EA040048B1 - PHARMACEUTICAL COMPOSITION AND METHOD FOR TREATMENT OF GLIOMA CHARACTERIZED BY THE PRESENCE OF HIGH LIF LEVELS - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to a method for the treatment of glioma characterized by the presence of high levels of LIF, comprising the introduction of a LIF inhibitory agent selected from an antibody or siRNA, and to a pharmaceutical composition containing a LIF inhibitory agent used in the proposed method. The invention also relates to an in vitro method for selecting patients suffering from glioma to be treated with a LIF inhibitory agent and an in vitro method for predicting the life expectancy of patients suffering from glioma.

Prior Art

Recently, a subpopulation of tumor cells with properties of stem cells has been discovered in malignant neoplasms. This cell population, termed malignant stem cells, is believed to be responsible for the occurrence, spread, and recurrence of tumors, suggesting that the most effective therapies come from therapies aimed at isolating tumor stem cells. Little is known about the molecular characteristics and regulatory mechanisms that control the biology of tumor stem cells. Glioma is one type of tumor in which tumor stem cells, the so-called glioma stem cells or glioma initiating cells (GICs), play an important role.

GICs are characterized by high oncogenic potential, the ability to self-renewal and the ability to differentiate into many cell lines. The number of cells similar to stem cells in a tumor is regulated by their ability to self-repair. GICs, and cancer stem cells in general, undergo symmetrical and asymmetric divisions, whereby the stem cells form two identical copies, or a copy of the stem cell and a copy of a more differentiated cell (asymmetric division). The self-repairing ability of a malignant tumor stem cell is regulated by the balance between symmetrical and asymmetric divisions, and deregulation of the mechanisms controlling this self-renewal is most likely involved in the occurrence of a tumor. Glioma is the most common primary brain tumor and can be classified into four clinical grades based on histology and prognosis. Grade IV gliomas (glioblastoma multiforme) are extremely aggressive and resistant to both radiotherapy and chemotherapy. Despite progress in understanding the molecular mechanisms involved in the genesis and progression of glioma, the prognosis and treatment of this type of tumors remain ineffective. The preferred treatment for gliomas is surgery. However, surgical treatment is usually followed by pharmacological adjuvant treatment or radiotherapy. Preferred drugs for the treatment of glioma include a combination called PCV including procarbazine, CCNU (lomustine) and vincristine, temozolomide in combination with radiation therapy.

GICs are thought to be responsible for the onset, spread, and recurrence of tumors, suggesting that the most effective therapies will be derived from therapies aimed at isolating glioma stem cells. The tumor will not be eradicated until the GICs are destroyed.

Therefore, there is a need to have alternative treatments that overcome the shortcomings of the treatments known in the art and that effectively eliminate GICs.

The essence of the invention

The first aspect of the present invention provides a method for the treatment of glioma characterized by the presence of high levels of LIF, comprising the introduction of an agent that inhibits LIF, selected from an antibody or siRNA.

According to the invention, the antibody acts by inhibiting the self-renewal of tumor stem cells.

According to the invention, glioma is caused by high activity of the JAK-STAT signaling pathway.

According to the invention, said glioma is grade IV glioma.

Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an LIF inhibitory agent, together with a pharmaceutically acceptable carrier, for use in the method of the invention, wherein the inhibitory agent is selected from an antibody and an siRNA.

Another aspect of the present invention provides a method of treating a patient suffering from glioma, which includes:

(a) quantifying LIF expression levels in said patient, (b) comparing expression levels with a control level, and (c) administering an LIF inhibitory agent to a patient having a LIF expression level greater than the control level, wherein the inhibitory agent is selected from anti- LIF antibodies and anti-LIF siRNA.

Another aspect of the present invention provides an in vitro method for selecting patients suffering from glioma to be treated with a LIF inhibitory agent, which comprises:

(a) quantifying LIF expression levels in a sample from said patient, and

- 1 040048 (b) comparing said expression levels with control levels, where if LIF expression levels in a sample from said patient are higher than control values, then said patient is selected to receive treatment with a LIF inhibitory agent, and where the inhibitory agent is selected from anti- LIF antibodies and anti-LIF siRNA.

According to the invention, said agent acts by inhibiting the self-renewal of tumor stem cells.

According to the invention, the antibodies are inhibitory antibodies specific for LIF or antibodies that block LIF receptors.

According to the invention, said glioma is caused by high activity of the JAKSTAT signaling pathway.

According to the invention, said glioma is grade IV glioma.

Another aspect of the present invention provides an in vitro method for predicting the lifespan of patients suffering from glioma, comprising quantifying expression levels of a gene encoding LIF or a protein encoded by said gene in a biological sample from said patient, wherein the increase in expression of a gene encoding LIF or protein , encoded by the specified gene, relative to the expression of the gene encoding LIF, or the protein encoded by the specified gene, in the control sample, indicates a reduced lifespan.

According to the invention, glioma is characterized by the presentation of high levels of LIF in a subset of patients.

