Classifications

• • 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
  • C12Q2600/154—Methylation markers

Definitions

• the present invention relates to a method of predicting/determining the responsiveness of a cancer disease to treatment with an inhibitor of CD95/CD95L signalling on the basis of methylation levels of specific DNA sequences.
• Treatment regimens used for cancers are of very limited efficacy [1 -3].
• alkylating chemotherapy has been the mainstay, although patients already had been exposed to temozolomide in conjunction with radiotherapy after diagnosis and surgery.
• Recent and ongoing trials often have used lomustine (CCNU) as control arm and tested anti-vascular endothelial growth factor (receptor) strategies [4,5], protein kinase C-beta inhibition with enzastaurin [6], or they embarked on various temozolomide regimens [7,8].
• CCNU lomustine
• receptor anti-vascular endothelial growth factor
• CD95 (Fas and APO-1 ) is a pleiotrophic receptor that regulates tissue homeostasis. During cancer progression, CD95 is frequently down-regulated or tumour cells are rendered apoptosis resistant. However, evidence exists that cancer cells, regardless of their CD95 apoptosis sensitivity, depend on constitutive activation of CD95 for optimal growth [1 1 ], stimulated by CD95L produced in an autocrine or paracrine manner.
• CD95 activation in glioblastoma leads to invasive growth and migration facilitated by increased expression of matrix metalloproteinases (MMP), which are key mediators of glioma invasiveness [13].
• MMP matrix metalloproteinases
• blocking of CD95 activation was demonstrated to inhibit increased invasiveness of sublethally irradiated glioblastoma cells as an adaptive evasive response to radiation [14,15]. This unwanted effect of radiotherapy is proposed to be mediated by stimulation of PI3K/AKT- dependent MMP-2 and MMP-9 activity [16,17] or alternative mechanisms [18].
• inhibitors of CD95/CD95L signalling and their use in the treatment of cancer diseases are known in the art.
• this object is achieved by a method of determining the methylation level of a DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling in a sample obtained from a patient and classifying the cancer disease according to said methylation level. This classification can then be used to predict/determine the responsiveness of the cancer disease to the treatment described above.
• the methylation level of specific DNA sequences located upstream of and/or in a gene involved in CD95/CD95L signalling correlates with the clinical outcome of treatment with an inhibitor of CD95/CD95L signalling, optionally in combination with radiotherapy.
• DNA methylation is a biochemical process which involves the addition of methyl groups to adenine or cytosine in the DNA.
• DNA methylation has been shown to play an important role, for example, in developmental processes and in regulation of gene expression.
• methylation of cytosines of CpG sites within so-called CpG islands is especially interesting.
• the term "CpG island” which is known to the person skilled in the art denotes DNA regions which exhibit a higher frequency of the di-nucleotide sequence CpG (a CpG site) compared to the corresponding frequency over the whole genome.
• CpG islands are several hundred base pairs long and mostly found in the 5' region of genes.
• One aspect of the present invention is a method of predicting/determining the responsiveness of a cancer disease to treatment with an inhibitor of CD95/CD95L signalling comprising the steps of
• the cancer disease is considered to be responsive to said treatment if the methylation at defined CpGs level is ⁇ 98%, ⁇ 95%, ⁇ 90%, ⁇ 85%, ⁇ 80%, or ⁇ 75%.
• a methylation level of 100% denotes that in a given sample in all DNA copies the respective CpG sites are methylated.
• the methylation level of a DNA sequence may be determined by any method known in the art.
• the methylation level can be determined by the MassARRAY technique (Sequenom, San Diego, CA, USA). This technique is based on detection of mass shifts introduced through sequence changes following bisulfite treatment.
• a further aspect of the present invention is a method of treating a cancer disease comprising the steps of
• the to be classified may be any malignant disease, in particular cancer disease or malignancy derived from tissues from epithelial or other than epithelial tissues known in the art.
• the cancer is characterized by increased CD95 and/or CD95L transcription and/or expression.
• the cancer disease may be an epithelial or hematological cancer.
