WO2009098656A2 - Method for predicting or diagnosing outcome of intracranial tumors in a subject - Google Patents

Abstract

The present invention relates to a method for diagnosing or predicting outcome of intracranial tumors in the cerebrospinal fluid of a subject. The present invention further relates to compositions and methods for treatment or prevention of intracranial tumors in a subject and to a kit useful for predicting same.

Description

METHOD FOR PREDICTING OR DIAGNOSING OUTCOME OF INTRACRANIAL TUMORS IN A

SUBJECT

FIELD OF THE INVENTION

The present invention relates to a method for diagnosing or predicting outcome of intracranial tumors in the cerebro-spinal fluid of a subject. The present invention further relates to compositions and methods for treatment or prevention of intracranial tumors in a subject and to a kit useful for predicting same.

BACKGROUND OF THE INVENTION

Glioblastoma is the most aggressive and most common type of primary brain tumors. Despite surgery, and combined chemo -radiotherapy patients ultimately recur with a median survival of 15 months. Thus, new avenues have to be taken to improve treatment strategies for this devastating disease.

The identification of molecular markers predictive for response to therapy would allow individually tailored treatment modalities, improving outcome and quality of life of the patients. In neuro-oncology the possibility of using easily and repeatedly accessible patient material for diagnostic purposes, such as plasma or cerebrospinal fluid (CSF) obtainable by marginally invasive procedures, would greatly facilitate patient management and follow up.

GDF- 15, also known as Macrophage-inhibitory cytokine- 1 (MIC-I), PLAB, prostate-derived factor, PTGF-β, PTGFB, PDF, NRG-I, and NAG-I, is a divergent member of the transforming growth factor β superfamily . The placenta is the only tissue that expresses large amounts of GDF- 15 under normal physiologic conditions. However, a wide variety of epithelial cells, including the neuroepithelium express low levels of GDF- 15 mRNA. GDF- 15 protein is synthesized as a 308-amino acid propeptide which, when secreted, binds to local extracellular matrix and subsequently becomes cleaved by a furin-like protease. The mature peptide, which is secreted by an alternate pathway, is a 112-amino acid protein which diffuses rapidly into the circulation. GDF- 15 has been shown by several groups to induce apoptosis and local GDF- 15 expression in the stroma of the malignant prostate gland, and has been linked to improved outcome.

Although changes in serum GDF- 15 levels are associated with a number of disease conditions, they are mostly strongly linked to cancer(Bauskin et al, 2006). Increased GDF-15 expression has been documented in a variety of epithelial cancer cell lines, including breast, pancreas, colorectal, and prostate cancers. Microarray studies have revealed increased expressions of GDF-15 in patients with breast cancer, and serum GDF-15 levels are the best single predictor of the presence of pancreatic carcinoma. In colon cancer, increasing GDF-15 expression is associated with the progression of colonic adenomas to invasive cancer and subsequent metastasis, with serum levels at presentation being an independent predictor of subsequent disease-free and overall survival.

GDF-15 has recently been described as one of the 20 best cancer biomarkers based on transcriptional profiling of a broad range of tumor types, in particular of epithelial origin, including renal cell carcinoma, colon adenocarcinoma, ovarian, esophageal cancers and melanoma (Basil et al., 2006), and protein concentrations in the plasma may have potential clinical utility in monitoring diagnosis and/or progression in colorectal (Brown et al., 2003), prostate (Brown et al., 2006), and pancreatic cancer . Quantification of GDF-15 mRNA expression and/or protein secretion in cancer patients has revealed a significant up-regulation in advanced or more aggressive tumors lesions of epithelial origin as compared to respective noncancerous tissues or less aggressive tumors.

GDF-15 was originally identified as macrophage inhibitory cytokine 1 (MIC-I) using a cDNA subtraction library enriched for genes associated with macrophage activation (Bootcov et al., 1997). It is a divergent member of the TGF-beta superfamily and encodes a 308-amino acid (40-kDa) precursor polypeptide. During protein processing, the newly synthesized polypeptide is cleaved by a furin-like protease releasing a 112-amino acid mature protein. The mature protein is secreted as a 25-kDa disulfϊde-linked homodimer (Bootcov et al., 1997). High expression has been reported from placenta and adult prostate tissue, lower levels are detectable in liver, kidney, pancreas and fetal brain. In the brain, immunochistochemical analysis has revealed that GDF-15 protein is synthesized by the neuro-epithelium of the choroid plexus and secreted into the CSF. There is a profound need to develop a more accurate marker of intracranial tumors and in particular glioblastoma which could improve the early detection and prognosis of this deadly disease. Furthermore, effective methods and compositions for treatment or prevention of this condition are needed. The main problem is that to date, no efficient methods or strategies have been developed to overcome this problem.

