 and has the following structure:
  • Rl and R2 are independently H or C(l-6)alkyl
  • R3 is H or a C(3-8)cycloalkyl
  • R4 is -OC(3-10)alkylO-, -OC(3-10)alkenylO- or -OC(3-10)alkynylO-,
  • R5 is H or C(3-8)cycloalkyl
  • R6 and R7 are independently H or C(l-6)alkyl; or
  • the compounds of Figure 1 can be considered dimeric.
  • the dimeric compounds described therein can be homodimers or heterodimers.
  • heterodimer describe dimers that contain two identical subunits or two different subunits, respectively.
  • the two subunits are linked by a linker moiety, i.e. R4, wherein the linker moiety is covalently bonded to each of the subunits at the indicated position.
  • Rl, R2 and R3 are the same as R6, R7 and R5, respectively, with the linker being R4.
  • one or more Rl, R2 and R3 are different than R6, R7 and R5, respectively.
  • one, two or all of Rl , R2 and R3 can be different than R6, R7 and R5, respectively.
  • IAP antagonist compounds useful in the present invention include: N1,N4- bis((3S,5S)-l-((S)-3,3-dimethyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-((R)- l,2,3,4-tetrahydronaphthalen-l-ylcarbamoyl)pyrrolidin-3-yl)terephthalamide (Aegera) - compound 3 from US7579320; (S)-l-((S)-2-cyclohexyl-2-((S)-2-
  • the IAP antagonist is selected from: (2S,2'S)-N,N'- ⁇ hexa- 2,4-diyne- 1 ,6-diylbis[oxy(l S,2R)-2,3-dihydro-lH-indene-2, 1 -diyl] ⁇ bis ⁇ 1 -[(2S)-2-cyclohexyl- 2- ⁇ [(2S)-2-(methylamino)propanoyl]amino ⁇ acetyl]pyrrolidine-2-carboxamide ⁇ ; (S,S,2S,2'S)- N,N * -((1 S, rS,2R,2'R)-2,2'-(Hexane- 1 ,6-diylbis(oxy))bis(2,3-dihydro-lH-indene-2, 1 - diyl))bis(l-((S)-2-cyclohexyl-2-((S)-2-((S)
  • the IAP antagonist is a dimer, i.e. it is a SMAC mimetic which mimics two adjacent AVPI protein motifs present in the mature SMAC homodimer.
  • a method for predicting a patient's responsiveness to an IAP antagonist comprising determining whether the TP53 gene in the patient's tumour cells is wild type or mutant and based thereon, predicting a patient's responsiveness to treatment with an IAP antagonist.
  • a method for determining the likelihood of effectiveness of an IAP antagonist treatment in a human patient affected with cancer comprising: determining whether the TP53 gene in the patient's tumour cells is wild type or mutant and based thereon, predicting a patient's responsiveness to treatment with an IAP antagonist. In a particular embodiment, if the patient's tumour cells comprise mutant TP53 the patient has an increased likelihood of effective treatment with ab IAP antagonist. In another embodiment, if the patient's tumour cells comprise mutant TP53 the patient is subsequently prescribed and/or treated with an IAP antagonist.
  • a gene status of wild-type is meant to indicate normal or appropriate expression of the gene and normal function of the encoded protein.
  • mutant status is meant to indicate abnormal or inappropriate gene expression, or expression of a protein with altered function, consistent with the known roles of mutant TP53 in cancer (see below). Any number of genetic or epigenetic alterations, including but not limited to mutation, amplification, deletion, genomic rearrangement, or changes in
  • methylation profile may result in a mutant status.
  • the gene status is regarded as wild-type.
  • variants that typically would not result in a functional mutant gene status include synonomous coding variants and common polymorphisms (synonymous or non-synonymous).
  • gene status can be assessed by a functional assay, or it may be inferred from the nature of detected deviations from a reference sequence.
  • the wild-type or mutant status of the two genes is determined by the presence or absence of non-synonymous nucleic acid variations in the genes. Observed non-synonymous variations corresponding to known common polymorphisms with no annotated functional effects do not contribute to a gene status of mutant.