• the cancer disease may be a cancer of lymphoid or myeloid origin. It may be any type of cancer, in particular solid tumor tissue.
• the cancer or other malignant disease can be selected from the group consisting of brain tumors, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, lung cancer, renal cancer, liver cancer or/and metastatic disease thereof.
• the malignant disease is glioma, in particular glioblastoma.
• the cancer can be newly diagnosed and/or progressive glioblastoma.
• Responsiveness of a cancer disease to a specific treatment may be evaluated by any method known in the art.
• a non-limiting prognostic factor to be considered when evaluating responsiveness of a cancer disease is tumor size.
• Objective radiological responses, increased progression-free survival or overall survival, set intervals thereof and objective clinical improvement are indicative of responsiveness of a cancer. Further definitions can be found in the examples section.
• the samples to be obtained from a patient can be obtained by any method known in the art such as initial surgery and/or biopsy and also samples from blood or cerebrospinal fluid.
• the samples are obtained from tumor tissue.
• the inventive method is an in vitro method.
• treatment of the cancer disease is effected by administration of an inhibitor of CD95/CD95L signalling, alone or in combination with other treatments and/or chemotherapeutic agents.
• Other treatments may be any treatments known in the art, such as radiotherapy and/or surgery.
• Examples for preferred chemotherapeutic agents are alkylating agents such as temozolomide and/or nitrosoureas.
• Other treatments are targeted based therapies like kinase inhibitors or antibody therapy, e.g. based on bevacizumab. Conventionally a combination of radiotherapy and temozolomide is used.
• treatment is preferably effected by the administration of an inhibitor of CD95/CD95L signalling in combination with radiotherapy.
• Radiotherapy increases the permeability of the blood-brain-barrier (BBB), which may lead to oedema and potentially worsening of neurological symptoms.
• BBB-permeability may facilitate agents entering the tumour stroma and the brain parenchyma surrounding the tumour, specifically the invasive front.
• CD95, CD95R and CD95 receptor may be used interchangeably.
• Further synonyms are Apo-1 or Fas which may be used interchangeably herein.
• CD95L, CD95 ligand and the corresponding synonyms FasL, Apo-1 L, CD178 or TNF-SF6 may be used interchangeably.
• CD95/CD95L signalling is known to a person skilled in the art. It refers to the CD95/CD95L signalling pathway and comprises any component or interaction of this signalling pathway.
• the components are not restricted to a specific class of molecules.
• such a component may be a protein but it may also be a nucleic acid or a small molecule.
• Non-limiting examples include the proteins CD95, CD95 ligand, Yes, FADD, PI3K, GSK-3 , ⁇ -Catenin, JNK, ERK1/2, AKT, NFKB or MMPs or the respective nucleic acid sequences.
• Genes involved in CD95/CD95L signalling are, e.g., genes which encode components of the CD95/CD95L signalling pathway as specified above.
• the "DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling” may be any type of DNA sequence.
• upstream of a gene refers to the 5' region of a gene.
• this DNA sequence may be part of a regulatory sequence and/or a CpG island, or it may comprise a regulatory sequence and/or a CpG island, as well as flanking regions.
• the DNA sequence may comprise or be comprised by a regulatory sequence or the DNA sequence may comprise or be comprised by a CpG island.
• the length of the DNA sequence may depend on the specific type of cancer disease and/or the specific gene involved in CD95/CD95L signalling.
• the DNA sequence may be > 100 nucleotides long, preferably > 50 nucleotides or > 10 nucleotides.
• the DNA sequence can also be from 1 -10 nucleotides in length.
• the DNA sequence to be methylated consists of one nucleotide.
• the DNA sequence is C at position 135 in SEQ ID NO: 2, denoted as CpG1 , and/or C at position 180 of SEQ ID NO: 2, denoted as CpG2 (based on Human Feb. 2009 (GRCh37/hg19) Assembly), ranging from chrl :172,628,000- 172,628,120 (reference genome GrCh37).