SUMMARY OF THE INVENTION

The aim of the present invention was to evaluate the utility of GDF- 15 (GDF 15; REFSEQ: accession NM_004864) as a potential molecular marker for intracranial tumors that can be measured repeatedly in easy accessible body fluids. Glioblastoma, the most malignant form of primary brain tumors was significantly associated with enhanced concentrations of GDF- 15 in the CSF. In addition, at recurrence GDF- 15 concentrations in the CSF were significantly higher than at initial diagnosis. Most interestingly, elevated GDF- 15 concentrations in the CSF were associated with poor prognosis. These data show that measurement of GDF- 15 concentrations in the CSF is of diagnostic value in patients with intracranial tumors and a prognostic value for glioblastoma patients, it allows measuring response to treatment, or relapse, and adapting therapy accordingly. Moreover, GDF- 15 measurements in the CSF require only a marginally invasive procedure that renders it an attractive biomarker for further development. In contrast, serum levels of GDF- 15 did not differ between brain tumor patients and the control cohort that was treated for non-cancerous diseases.

Human growth differentiation factor- 15 (GDF- 15) expression in cerebro-spinal fluid (CSF) of glioblastoma (GBM) patients has been evaluated and it unexpectedly results that elevated levels of GDF- 15 in the CSF of glioblastoma patients is associated with worse outcome Thus, the present invention has surprisingly shown that GDF- 15 in the CSF is a prognostic factor in patients with glioblastoma.

Thus, in a first aspect, the present invention provides a method of diagnosis or prognosis of intracranial tumors in a subject, the method comprising detecting an elevated amount of GDF- 15 in the CSF from said subject.

In a second aspect, the method provides a method of treatment of intracranial tumors and in particular glioblastoma in a subject said method comprising administering to said subject an effective amount of an agent which inhibits the activity or expression of GDF-15. In a third aspect, the present invention provides a method of treatment of intracranial tumors and in particular glioblastoma in a subject, said method comprising administering to said subject an effective amount of an agent which enhances or increases the activity or expression of GDF-15.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1

Concentrations of GDF-15 in CSF and plasma of patients. GDF-15 protein concentrations were measured by ELISA in CSF and plasma of patients with intracranial tumors or patients treated for non-neoplastic diseases ("normal"). The frequency and concentrations are shown separately for each tumor type. The broken lines indicate the limit of detection for GDF-15 at 156 pg/ml.

Figure 2

Association of GDF-15 concentration in the CSF of glioblastoma patients and outcome.

The presence of elevated concentrations of GDF-15 in the CSF at the time of operation is associated with decreased overall survival. The cut-off was set at the limit of detection of 156 pg/ml.

Figure 3

GDF-15 immunohistochemistry on glioblastoma tissue.

GDF-15 exhibits immunostaining on a subtype of infiltrative macrophages in human glioblastoma (A, B). Positively stained macrophages (A) here located in the perinecrotic region, close to pseudopalisades, both hallmarks of glioblastoma. Tumor cells appear negative.

Colon carcinoma (C) was used as positive control.

Figure 4

Gene expression levels of GDF-15. Expression of the GDF-15 transcript determined by qRT- PCR is relatively low in glioblastoma tissues as compared to prostate (A). Freshly isolated monocytes, and undifferentiated monocytoid cells (U937) exert low expression of GDF-15 which markedly increases upon differentiation using 65 ng/niL PMA for 48 hours. GDF-15 expression was normalized with RPOLII expression.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs.

As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The term "a protein" includes a plurality of proteins.

As used herein, the terms "peptide", "protein", "polypeptide", "polypeptidic" and "peptidic" are used interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Glioblastoma are notorious for resistance to therapy which has been attributed to DNA repair proficiency, a multitude of deregulated molecular pathways, and more recently to the particular biological behavior of tumor stem- like cells.

The present invention is useful to guide a rational choice of agents, targets, trial design, and appropriate patient selection, incorporating biomarkers defining mechanisms of response and resistance.

One object of the invention is to provide a method for diagnosing or predicting outcome of intracranial tumors in the cerebro-spinal fluid of a subject comprising:

(a) measuring the expression level of GDF-15, a proteotypic peptide derived thereof, or an immunogenic fragment thereof in the cerebro-spinal fluid of said subject,

(b) comparing the expression level of said GDF-15; a proteotypic peptide derived thereof, or immunogenic fragment thereof to threshold value, wherein the elevated levels of said GDF-15; a proteotypic peptide derived thereof, or immunogenic fragment thereof indicate worse outcome.

