WO2012052758A1 - Response biomarkers for iap antagonists in human cancers - Google Patents

Abstract

The invention is directed at a method for predicting a patient's responsiveness to an IAP antagonist. The method includes analysing a tumour cell containing sample from a test patient for the gene status of the TP53 gene, wherein tumours that possess a mutant TP53 gene are more likely to respond favourably to treatment with an IAP antagonist and can thus be selected for, and treated with, an IAP antagonist.

Description

RESPONSE BIOMARKERS FOR lAP ANTAGONISTS IN

HUMAN CANCERS

Field of the Invention

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.

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.

Inhibitor of apoptosis proteins (IAPs) were initially identified in baculoviruses, where they play a role in replication by preventing infected cells from undergoing apoptosis. Two motifs present in the baculo virus IAP protein have been identified in cellular IAPs. The baculo virus IAP repeat (BIR) domain is approximately 70-80 amino acids long and contains a Zn-binding motif. The presence of a BIR domain is what defines a protein as a member of the IAP family. BIR domains facilitate protein-protein interactions involved in IAP function. 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. (Riedl, S. J. and Y Shi, 2004, Molecular Mechanisms of Caspase

Regulation During Apoptosis, Nat. Rev. Mol. Cell Biol, 5: 897-907) 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

Proteins, Cell, 83 : 1243-1252). 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

Mitochondrial Activator of Caspases, J. Biol. Chem. 281 : 1080-1090). In the activated TNFR complex, ubiquitination of RIPK1 by the RING domain of cIAPl and 2 is a key step in activating TAK1 signaling downstream of the TNFR and leads to activation of the

prosurvival, canonical NF-κΒ pathway and synthesis of the caspase-8 inhibitor FLIP

(Varfolomeev E., et al. 2008 c-IAPl and C-IAP2 are Critical Mediators of Tumor Necrosis Factor a (TNFa)-induced NF-κΒ Activation, J. Biol. Chem., 283 : 24295-24299.). cIAPl also acts to negatively regulate the non-canonical NF-κΒ pathway by ubiquitination and subsequent proteosomal degradation of NIK. Like XIAP, 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.).

A cellular antagonist of the BIR2 and 3 domains of XIAP, cIAPl and cIAP2, along with the single BIR domain of Livin, is called the second mitochondrial activator of caspases (SMAC). SMAC is a homodimeric protein synthesized in the cytoplasm and then imported into the mitochondria where the N-terminal 55 amino acids are cleaved from each of the two identical subunits. Upon loss of mitochondrial integrity, as can occur upon DNA damage or treatment with agents that lead to apoptosis, mitochondrial SMAC enters the cytoplasm where it binds to XIAP, cIAPl, cIAP2 and Livin. SMAC binding to these IAPs is facilitated by the binding of the N-terminal 4 amino acids of each subunit, AVPI, to the BIR2 and/or 3 domains of cIAPl, cIAP2, XIAP and the single BIR domain of ML-IAP (Hunter et al).

Within a cell, 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

autoubiquitination and proteosome-mediated degradation of cIAPl and cIAP2. Loss of cIAPl and 2 inhibits signaling downstream of the TNFR through the canonical NF-KB pathway. In cells in which an active complex of TNF-a and TNFR occurs, it also leads to caspase-8 activation through the formation of a complex between TRAD, RIPK1 and procaspase-8. The formation of active caspase-8 and the absence of FLIP, due to inactivation of the canonical NF-κΒ pathway, leads to apoptosis in these tumor cells.

Structural studies have shown that the 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. These 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. When cancer cells in culture are exposed to cell permeable dimeric SMAC mimetics, some cell lines undergo apoptosis after exposure to very low concentrations of SMAC mimetics, while other lines are completely insensitive to IAP inhibitor exposure. For example, when a panel of 50 lung cancer cell lines were tested for sensitivity to a SMAC mimetic, only 14% of the lines were determined to be sensitive to treatment with this compound (Petersen et al, 2007, Autocrine TNFa signalling renders human cancer cells susceptible to smac-mimetic-induced apoptosis, Cancer Cell 12:445-456.) Therefore, biomarkers that predict tumor cell sensitivity to SMAC mimetics would be of clear benefit, particularly if they could be used in the clinical development of these molecules to select patients with a tumor that has the potential to respond to treatment with these agents. Treatment with SMAC mimetics can sensitize tumor cells to the apoptotic effects of tumor necrosis factor alpha (TNFa) (Gaither et al, 2007, A Smac mimetic rescue screen reveals roles for inhibitor or apoptosis proteins in tumor necrosis factor alpha signalling, Cancer Research 67: 11493-1 1498). In addition, recent studies have shown that cell lines that are sensitive to treatment with SMAC mimetics have either high basal secretion of TNFa in culture or they secrete TNFa as a direct response to treatment with SMAC mimetics (Probst et al 2010, Smac mimetics increase cancer cell response to chemotherapeutics in a TNF-a-dependent manner, Cell Death and Differentiation epublished April 30, doi: 10.1038/cdd.2010.44; Vince et al, 2007, IAP Antagonists Target cIAPl to induce TNFa-dependent apoptosis; Cell 131 :682- 693.). 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. However, no specific biomarkers exist to identify tumors that have low or undetectable levels of TNFa but will secrete TNFa after treatment with a SMAC mimetic.