• inhibitor of CD95/CD95L signalling or “inhibitor of the CD95/CD95L signalling pathway” in terms of the present invention may be used interchangeably and may be any compound which interferes or blocks at least partially the CD95/CD95L signalling pathway. According to a preferred embodiment an "inhibitor of CD95/CD95L signalling” blocks the CD95/CD95L signalling pathway.
• Methods for determining and/or assessing CD95/CD95L signalling pathway activity are known to the person skilled in the art and are, for example, described by Lavrik et. al. (Cell Death Differ. 2012 Jan;19(1 ):36-41 Regulation of CD95/Fas signalling at the DISC).
• An inhibitor used in a method according to the invention may act on the protein level and/or the nucleic acid level.
• Inhibitors acting on the protein level may be selected from antibodies, proteins and/or small molecules.
• Inhibitors acting on the nucleic acid level are for example antisense molecules, RNAi molecules and/or ribozymes.
• the inhibitor binds to the CD95 receptor (CD95) and/or the CD95 ligand (CD95L).
• CD95 CD95 receptor
• CD95L CD95 ligand
• the inhibitor used in a method according to the invention is an antibody or a functional fragment thereof.
• the inhibitor being an antibody may bind to CD95, but, of course, also to CD95L.
• An example for an antibody binding CD95L is Nok-1 .
• the antibody may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a multi-specific antibody, or an antibody fragment thereof (e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody, or a single chain antibody molecule).
• the antibody can be of the lgG1 -, lgG2-, lgG3- or lgG4-type.
• the antibody may be used with or without modification, and may be labelled, either covalently or non-covalently, with, for example, a reporter group or an effector group.
• an "antibody fragment” used in a method according to the invention presents essentially the same epitope binding site as the corresponding antibody does and/or has substantially the same CD95 and/or CD95L inhibiting activity as the corresponding antibody has.
• CD95 ligand inhibitors may be selected from (a) an inhibitory anti-CD95 ligand-antibody or a fragment thereof as outlined above; (b) a soluble CD95 receptor molecule or a CD95 ligand-binding portion thereof; and (c) a CD95 ligand inhibitor selected from FLINT, DcR3 or fragments thereof.
• Soluble CD95 receptor molecules e.g. a soluble CD95 receptor molecule without transmembrane domain are described in EP-A-0 595 659 and EP-A- 0 965 637 or CD95 receptor peptides as described in WO 99/65935
• the Fas ligand inhibitor FLINT or DcR3 or a fragment, e.g. soluble fragment thereof, for example the extracellular domain optionally fused to a heterologous polypeptide, particularly a Fc immunoglobulin molecule is described in WO 99/14330 or WO 99/50413.
• FLINT and DcR3 are proteins which are capable of binding the CD95 ligand.
• the inhibitor is a fusion protein, in particular a fusion protein that binds to a CD95L.
• the inhibitor of CD95/CD95L signalling comprises a fusion protein comprising at least an extracellular CD95 domain or a functional fragment thereof and at least a Fc domain or a functional fragment thereof, an anti-CD95L specific antibody or a CD95L recognising fragment thereof, an anti-CD95 specific antibody or a CD95 recognising fragment thereof, a small molecule and/or combinations thereof.
• the fusion protein is selected from APG101 as set forth in SEQ ID NO: 1 , polypeptides having at least 70% sequence identity to APG101 and/or functional fragments thereof.
• Fusion proteins comprising the extracellular domain of the death receptor CD95 (Apo-1 ; Fas) fused to an immunoglobulin Fc domain are described in PCT/EP04/03239, the disclosure of which is included herein by reference.
• Fusion protein includes a mixture of fusion protein isoforms, each fusion protein comprising at least an extracellular CD95 domain (APO-1 ; Fas) or a functional fragment thereof and at least a second domain being an Fc domain or a functional fragment thereof distributing within a pi range of about 4.0 to about 8.5. Accordingly, the extracellular CD95 domain as used herein may be also called “first domain”, while the Fc domain may be called "second domain”.
• the mixture of fusion proteins can be provided in a composition.
• the first protein domain is an extracellular CD95 domain, preferably a mammalian extracellular domain, in particular a human protein, i.e. a human extracellular CD95 domain.