In the context of the invention, "proteotypic peptides" and "immunogenic fragments" refer to a part of a sequence containing less amino acids in length than the sequence of the peptide of the invention. This sequence can be used as long as it comprises sequences that are unique to the native sequence from which it derives, known as "proteotypic peptides" (Craig et al, 2005), or exhibits similar immunogenic properties "immunogenic fragments" as the native sequence from which it derives. Immunogenic fragments are fragments of the GDF-15 that can be used to make anti-bodies for detection. Preferably this sequence contains less than 30%, preferably less than 60%, in particular less than 90% amino acids in length than the respective sequence of the peptide of the invention. Preferably also these sequences contain at least 7, most preferably 25, more preferably 40, even more preferably 50 and still even more preferably 88 contiguous amino acids in length in common with sequence of the peptide of the invention. These fragments can be prepared by a variety of methods and techniques known in the art such as for example chemical synthesis. Preferably the intracranial tumor is a glioblastoma or brain metastasis and most preferably the intracranial tumor is a glioblastoma.

In the context of the invention, the measuring of the expression levels of GDF- 15 is preferably obtained through Western blot, immunoprecipitation, ELISA, Radio Immuno Assay, proteomics methods comprising but not limited to Mass Spectroscopy, or any other quantitative method detecting GDF- 15 that is known by the skilled in the art.

Optionally, the method according to the invention allows evaluating the medical prognosis of said subject based on the comparison of step (b), and/or adapting the treatment of said subject.

The term "adapting the treatment" generally refers to the choice of a treatment among different options, based on the specificities of the disease, concomitant pathologies or patient conditions, or the switch from one treatment to another in the course of the therapy because of the non-response, progression or resistance of the disease to the initial treatment, with the intent to offer to the patients the beast treatment for his diseases under the given circumstances.

The biological sample, used in the method of the invention, is a bio-fluid comprising cerebrospinal fluid, blood, urine, or biopsy of brain tumor.

Preferably, the biological sample is cerebrospinal fluid sample.

In the method of the present invention, the subject is a mammal and preferably a human.

As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment. However, in other embodiments, the subject can be a normal subject.

The present invention also relates to a method for treatment of intracranial tumors in a subject. The present invention encompasses the use of modulators of expression of GDF- 15 or a biologically active fragment thereof in the preparation of a medicament for the treatment or prevention of intracranial tumors in a subject.

GDF- 15 may be like other members of the TGF-beta superfamily, acting as a tumor suppressor in the early stages, but acting pro-tumorigenic at the later stages of tumor progression. The expression of GDF- 15 can be increased by treatment with drugs and chemicals documented to prevent tumor formation and development. GDF 15 is induced by multiple types of cellular stress, such as anoxia, DNA damaging agents, and NSAIDs, independent of p53 or HIF-I alpha. However, Applicants did not observe increased GDF 15 CSF concentrations in glioblastoma patients receiving alkylating agent chemotherapy compared to patients with measurements at the onset of surgery (chemo-naϊve). Applicants analyses of glioblastoma tissues show that inflammatory cells are the major source of GDF 15 in glioblastoma, in particular macrophages. Macrophages are increasingly recognized as a major contributor to progression of tumors via alternative activation of the NF-kappa-B (nuclear factor of kappaB) pathway. As GDF 15 may promote or suppress tumor growth in different situations, this also occurs in the brain and elevated CSF GDF 15 concentration is a marker of macrophage activation assisting tumor progression.

"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.

"Prevention" as used herein means that the administration of the modulator(s) as described results in a reduction in the likelihood that a subject at high risk for intracranial tumor, relapse and/or metastatic progression after targeted anti-tumor therapy, radiotherapy, chemotherapy, or combination thereof is applied. Preferably, in the context of the present invention, this phrase means that the administration of the modulator(s) results in the reduction of the likelihood or probability that a subject at risk will indeed develop intracranial tumors like for example glioblastoma. "Biologically active" means affecting any physical or biochemical properties of a living organism or biological process. Biologically Active Substance refers to any molecule or mixture or complex of molecules that exerts a biological effect in vitro and/or in vivo, including pharmaceuticals, drugs, proteins, peptides, polypeptides, hormones, vitamins, steroids, polyanions, nucleosides, nucleotides, nucleic acids (e.g. DNA or RNA), nucleotides, polynucleotides, etc.

In the present invention, said modulators of expression of GDF- 15 or biologically active fragment thereof are preferably inhibitors or competitors, which preferably comprise an antibody, or an immunologically active fragment thereof, that binds to a GDF- 15 protein or biologically active fragment thereof.

The present invention also relates to the use of modulators of the biological activity of GDF- 15 or a biologically active fragment thereof in the preparation of a medicament for treatment or prevention of intracranial tumors in a subject.

The competitors are compounds able to disturb interaction between on one hand a GDF- 15 protein, variants thereof, or a biologically active fragment thereof and on the other hand a receptor thereof.