Treatment with SMAC mimetics can sensitize tumor cells to the apoptotic effects of Trail ligand. When twenty seven patient-derived chronic lymphocytic leukemia samples were screened for ex vivo sensitivity to Trail ligand and IAP inhibitor, the data showed that increased apoptosis due to the combination vs Trail treatment alone was more commonly observed in the TP53 wild type samples compared to the TP53 mutant samples (Loeder et al, 2009, A novel paradigm to trigger apoptosis in chronic lymphocytic leukemia, Cancer Research 69: 8977-8986). The same study reported that samples with unmutated Vh genes are more sensitive to the combination of Trail ligand and IAP inhibitor compared to samples with Vh gene mutations.

WO 2008/137930 (Tetralogic Pharmaceuticals Corp.) teaches the use of TNFalpha gene expression as a biomarker of sensitivity to IAP antagonists.

US 2008/0194613 (Novartis) teaches that p53 (TP53) wild-type can be used as a biomarker for determining sensitivity to treatment with mTOR inhibitors in combination with a cytotoxic agent.

US 2009/0226429 (Human Genome Sciences) is directed to specific antibody molecules that bind immunospecifically to trail receptor 4. They propose that these molecules can be used in combination with IAP inhibitors in the treatment of many types of cancers.

There is a clear need for biomarkers that will enrich for or select patients whose tumors will respond to SMAC mimetic treatment. Patient selection biomarkers that identify the patients most likely to respond to an agent are ideal in the treatment of cancer, since they reduce the unnecessary treatment of patients with non-responding tumors to the potential side effects of such agents.

Background of the Invention

A biomarker can be described as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or

pharmacologic responses to a therapeutic intervention". 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. Typically 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.

In order to optimally design clinical trials and to gain the most information from these trials, 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.

Thus, 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.

As detailed in the examples herein, it was found that cells that possess a mutant TP53 gene are generally more susceptible to cell killing by the IAP antagonist.

Summary of the Invention

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. Specifically 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.

Furthermore 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.

In another embodiment, 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. As a single gene biomarker test, 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.

Definitions:

"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

transformation to a neoplastic phenotype. Such cellular transformation often involves genetic mutation. "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. A catalog of human polymorphisms and associated annotation, including validation, observed frequencies, and disease association, is maintained by 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.

"Overall survival" (OS) is defined as the time from the initiation of drug

administration to death from any cause. "Progression-free survival" (PFS) is defined as the time from the initiation of drug administration to first appearance of progressive disease or death from any cause.

According to one aspect of the invention there is provided a method for selecting a patient for treatment with an IAP antagonist, the method 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. In one embodiment, if the TP53 gene in the patient's tumour cell containing sample is mutant the patient is selected for treatment with an IAP antagonist. In another embodiment, if the TP53 gene in the patient's tumour cell containing sample is mutant the patient is selected for monotherapy treatment with an IAP antagonist. In another embodiment, if 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. In a particular embodiment the other therapeutic agent is a small molecule compound.

The method may include or exclude the actual patient sample isolation step. Thus, according to one aspect of the invention there is provided a method for selecting a patient for treatment with an IAP antagonist, the method 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.

In one embodiment, the patient is selected for treatment with the IAP antagonist if the tumour cell has a mutant TP53 gene.

IAP antagonists

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). In some embodiments, the IAP antagonist binds to cIAP-1 and cIAP-2 with greater affinity than it binds to XIAP. In some embodiments, 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. In other embodiments the IAP antagonist binds XIAP with greater affinity than cIAP-1 or cIAP-2. There are many IAP antagonists known in the art. 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

US20050197403, US7244851, US 7309792, US 7517906, US7579320, US 7547724, WO2004/007529, WO 2005/069888, WO 2005/069894, WO2005097791, WO 2006/010118, WO 2006/122408, WO 2006/017295, WO 2006/133147, WO 2006/128455, WO

2006/091972, WO 2006/020060, WO 2006/014361, WO 2006/097791, WO 2007/021825, WO 2007/106192, WO2007/101347, WO 2008/045905, WO 2008/016893, WO2008/128121, WO2008/128171, WO 2008/134679, WO 2008/073305, WO 2009/060292, WO 2007/104162, WO 2007/130626, WO 2007/131366, WO 2007/136921, WO 2008/014229, WO 2008/014236, WO 2008/014238, WO 2008/014240, WO 2008/134679, WO

2009/136290, WO 2008/014236 and WO 2008/144925.

Another suitable 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:

The Examples herein, utilise three compounds from WO 2010/142994. Thus, any of the compounds disclosed in WO 2010/142994 are considered to be suitable for use in the present invention.

Thus, the various aspects of the invention can be used in conjunction with a compound of formula I:

I

wherein:

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; and

R6 and R7 are independently H or C(l-6)alkyl; or

a salt thereof.

The compounds of Figure 1 can be considered dimeric. The dimeric compounds described therein can be homodimers or heterodimers. The terms homodimer and

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. Accordingly, in homodimers of the compounds described in WO 2010/142994, Rl, R2 and R3 are the same as R6, R7 and R5, respectively, with the linker being R4. In heterodimers of the described compounds, one or more Rl, R2 and R3 are different than R6, R7 and R5, respectively. In heterodimers, one, two or all of Rl , R2 and R3 can be different than R6, R7 and R5, respectively.