According to the invention, the glioma is a glioblastoma.

According to the invention, quantitative analysis of expression levels of a gene encoding LIF includes quantitative analysis of messenger RNA (mRNA) of said gene, fragment of said mRNA, complementary DNA (cDNA) of said gene, fragment of said cDNA, or mixtures thereof.

According to the invention, the quantitative analysis of expression levels of the gene encoding LIF is carried out by quantitative polymerase chain reaction (PCR).

According to the invention, quantitative analysis of protein levels is carried out by Western blotting, immunohistochemistry or ELISA.

According to the invention, the quantitative analysis of protein levels is carried out by determining the activity of said protein.

Brief description of the figures

Fig. 1. Effect of TGFe on the self-renewal of patient-derived GICs.

(A) Representative images of PCTS and GBM neurospheres obtained from samples from 3 different GBM patients (GBM1, GBM2, GBM3).

(B) Musashi-1 (Msi-1), Sox2, Nestin, and β-actin were determined by RT-PCR analysis of PCTS and neurospheres 3 of human GBM samples.

(C) 100,000 neurospheres (nsph.) or PCTC cells derived from GBM1, GBM2 and GBM3 tumor samples were inoculated intracranially in each case into three Balbc nu/nu mice. Magnetic resonance imaging (MRI) studies were performed in each mouse on day 30-40. The images represent an example of mice inoculated with neurosphere or PCTC cells derived from GBM1. Mice were weighed twice a week. The graph shows mice inoculated with cells derived from GBM1.

(D and E) Neurosphere cells of the indicated GBMs were incubated in the absence of growth factors with 100 pM TGFe1 and/or 2 μM TeRI inhibitor for 7 days and the number of newly formed neurospheres (D) and the total number of cells (E) were determined.

(F) Representative images of GBM1 neurospheres processed as indicated in D and E.

Fig. 2. Immunohistochemistry of these markers in GBM1-derived neurospheres and in neurospheres differentiated in GBM1-derived serum.

Fig. 3. TGFe induces LIF expression in PCTS and GBM neurospheres.

(A) PCTC cells from 11 samples of indicated GBMs (GBM1-11) were treated with 100 pM TGFe1 for 3 h in medium-free medium or left untreated, and LIF expression levels were determined by qPCR-RT. β-actin was determined as an internal control for normalization.

(B) GBM neurosphere cells were treated as in A and LIF expression levels were determined by qPCR-RT as in A.

(C) GBM neurospheres were incubated with 100 pM TGFe1 and/or 2 μM TeRI inhibitor for 3 h in serum-free medium and LIF mRNA levels were determined by qPCR-RT as in A.

(D) GBM1 neurospheres were incubated with 100 pM TGFe1, TGFe2 and TGFe3 for 3 h in serum free medium and LIF mRNA levels were determined by qPCR-RT as in A.

(E) LIF secreted protein levels were determined by ELISA in GBM1 neurospheres after 48 h of treatment with 100 pM TGFe1.

Fig. 4. TGFe induces transcription of LIF via activated Smad complex.

(A) Schematics of luciferase-LIF indicator constructs.

- 2 040048 (B) A172 glioma cells were transfected with the indicator construct luciferase-LIF (-534/+32), (-276/+32), (-275/+32) mutSBE or (-73/+32). 16 h after transfection, cells were treated with 100 pM

TGFe1 for 20 h and analyzed the activity of luciferase.

(C) U373MG cells were treated with 100 pM TGFe1 for 3 h and ChIP assays were performed with the indicated antibodies and the indicated PCR primers.

(D, E) LIF levels were determined by qRT-PCR in U373MG (D) or GBM neurospheres (E) treated with 100 pM TGFe1 for 3 h after siRNA-mediated silencing of the indicated members of the indicated Smad family. Western blotting was performed using antibodies specific for Smad or quantitative PCR-RT Smad4. β-actin was determined as an internal control for normalization.

Fig. 5. TGFe induces the LIF-JAK-STAT signaling pathway in patient-derived GBM neurospheres.

(A) p-STAT3 and STAT3 levels were determined by Western blotting of the neurospheres of a GBM1 sample treated with 20 ng/ml LIF for the indicated time periods.

(B) Sample GBM1 neurospheres were treated with 20 ng/ml LIF and/or 0.5 μM P6 for 15 min and total p-STAT3 and STAT3 levels were determined by Western blotting.

(C) Sample GBM1 neurospheres were treated with 100 pM TGFe1 or 2 μM TeRI inhibitor for 4 h in the absence of EFG and FGF, and p-STAT3, STAT3, p-Smad2, Smad2 and α-tubulin levels were determined by Western blotting.

(D) GBM1 sample neurospheres were treated with 100 pM TGFe1 or 2 μM TeRI inhibitor or anti-LIF blocking antibody for 4 h in the absence of EFG and FGF, and total p-STAT3 and STAT3 levels were determined by Western blotting.

Fig. 6. LIF mediates an increase in GIC self-renewal via TGFe.