  • TP53 gene that signify mutant status
  • splice site variations that decrease recognition of an intron/exon junction during processing of pre- mR A to mR A. This can result in exon skipping or the inclusion of normally intronic sequence in spliced mRNA (intron retention or utilization of cryptic splice junctions). This can, in turn, result in the production of aberrant protein with insertions and/or deletions relative to the normal protein.
  • the gene has a mutant status if there is a variant that alters splice site recognition sequence at an intron/exon junction.
  • tumour suppressor protein p53 was first identified as a tumour suppressor (Levine et al, 1990, Tumor suppressor genes: The p53 and Rb sensitivity genes and gene products, Biochemica Biophysica Acta 1032: 119-136).
  • the TP53 gene contains 11 exons, is located in chromosome 17p 13.1 and the coded protein is approximately 53 kDa in size, containing some 393 amino acids.
  • the p53 protein consists of an acidic N-terminus with a transactivation domain, a hydrophobic central DNA-binding core and a basic C-terminus with regulatory and oligomerisation domains (Joerger and Fersht, Advances in Cancer Research 97: 1-23, 2008).
  • p53 is not functional or functions incorrectly in most human cancers, and that it plays a crucial role in the prevention of tumour development (Joerger and Fersht, 2008, supra).
  • the importance of a functional p53 protein is emphasized by the fact that p53 -deficient mice show a very high incidence of multiple, spontaneous tumours at an early age (Donehower and Lozano, Nature Reviews in Cancer 9:831-841, 2009).
  • the key functions of p53 are cell cycle arrest, DNA repair and triggering of programmed cell death. Because these processes ensure genomic integrity or destroy the damaged cell, p53 has been called the "guardian of the genome" (Vousden and Lane, Nature Reviews in Molecular and Cellular Biology 8:275-283, 2007).
  • the p53-induced activation of target genes may result in the induction of growth arrest, enabling the repair of damaged DNA.
  • programmed cell death which is often referred to as apoptosis according to its morphological appearance, the cells damaged beyond repair are eliminated thus preventing the fixation of DNA damage as mutations.
  • p53 can be inactivated indirectly through binding to viral proteins, as a result of alterations in the mdm.2 or pl9 ARV genes or by localization of the p53 protein to the cytoplasm (Joerger and Fersht, 2008, supra).
  • the most common aberration of p53 in human cancers is, however, mutation of the TP53 gene.
  • Databases devoted to cataloging and characterizing TP53 mutations include the IARC TP53 Database (http://www-p53.iarc.fr/) and the UMD TP53 Mutation Database (http://p53.free.fr/Database/p53_database.html).
  • TP53 most, but not all, of the mutations in the TP53 gene occur in the exons 4-9, the coding region for the DNA-binding central domain of the protein.
  • a large proportion of all mutations in TP53 are single base substitutions and are located in the DNA-binding part of the protein, modifying the ability of the protein to activate target genes that mediate growth arrest and apoptosis after stressors such as DNA damage (Reviewed in Joerger and Fersht, 2008, supra).
  • reference sequences are available for the gene (GenBank accession number: NG 017013), predominant mRNA (GenBank accession number: NM 000546), and primary protein isoform (GenBank accession number: NP 000537 or Swiss-Prot accession number: P04637).
  • the reference gene (genomic region) sequence includes 5000 bases of upstream sequence and 2000 bases of downstream sequence.
  • wild type TP53 gene, mRNA, and protein sequence are as disclosed in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • sequences with SEQ ID NOs 1-3 correspond to GenBank records with compound accession numbers NG_017013.1, NM_000546.4, NP_000537.3, respectively.
  • TP53 SEQ ID NO: 1 and SEQ ID NO: 2 are each a representative sequence. In normal individuals there are two copies of each gene, a maternal and paternal copy, which will likely have some sequence differences, moreover within a population there will exist numerous allelic variants of the gene sequence.
  • Other sequences regarded as wild type include those that possess one or more synonymous changes to the nucleic acid sequence, which changes do not alter the encoded protein sequence, non-synonymous common polymorphisms, which alter the protein sequence but do not affect protein function, and intronic non-splice-site sequence changes.
  • a method for determining the likelihood of effectiveness of an IAP antagonist treatment in a human patient affected with cancer comprising: detecting the presence or absence of at least one non- synonymous nucleic acid variance in the TP53 gene of said patient relative to the wild type genes, wherein the presence of the at least one non-synonymous nucleic acid variance in the TP53 gene indicates that the IAP antagonist treatment is likely to be effective.