• the first domain, i.e. the extracellular CD95 domain, of the fusion protein preferably comprises the amino acid sequence up to amino acid 170, 171 , 172 or 173 of human CD95 (SEQ ID NO. 1 ).
• a signal peptide (e.g. position 1 -25 of SEQ ID NO: 1 ) may be present or not. Particularly for therapeutic purposes the use of a human protein is preferred.
• the fusion protein can comprise one or more first domains which may be the same or different.
• One first domain i.e. one extracellular CD95 domain is preferred to be present in the fusion protein.
• the Fc domain or functional fragment thereof i.e. the second domain of the fusion protein used in method according to the invention, comprises the CH2 and/or CH3 domain, and optionally at least a part of the hinge region, or a modified immunoglobulin domain derived therefrom.
• the immunoglobulin domain may be an IgG, IgM, IgD, or IgE immunoglobulin domain or a modified immunoglobulin domain derived, therefrom.
• the second domain comprises at least a portion of a constant IgG immunoglobulin domain.
• the IgG immunoglobulin domain may be selected from lgG1 , lgG2, lgG3 or lgG4 domains or from modified domains therefrom.
• the second domain is a human Fc domain, such as a IgG Fc domain, e.g. a human lgG1 Fc domain.
• the fusion protein can comprise one or more second domains which may be the same or different.
• One second domain, i.e. one Fc domain is preferred to be present in the fusion protein.
• both the first and second domains are preferably from the same species.
• the first domain i.e. the extracellular CD95 domain or the functional fragment thereof may be located at the N- or C-terminus.
• the second domain i.e. the Fc domain or functional fragment may also be located at the C- or N-terminus of the fusion protein.
• the extracellular CD95 domain at the N-terminus of the fusion protein is preferred.
• the fusion protein is APG101 (CD95-FC, position 26-400 in SEQ ID NO: 1 ).
• APG101 is a CD95L-binding protein consisting of the extracellular domain of human CD95 fused to the Fc region of human lgG1 . It interferes with CD95-dependent signalling by binding to CD95L, thereby blocking subsequent CD95-dependent activation [19].
• APG101 can be a fusion protein comprising a human extracellular CD95 domain (amino acids 26-172) and a human lgG1 Fc domain (amino acids 172-400), further optionally comprising an N- terminal signal sequence (e.g. amino acids 1 -25 of SEQ ID NO: 1 ).
• the presence of the signal peptide indicates the immature form of APG101 .
• the signal peptide is cleaved off.
• the signal sequence is cleaved off.
• APG101 with the signal sequence being cleaved off is also comprised by the term "unmodified APG101 ".
• the fusion protein is a polypeptide having at least 70% identity, more preferably 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity with APG101 .
• identity relates to the extent to which two amino acid sequences being compared are invariant, in other words share the same amino acids in the same position.
• APG101 includes a fusion protein of position 26-400 of SEQ ID NO: 1 , with and without a signal peptide.
• APG101 also includes fusion proteins containing N-terminally truncated forms of the CD95 extracellular domain.
• the fusion protein used in a method according to the invention is a functional fragment of APG101 .
• fragment generally designates a "functional fragment", i.e. a fragment or portion of a wild-type or full-length protein which has essentially the same biological activity and/or properties as the corresponding wild-type or full-length protein has.
• designing a fusion protein of the present invention comprises a selection of the terminal amino acid(s) of the first domain and of the second domain in order to create at least one amino acid overlap between both domains.
• the overlap between the first and the second domain or between the two first domains has a length of preferably 1 , 2 or 3 amino acids. More preferably, the overlap has a length of one amino acid. Examples for overlapping amino acids are S, E, K, H, T, P, and D.
• fusion protein includes a mixture of isoforms.
• isoform designates different forms of the same protein, such as different forms of APG101 , in particular APG101 without signal sequence.
• Such isoforms can differ, for example, by protein length, by amino acid, i.e. substitution and/or deletion, and/or post- translational modification when compared to the corresponding unmodified protein, i.e. the protein which is translated and expressed from a given coding sequence without any modification.