(A, B) GBM1, GBM2 and GBM3 neurosphere cells were treated with 100 pM TGFe1, 20 ng/ml LIF and/or a neutralizing antibody to LIF and 0.5 μM P6 in the absence of EGF and FGF and the number of newly formed neurospheres was determined (A) or the total number of cells (IN).

(C) Representative images of GBM1 neurospheres processed as indicated in A and B.

Fig. 7. TGFe and LIF prevent differentiation of GBM neurospheres.

(A) Immunohistochemistry of these proteins was performed on GBM1 derived neurospheres treated with 100 pM TGFe1 or 20 ng/ml LIF for 7 days in the absence of growth factors and for the last 3 days on polyA-lysine coated slides.

(B) Musashi-1 (Msi-D), Sox2 and Nestin mRNA levels were determined in GBM1 neurospheres 7 days after said treatments without growth factors. 18S RNA levels were used as an internal control for normalization.

Fig. 8. Effects of TGFe on the self-renewal ability of normal human neuroprogenitors. (A) Immunohistochemistry of these markers was performed in human neuroprogenitor nephrospheres. (B) Sample GBM1 neurospheres and human neuroprogenitors were incubated with the indicated members of the TGFe family at 100 pM for 3 hours and LIF mRNA levels were determined. (C, D) Normal human neuroprogenitor neurosphere cells were incubated under the same conditions as described above in FIG. 1D in the presence of 100 pM TGFe1 or 20 ng/ml LIF for 7 days and the number of newly formed neurospheres (C) and the total number of cells (D) were determined.

Fig. 9. Expression of LIF in human glioma tumors.

(A) Transcripts of LIF, TGFe2, Musashi-1 (Msi-D), Sox2 and Nestin were determined by qPCR-RT analysis in 39 samples obtained from human patients with glioma.

(B) Correlations between LIF and TGFe2, Musashi-1 (Msi-1), Sox2, or Nestin. Spearman's rank correlation coefficient (Rho), two-tailed significance.

(C) TGFe promotes self-renewal of GSCs through induction of LIF, which leads to an increase in the number of stem-like cell groups in the tumor mass.

Fig. 10. LIF mRNA levels in glioma patients are associated with mean lifespan. Kaplan-Meier curves demonstrate that the overall survival of glioma patients with positively regulated LIF mRNA levels, >2-fold, is significantly lower than the rest of the patients (P=7.2E-8) according to the log-rank test. Data obtained from the REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) program of the National Cancer Institute.

Fig. 11. LIF mRNA levels in patients with glioblastoma (GBM) are associated with mean lifespan. The Kaplan-Meier curves show that the overall survival of GBM patients with positively regulated LIF mRNA levels, >9-fold, is significantly lower than that of the rest of the patients (p=6.9E-4) by log-rank test. Data obtained from the REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) program.

- 3 040048

National Cancer Institute.

Fig. 12. Some patients with certain tumor types have abnormally high levels

L.I.F. LIF mRNA levels in various tumor types showed that some patients (circles) have aberrantly high LIF levels. Data obtained from the GeneSapiens bioinformatics team (www.genesapiens.org).

Number designations: 1) pre-B-cell acute lymphoblastic leukemia (B-ALL); 2) pre-T-cell acute lymphoblastic leukemia (T-ALL); 3) B-cell chronic lymphocytic leukemia (B-CLL); 4) acute myeloid leukemia (AML); 5) plasma cell leukemia; 6) myeloma; 7) B-cell lymphoma; 8) Burkitt's lymphoma; 9) T-cell lymphoma; 10) chondrosarcoma; 11) osteosarcoma; 12) Ewing's sarcoma; 13) synovial sarcoma; 14) leiomyosarcoma; 15) rhabdomyosarcoma; 16) liposarcoma; 17) sarcoma, not specified (NOS); 18) cutaneous squamous cell carcinoma; 19) melanoma; 20) glioma; 21) neuroblastoma; 22) malignant tumor of peripheral nerve sheaths (MNM); 23) chordoma; 24) squamous cell carcinoma of the oral cavity; 25) squamous cell carcinoma of the laryngopharynx; 26) carcinoma of the parotid gland; 27) lung adenocarcinoma; 28) large cell lung cancer; 29) small cell lung cancer; 30) squamous cell carcinoma of the lung; 31) carcinoid tumor of the lung; 32) mesothelioma; 33) adenocarcinoma of the esophagus; 34) adenocarcinoma of the stomach; 35) gastrointestinal stromal tumor (GIST); 36) adenocarcinoma of the small intestine; 37) colorectal carcinoma; 38) liver cancer; 39) pancreatic cancer; 40) tumors of the adrenal glands; 41) thyroid carcinoma; 42) kidney oncocytoma; 43) kidney cancer; 44) nephroblastoma; 45) bladder cancer; 46) testicular seminoma; 47) testicular non-seminoma; 48) prostate adenocarcinoma; 49) ductal breast cancer; 50) lobular (lobular) breast cancer; 51) medullary breast cancer; 52) other breast cancer; 53) breast carcinoma, NOS; 54) clear cell carcinoma of the ovaries; 55) endometrioid ovarian cancer; 56) ovarian germinoma; 57) mucus-forming ovarian carcinoma; 58) serous ovarian carcinoma; 59) ovarian adenocarcinoma, not otherwise specified (NOS); 60) another ovarian tumor; 61) peritoneal adenocarcinoma; 62) uterine sarcoma; 63) adenocarcinoma of the uterus; 64) squamous cell carcinoma of the uterus; 65) Mullerian tumor of the uterus; 66) adenocarcinoma of the cervix; 67) squamous cell carcinoma of the cervix; 68) carcinoma of the vagina/vulva.