A variant is a peptide having an amino acid sequence that differs to some extent from a native sequence peptide that is an amino acid sequence that varies from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles. The amino acid sequence variants possess substitutions, deletions, side-chain modifications and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, GIy II. Polar, positively charged residues: His, Arg, Lys III. Polar, negatively charged residues: and their amides: Asp, Asn, GIu, GIn

IV. Large, aromatic residues: Phe, Tyr, Trp

V. Large, aliphatic, nonpolar residues: Met, Leu, He, VaI, Cys.

It is to be understood that some non-conventional amino acids may also be suitable replacements for the naturally occurring amino acids. For example Lys residues may be substituted by ornithine, homoarginine, nor-Lys, N-methyl-Lys, N, N-dimethyl-Lys and N, N, N- trimethyl-Lys. Lys residues can also be replaced with synthetic basic amino acids including, but not limited to, N-I- (2-pyrazolinyl)-Arg, 2- (4-piperinyl)-Gly, 2- (4- piperinyl)-Ala, 2- [3- (2S) pyrrolininyl]-Gly and2- [3- (2S) pyrolininyl]-Ala. Tyr residues may be substituted with 4- methoxy tyrosine (MeY), meta-Tyr,ortho-Tyr, nor-Tyr, 1251 -Tyr, mono -halo -Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, and nitro-Tyr.

Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho-and O-phospho derivatives. Tyr residues can also be replaced with synthetic hydroxyl containing amino acids including, but not limited to4-hydroxymethyl-Phe, 4-hydroxyphenyl- GIy, 2, 6-dimethyl-Tyr and 5-amino-Tyr. Aliphatic amino acids may be substituted by synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 where n is a number from 1 up to and including 8. Examples of suitable conservative substitutions by non-conventional amino acids are given in W002/064740.

Insertions encompass the addition of one or more naturally occurring or non conventional amino acid residues.

Deletion encompasses the deletion of one or more amino acid residues.

Furthermore, since an inherent problem with native peptides (in L-form) is the degradation by natural proteases, the physiological active protein of the invention may be prepared in order to include D-forms and/or "retro -inverso isomers" of the peptide. Preferably, retro-inverso isomers of short parts, variants or combinations of the physiological active protein of the invention are prepared. Retro-inverso peptides are prepared for peptides of known sequence as described for example in SeIa and Zisman, in a review published in FASEB J. 1997 May;l l(6):449-56.

By "retro-inverso isomer" is meant an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted; thus, there can be no end-group complementarity.

The invention also includes analogs in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases. Where proteolytic degradation of the peptides following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as an active substance. Such mimetics, and methods of incorporating them into peptides, are well known in the art.

The term "inhibitor" or "antagonist" refers to molecules that inhibit the function of the protein or polypeptide by binding thereto.

The term "competitors" refers to "inhibitors" or "antagonists" that directly inhibit the interaction between a protein or polypeptide (i.e. receptor) and its natural ligand resulting in disturbed biochemical or biological function of the receptor. Competitive inhibition is a form of inhibition where binding of the inhibitor prevents binding of the ligand and vice versa. In competitive inhibition, the inhibitor binds to the same active site as the natural ligand, without undergoing a reaction. The ligand molecule cannot enter the active site while the inhibitor is there, and the inhibitor cannot enter the site when the ligand is there.

The "biological activity" of a protein refers to the ability to carry out diverse cellular functions and to bind other molecules specifically and tightly.

The present invention also includes vaccines and vaccination methods. For example, methods of treating or preventing intracranial tumors in a subject can involve administering to the subject a vaccine composition comprising one or more antibody directed against GDF- 15 or a biologically active fragment thereof and/or combinations thereof or immunologically active fragments of such polypeptides.

In the context of the present invention, an immunologically active fragment is a polypeptide that is shorter in length than the full-length naturally-occurring protein yet which induces an immune response analogous to that induced by the full-length protein. For example, an immunologically active fragment should be at least 8 residues in length and capable of stimulating an immune cell, for example, a T cell or a B cell. Immune cell stimulation can be measured by detecting cell proliferation, elaboration of cytokines (e.g., IL-2), or production of an antibody. See, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, 1998, Cold Spring Harbor Laboratory Press; and Coligan, et al, Current Protocols in Immunology, 1991-2006, John Wiley & Sons.

Usually the inhibitor of the biological activity of said protein is an antibody or an immunologically fragment thereof that binds to a GDF- 15 protein or biologically active fragment thereof.