Specific 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-

(methylamino)propanamido)acetyl)-N-((R)- 1 ,2,3 ,4-tetrahydronaphthalen- 1 -yl)pyrrolidine-2- carboxamide (Abbott)- Example 3 from WO2006/017295; (S)-N-((S)-l-cyclohexyl-2-((S)-2- (4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-l-yl)-2-oxoethyl)-2-(methylamino)propanamide [also known as LCL-161 (Novartis) - Example 1 from WO2008/016893]; (S)-N-((S)-1- cyclohexyl-2-oxo-2-((3aR,7aS)-6-phenethyloctahydro-lH-pyrrolo[2,3-c]pyridin-l-yl)ethyl)- 2-(methylamino)propanamide [also known as (LBW-242 (Novartis) - Example 1 from

WO2005/097791]; and, (5S,8S,10aR)-N-benzhydryl-5-((S)-2-(methylamino)propanamido)-3- (3-methylbutanoyl)-6-oxodecahydropyrrolo[l,2-a][l,5]diazocine-8-carboxamide [also known as AT-406 (Ascenta) - Example 16 from WO2008/128171].

In particular embodiments 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-(methylamino)propanamido)acetyl)pyrrolidine-2- carboxamide) as a bis hydrochloride salt; (S,S,2S,2'S)-N,N^*-((lS,rS,2S,2^,S)-2,2^,-(hexa-2,4- diyne-l,6-diylbis(oxy))bis(2,3-dihydro-lH-indene-2,l-diyl))bis(l-((S)-2-cyclohexyl-2-((S)-2^ (methylamino)propanamido)acetyl)pyrrolidine-2-carboxamide); N 1 ,N4-bis((3 S,5 S)- 1 -((S)- 3,3-dimethyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-((R)-l, 2,3,4- tetrahydronaphthalen- 1 -ylcarbamoyl)pyrrolidin-3 -yl)terephthalamide; (S)- 1 -((S)-2- cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-((R)- 1 ,2,3 ,4- tetrahydronaphthalen- 1 -yl)pyrrolidine-2-carboxamide; (S)-N-((S)- 1 -cyclohexyl-2-((S)-2-(4- (4-fluorobenzoyl)thiazol-2-yl)pyrrolidin- 1 -yl)-2-oxoethyl)-2-(methylamino)propanamide; (S)- N-((S)-l-cyclohexyl-2-oxo-2-((3aR,7aS)-6-phenethyloctahydro-lH-pyrrolo[2,3-c]pyridin-l- yl)ethyl)-2-(methylamino)propanamide; and, (5S,8S,10aR)-N-benzhydryl-5-((S)-2- (methylamino)propanamido)-3 -(3 -methylbutanoyl)-6-oxodecahydropyrrolo [ 1 ,2- a][l,5]diazocine-8-carboxamide. The compound: (2S,2'S)-N,N'-{hexa-2,4-diyne-l,6- diylbis[oxy(lS,2R)-2,3-dihydro-lH-ind^

(methylamino)propanoyl]amino}acetyl]pyrrolidine-2-carboxamide} is a particular

embodiment.

In a particular embodiment 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.

According to another aspect of the invention there is provided a method for predicting a patient's responsiveness to an IAP antagonist, the method 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.

According to another aspect of the invention there is provided 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.

For the purpose of this invention, a gene status of wild-type is meant to indicate normal or appropriate expression of the gene and normal function of the encoded protein. In contrast, 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. However, if such alterations nevertheless result in appropriate expression of the normal protein, or a functionally equivalent variant, then the gene status is regarded as wild-type. Examples of variants that typically would not result in a functional mutant gene status include synonomous coding variants and common polymorphisms (synonymous or non-synonymous). As discussed below, gene status can be assessed by a functional assay, or it may be inferred from the nature of detected deviations from a reference sequence.

In certain embodiments 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.

Other variations in the TP53 gene that signify mutant status include 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. Thus, in other embodiments, the gene has a mutant status if there is a variant that alters splice site recognition sequence at an intron/exon junction.

The 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). In humans, 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). It is nowadays known that 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. By 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.

There are many ways in which the p53 function may be altered in human cancers. 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). 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).

For TP53, 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.

The person of skill in the art will be able to determine the TP53 gene status, i.e.

whether a particular TP53gene is wild type or mutant.

For purposes of this invention reference 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.

The 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.

It will be apparent that the gene sequences disclosed for 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.

According to another aspect of the invention there is provided 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.

According to another aspect of the invention there is provided a method for assessing the susceptibility of an individual to treatment with an IAP antagonist, which method comprises:

(i) determining the non- synonymous mutation status of the TP53 gene in a tumour cell containing sample from the individual; and,

(ii) determining the likely susceptibility of the individual to treatment with an IAP

antagonist by reference to the non-synonymous mutation status of the TP53 gene in the tumour cells.