• Different isoforms can be distinguished, for example, by electrophoresis, such as SDS- electrophoresis, and/or isoelectric focussing which is preferred according to the present invention.
• Isoforms differing in protein length can be, for example, N- terminally and/or C-terminally extended and/or shortened when compared with the corresponding unmodified protein.
• a mixture of APG101 isoforms used in a method according to the invention can comprise APG101 in unmodified form as well as N- terminally and/or C-terminally extended and/or shortened variants thereof.
• the mixture used in a method according to the invention comprises N-terminally and/or C-terminally shortened variants of APG101 .
• a mixture of fusion protein isoforms comprising N- terminally shortened fusion proteins.
• Such N-terminally shortened fusion proteins may comprise -1 , -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, -13, -14, -15, -16, -17, -18, -19, -20, -21 , -22, -23, -24, -25, -26, -27, -28, -29, -30, -35, -40, -45 and/or -50 N-terminally shortened variants of unmodified APG101 .
• Particularly preferred are -17, -21 and/or -26 N-terminally shortened variants.
• the numbering refers to the APG101 protein including signal sequence according to SEQ ID NO: 1 .
• the shortened fusion proteins can comprise a sequence SEQ ID NO: 1 N-terminally truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 and/or 50 amino acids.
• Preferred shortened fusion proteins have SEQ ID NO: 1 N-terminally truncated by 16, 20, or 25 amino acids.
• APG101 isoforms An example for a C-terminal shortening of APG101 isoforms is C-terminal Lys-clipping.
• the mixture of fusion proteins used in a method according to the present invention preferably comprises 50 mol-% unmodified APG101 in relation to modified isoforms, more preferably 40 mol-% unmodified APG101 , more preferably 30 mol-% unmodified APG101 , more preferably 20, more preferably 10 mol- % unmodified APG101 , more preferably 5 mol-% unmodified APG101 and even more preferably 3 mol-% unmodified APG101 and most preferably 1 mol-% and/or less unmodified APG101 .
• Most preferred is an embodiment comprising a mixture of fusion protein isoforms that does not comprise any unmodified APG101 .
• isoforms can also differ by amino acid substitution, amino acid deletion and/or addition of amino acids.
• Such a substitution and/or deletion may comprise one or more amino acids.
• the substitution of a single amino acid is preferred according to this embodiment.
• Post-translational modification may involve, without being limited thereto, the addition of hydrophobic groups, in particular for membrane localisation such as myristoylation, palmitoylation, isoprenylation or glypiation, the addition of cofactors for enhanced enzymatic activity such as lipoyation, the addition of smaller chemical groups such as acylation, formylation, alkylation, methylation, amidation at the C-terminus, amino acid addition, ⁇ - carboxylation, glycosylation, hydroxylation, oxidation, glycilation, biotinylation and/or pegylation.
• hydrophobic groups in particular for membrane localisation such as myristoylation, palmitoylation, isoprenylation or glypiation
• cofactors for enhanced enzymatic activity such as lipoyation
• the addition of smaller chemical groups such as acylation, formylation, alkylation, methylation, amidation at the C-terminus, amino acid addition, ⁇ - carboxylation
• sialic acids Fc-based glycosylation, in particular Fc-based N-terminal glycosylation, and/or pyro- Glu-modification are preferred embodiments of post-translational modification.
• the fusion proteins used in a method according to the invention may comprise further domains such as further targeting domains, e.g. single chain antibodies or fragments thereof and/or signal domains.
• the fusion protein used in a method according to the invention may comprise an N- terminal signal sequence, which allows secretion from a host cell after recombinant expression.
• the signal sequence may be a signal sequence which is homologous to the first domain of the fusion protein.
• the signal sequence may also be a heterologous signal sequence.
• the fusion protein is free from an additional N-terminal sequence, such as a signal peptide.
• the fusion protein as described herein may be an N-terminally blocked fusion protein, which provides a higher stability with regard to N-terminal degradation by proteases, as well as a fusion protein having a free N- terminus, which provides a higher stability with regard to N-terminal degradation by proteases.