As used herein, the term "antibody" refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto. Furthermore, an antibody can be a fragment of an antibody or a modified antibody, so long as it binds to one or more of the proteins encoded by the marker genes. For instance, the antibody fragment can be Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston J. S. et al., (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83.). More specifically, an antibody fragment can be generated by treating an antibody with an enzyme, including papain or pepsin. Alternatively, a gene encoding the antibody fragment can be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co M. S. et al., (1994) J. Immunol. 152:2968-76; Better M. and Horwitz A. H. (1989) Methods Enzymol. 178:476-96.; Pluckthun A. and Skerra A. (1989) Methods Enzymol. 178:497-515.; Lamoyi E. (1986) Methods Enzymol. 121 :652- 63.; Rousseaux J. et al, (1986) Methods Enzymol. 121 :663-9.; Bird R. E. and Walker B. W. (1991) Trends Biotechnol. 9:132-7.). An antibody can be modified by conjugation with a variety of molecules, for example, polyethylene glycol (PEG). The present invention provides such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.

Alternatively, an antibody can comprise a chimeric antibody having a variable region from a nonhuman antibody and a constant region from a human antibody, or a humanized antibody, comprising a complementarity determining region (CDR) from a nonhuman antibody, a frame work region (FR) and a constant region from a human antibody. Such antibodies can be prepared by using known technologies. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see, e.g., Verhoeyen et ah, (1988) Science 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species.

Fully human antibodies comprising human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom & Winter, (1992) J. MoI. Biol. 227:381-8). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in U.S. Patent Nos. 6,150,584; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016. Such antibodies can be prepared by using known technologies.

The present invention also relates to a pharmaceutical composition for the treatment or prevention of an intracranial tumors in a subject, said composition comprising a pharmaceutically effective amount of an antibody or an immunologically fragment thereof that binds to a GDF- 15 protein, biologically active fragment thereof and/or combinations thereof. The present invention further relates to a pharmaceutical composition for the treatment or prevention of intracranial tumors in a subject, said composition comprising a pharmaceutically effective amount of a compound able to modulate (by disturbing or enhancing) the interaction between a GDF- 15 protein or a biologically active fragment thereof and its receptor.

"A pharmaceutically effective amount" refers to a chemical material or compound which, when administered to a human or animal organism induces a detectable pharmacologic and/or physiologic effect.

The respective pharmaceutically effective amount can depend on the specific patient to be treated, on the disease to be treated and on the method of administration. Further, the pharmaceutically effective amount depends on the specific protein used, especially if the protein additionally contains a drug as described or not. The treatment usually comprises a multiple administration of the pharmaceutical composition, usually in intervals of several hours, days or weeks. The pharmaceutically effective amount of a dosage unit of the polypeptide usually is in the range of 0.001 ng to 100 mg per kg of body weight of the patient to be treated.

For systemic administration, a therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

Initial doses can also be estimated from in vivo data, e.g. animal models, using techniques that are well known in the art. One ordinarily skill in the art could readily optimise administration to humans based on animal data and will, of course, depend on the subject being treated, on the subject's weight, the severity of the disorder, the manner of administration and the judgement of the prescribing physician.

"Administering", as it applies in the present invention, refers to contact of the pharmaceutical compositions to the subject, preferably a human. The pharmaceutical composition may be dissolved or dispersed in a pharmaceutically acceptable carrier well known to those skilled in the art, for parenteral administration by, e. g., intravenous, subcutaneous or intramuscular injection or by intravenous drip infusion.

As to a pharmaceutical composition for parenteral administration, any conventional additives may be used such as excipients, adjuvants, binders, disintegrants, dispersing agents, lubricants, diluents, absorption enhancers, buffering agents, surfactants, solubilizing agents, preservatives, emulsifϊers, isotonizers, stabilizers, solubilizers for injection, pH adjusting agents, etc.

Acceptable carriers, diluents and adjuvants which facilitates processing of the active compounds into preparation which can be used pharmaceutically are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as

EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

The form of administration of the pharmaceutical composition may be systemic or topical. For example, administration of such a pharmaceutical composition may be various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, buccal routes or via an implanted device, and may also be delivered by peristaltic means.

The pharmaceutical composition comprising an active ingredient of the present invention may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support. In addition the matrix may be comprised of a biopolymer.

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.

It is understood that the suitable dosage of a peptide of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.

The appropriate dosage form will depend on the disease, the protein, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots.

The present invention further provides a method of treatment of intracranial tumors and in particular glioblastoma in a subject, said method comprising administering to said subject an effective amount of an agent which enhances or increases the activity or expression of GDF- 15. Examples of an agent which enhances or increases the activity or expression of GDF- 15 comprise agents intervening in p53 and hypoxia inducible factor 1 (HIF-I) independent pathways or EGR-I related pathways, inhibitors of cyclooxygenases, or biologically active fragments of GDF- 15, and/or combinations thereof. The present invention further relates to a kit useful for detecting or diagnosing the intracranial tumors in a subject or predicting outcome of a subject with a glioblastoma, said kit comprises a set of: a) antibodies adapted to detect GDF- 15 and/or immunogenic fragments thereof, b) a control reagent and/or a detectable label, and optionally instructions to use.