As noted above, it is well established that 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

(http://p53.free.fr/Data.base/p53 database.html) catalog and curate the various known TP53 gene mutations, characterise their effect of p53 function, and indicate mutation frequency in different tumor tissue or disease types (Petitjean et al. Hum Mutat. 28(6):622-9, Jun 2007; Hamroun D et al., Hum Mutat. 27(1): 14-20, Jan 2006; Caron de Fromentel C, Soussi T. Genes Chromosomes Cancer.;4(l): l-15, Jan 1992). The spectrum of TP53 mutations in human cancer are also described in recent reviews (Robles Al, Harris CC. Clinical outcomes and correlates of TP53 mutations and cancer. Cold Spring Harb Perspect Biol. 2010

Mar;2(3):a001016; Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb Perspect Biol. 2010

Jan;2(l):a001008) There are numerous techniques available to the person skilled in the art to determine the gene status of TP53. The gene status 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.

An alternative means for determining whether or not the TP53 gene is wild type or mutant is to assess the function of the transcribed gene. 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).

Samples

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.

It will be appreciated that 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. In particular embodiments 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 methods of the invention can be applied using cells from any tumour. For example, 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.

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

Table 1.

Endocrine glands, NOS 6

Cervix uteri 4

Nerves 4

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.

The frequency of 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.).

Methods for Detection of Nucleic Acids

The detection of 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.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of variant nucleotides at one or more positions in a gene. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. List 1 recites a number of mutation detection techniques, some based on the PCR. These may be used in combination with a number of signal generation systems, a selection of which is listed in List 2. Many current methods for the detection of allelic variation are reviewed by Nollau et al., Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2^nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Table 2. Abbreviations:

ALEX™ Amplification refractory mutation system linear extension

APEX Arrayed primer extension

ARMS™ Amplification refractory mutation system

b-DNA Branched DNA

bp base pair

CMC Chemical mismatch cleavage

COPS Competitive oligonucleotide priming system

DGGE Denaturing gradient gel electrophoresis

FRET Fluorescence resonance energy transfer

HMG-CoA 3 -hydroxy-3 -methylglutaryl-coenzyme A

LCR Ligase chain reaction

MASDA Multiple allele specific diagnostic assay

NASBA Nucleic acid sequence based amplification

OATP Na+-independent organic anion transporting polypeptide

OLA Oligonucleotide ligation assay

PCR Polymerase chain reaction

PTT Protein truncation test

RFLP Restriction fragment length polymorphism

SDA Strand displacement amplification

SNP Single nucleotide polymorphism

SSCP Single-strand conformation polymorphism analysis

SSR Self sustained replication

TGGE Temperature gradient gel electrophoresis List 1. Examples of mutation detection techniques include:

General: DNA sequencing, Sequencing by hybridisation, Pyrosequencing™

Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage

* Note: not useful for detection of promoter polymorphisms.

Hybridisation Based

Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays such as the p53 Amplichip™ (DNA Chips).

Solution phase hybridisation: Taqman™ - US-5210015 & US-5487972 (Hoffmann-La Roche), Molecular Beacons - Tyagi et al (1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public Health Inst., New York).

Extension Based: ARMS™, ALEX™ - European Patent No. EP 332435 Bl (Zeneca

Limited), COPS - Gibbs et al (1989), Nucleic Acids Research, 17, 2347.

Incorporation Based: Mini-sequencing, APEX.

Restriction Enzyme Based: RFLP, Restriction site generating PCR.

Ligation Based: OLA.

Other: Invader assay.

List 2. Examples of Signal Generation or Detection Systems include:

Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998 (Zeneca Limited)

Other: Chemiluminescence, Electrochemilummescence, Raman, Radioactivity, Colorimetric, Hybridisation protection assay, Mass spectrometry

As noted above, 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 ARMS™, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.

In particular embodiments 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.

In particular embodiments, 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. Such 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.

Thus, in one embodiment, 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.

In another embodiment, 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

complementary nucleotide base to the mutation of interest in the centre of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-248) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238).

In yet another embodiment, 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.

Alternatively, the presence or absence of at least one mutation comprises mass spectrometric determination of at least one nucleic acid sequence.

In one embodiment, the detection of the presence or absence of at least one nucleic acid variance comprises performing a polymerase chain reaction (PCR). 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. Alternatively, 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.,

BioTechniques 26(6): 1134-1148 (June 1999) are available commercially.

In a particular example, 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.

Generation of 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. Preferably, the amplification according to the invention is an exponential amplification, as exhibited by for example the polymerase chain reaction.

Many target and signal amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U. , et al, Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10: 1, 54-55 (1990). These amplification methods can be used in the methods of our invention, and include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, QP

bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based

amplification (NASBA) and in situ hybridisation. Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.

Polymerase Chain Reaction (PCR) 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 (3SR) 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.

In Qbeta Replicase, 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. First, the target DNA is hybridised to a primer including a T7 promoter and a QP 5' sequence region. From this primer, 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 10⁷ fold amplification in approximately 20 minutes.

A further technique, strand displacement amplification (SDA; Walker et al., (1992) PNAS (USA) 80:392) begins with a specifically defined sequence unique to a specific target. However, unlike other techniques which rely on thermal cycling, SDA is an isothermal process that utilises a series of primers, DNA polymerase and a restriction enzyme to exponentially amplify the unique nucleic acid sequence.

Chemical mismatch cleavage (CMC) is based on the recognition and cleavage of DNA mismatched base pairs by a combination of hydroxylamine, osmium tetroxide and piperidine. Thus, 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.