  • a method for assessing the susceptibility of an individual to treatment with an IAP antagonist which method comprises:
  • the TP53 gene can be mutated in numerous different ways (including, substitutions, additions, deletions, splice variations etc.).
  • the IARC TP53 Database http://www-p53.iarc.fr and UMD TP53 Mutation Database
  • the gene status of TP53 can be determined by determination of the nucleic acid sequence. This could be via direct sequencing of the full-length gene or analysis of specific sites within the gene, e.g. commonly mutated sites.
  • TP53 gene is wild type or mutant.
  • Functional mutation of the TP53 gene is classified based on changes in one or both of the following functions of the p53 protein: 1. Decreased ability to bind to the p53 response element and cause transactivation of p53 -dependent genes when compared to wild type p53 (Kakudo Y, Shibata H, Otsuka K, Kato S and Ishioka C, Lack of Correlation between p53-dependent transcriptional activity and the ability to induce apoptosis among 179 mutant p53s. Cancer Research 65: 2108-2114); 2. Decreased ability to cause the induction of apoptosis when the protein is overexpressed in a cell that lacks endogenous p53 when compared to wild type p53 (Kakudo etal, above).
  • the patient's sample to be tested for the TP53 gene status can be any tumour tissue or tumour-cell containing sample obtained or obtainable from the individual.
  • the test sample is conveniently a sample of blood, mouth swab, biopsy, or other body fluid or tissue obtained from an individual.
  • Particular examples include: circulating tumor cells, circulating DNA in the plasma or serum, cells isolated from the ascites fluid of ovarian cancer patients, lung sputum for patients with tumours within the lung, a fine needle aspirate from a breast cancer patient, urine, peripheral blood, a cell scraping, a hair follicle, a skin punch or a buccal sample.
  • the test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. polymerase chain reaction (PCR), before analysis.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA.
  • the RNA is whole cell RNA and is used directly as the template for labelling a first strand cDNA using random primers or poly A primers.
  • the nucleic acid or protein in the test sample may be extracted from the sample according to standard methodologies (Sambrook et al. "Molecular Cloning- A Laboratory manual", second edition. Cold Spring Harbor, NY (1989)).
  • the diagnostic methods of the invention can be undertaken using a sample previously taken from the individual or patient. Such samples may be preserved by freezing or fixed and embeded in formalin-paraffin or other media. Alternatively, a fresh tumour cell containing sample may be obtained and used.
  • the tumour can be a non-solid tumour such as leukaemia, multiple myeloma or lymphoma, or can be a solid tumour, for example bile duct, bone, bladder, brain/CNS, breast, ovary, small bowel, colorectal, cervical, endometrial, gastric, head and neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural/peritoneal membranes, prostate, renal, skin, testicular, thyroid, uterine, vulval tumours and other tumour types such as melanoma, and sarcomas including fibrosarcoma, mesothelioma, and osteosarcoma, and endocrine tumours such as islet cell tumours and thyroid tumours.
  • a non-solid tumour such as leukaemia, multiple myeloma or lymphoma
  • a solid tumour for example bile duct, bone, bladder, brain/CN
  • TP53 mutations in TP53 are found broadly in clinical tumours, but the prevalence of mutations in each gene varies significantly by tumour tissue type. For example, TP53 mutations are very common in ovarian and lung tumours, but observed less frequently in breast tumours or haematopoietic malignancies
  • TP53 Prevalence of TP53 mutations in clinical samples.
  • Source for TP53 information is the IARC TP53 database (release R15, November 2010) using the data based on full gene scans (all coding exons) only and filtering to tissues represented by at least 100 samples.
  • TP53 mutations also varies between clinical or molecular subtypes of disease. For example, TP53 mutations are prevalent (80-97%) in the high- grade serous subtype of ovarian cancer, but the mutation profile is very different in other subtypes like clear cell carcinoma (see Kuo, KT et al, Am J Pathol. ;174(5): 1597-601, May 2009; Ahmed AA et al, J Pathology, 221 :49-56, 2010).