The detection reagents can be packaged together in the form of a kit. For example, the detection reagents can be packaged in separate containers, e.g., antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix), a control reagent (positive and/or negative), and/or a detectable label. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay can also be included in the kit. The assay format of the kit can be a sandwich ELISA, both of which are known in the art. See, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Edition, 2001, Cold Spring Harbor Laboratory Press; and Harlow and Lane, Using Antibodies, supra.

An advantage of the present invention is that for patients with brain tumors, identification of diagnostic and prognostic markers in easy accessible biological material, such as plasma or cerebro-spinal fluid (CSF), greatly facilitates patient management. GDF-15 (growth differentiation factor 15) encodes a secreted protein of the TGF-beta superfamily and emerged as a candidate marker from Applicant's gene expression studies, with increasing expression during malignant progression of glioma.

In Applicant's experiences, GDF-15 protein levels were determined by ELISA in the CSF of 94 patients operated for intracranial tumors. Respective plasma samples were available for 62 of the patients. As control Applicant analyzed the CSF and plasma of a patient cohort (n=53 and 50, respectively) treated for non-neoplastic diseases.

The results have shown that concentrations of GDF-15 in the CSF were significantly augmented in glioblastoma patients (median, 229 pg/ml) as compared to the control cohort

(p<0.0001, Wilcoxon rank sum test). In contrast, no elevated levels were observed in the plasma of these patients. Most interestingly, among patients with glioblastoma, high levels of GDF- 15 in the CSF was associated with shorter survival (p=0.007, log rank test). Molecular studies suggest that the source of GDF- 15 is likely a subpopulation of tumor infiltrating macrophages immunoreactive for GDF- 15. This is in accordance with hyper-methylation of the GDF-15 promoter in glioblastoma and increased GDF-15 expression observed upon differentiation of the macrophage-like cell-line U937.

In conclusion, GDF-15 protein measured in the CSF has a diagnostic value in patients with intracranial tumors, and has surprisingly been detected as being a prognostic marker in glioblastoma patients.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

EXAMPLES

Materials and Methods

Cerebrospinal fluid and plasma samples Applicant has assembled a collection of 94 frozen CSF samples at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, for the identification of prognostic factors in brain tumors, approved by the local ethics committee (protocol 39/04). CSF (n=64) had been collected during brain tumor surgery by lumbar puncture as part of a routine procedure aimed at controlling brain pressure during surgery. A second set of 30 CSF was collected postoperative during chemotherapy of glioma patients as described (Ostermann et al., 2004). Respective plasma samples were available for 66 of the brain tumor patients. Control CSF (n=53) and respective plasma (n=50) were obtained from the CSF-bank of the department of Neurology selecting patients treated for migraines or lumbar sciatic problems. Diagnosis of brain tumor patients was taken from respective pathology reports. For survival analysis only adult glioblastoma patients with CSF taken at the time of surgery were included (n=33, median age 58 years, 24 men and 9 women).

GDF-15 ELISA GDF-15 protein levels were measured using a sandwich ELISA with 26G6H6 antibody as previously described (Brown et al., 2002; Moore et al., 2000). ELISA plates (Maxisorb; Nunc) were coated with 26G6H6 (1 :500) in bicarbonate buffer (pH 9.4-9.8). Recombinant GDF-15 was diluted 1 :1000 with eight doubling dilutions (1000-7.8 pg/ml) and used as the standard curve for each plate in the assay. Coated plates were washed three times and then blocked by incubation with 250 μl of 1% BSA w/v at 37°C for 1 hour. The detection antibody, 233-BP (1 :25,000), was added to the wells and incubated for 16 hours at 4°C. Visualization was achieved with donkey, antisheep, biotinylated IgG (Jackson's Laboratories), Streptavidin-large volume kit conjugate (Genzyme), and o-phenylenediamine substrate (Sigma). The reaction was terminated with 2N H₂SO₄. Absorbance was measured at 490 nm. Each sample was assayed at minimum in duplicate and the coefficients of variations were always <10%. The limit of detection was 156 pg/ml of the diluted sample. Using this technology the limit of detection was used as cut-off for diagnosis or prediction outcome in glioblastoma patients.

GDF-15 Immunohistochemistry GDF-15 expression was determined by immunohistochemistry on paraffin embedded glioblastoma samples using a high temperature epitope retrieval method (5 min high pressure cooker, citrate buffer pH 6.0; anti-GDF-15, R&D systems, dilution 1 :100). Isolation of human monocytes and cell culturing.