Detailed Description of the Invention Therapeutic Methods of the Invention

Surprisingly, the present inventors have found that the IL-6-type cytokine, more specifically LIF, is involved in the activation of the JAK-STAT cascade mediated by TGFe, thus leading to the induction of cell proliferation and an increase in tumor stem cells ( malignant tumor stem cells). Based on this fact, the authors discovered a new therapeutic route for the treatment of diseases associated with unwanted cell proliferation, such as malignancies, and especially for the treatment of malignancies caused by high activity of the JAK-STAT signaling pathway, where this therapy is based on the use of inhibitors IL-6-type cytokines. The identification of LIF as an element downstream of TGFe upon activation of JAK-STAT further enables more effective inhibition of said JAK-STAT cascade, because it prevents activation not only upon activation by TGFe, but also upon activation by other stimuli such as interleukins, erythropoietin, growth hormone prolactin, and the like.

Therefore, in one aspect, the invention relates to an agent that inhibits the expression and/or activity of IL-6-type cytokines for the treatment of diseases associated with unwanted cell proliferation.

Without wishing to be bound by theory, it is believed that the effect of LIF and its inhibitors on tumor proliferation lies in the ability of LIF to promote tumor stem cell proliferation. Treatment with LIF inhibitors will therefore be particularly required in those tumors in which there is high expression of the IL-6-type cytokine, and more specifically LIF, and treatment of chemotherapy-resistant tumors will also be of interest given the known ability of tumor stem cells to be resistant to chemotherapy. Finally, given that tumor stem cells appear to be responsible for relapses, the use of LIF inhibitors for the treatment of diseases associated with unwanted cell proliferation would be particularly useful in preventing relapses.

Therefore, in a first aspect, the invention relates to an agent that inhibits the expression and/or activity of IL-6-type cytokines for the treatment of diseases associated with unwanted cell proliferation.

In another aspect, the invention relates to a method for treating diseases associated with unwanted cell proliferation, comprising administering an agent that inhibits the expression and/or activity of an IL-6-type cytokine. In another aspect, the invention relates to an agent that inhibits the expression and/or activity of an IL-6-type cytokine for the manufacture of a pharmaceutical composition for the treatment of diseases associated with unwanted cell proliferation. In the context of the present invention, an IL-6-type cytokine is understood to be a cytokine member of the IL-6 family, which includes IL-6, IL-11, leukemic inhibitory factor (LIP), oncostatin M (OSM), cardiotrophin-1 (CT-1 ), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine (CLC), which activates the JAK-STAT signaling pathway. These cytokines participate in the same receptor complex in which the glycoprotein 130 (gp130) receptor subunit is a common component.

Therefore, in a specific embodiment of the invention, the IL-6-type cytokine is selected from LIF, IL-6, IL-11, oncostatin M, carcinotrophin-1, CNTF and CLC. In yet another specific embodiment, the IL-6-type cytokine is LIF.

In the context of the present invention, an inhibitory agent is understood to be any substance or compound that is capable of preventing or blocking the transcription and translation of a gene encoding an IL-6-type cytokine (i.e. preventing or blocking the expression of said gene), or that is capable of preventing a protein from encoded by the specified gene, its function (activity), i.e. prevent the ability of the IL-6-type cytokine to induce activation of the JAK-STAT signaling pathway. Assays for determining whether a compound is an IL-6 type cytokine inhibitory agent are well known in the art. For example, Mezt S. et al. (J. Biol. Chem, 2007, vol. 282:12381248) describe an assay based on the ability of an inhibitor to block the expression of a reporter gene that is under the control of an IL-6 cytokine sensitive promoter. In the specific case of LIF, assays to identify inhibitory agents include inhibition of differentiation of murine myeloid leukemia Ml cells in the absence of LIF (WO 2005/30803), inhibition of stimulation of calcium release from Jurkatt cells (US5980894), measurement of STAT-3 phosphorylation by IL-6-type cytokine (see, for example, Example 2, section 2.4 examples of this description), etc.