  • High-grade serous ovarian cancer subtype is therefore naturally enriched for TP53 mutation and thus, whilst it would be possible to perform the patient selection methods of the invention in such a population, it may be desirable to forego the patient selection method and treat any or all high-grade serous ovarian cancer patients with an IAP antagonist without utilising the patient selection method.
  • the patient selection methods of the invention may be particularly useful in the disease (tissue) segments where there is low prevalence of TP53 mutations (e.g. breast, skin, prostate, haematiopoietic, kidney etc.).
  • TP53 mutations e.g. breast, skin, prostate, haematiopoietic, kidney etc.
  • mutant TP53 nucleic acids nucleic acids can be employed, in the context of the present invention, to predict the response to drug treatment. Since mutations in these genes generally occur at the DNA level, the methods of the invention can be based on detection of mutations or variances in genomic DNA, as well as transcripts and proteins themselves. It can be desirable to confirm mutations in genomic DNA by analysis of transcripts and/or polypeptides, in order to ensure that the detected mutation is indeed expressed in the subject.
  • TGGE Temperature gradient gel electrophoresis List 1 Examples of mutation detection techniques include:
  • Solid phase hybridisation Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays such as the p53 AmplichipTM (DNA Chips).
  • Fluorescence Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998 (Zeneca Limited)
  • determining the presence or absence of a particular variance or plurality of variances in the TP53 gene in a patient with cancer can be performed in a variety of ways. Such tests are commonly performed using DNA or RNA collected from biological samples, e.g., tissue biopsies, urine, stool, sputum, blood, cells, tissue scrapings, breast aspirates or other cellular materials, and can be performed by a variety of methods including, but not limited to, PCR, hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatches, mass spectrometry or DNA sequencing, including minisequencing. Suitable mutation detection techniques include ARMSTM, ALEXTM, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.
  • the method employed for determining the nucleotide(s) within a biomarker gene is selected from: allele-specific amplification (allele specific PCR) - such as amplification refractory mutation system (ARMS), sequencing, allelic discrimination assay, hybridisation, restriction fragment length polymorphism or oligonucleotide ligation assay.
  • allele-specific amplification allele specific PCR
  • ARMS amplification refractory mutation system
  • sequencing allelic discrimination assay
  • hybridisation restriction fragment length polymorphism
  • restriction fragment length polymorphism or oligonucleotide ligation assay.
  • hybridization with allele specific probes can be conducted by: (1) allele specific oligonucleotides bound to a solid phase (e.g. glass, silicon, nylon membranes) with the labelled sample in solution, for example as in many DNA chip applications; or, (2) bound sample (often cloned DNA or PCR amplified DNA) and labelled oligonucleotides in solution (either allele specific or short so as to allow sequencing by hybridization). Diagnostic tests may involve a panel of variances, often on a solid support, which enables the simultaneous determination of more than one variance.
  • a solid phase e.g. glass, silicon, nylon membranes
  • Diagnostic tests may involve a panel of variances, often on a solid support, which enables the simultaneous determination of more than one variance.
  • hybridization probes are well known in the art (see, e.g., Sambrook et al, Eds., (most recent edition), Molecular Cloning: A Laboratory Manual, (third edition, 2001), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and may span two or more variance sites.
  • the detection of the presence or absence of at least one mutation provides for contacting TP53 nucleic acid containing a putative mutation site with at least one nucleic acid probe.
  • the probe preferentially hybridizes with a nucleic acid sequence including a variance site and containing complementary nucleotide bases at the variance site under selective hybridization conditions.
  • Hybridization can be detected with a detectable label using labels known to one skilled in the art. Such labels include, but are not limited to radioactive, fluorescent, dye, and enzymatic labels.
  • the detection of the presence or absence of at least one mutation provides for contacting TP53 nucleic acid containing a putative mutation site with at least one nucleic acid primer.
  • the primer preferentially hybridizes with a nucleic acid sequence including a variance site and containing complementary nucleotide bases at the variance site under selective hybridization conditions.
  • Oligonucleotides used as primers for specific amplification may carry the
  • the detection of the presence or absence of at least one mutation comprises sequencing at least one nucleic acid sequence and comparing the obtained sequence with the known wild type nucleic acid sequence.
  • the presence or absence of at least one mutation comprises mass spectrometric determination of at least one nucleic acid sequence.