• Modifications blocking the N-terminus of protein are known to a person skilled in the art.
• a preferred post-translational modification according to the present invention blocking the N-terminus is the pyro-Glu- modification.
• Pyro-Glu is also termed pyrrolidone carboxylic acid.
• Pyro-Glu- modification according to the present invention relates to the modification of an N-terminal glutamine by cyclisation of the glutamine via condensation of the a-amino group with a side chain carboxyl group. Modified proteins show an increased half-life. Such a modification can also occur at a glutamate residue.
• Particularly preferred is a pyro-Glu-modification, i.e. a pyrrolidone carboxylic acid, with regard to the N-terminally shortened fusion protein -26.
• a mixture as described herein may comprise 80-99 mol-% N-terminally blocked fusion proteins and/or 1 -20 mol-% fusion proteins having a free N- terminus.
• the mixture as described herein comprises 0.0 to 5.0 mol-%, more preferably 0.0 to 3.0 mol-% and even more preferably 0.0 to 1 .0 mol-%, of fusion protein high molecular weight forms such as aggregates.
• the mixture does not comprise any aggregates of fusion protein isoforms, in particular no dimers or aggregates of APG101 . Dimers or aggregates are generally undesired because they have a negative effect on solubility.
• the inhibitor is a nucleic acid effector molecule.
• the nucleic acid effector molecule may be DNA; RNA, PNA or an DNA-RNA-hybrid. It may be single stranded or double stranded.
• Expression vectors derived from retroviruses, adenovirus, herpes or vaccina viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
• the nucleic acid effector molecule may be in particular selected from antisense molecules, RNAi molecules and ribozymes which are preferably capable of inhibiting the expression of the CD95R and/or CD95L gene.
• An inhibitor of the CD95 signalling pathway used in a method according to the present invention course may be provided as a pharmaceutical composition.
• This composition may comprise pharmaceutically acceptable carriers, diluents and/or adjuvants, etc.
• compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
• the determination of an effective dose is well within the capability of those skilled in the art.
• a therapeutically effective dose refers to that amount of active ingredient, for example a nucleic acid or a protein of the invention or an antibody, which is sufficient for treating a specific condition.
• the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment.
• Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.
• a person skilled in the art is aware of further methods to provide sufficient levels of the active moiety and/or to maintain the desired effect.
• Factors which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
• the total amount of the inhibitor of the CD95 signalling pathway according to the present invention to be administered for a patient suffering from MDS is from about 50 to about 600 mg/week, preferably from about 50 to about 400 mg/week and more preferably about 100 to about 200 mg/week.
• the preferred weekly dose can be administered as a single dose or as several doses. Especially preferred is a single dose particularly from 100 to 200 mg/week which is administered intravenously as a single dose.
• the treatment can last for several weeks. In each individual case, the duration of the treatment is determined by the supervising doctor and is e.g. based on the success of the treatment, the occurrence of side effects etc.
• a further aspect of the present invention is a CD95L inhibitor for use in the treatment of a cancer disease in a subject selected from the group consisting of patients exhibiting a methylation level of a DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling in a sample obtained from the patient of ⁇ 98%, ⁇ 95%, ⁇ 90%, ⁇ 85%, ⁇ 80%, or ⁇ 75%.
• a still further aspect of the present invention is a method of selecting a treatment for a cancer disease, comprising the steps of
• a treatment with an inhibitor of CD95/CD95L signalling is selected, if the methylation level is ⁇ 98%, ⁇ 95%, ⁇ 90%, ⁇ 85%, ⁇ 80%, or ⁇ 75%.
• the methylation level of CpGs contained in SEQ ID NO: 2 located at chrl :172,628,020 (CpG2) and chrl :172,628,065 (CpG1 ) (GRCh37/hg19) are used as a biomarker for the prediction/determination of diseases.
• diseases are cancer diseases such as epithelial or hematological cancer.
• the cancer disease may be a cancer of lymphoid or myeloid origin. It may be any type of cancer, in particular solid tumor tissue.
• the cancer disease can be selected from the group consisting of brain cancer, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, lung cancer, renal cancer, liver cancer or/and metastatic disease thereof.