By way of illustration, LIF expression-inhibiting agents suitable for use in the present invention are, for example, antisense oligonucleotides, interfering RNA (siRNA), catalytic RNA or specific ribozymes, RNA with decoy activity, i.e. with the ability to specifically bind a factor (usually a protein) important for gene expression, etc. In addition, inhibitory agents capable of preventing the function of the protein encoded by the specified gene encoding the IL-6 type cytokine are, for example, inhibitory peptides of the protein, antibodies directed specifically against those epitopes of the protein that are necessary for the implementation of its function, or antibodies against IL-6-type cytokine receptors and the like.

Therefore, in a specific embodiment of the invention, the inhibitory agent is selected from the group consisting of siRNAs, antisense oligonucleotides, specific ribozymes, antibodies, polypeptides, and IL-6-type cytokine receptor inhibitors.

miRNA

Small interfering RNAs, or siRNAs (eng. siRNA), are agents that are able to inhibit the expression of a target gene through RNA interference. siRNAs can be synthesized chemically, can be produced by in vitro transcription, or can be synthesized in vivo by the target cell. miRNAs typically consist of double-stranded RNA 15 to 40 nucleotides in length, which may contain an overhang at the 3' and/or 5' end of 1 to 6 nucleotides in length. The length of the protruding region does not depend on the total length of the miRNA molecule. siRNA acts through degradation or post-translational silencing of the target messenger.

miRNA can be called shRNA (short hairpin RNA, English shRNA), which differs in that the antiparallel strands that form the miRNA are connected by a loop or hairpin region. These siRNAs are compounds of a short antisense sequence (19 to 25 nucleotides) followed by a loop of 5 to 9 nucleotides in length followed by a sense strand. shRNAs may be encoded by plasmids or viruses, especially retroviruses, and more particularly retroviruses, and may be under the control of promoters such as the U6 promoter of RNA polymerase III.

miRNAs of the invention are essentially homologous to mRNAs of the gene encoding the IL-6 type cytokine or the genomic sequence encoding said protein. Essentially being homologous is understood as having a sequence that is sufficiently complementary to or similar to the target mRNA such that the miRNA is capable of causing degradation of the latter via RNA interference. miRNAs that are suitable for causing this interference include miRNAs formed from RNA, as well as miRNAs containing various chemical modifications, such as miRNA, in which the bonds between nucleotides are different from those found in nature, an example is phosphorothioate bonds, conjugates of an RNA chain with a functional reagent such as a fluorophore. Modification of the ends of RNA strands, especially the 3' end, by modification with various hydroxyl functional groups in the 2' position.

Nucleotides with modified sugars, such as O-alkylated residues in the 2' position, an example is 2'-O-methylribose-p 2'-O-fluororibose.

Base-modified nucleotides such as halogenated bases (eg 5-bromouracil and 5-ioduracil), alkylated bases (eg 7-methylguanosine).

siRNA and shRNA of the invention can be obtained using a variety of methods known

- 5 040048 specialist in this field. For example, miRNA can be chemically synthesized from phosphoramidite-protected ribonucleosides in a conventional DNA/RNA synthesizer. Alternatively, siRNA can be produced recombinantly from a plasmid and a viral vector, in which case the region coding for the siRNA-forming strand or strands is under the functional control of RNA polymerase III promoters. In cells, RNase Dicer processes shRNA into functional siRNA.

The region of the nucleotide sequence that is taken as the basis for the design of the siRNA is not limited and may contain a region of the coding sequence (between the start codon and the last codon) or may otherwise contain sequences of the 5' or 3' untranslated region, preferably from 25 up to 50 nucleotides and in any position in the 3' direction relative to the start codon. One way to design siRNAs involves identifying AA(N ₁₉ )TT motifs in which N can be any nucleotide in the IL-6-type cytokine coding sequence and selecting those that have a high G/C content. If the indicated motif is not found, it is possible to identify the NA(N ₂₁ ) motif, in which N can be any nucleotide.

Antisense oligonucleotides

In a further aspect, the invention relates to the use of isolated antisense nucleic acids to inhibit expression, for example, to inhibit transcription and/or translation of a nucleic acid encoding an IL-6-type cytokine whose activity is inhibited. Antisense nucleic acids can bind a potential drug target through normal base complementarity, or, for example, in the case of double-stranded DNA binding, through specific interactions in the major groove of the double helix. These methods generally fall within the range of methods commonly used in the art and include any method that relies on specific binding to oligonucleotide sequences.

The antisense construct of the present invention can be delivered, for example, by an expression plasmid that, when transcribed in a cell, produces an RNA complementary to at least one portion of a cellular mRNA encoding an IL-6-type cytokine. Alternatively, the antisense construct is an oligonucleotide probe that is prepared ex vivo and that, when introduced into a cell, inhibits expression by hybridization with the mRNA and/or genomic sequences of the target nucleic acid. Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, eg exonucleases and/or endonucleases, and which are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothionate, and methylphosphonate DNA analogs (see also US Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). General approaches in the design of oligomers useful in antisense therapy have been further discussed in, for example, Van der Krol et al., BioTechniques 6: 958-976, 1988; and Stein et al., Cancer Res 48: 2659-2668, 1988.