Techniques based on restriction fragment polymorphisms (RFLPs) can also be used.

Although many single nucleotide polymorphisms (SNPs) do not permit conventional RFLP analysis, primer-induced restriction analysis PCR (PIRA-PCR) can be used to introduce restriction sites using PCR primers in a SNP-dependent manner. Primers for PIRA- PCR which introduce suitable restriction sites can be designed by computational analysis, for example as described in Xiaiyi et al., (2001) Bioinformatics 17:838-839.

Furthermore, techniques based on WAVE analysis can be used (Methods Mol. Med. 108: 173-88, 2004). This system of DNA fragment analysis can be used to detect single nucleotide polymorphisms and is based on temperature-modulated liquid chromatography and a high-resolution matrix (Genet Test. l(3):201-6, 1997) Real-time PCR (also known as Quantitative PCR, Real-time Quantitative PCR, or RTQ-PCR) is a method of simultaneous DNA quantification and amplification (Expert Rev. Mol. Diagn. 2:209-19, 2005). DNA is specifically amplified by polymerase chain reaction. After each round of amplification, the DNA is quantified. Common methods of quantification include the use of fluorescent dyes that intercalate with double-strand DNA and modified DNA oligonucleotides (called probes) that fluoresce when hybridised with a complementary DNA.

An allele specific amplification technique such as Amplification Refractory Mutation System (ARMS™) (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. Alternatively, 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.

The methods of nucleic acid isolation, amplification and analysis are routine for one skilled in the art and examples of protocols can be found, for example, in the Molecular Cloning: A Laboratory Manual (3-Volume Set) Ed. Joseph Sambrook, David W. Russel, and Joe Sambrook, Cold Spring Harbor Laboratory; 3rd edition (January 15, 2001), ISBN:

0879695773. Particularly useful protocol source for methods used in PCR amplification is PCR (Basics: From Background to Bench) by M. J. McPherson, S. G. Mailer, R. Beynon, C. Howe, Springer Verlag; 1st edition (October 15, 2000), ISBN: 0387916008.

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. Preferably, 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.

In one embodiment, 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.

In another aspect, 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.

In another aspect, 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. In another aspect of the invention there is provided a method of making a marketable drug, the method 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.

As used herein, the terms "effective" and "effectiveness" includes both

pharmacological effectiveness and physiological safety. 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

therapeutically greater level of adverse physiological effects.

According to another aspect of the invention there is provided the use of an IAP antagonist to treat a cancer patient whose tumour cells have been identified as possessing a mutant TP53 gene.

According to another aspect of the invention there is provided the use of 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.

In still further embodiments, 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.

The pharmaceutical 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.

In a particular embodiment, 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. Typically, for intravenous administration the active compounds are solutions in sterile isotonic aqueous buffer. Where necessary, 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. Generally, 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. Where 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. Where the active compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to

administration.

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. Thus, 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.

For oral administration, the 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). The tablets can be coated by methods well known in the art. 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.

In some embodiments, 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.

The invention will be further described by virtue of the following non-limiting examples and figures wherein

Fig. 1 depicts a bar chart showing the Compound 1 response profile for 163 cell lines in the proliferation assay with TP53 status indicated. Overall, 54 of 191 cell lines (28.3%) and 47 of 163 with defined TP53 status (28.8%>) were classified as sensitive (GI50 <= 1 μΜ). The sensitive lines (upward bars) exhibit a higher prevalence of TP53 mutations.

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. For this plot, all GI50 values above 5 μΜ were set to 5 μΜ. Cell lines are ordered on the X-axis by Compound 1 GI50. There is general agreement between responses for the two compounds.

All publications (e.g. scientific articles and patent publications) disclosed herein are incorporated by reference.

Example 1:

The test compound (IAP antagonist) used in the following example, Compound 1 = ((2S,2'S)- Ν,Ν'- {hexa-2,4-diyne- 1 ,6-diylbis[oxy( 1 S,2R)-2,3-dihydro- 1 H-indene-2, 1 -diyl] }bis { 1 - [(2S)-2- cyclohexyl-2- {[(2S)-2-(methylamino)propanoyl]amino}acetyl]pyrrolidine-2-carboxamide}), is disclosed in Example 1 of WO 2010/142994 and USSN 12/796089 and has the following structure:

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. In order to determine the GI50 (concentration at which growth of cells was inhibited by 50%) 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.

Additional dimeric IAP inhibitors were tested in the proliferation assay in selected tumor cell lines. The compounds are Compound 2 = (S,S,2S,2^,S)-N,N^*-((lS,rS,2R,2^,R)-2,2'- (Hexane-l,6-diylbis(oxy))bis(2,3-dihydro-lH-indene-2,l-diyl))bis(l-((S)-2-cyclohexyl-2- ((S)-2-(methylamino)propanamido)acetyl)pyrrolidine-2-carboxamide) as a bis hydrochloride salt which is Example 3 in WO 2010/142994; Compound 3 = (S,S,2S,2'S)-N,N^*- ((lS,rS,2S,2'S)-2,2^,-(hexa-2,4-diyne-l,6-diylbis(oxy))bis(2,3-dihydro-lH-indene^^ diyl))bis(l-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidine-2- carboxamide) which is Example 4 in WO 2010/142994; and, Compound 4 = (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) another dimeric IAP inhibitor referred to as compound 3 in US Patent Serial Number

7,579,320.