• the cancer disease is brain tumor such as but not limited to glioblastoma, glioma, astrocytoma, oligodendroglioma, ependymoma, in particular glioblastoma.
• the cancer can be newly diagnosed and/or progressive glioblastoma.
• Figure 1 Trial design and CONSORT flow chart. Patients were
• Figure 2 Kaplan-Meier survival estimates. Data of PFS (panel a) or OS (panel b) were analyzed by treatment arm.
• Figure 3 Supplementary Figure 1. Overview of the genomic position of cg10161121 , cg06983746 and CD95 ligand.
• APG 101 was given at 400 mg weekly as a 30-minute i.v. infusion. Neuropathological Methods
• Archived tumour tissue was available from 81 patients. This tumour tissue was used to examine the neuropathological markers IDH1 , MGMT, CD95 and CD95L, as well as genome-wide methylation levels in a discovery set of 20 patients.
• CD95L promoter methylation at probes interrogating two CpGs located at chrl 172.628,020 (CpG2) and chrl :172,628,065 (CpG1 ) (GRCh37/hg19) was then examined in a validation set of 40 patients by MassARRAY. For clinical data evaluations, a median based cut-off for each CpG was used (see Supplementary Methods and Tables S1-4).
• the primary endpoint was the proportion of patients free of progression based on the central assessment and alive at six months (PFS-6), calculated in days from randomisation.
• the sample size of the study was planned according to the optimal two- stage design of Simon 24 for the rRT+APG101 arm with a PFS-6 target rate of 30%, a non-interesting rate of 15%, first-type error rate of 0.05 and a power of 80%.
• a control arm of patients treated with rRT alone was added to the Simon design to calibrate the PFS-6 rate.
• the sample size of the control arm was defined as 50% of the investigational treatment arm.
• the Simon design required the recruitment of 55 patients for the rRT+APG101 arm (19 patients in Stage 1 and 36 patients in Stage 2). With the addition of 28 patients in the control arm, the study was hence planned with a total sample size of 83 patients.
• the study was considered positive if 13 PFS- 6 responses were observed among the 55 patients treated with rRT+APG101 , based on the assumption that five PFS-6 responses would be seen in the control arm.
• Secondary efficacy endpoints were: objective response rates, OS, PFS, quality-of-life as determined by EORTC PAL QLQ-C15 and BN-20, and cognitive function determined by MMSE.
• Safety and tolerability of APG101 were assessed by adverse events with intensity mild/moderate/severe.
• PFS was defined as time from randomisation to next progression for patients with progression or, respectively, as time to death of any cause for patients without progression described with Kaplan-Meier estimates. Patients without progression or death were censored at the day of the last assessment of tumour response. The significance level for remarkable findings was set to 0.05 for all tests in this study.
• the ITT population included 84 patients who were randomised and received at least one dose of APG101 or rRT.
• the per-protocol population consisted of 72 patients (Fig. 1 ).
• median follow-up was 1 1 .4 months in both treatment arms. Baseline patient and disease characteristics were well balanced (Table 1 ).
• Tumour tissue was analysed for IDH1 R132H mutation (7/84, 8.4%), MGMT promoter methylation (57/84, 67.9%), expression of the APG101 target pathway, CD95 and CD95L, as well as CpG methylation analysis upstream of CD95L (Supplementary Figure 1).
• CD95L is the target of APG101 .
• APG101 to rRT produces a relevant number of patients with first or second progression of a glioblastoma without tumour progression at six months after randomisation.
• CD95L promoter methylation can be used as a selection marker. Expression of CD95L seems to be associated with impaired prognosis in other malignancies as well [24].
• glioblastoma The main reason for a poor outcome in glioblastoma is the therapy resistance, the highly invasive behaviour of the tumour as well as the local immunosuppression. While for patients with newly diagnosed glioblastoma the current standard of care is radiochemotherapy with temozolomide, no such standard exists for progressive disease. Therapeutic options at recurrence depend on the individual disease situation and include reoperation, rRT, alkylating chemotherapy with temozolomide or nitrosoureas, bevacizumab and experimental agents within clinical trials. By targeting the invasive growth, APG101 addresses a pathological hallmark of glioblastoma different from all previous approaches.