With respect to antisense DNA, oligodeoxyribonucleotide regions derived from the translation initiation site, eg between -10 and +10 of the target gene, are preferred. Antisense approaches involve designing oligonucleotides (either DNA or RNA) that are complementary to the mRNA encoding the target polypeptide. Antisense oligonucleotides will bind mRNA transcripts and prevent translation. Complete complementarity is not required, although it is preferred. In the case of double-stranded antisense nucleic acids, single-strand double-stranded DNA, or the formation of triple-stranded DNA can be tested. The hybridization capacity will depend on the degree of complementarity and on the length of the antisense nucleic acid. Generally, the longer the nucleic acid hybridizes, the more RNA mismatches it can contain while still forming a stable duplex (or triplex, which can be formed as the case may be). One of ordinary skill in the art can determine the tolerable degree of mismatching by applying standard processes for determining the melting temperature of a hybridization complex. Oligonucleotides that are complementary to the 5' end of the mRNA, such as the untranslated 5' sequence up to and including the AUG start codon, should function as efficiently as possible to inhibit translation. However, it has recently been shown that sequences complementary to untranslated 3' mRNA sequences are also effective in inhibiting mRNA translation (Wagner, Nature 372: 333, 1994). Therefore, oligonucleotides complementary to either the 5' or 3' untranslated, non-coding regions of a gene can be used in an antisense approach to inhibit translation of a given RNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA must include a fragment complementary to the AUG start codon. Oligonucleotides complementary to mRNA coding regions are less effective inhibitors of translation, but they can also be used in accordance with the invention. If antisense nucleic acids are designed to hybridize to the 5', 3', or mRNA coding region, then they should be at least six oligonucleotides in length, and preferably not less than about 100 or more, preferably not less than about 50 , 25, 17 or 10 nucleotides.

Preferably, in vitro assays are first conducted to quantify the ability of antisense oligonucleotides to inhibit gene expression. Preferably, these assays use controls that distinguish between antisense inhibition of genes and non-specific biological effects of oligonucleotides. Preferably, these assays compare levels of RNA or target protein with levels of an internal RNA or protein control. The results obtained with antisense oligonucleotides can be compared with the results obtained with control oligonucleotides. Preferably, the control oligonucleotide is the same length as the oligonucleotide under analysis, and if the oligonucleotide sequence differs from the antisense sequence by no more than necessary to prevent specific hybridization to the target sequence.

Oligonucleotide sequences can be DNA or RNA or chimeric mixtures or modified derivatives or versions thereof, single or double stranded. The oligonucleotide may be modified in the base group, in the sugar group, or in the phosphate backbone, for example, to improve the stability of the molecule, hybridization, and the like. Oligonucleotides may include other linking groups such as peptides (for example, to direct them to host cell receptors), or agents to create easier transport across the cell membrane (see, for example, Letsinger et al., Proc. Natl. Acad. Sci 86: 6553-6556, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. 84: 648-652, 1987; PCT Publication No. WO 88/09810) or through the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134), cleavage agents switched by hybridization (see e.g. Krol et al., BioTechniques 6: 958-976, 1988), intercalators (see e.g. Zon, Pharm. Res. 5 : 539-549, 1988). For this purpose, the oligonucleotide can be conjugated to another molecule, such as a hybridization-switched peptide, a crosslinker, a transfer agent, a hybridization-switched cleavage agent, and the like.

The antisense oligonucleotides may contain at least one modified base selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxyethyl )uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-O-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- hydroxyacetic acid (v), vibutoxosin, vseudouracil, queosin, 2-tocytosine, 5methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid ( v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-car boxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also contain at least one modified sugar group selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. The antisense oligonucleotide may also contain a backbone like a neutral peptide. Such molecules are referred to as peptide-nucleic acid (PNA) oligomers, as described, for example, in Perry-O' Keefe et al., Proc. Natl. Acad. sci. USA 93: 14670, 1996, and in Eglom et al., Nature 365: 565, 1993. The advantage of PNA oligomers is their ability to bind complementary DNA in a manner that is virtually independent of the ionic strength of the medium due to the neutral backbone of the DNA. In yet another embodiment, the antisense oligonucleotide contains at least one modified phosphate backbone selected from the group consisting of phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphorodiamidate, methylphosphonate, alkyl phosphotriester, and formaacetal or an analogue thereof.

In yet another embodiment, the antisense oligonucleotide is an alpha anomeric oligonucleotide. The alpha-anomeric oligonucleotide forms specific double-stranded hybrids with a complementary RNA in which, in contrast to the typical anti-parallel orientation, the strands are parallel to each other (Gautier et al., Nucl. Acids Res. 15: 6625-6641, 1987). The oligonucleotide is a 2'-O-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15: 6131-6148, 1987) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215: 327-330, 1987) .

Although antisense oligonucleotides complementary to the coding region of the target mRNA sequence can be used, complementarity to the untranslated transcribed region can also be used.