  • the detection of the presence or absence of at least one nucleic acid variance comprises performing a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the target nucleic acid sequence containing the hypothetical variance is amplified and the nucleotide sequence of the amplified nucleic acid is determined.
  • Determining the nucleotide sequence of the amplified nucleic acid comprises sequencing at least one nucleic acid segment.
  • amplification products can be analyzed using any method capable of separating the amplification products according to their size, including automated and manual gel electrophoresis, and the like.
  • Mutations in genomic nucleic acid are advantageously detected by techniques based on mobility shift in amplified nucleic acid fragments. For instance, Chen et al., Anal Biochem 1996 Jul 1 5;239(l):61-9, describe the detection of single-base mutations by a competitive mobility shift assay. Moreover, assays based on the technique of Marcelino et al.,
  • capillary heteroduplex analysis may be used to detect the presence of mutations based on mobility shift of duplex nucleic acids in capillary systems as a result of the presence of mismatches.
  • nucleic acids for analysis from samples generally requires nucleic acid amplification.
  • Many amplification methods rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self-sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned.
  • the amplification according to the invention is an exponential amplification, as exhibited by for example the polymerase chain reaction.
  • bacteriophage replicase transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based
  • NASBA NASBA
  • Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
  • PCR Polymerase Chain Reaction
  • PCR is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.
  • PCR consists of repeated cycles of DNA polymerase generated primer extension reactions.
  • the target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridised. These oligonucleotides become primers for use with DNA polymerase.
  • the DNA is copied by primer extension to make a second copy of both strands. By repeating the cycle of heat denaturation, primer hybridisation and extension, the target DNA can be amplified a million fold or more in about two to four hours.
  • PCR is a molecular biology tool, which must be used in conjunction with a detection technique to determine the results of amplification.
  • An advantage of PCR is that it increases sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in approximately 4 hours.
  • PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., Gynaecologic Oncology, 52: 247-252, 1994).
  • Self- Sustained Sequence Replication is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874). Enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation. RNase H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent 6 9 additions. Following this process, amplifications of 10 to 10 have been achieved in one hour at42°C.
  • Ligation Amplification (LAR/LAS) reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridise to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated.
  • RNA replicase for the bacteriophage QP which replicates single- stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6: 1197.
  • the target DNA is hybridised to a primer including a T7 promoter and a QP 5' sequence region.
  • reverse transcriptase produces a cDNA connecting the primer to its 5' end in the process.
  • the heteroduplex product is denatured by heating and then a a second primer containing a QP 3' sequence region is used to initiate a second round of cDNA synthesis.
  • This resulting product is a double stranded DNA containing both 5' and 3' ends of the QP bacteriophage as well as an active T7 RNA polymerase binding site.
  • T7 RNA polymerase then transcribes the double-stranded DNA into new RNA, which mimics the QP. After washing to remove any unhybridised probe, the new RNA is eluted from the target and replicated by QP replicase. The latter reaction creates 10 7 fold amplification in approximately 20 minutes.
  • SDA strand displacement amplification
  • Chemical mismatch cleavage is based on the recognition and cleavage of DNA mismatched base pairs by a combination of hydroxylamine, osmium tetroxide and piperidine.
  • CMC Chemical mismatch cleavage
  • both reference DNA and mutant DNA are amplified with fluorescent labelled primers.
  • the amplicons are hybridised and then subjected to cleavage using Osmium tetroxjde, which binds to a mismatched T base, or Hydroxylamine, which binds to mismatched C base, followed by Piperidine which cleaves at the site of a modified base. Cleaved fragments are then detected by electrophoresis.
  • RFLPs restriction fragment polymorphisms
  • PIRA-PCR primer-induced restriction analysis PCR
  • An allele specific amplification technique such as Amplification Refractory Mutation System (ARMSTM) (Newton et al, Nucleic Acids Res. 17:2503-2516, 1989) can also be used to detect single base mutations. Under the appropriate PCR amplification conditions a single base mismatch located at the 3 '-end of the primer is sufficient for preferential amplification of the perfectly matched allele (Newton et al., 1989, supra), allowing the discrimination of closely related species.
  • the basis of an amplification system using the primers described above is that oligonucleotides with a mismatched 3 '-residue will not function as primers in the PCR under appropriate conditions. This amplification system allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis.