Mutation Correlation Analysis

Pharmacology data measuring growth inhibition in response to treatment with

Compound 1 was obtained for a collection of 191 cancer cell lines from a variety of tissues from multiple sources. Each cell line was classified as sensitive (GI50 <= 1.0 μΜ)θΓ resistant (GI50 > Ι .Ο μΜ).

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;

http://www.sanger.ac.uk/genetics/CGP/cosmic/; Forbes SA, et al. COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer. Nucleic Acids Res. 2010 Jan;38(Database issue):D652-7; Forbes SA, et al. The Catalogue of Somatic Mutations in Cancer (COSMIC). Curr Protoc Hum Genet. 2008 Apr; Chapter 10:Unit 10.11.), as well as data for TP53 from the IARC TP53 database (release R15;

http://www-p53.iarc.fr/) and UMD TP53 Mutation Database (release 2010_R1;

http ://p53. free. fr/Database/p53_database .html). Silent coding region mutations (synonymous variants) and non-synonymous polymorphisms were excluded, and, for the purpose of this analysis, the zygosity of mutations was ignored. For each combination of cell line and gene, status was summarized as mutant (MUT), wild-type (WT), none detected (ND), inconsistent (INCON), or unknown. In cases where a gene was screened and no mutations were observed, the WT label was used if the screen was annotated as having covered the whole gene and the ND label was used otherwise. Some initially inconsistent cases (independent WT and MUT observations for the same gene in the same cell line) were resolved by weighting internal observations and those for the Cancer Cell line Project (CCLP) subset of COSMIC, or by selecting a status after manual review. In cases where inconsistent observations could not be resolved, the INCON label was retained and the gene status was regarded as unknown during analysis.

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.

Results and Discussion

Associations between mutation status and response were identified as describe in Methods. The cell line response to Compound 1 and the corresponding genetic status for both

TP53 gene are shown in Table 3.