In some cases, it may be difficult to achieve sufficient intracellular concentrations of the antisense molecule to suppress translation of the endogenous mRNA. Therefore, the preferred approach is to use recombinant DNA constructs in which the antisense oligonucleotide is placed under the control of a strong polymerase III or polymerase II promoter. The use of such a construct for transfection of target cells will result in the transcription of sufficient amounts of single-stranded RNAs that will form complementary base pairs with potential drug-targeted endogenous transcripts and will therefore prevent translation. For example, a vector can be introduced so that when it is captured by a cell, trans

- 7 040048 antisense RNA encryption. Such a vector may remain episomal, or may be integrated into the chromosome, while being transcribed to produce the desired antisense RNA. Such vectors can be constructed using art-standard recombinant DNA techniques. The vectors may be viral plasmids, or other plasmids known in the art, used for replication and expression in mammalian cells. Expression of antisense RNA encoding sequences can be mediated by any promoter known in the art that acts on mammalian cells, preferably human cells. Such promoters may be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early region promoter (Bernoist and Chambon, Nature 290: 304-310, 1981), the promoter contained in the 3' portion of the Payca sarcoma virus long terminal repeat (Yamamoto et al., Cell 22: 787- 797, 1980), herpes virus thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981), metallothionein gene regulatory sequences (Brinster et al., Nature 296: 39-42 , 1982), etc. Any type of plasmid, cosmid, YAC and viral vector can be used to prepare a recombinant DNA construct that can be introduced directly into a tissue site.

Expression of a target gene may otherwise be expressed by targeting complementary deoxyribonucleotide sequences to the gene's regulatory region (i.e., promoter and enhancer) to form triple helical structures that prevent transcription of the gene in target cells in the body (see, in general terms, Helene, Anticancer Drug Des, 6(6): 569-84, 1991; Helene et al., Ann. N. E. Acad. Sci., 660: 27-36, 1992; and Maher, Bioassays 14(12): 807-15, 1992 ).

The nucleic acid molecules to be used in the formation of triple helices to inhibit transcription are preferably single stranded and formed from deoxyribonucleotides. The base composition of these oligonucleotides should enhance triple helix formation according to Hoogsteen's rules for base pairing, which generally require the presence of somewhat large portions of purines or pyrimidines in the duplex chain. Nucleotide sequences can be based on pyrimidines which will give TAT and CGC in the three linked strands of the resulting triple helix. Pyrimidine-rich molecules provide base complementarity to a region rich in single-stranded purines of the duplex in an orientation parallel to said strand. Moreover, nucleic acid molecules that are enriched in purines can be selected, for example, which contain a region of G residues. These molecules form a triple helix with double-stranded DNA that is enriched in GC pairs, in which most of the purine residues are located in the same strand of the target duplex. , which results in the formation of CGC triplets by the three strands of the triplet.

Potential target sequences that can be chosen to form triple helices can otherwise be increased by the formation of a nucleic acid molecule called a hairpin. Hairpin molecules are synthesized in a variable 5'-3', 3'-5' form, so that they base pair first with the duplex strand and then with the other strand, obviating the need for a fairly large patch of purines or pyrimidines in the duplex strand.

In some embodiments, the antisense oligonucleotides are antisense morpholines. Morpholines are synthetic molecules that are products of the rearrangement of the natural structure of nucleic acids. Typically 25 bases long, they link complementary RNA sequences by standard nucleic acid base pairing. Structurally speaking, the difference between morpholines and DNA is that although morpholines have standard nucleic acid bases, these bases are linked to morpholine rings rather than deoxyribose rings, and they are linked by phosphorodiamidate groups rather than phosphate ones. Switching from anionic phosphates to neutral phosphorodiamidate groups eliminates ionization in the normal physiological pH range so that morpholines in cells or organisms are uncharged molecules. Morpholines are not chimeric oligomers; the whole backbone of morpholine is made from such modified subunits. Morpholines are more commonly used as single-stranded oligomers, although heteroduplexes of the morpholine chain and complementary DNA strand can be used in combination with cationic cytosolic distribution reagents.

Unlike many antisense structural types (eg, phosphorothiodates), morpholines do not degrade their target RNA molecules. Conversely, morpholines act through steric mismatch by binding to a target sequence in RNA and simply placing themselves in the path of molecules that would otherwise interact with RNA. Morpholine oligomers are widely used in the study of the role of a particular RNA transcript in an embryo, such as eggs, or embryos of zebrafish, African clawed frog (Xenopus), in chickens and in sea urchins, to obtain morphant (English morphant) embryos. With a suitable distribution system in the cytosol, morpholines are effective in cell culture.

Morpholines were developed by AVI BioPharma Inc. like medicines called NeuGenes. They have been used in mammals ranging from mice to humans, and some are currently being tested in clinical trials.