  • Analysis of amplification products can be performed using any method capable of separating the amplification products according to their size, including automated and manual gel electrophoresis, mass spectrometry, and the like.
  • the amplification products can be separated using sequence differences, using SSCP, DGGE, TGGE, chemical cleavage or restriction fragment polymorphisms as well as hybridization to, for example, a nucleic acid arrays.
  • Systematic hybridization-based identification of sequence variants for TP53 can be achieved with the AmpliChip p53 Test, under development at Roche Molecular Systems, which uses the AmpliChip p53 Microarray manufactured by Affymetrix,
  • the array consists of over 220,000 oligonucleotide probes designed to interrogate the sequence at each position in exons 2-11 of TP53.
  • For each nucleotide position there are at least twenty four probe sets, each containing five probes.
  • the five probes within each probe set are all perfectly complimentary to the reference sequence except for the position being interrogated: one probe is specific for the reference sequence, three correspond to the three possible nucleotide substitutions, and one corresponds to a deletion of the base at the target position.
  • Relative normalized intensities for the different probe types is used to determine the allele or alleles present at each position within a sample.
  • the present invention also provides predictive and diagnostic kits comprising degenerate primers to amplify a target nucleic acid in the TP53 gene and instructions comprising; amplification protocol and analysis of the results.
  • the kit may alternatively also comprise buffers, enzymes, and containers for performing the amplification and analysis of the amplification products.
  • the kit may also be a component of a screening, or diagnostic kit comprising other tools such as DNA microarrays, or other supports.
  • the kit also provides one or more control templates, such as nucleic acids isolated from normal tissue sample, and/or a series of samples representing different variances in the reference genes.
  • the kit provides two or more primer pairs, each pair capable of amplifying a different region of the reference (TP53) gene (each region a site of potential variance) thereby providing a kit for analysis of expression of several gene variances in a biological sample in one reaction or several parallel reactions.
  • Primers in the kits may be labelled, for example fluorescently labelled, to facilitate detection of the amplification products and consequent analysis of the nucleic acid variances.
  • the kit may also allow for more than one variance to be detected in one analysis.
  • a combination kit will therefore comprise of primers capable of amplifying different segments of the reference gene.
  • the primers may be differentially labelled, for example using different fluorescent labels, so as to differentiate between the variances.
  • the invention provides a method of treating a patient suffering from cancer comprising: determining the mutant or wild type status of the TP53 gene in the patient's tumour cells and if the TP53 gene is mutant, administering to the patient an effective amount of an IAP antagonist.
  • the invention provides a method of treating a patient suffering from cancer comprising: providing a tumour cell containing sample from a patient; determining whether the TP53 gene in the patient's tumour cells is wild type or mutant; and administering to the patient an effective amount of an IAP antagonist if the tumour cells possess a mutant TP53 gene.
  • a method of making a marketable drug comprising preparing a package containing an IAP antagonist; and including in the package a label or printed inset recommending use of the IAP antagonist for the treatment of cancer in a patient whose tumour cells have been determined to comprise mutant TP53 gene.
  • Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient.
  • Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment. "Less effective” means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a
  • an IAP antagonist to treat a cancer patient whose tumour cells have been identified as possessing a mutant TP53 gene.
  • an IAP antagonist in the manufacture of a medicament for treating a cancer patient whose tumour cells have been identified as possessing a mutant TP53 gene.
  • the invention relates to pharmaceutical composition comprising an IAP antagonist for use in the prevention and treatment of cancer with tumour cells identified as harbouring mutant TP53 gene.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained- release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • Administration may be topical, i.e., substance is applied directly where its action is desired, enteral or oral, i.e., substance is given via the digestive tract, parenteral, i.e., substance is given by other routes than the digestive tract such as by injection.
  • the active compound (IAP antagonist) and optionally another therapeutic or prophylactic agent are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings.
  • the active compounds are solutions in sterile isotonic aqueous buffer.
  • the compositions can also include a solubilizing agent.
  • Compositions for intravenous administration can optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule.
  • the active compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to
  • compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • a time delay material such as glycerol monostearate or glycerol stearate can also be used.
  • Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such vehicles are of pharmaceutical grade in particular embodiments.
  • compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the IAP antagonist compound and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or the nose) or oral, parenteral or mucosol (such as buccal, vaginal, rectal, sublingual)
  • administration In one embodiment, local or systemic parenteral administration is used.
  • compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch or sodium
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • compounds or salts disclosed herein or can be administered as a pharmaceutical composition in which the pharmaceutical composition comprises between 0.1-lmg, 1-10 mg, 10-50mg, 50-100mg, 100-500mg, or 500mg to 5 g of said IAP antagonist compound or salt.
  • Fig. 2 depicts the relationship between the Compound 1 response profile and that for two other dimer IAP inhibitors (Compound 2 and Compound 3). Cell lines tend to exhibit comparable sensitivity to all three compounds.
  • Fig. 3 depicts the relationship between the Compound 1 response profile and that for the other dimer IAP inhibitor Compound 4.
  • all GI50 values above 5 ⁇ were set to 5 ⁇ .
  • Cell lines are ordered on the X-axis by Compound 1 GI50. There is general agreement between responses for the two compounds.
  • the sensitivity of cancer cell lines to the effects of Compound 1 were determined in a standard proliferation assay.
  • Cells were plated in 96 well plates at densities of 1000-6000 cells per well in RPMI media containing 10% fetal bovine serum. After incubation at 37°C for 16 hours, various concentrations of Compound 1 were added to the assay plates. After incubation for an additional 72h, the viable cells were determined by the addition of MTS reagent (Promega) to each well for 2h.
  • MTS is a tetrazolium salt that is bioreduced by metabolically active cells in the presence of an electron coupling reagent to formazan. The formazan product is then quantitated by absorbance at 490 nm, as an indicator of the relative number of live cells.
  • the relative number of cells present at the time of drug addition was determine with the MTS readout before the drug was added, and this value was subtracted from the 72h value of untreated cells as a measure of cell growth during the assay.
  • Mutation status for genes in each cell line was obtained by integrating results from internal (AstraZeneca) and public sources.
  • Public data included all cell line data from the Catalogue of Somatic Mutations In Cancer (COSMIC) database (release v55 for selected genes including TP53 and v49 for other genes;
  • associations between mutation status and response were identified by constructing contingency tables for each gene and determining corresponding odd-ratios and two-tailed Fisher's exact test p-values. For mutation status, MUT or WT findings were counted, and the ND cases were regarded as WT. All genes for which at least one mutation is known to exist in any of the 191 cell lines were included in the analysis. In total, 761 genes had at least one cell line with MUT status (in addition to cell lines with WT status) across the set of 191 cell lines with response data, and thus were capable of showing an association with response. Most of these genes, all but 42, had a mutation in only one or two of the cell lines.
  • TP53 gene are shown in Table 3.
  • Table 3 The Compound 1 pharmacology data, response classification and the mutation status of the TP53 gene for the cell lines used in this study. GI50 values are indicated in micromolar units. A qualifier indicates the true GI50 was either below the minimum concentration tested or above the highest concentration tested. Asterisks denote cases where no mutations were detected (ND) but where there was no annotation that the whole gene had been surveyed in any screen. Status in those cases was treated asWT. .
  • the gene for which mutations were most strongly correlated with sensitivity to Compound 1 was TP53. Only 6 of 46 cell lines ( 13.0%) that were WT for TP53 were sensitive to Compound 1, whereas 41 of 117 TP53 mutant cell lines ( 35.0%) were sensitive, corresponding to an odd-ratio of 3.60 and a p-value of 0.007 (see Table 4).

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Abstract

L'invention concerne une méthode permettant de prévoir la réponse d'un patient à un antagoniste de IAP. Cette méthode consiste à analyser un prélèvement contenant des cellules tumorales effectué sur un patient de test pour déterminer le statut du gène TP53, les tumeurs possédant un gène TP53 mutant étant plus susceptibles de répondre favorablement à un traitement à base d'antagoniste de IAP et pouvant ainsi être sélectionnées pour être traitées au moyen d'un antagoniste de IAP.
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WO2015188107A1 (fr) * 2014-06-06 2015-12-10 Arizona Board of Regents of behalf of Arizona State University Polymères uniques, autoassemblés de polyamidoamine et leur réactivité électrochimique
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