RESPONSE

TISSUE TP53 QUAL GI50 (μΜ) CLASSIFICATION

breast MUT 8.000E-05 sensitive

breast MUT 1.500E-04 sensitive

stomach WT 1.000E-03 sensitive

pancreas MUT 1.000E-03 sensitive

breast WT 1.000E-03 sensitive

bladder < 1.289E-03 sensitive

bladder MUT 1.860E-03 sensitive

bladder MUT 2.035E-03 sensitive

ovary MUT < 4.870E-03 sensitive

breast MUT 7.549E-03 sensitive

pancreas MUT < 8.740E-03 sensitive

hematopoietic < 8.740E-03 sensitive

hematopoietic MUT < 8.740E-03 sensitive

hematopoietic MUT < 8.740E-03 sensitive

hematopoietic < 8.740E-03 sensitive

ovary MUT 1.000E-02 sensitive

hematopoietic MUT 1.456E-02 sensitive

hematopoietic 1.492E-02 sensitive

liver INCON 1.637E-02 sensitive

breast MUT 1.965E-02 sensitive

stomach MUT 2.000E-02 sensitive

stomach MUT 2.000E-02 sensitive

lung WT 2.800E-02 sensitive

lung MUT 2.800E-02 sensitive

lung MUT 2.800E-02 sensitive

lung MUT 2.800E-02 sensitive

colon WT 2.800E-02 sensitive

lung 3.000E-02 sensitive

liver WT 5.392E-02 sensitive breast MUT 5.423E-02 sensitive ovary MUT 6.200E-02 sensitive colon 8.000E-02 sensitive colon MUT 8.000E-02 sensitive breast MUT 8.000E-02 sensitive hematopoietic MUT 8.095E-02 sensitive ovary MUT 8.100E-02 sensitive colon MUT l .OOOE-01 sensitive lung MUT 1.540E-01 sensitive breast MUT 2.000E-01 sensitive colon MUT 2.000E-01 sensitive colon MUT 2.950E-01 sensitive lung MUT 2.998E-01 sensitive lung MUT 3.000E-01 sensitive lung MUT 3.200E-01 sensitive lung MUT 3.210E-01 sensitive lung MUT 3.300E-01 sensitive colon MUT 4.000E-01 sensitive liver MUT 5.871E-01 sensitive colon MUT 6.430E-01 sensitive stomach MUT 6.726E-01 sensitive ovary WT* 8.000E-01 sensitive stomach MUT 8.903E-01 sensitive colon MUT l .OOOE+00 sensitive lung MUT l .OOOE+00 sensitive stomach MUT 1.021E+00 resistant bladder MUT 1.122E+00 resistant liver WT 1.243E+00 resistant lung WT 1.300E+00 resistant liver MUT 1.496E+00 resistant lung MUT 1.900E+00 resistant liver INCON 1.972E+00 resistant hematopoietic MUT 2.092E+00 resistant bladder MUT 2.319E+00 resistant colon WT 2.461E+00 resistant lung WT 2.481E+00 resistant colon MUT 2.500E+00 resistant stomach MUT 2.520E+00 resistant ovary WT 2.800E+00 resistant colon MUT 2.888E+00 resistant bladder MUT 2.904E+00 resistant hematopoietic MUT 2.996E+00 resistant lung MUT 3.000E+00 resistant colon WT 3.000E+00 resistant colon WT 3.000E+00 resistant lung MUT 3.000E+00 resistant lung 3.000E+00 resistant lung MUT 3.000E+00 resistant colon 3.000E+00 resistant colon MUT 3.000E+00 resistant colon WT 3.000E+00 resistant colon MUT 3.001E+00 resistant stomach WT 3.043E+00 resistant breast MUT > 3.243E+00 resistant breast MUT > 3.243E+00 resistant breast MUT > 3.243E+00 resistant breast MUT > 3.243E+00 resistant breast > 3.243E+00 resistant breast WT* > 3.243E+00 resistant breast WT > 3.243E+00 resistant breast MUT > 3.243E+00 resistant breast MUT > 3.243E+00 resistant lung MUT 3.345E+00 resistant lung WT 3.362E+00 resistant stomach MUT 3.402E+00 resistant lung (SCLC) MUT 3.420E+00 resistant hematopoietic WT 3.519E+00 resistant colon WT 3.539E+00 resistant stomach WT 3.759E+00 resistant bladder MUT 4.106E+00 resistant prostate WT > 4.160E+00 resistant liver WT 4.193E+00 resistant liver MUT 4.213E+00 resistant hematopoietic MUT 4.341E+00 resistant liver WT 4.422E+00 resistant stomach MUT 4.443E+00 resistant ovary 4.500E+00 resistant breast MUT 4.500E+00 resistant stomach MUT 4.505E+00 resistant hematopoietic MUT 4.506E+00 resistant stomach INCON 4.528E+00 resistant liver WT 4.538E+00 resistant stomach MUT 4.551E+00 resistant lung 4.568E+00 resistant bladder 4.622E+00 resistant bladder MUT 4.645E+00 resistant stomach MUT 4.676E+00 resistant bladder WT 4.700E+00 resistant breast MUT 4.793E+00 resistant lung MUT 4.801E+00 resistant hematopoietic 4.817E+00 resistant stomach MUT 4.839E+00 resistant colon WT 4.879E+00 resistant lung WT* 4.900E+00 resistant colon MUT 4.945E+00 resistant stomach INCON 4.950E+00 resistant hematopoietic WT 4.990E+00 resistant ovary MUT 5.100E+00 resistant hematopoietic MUT 5.111E+00 resistant hematopoietic 5.118E+00 resistant bladder MUT 5.249E+00 resistant bladder WT 5.286E+00 resistant stomach WT 5.351E+00 resistant liver INCON 5.390E+00 resistant breast MUT 5.400E+00 resistant skin MUT 5.487E+00 resistant lung WT* 5.507E+00 resistant liver WT 5.574E+00 resistant hematopoietic MUT 5.648E+00 resistant bladder MUT 5.858E+00 resistant lung MUT 6.025E+00 resistant hematopoietic MUT 6.410E+00 resistant stomach INCON 6.411E+00 resistant lung MUT 6.664E+00 resistant liver MUT 6.717E+00 resistant bladder WT 6.733E+00 resistant lung MUT 6.797E+00 resistant lung WT 6.881E+00 resistant bladder MUT 6.973E+00 resistant colon MUT 7.135E+00 resistant hematopoietic 7.397E+00 resistant bladder MUT 7.471E+00 resistant lung MUT 7.600E+00 resistant hematopoietic 7.941E+00 resistant stomach WT 8.023E+00 resistant stomach MUT 8.233E+00 resistant stomach MUT 8.537E+00 resistant hematopoietic MUT 8.551E+00 resistant liver INCON 8.618E+00 resistant hematopoietic 8.664E+00 resistant liver WT 9.098E+00 resistant bladder MUT 9.900E+00 resistant lung MUT 1.032E+01 resistant

bladder 1.042E+01 resistant

stomach WT 1.042E+01 resistant

prostate MUT 1.051E+01 resistant

colon MUT 1.089E+01 resistant

ovary MUT l . lOOE+01 resistant

liver WT 1.109E+01 resistant

colon WT 1.208E+01 resistant

lung WT* 1.239E+01 resistant

liver MUT 1.240E+01 resistant

ovary MUT 1.240E+01 resistant

colon WT* 1.260E+01 resistant

lung (SCLC) MUT 1.274E+01 resistant

colon 1.412E+01 resistant

liver INCON 1.416E+01 resistant

colon WT 1.799E+01 resistant

hematopoietic WT 1.925E+01 resistant

hematopoietic MUT 1.943E+01 resistant

liver MUT 1.971E+01 resistant

prostate MUT 2.056E+01 resistant

liver MUT 2.073E+01 resistant

bladder WT 2.090E+01 resistant

colon MUT 2.246E+01 resistant

liver MUT 2.564E+01 resistant

ovary WT* 2.600E+01 resistant

hematopoietic MUT 2.859E+01 resistant

breast MUT 2.980E+01 resistant

colon MUT 3.000E+01 resistant

breast > 3.243E+01 resistant

breast MUT > 3.330E+01 resistant

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).

Odds-Ratio: 3.60

p-value: 0.007

Table 4. Contingency table for TP53 mutation status and response to Compound 1.