Human peripheral blood monocytes were obtained from whole blood of healthy volunteers by dextran-sedimentation of the leukocytic fraction according to the Nycoprep protocol (AXIS- SHIELD, Oslo, N). Purified monocytes were cultured in Macrophage Serum Free Medium (Invitrogen). One million U937 cells were seeded on a lOcm-diameter Petri dish and differentiated using 65 ng/ml Phorbol 12-myristate 13-acetate (PMA) for 48 hours.

Quantitative assessment of GDF- 15 mRNA levels by real-time RT-PCR

Total RNA was extracted from glioblastoma tissues and cultured cells (monocytoid cell line U937 or freshly isolated monocytes) using RNeasy total RNA extraction kit (Qiagen) according to the manufacturers' instructions. cDNA was synthesized using Superscript RT II

(Invitrogen). Quantitative PCR was performed with gene specific primers for GDF- 15 (Qiagen

QuantiTect Primers; Assay Hs_GDF15-l-SG) and for RPOLII (5'-

CTGCCAAC AC AGCCATCTAC-3 ' SEQ ID N°l (forward); 5'- TCACCCATTCCTGATCCTCT-3' SEQ ID N° 2 (reverse)) using the QuantiTect SYBR

Green PCR kit (Qiagen) on a LightCycler 2.0 Instrument (Roche Applied Science). The PCR conditions were as follows: 95° C, 15 minutes; 45 cycles at 94° C, 30 seconds; 50° C, 30 seconds; 72° C, 20 seconds. The measured GDF- 15 transcript abundance was normalized to the expression level of RPOLII (RNA Polymerase II) for all samples. GDF-15 qPCR from glioblastoma tissues was performed in triplicates. Three independent experiments were carried out with cultured cells.

Statistical analysis

All analyses were carried out with R, a free software environment available at http://www.r- project.org/, SAS (V9.1.3) or using Stat View software (version 5.0, SAS Institute, Cary, NC). Survival curves were prepared according to the method of Kaplan and Meier, and statistical difference between the curves was tested using the logrank test.

Results

GDF- 15 CSF concentrations are increased in glioblastoma patients. Applicant measured GDF- 15 protein levels in the CSF and plasma of patients with intracranial tumors, including glioblastoma, astrocytoma (WHO grade II and III), meningioma (WHO grade I and II), and metastasis, and compared them to a control cohort of patients treated in the neurology department for disorders unrelated to cancer. The distribution of the measurements is depicted in Figure 1. The median concentration of GDF- 15 in the CSF of glioblastoma patients (229 pg/ml) was significantly higher than in the control cohort in which most measurements were below the limit of detection of 156 pg/ml (p<0.0001, Wilcoxon rank sum test with continuity correction; Table 1). CSF MIC-1/GDF15 concentrations of patients with newly diagnosed glioblastoma were significantly lower than from those with recurrent glioblastoma (p=0.01; 220 vs. 502 pg/ml, respectively). In patients with other intracranial tumors the concentration was not significantly different from the control after correction with multiple testing. However, a trend towards higher CSF levels was observed in patients with brain metastasis (Table 1). Plasma concentrations of GDF- 15 were generally higher than in the CSF, but did not differentiate patients with intracranial tumors from the control cohort with a median concentration of 612 pg/ml (Figure 1, Table 1).

TABLE 1 Concentration of MIC-1/GDF15 protein in CSF and plasma of patients with brain tumorscompared to patients evaluated for non-neoplastic, non-inflammatory diseases.

< Cerebro Spinal Fluid [pg/ml] Plasma

³adj p-

Samples # Samples #

Patients < ¹LoD Median ²p-val val < LoD Median ²p-val

Normal 53 35 < LoD 50 2 612

Glioblastoma 61 22 229 5.03E-05 2.12E-04 41 2 665 0.76

Astrocytoma 10 7 < LoD 0.99 1 5 0 481 0.61

Meningioma 18 10 < LoD 0.31 1 11 1 868 0.3

Metastasis 5 1 273 0.038 0.15 5 0 975 0.18

¹ LoD, limit of detection : 156 pg/ml

² p-value, Mann- Whitney test, comparison with patients treated for non-neoplastic diseases ("normal")

³ adjusted p-value, after Bonferroni correction for multiple testing

Enhanced CSF concentrations of GDF- 15 are associated with worse outcome in glioblastoma patients. Next, Applicant evaluated whether the GDF- 15 concentration measured in the CSF of glioblastoma patients at the time of surgery was associated with overall survival. As displayed by Kaplan-Meier survival curves (Fig. 2), glioblastoma patients with higher CSF concentrations (cut-off defined as limit of detection) of MIC-1/GDF15 had a decreased overall survival (P=O.007, n=33) which remained significant after adjustment for age (>50 years, P=O.02). This association also remained significant, when excluding the 10 patients treated for recurrent disease (P=O.012). According to a Cox proportional hazards model the hazard ratio for elevated levels of CSF MIC-1/GDF15 concentrations (>limit of detection) was estimated at HR: 4.07, 95% CI: 1.37-12.10, p-value=0.011. The multivariate Cox proportional hazards model for MIC-I /GDF 15, considering MIC-1/GDF15 expression and age (>50 years), estimated the hazard ratio at 3.85 (95% CI: 1.23-12.11, p-value 0.021), while age was not significant (HR: 1.15, 95% CI 0.44-3.05, p-value 0.77).