• the present study was designed to identify a therapeutic effect of APG101 when combined with rRT in the treatment of progressive glioblastoma.
• the study population was selected with regard to tumour size, prior first-line therapy, and the long time frame between first-line therapy and first or second progression, as needed for a second RT.
• the latter is documented by the high number of patients with MGMT promoter methylation (Table 1 ).
• MRI and RT dummy runs had to be completed by all sites, and all sites had to obtain central approval prior to participation in the study.
• MRIs were assessed centrally in a blinded fashion and a strict algorithm to identify pseudoprogression was used. All measures ensured the accuracy of the observed therapeutic effects, however, the effect of rRT on PFS is at the lower end of what was reported in scientific publications.
• tumour tissue for the expression of CD95 and CD95L had been specified in the study protocol. Methylation analyses were also carried out. Both tests were carried out on identical archived tumour samples obtained during surgery at the time of diagnosis, and provided quantitative data with an easy to reproduce PCR-based assay. Patients with low methylation levels at two CpG upstream of CD95L showed the best response to treatment with APG101 .
• rRT+APG101 represents a therapeutic chance, especially for the subset of patients with the option for second RT.
• CD95/CD95L inhibition may also be exploited in newly diagnosed glioblastoma patients.
• Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Res 61 :2759-2765.
• Non-apoptotic Fas signaling regulates invasiveness of glioma cells and modulates MMP-2 activity via NFkappaB-TIMP-2 pathway.
• Sublethal irradiation promotes migration and invasiveness of glioma cells: implications for radiotherapy of human glioblastoma. Cancer Res 61 :2744- 2750.
• CD95L CD95 ligand
• IDH isocitrate dehydrogenase
• MGMT O6- methylguanine-DNA methyltransferase
• Cutaneous AE Dermatitis, 2 2 0 12 5 allergic rash, alopecia
• n Cutaneous AE (dermatitis, 2 2 0 12 5 allergic rash, alopecia), n
• tumour size considering the largest cross-sectional area or contrast-enhancement in the radiation field of >25% in the first or second scan post RT was called pseudoprogression and not deemed a progression until further confirmation on follow up. Further progression resulted in backdating to the scan of the initial suspicion of a progression and stable disease on follow-up in retrospective rating as stable. Stable or decreasing contrast enhancement resulted in a continuation of trial treatment and/or follow up.
• mutated IDH1 R132H protein was determined by immunohistochemistry [1 ].
• the MGMT promoter methylation status was analysed after bisulfite treatment by methylation-specific PCR [2].
• Expression of CD95 and CD95L was determined by immunohistochemistry. All CD95- and CD95L-stained slides were evaluated slide-by-slide in a single session by a board-certified neuropathologist (C.H.). Vital tumour tissue of each slide was evaluated regarding the CD95 and CD95L staining intensities 'high', 'moderate', 'low' and 'absent'. CD95 and CD95L calibration figures were used to standardize the evaluation.
• Unsupervised hierarchical clustering was performed after removing probes (i) targeting the X and Y chromosomes, (ii) containing a single nucleotide polymorphism within 5 base pairs of and including the CpG site and (iii) not mapping uniquely to the human reference genome (hg19), allowing for one mismatch. Student's t test assuming unequal variances was used to detect probes with significantly different mean methylation between the two groups.
• o APG101 was given i.v. once per week at 400 mg as an 30 min infusion
• Unsupervised hierarchical clustering of the discovery cohort identified 4 of the 10 responders to carry a hypermethylator phenotype (G-CIMP) [4]. However, the remaining 16 patients did not cluster distinctively depending on treatment response. As G-CIMP positive tumors most likely represent an epigenetically distinct entity [5], we excluded these 4 patients from further analysis.
• G-CIMP positive tumors most likely represent an epigenetically distinct entity [5].
• HR hazard ratio
• RT radiotherapy
• CI confidence interval

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