The association between TP53 mutant status and sensitivity was also observed in subsets of the full cell line collection. Table 5 lists several subsets with at least ten cell lines in which the response rate for TP53 mutants was greater than that for those without mutations in TP53. The trend was strongest in the lung (NSCLC) and colon subsets.

Table 5. Association summary for TP53 mutation status and response to Compound 1 in different cell line subsets. MUT S: TP53 mutant and sensitive; MUT R: TP53 mutant and resistant; WT S: TP53 wild-type and sensitive; WT R: TP53 wild-type and resistant

Claims

Claims:

1. A method for selecting a patient for treatment with an IAP antagonist, the method comprising providing a sample from a patient containing tumor-derived DNA or tumor cells; determining whether the TP53 gene in the patient's tumour cells or tumour-derived DNA are wild type or mutant; and if the TP53 gene is mutant selecting the patient for treatment with an IAP antagonist.

2. The method according to claim 1, wherein the IAP antagonist is a dimer.

3. The method according to claims 1 or 2, wherein the sample is a tumour sample or contains tumour cells or DNA from tumor cells selected from: circulating tumor cells, circulating DNA in the plasma or serum, cells isolated from the ascites fluid of ovarian cancer patients, lung sputum, a fine needle aspirate, urine, peripheral blood, a cell scraping, a hair follicle, a skin punch or a buccal sample.

4. The method according to any of claims 1 - 3, wherein the IAP antagonist is selected from the group consisting of: (2S,2'S)-N,N'-{hexa-2,4-diyne-l,6-diylbis[oxy(lS,2R)-2,3- dihydro- 1 H-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, 1 ^*S,2R,2'R)-2,2^,-(Hexane- 1 ,6-diylbis(oxy))bis(2,3-dihydro- 1 H-indene-2, 1 -diyl))bis( 1 - ((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidine-2-carboxamide) as a bis hydrochloride salt; (S,S,2S,2'S)-N,N^*-((lS,rS,2S,2^,S)-2,2^,-(hexa-2,4-diyne-l,6- diylbis(oxy))bis(2,3-dihydro-lH-indene-2,l-diyl))bis(l-((S)-2-cyclohexyl-2-((S)-2- (methylamino)propanamido)acetyl)pyrrolidine-2-carboxamide); N 1 ,N4-bis((3 S,5 S)- 1 -((S)- 3,3-dimethyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-((R)-l, 2,3,4- tetrahydronaphthalen- 1 -ylcarbamoyl)pyrrolidin-3 -yl)terephthalamide; (S)- 1 -((S)-2- cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-((R)- 1 ,2,3 ,4- tetrahydronaphthalen- 1 -yl)pyrrolidine-2-carboxamide; (S)-N-((S)- 1 -cyclohexyl-2-((S)-2-(4- (4-fluorobenzoyl)thiazol-2-yl)pyrrolidin- 1 -yl)-2-oxoethyl)-2-(methylamino)propanamide; (S)- N-((S)-l-cyclohexyl-2-oxo-2-((3aR,7aS)-6-phenethyloctahydro-lH-pyrrolo[2,3-c]pyridin-l- yl)ethyl)-2-(methylamino)propanamide; and, (5S,8S,10aR)-N-benzhydryl-5-((S)-2- (methylamino)propanamido)-3 -(3 -methylbutanoyl)-6-oxodecahydropyrrolo [ 1 ,2- a] [ 1 , 5 ] diazocine- 8 -carboxamide .

5. The method according to any of the preceding claims, wherein the IAP antagonist is (2S,2'S)-N,N'- {hexa-2,4-diyne- 1 ,6-diylbis[oxy( 1 S,2R)-2,3-dihydro- 1 H-indene-2, 1 -diyl] }bis { 1 - [(2S)-2-cyclohexyl-2-{[(2S)-2-(methylamino)propanoyl]amino}acetyl]pyrrolidine-2- carboxamide} .

6. The method according to any of the preceding claims wherein the mutant or wild type status of the genes is determined by sequencing or by determining by the presence or absence of a variant nucleic acid sequence.

7. The method according to claim 6, wherein said existence of said variant nucleic acid sequences is determined by amplifying a segment of nucleic acid and identifying whether or not said amplified region contains a mutant sequence.

8. The method according to any of the preceding claims wherein the detection of the presence or absence of said mutant sequence comprises mass spectrometric determination of at least one nucleic acid sequence.

9. The method of any of claims 1 to 6, wherein the detection of the presence or absence of said mutant sequence comprises performing a polymerase chain reaction (PCR) to amplify nucleic acid comprising TP53 coding sequence, and determining nucleotide sequence of the amplified nucleic acid.

10. The method of claim 9, wherein the polymerase chain reaction is carried out using allele specific primers that detect single base mutations, small in- frame deletions or base substitutions.

11. The method according to any of the preceding claims, wherein the presence of a mutation is determined by comparison of the gene sequence from the tumour cell containing sample with the gene sequence obtained from a non-tumour cell containing sample from said individual or by comparison to a wild-type TP53 reference sequence.

12. The method according to claim 11, wherein the wild-type TP53 reference sequence is one selected from the sequence disclosed in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

13. 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 are 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.

14. Use of 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.

15. An IAP antagonist for treating cancers with tumour cells identified as harbouring a mutant TP53 gene.

16. The IAP antagonist according to claim 15, which is a SMAC mimetic dimer.