Reference list

Basil, C. F., Zhao, Y., Zavaglia, K., Jin, P., Panelli, M. C, Voiculescu, S., Mandruzzato, S., Lee, H.M., Seliger, B., Freedman, R.S., et al. (2006). Common cancer biomarkers. Cancer Res 55, 2953-2961.

Bauskin, A.R., Brown, D.A., Kuffner, T., Johnen, H., Luo, X.W., Hunter, M., and Breit, S.N. (2006). Role of macrophage inhibitory cytokine- 1 in tumorigenesis and diagnosis of cancer. Cancer Res 66, 4983-4986.

Bootcov, M.R., Bauskin, A.R., Valenzuela, S.M., Moore, A.G., Bansal, M., He, X.Y., Zhang, H.P., Donnellan, M., Mahler, S., Pryor, K., et al. (1997). MIC-I, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci U S A 94, 11514-11519.

Brown, D.A., Bauskin, A.R., Fairlie, W.D., Smith, M.D., Liu, T., Xu, N., and Breit, S.N. (2002). Antibody-based approach to high- volume genotyping for MIC-I polymorphism. Biotechniques 33, 118-120, 122, 124 passim.

Brown, D.A., Stephan, C, Ward, R.L., Law, M., Hunter, M., Bauskin, A.R., Amin, J., Jung, K., Diamandis, E.P., Hampton, G.M., et al. (2006). Measurement of serum levels of macrophage inhibitory cytokine 1 combined with prostate-specific antigen improves prostate cancer diagnosis. Clin Cancer Res 12, 89-96. Brown, D.A., Ward, R.L., Buckhaults, P., Liu, T., Romans, K.E., Hawkins, N.J., Bauskin,

A.R., Kinzler, K. W., Vogelstein, B., and Breit, S.N. (2003). MIC-I serum level and genotype: associations with progress and prognosis of colorectal carcinoma. Clin Cancer Res 9, 2642- 2650.

Craig, R., Cortens, J.P., and Beavis, R.C. (2005). The use of proteotypic peptide libraries for protein identification. Rapid Commun Mass Spectrom 19, 1844-1850.

Moore, A.G., Brown, D.A., Fairlie, W.D., Bauskin, A.R., Brown, P. K., Munier, M.L., Russell, P.K., Salamonsen, L.A., Wallace, E.M., and Breit, S.N. (2000). The transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine- 1 is present in high concentrations in the serum of pregnant women. J Clin Endocrinol Metab 85, 4781-4788. Ostermann, S., Csajka, C, Buclin, T., Leyvraz, S., Lejeune, F., Decosterd, L.A., and Stupp, R. (2004). Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients. Clin Cancer Res 10, 3728-3736.

Claims

1. A method for diagnosing or predicting outcome of intracranial tumors in the cerebro-spinal fluid of a subject comprising:

(a) measuring the expression level of GDF-15, a proteotypic peptide or an immunogenic fragment thereof in the cerebro-spinal fluid of said subject,

(b) comparing the expression level of said GDF-15, a proteotypic peptide or immunogenic fragment thereof to threshold value, wherein the elevated levels of said GDF-15, a proteotypic peptide derived thereof or immunogenic fragment thereof indicate worse outcome.

2. The method of claim 1, wherein the intracranial tumor is a glioblastoma or brain metastasis.

3. The method of claim 2, wherein the intracranial tumor is a glioblastoma.

4. The method of claims 1 to 3, wherein the subject is a mammal.

5. The method of claim 4, wherein said mammal is a human.

6. The method of claim 1, wherein the measuring of the expression levels of GDF-15, a proteotypic peptide or immunogenic fragment thereof is obtained through Western blot, immunoprecipitation, immunohistochemistry, ELISA, Radio Immuno Assay, proteomics methods.

7. A kit useful for predicting or diagnosing an intracranial tumor in cerebro-spinal fluid of a subject, said kit comprises a set of: a) antibodies adapted to detect GDF-15 and/or immunogenic fragments thereof, b) a control reagent and/or a detectable label, and optionally instructions to use.