- 8 040048

By binding to the 5'-untranslated region of messenger RNA (mRNA), morpholines can prevent the progression of the ribosome initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the target transcript (so-called gene expression silencing). Morpholines provide a suitable environment for protein expression silencing and information about how this reduction alters cells or organisms. Some morpholines silence expression so effectively that after degradation of pre-existing proteins, target proteins become undetectable by Western blotting.

Morpholines can also interfere with pre-mRNA processing steps, typically by preventing the RNPnp complexes that direct splicing from binding to their targets within intron boundaries in the preRNA helix. Prevention of U1 binding (on the donor side) or U2/U5 binding (at the polypurimidine group and acceptor site) can induce modified splicing, typically leading to exclusion of mature mRNA exons. The control of some splicing targets causes the inclusion of introns, while the activation of cryptic splicing sites can result in partial inclusion or exclusion. U11/U12 RNPnp targets can also be blocked. Splicing modification can be suitably analyzed by reverse transcription polymerase chain reaction (RT-PCR) and appears as band migration after gel electrophoresis of RT-PCR products.

Morpholines are also used to block miRNA activity, ribozyme activity, intron splicing silencers, and splicing enhancers. U2 and U12 RNPnp functions are inhibited by morpholines. Morpholines directed against slippery RNA sequences within the coding regions of proteins can induce changes in reading frame during translation. The activities of morpholines against this variety of targets suggest that morpholines can be used as a major multipurpose tool for blocking protein and nucleic acid interactions with mRNA.

Examples of LIF-specific antisense oligonucleotides are described in Kamohara et al. (Int J Oncol, 2007, 30: 977-983) and Cheng et al. (Biol Reprod, 2004, 70: 1270-1276).

DNA enzymes

Another aspect of the invention relates to the use of DNA enzymes to inhibit the expression of genes encoding the IL-6 type cytokine of the invention. DNA enzymes include some of the mechanistic characteristics of both antisense and ribozyme technologies. DNA enzymes are designed to recognize a specific target nucleic acid sequence, like an antisense oligonucleotide, however, like ribozymes, they are catalytic and specifically cleave the target nucleic acid.

There are currently two types of DNA enzymes, both of which have been discovered by Santoro and Joyce (see eg US Pat. No. 6,110,462). DNA enzyme 10-23 contains a loop structure connecting two arms. The two arms provide specificity by recognizing a particular target nucleic acid sequence, while the loop structure provides a catalytic function under physiological conditions.

Briefly, to develop an ideal DNA enzyme that specifically recognizes and cleaves a target nucleic acid, one skilled in the art must first find a unique target sequence. This can be done in the same manner as described for antisense oligonucleotides. Preferably, the unique or virtually unique sequence is a G/C rich sequence of about 18-22 nucleotides in length. The high content of G/C helps in providing a stronger interaction between the DNA enzyme and the target sequence. When a DNA enzyme is synthesized, the specific antisense recognition sequence that directs the enzyme to the messenger is split so that it is contained in both arms of the DNA enzyme, and the DNA enzyme loop is located between the two specific arms. Methods for the manufacture and introduction of DNA enzymes can be found, for example, in US Pat. US 6,110,462. Similarly, methods for delivering DNA ribozymes in vitro or in vivo include methods for delivering RNA ribozymes as detailed above. Otherwise, one skilled in the art will appreciate that, like the antisense nucleotide, DNA enzymes may optionally be modified to improve stability and to improve resistance to degradation.

The antisense RNA and DNA, ribozymes, mRNA and triple helix molecules of the invention can be prepared by any method known in the art for synthesizing DNA and RNA molecules. These include methods well known in the art for the chemical synthesis of oligonucleotides and oligoribonucleotides, such as, for example, solid phase chemical synthesis of phosphoramidite. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding antisense RNA molecules. Such sequences can be included in a variety of vectors carrying suitable RNA polymerase promoters, such as promoters for T7 or SP6 polymerases. Otherwise, antisense cDNA constructs constitutively or inducibly synthesizing antisense RNA, depending on the promoter used, can be stably introduced into cell lines. Moreover, nucleic acid molecules can

- 9 040048 be introduced several known modifications as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking ribonucleotide or deoxyribonucleotide sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl bonds rather than phosphodiesterase in the backbone of the oligodeoxyribonucleotide.

Ribozymes

Ribozyme molecules designed to catalytically cleave a target mRNA can also be used to prevent translation of an mRNA encoding an IL-6-type cytokine whose activity is inhibited. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (For a review, see Rossi, Current Biology 4: 469-471, 1994). The mechanism of action of a ribozyme involves sequence-specific hybridization of the ribozyme molecule with a complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules preferably includes one or more sequences complementary to the target mRNA and a well-known mRNA cleavage sequence or functionally equivalent sequence (see, for example, US Pat. No. 5,093,246, incorporated herein by reference in its entirety).

Although ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy target mRNAs, the use of hammer-action ribozymes is preferred. Cleavage of mRNA by hammer-action ribozymes is directed by flanking regions that form complementary pairs with bases of the target mRNA. Preferably, the target mRNA has the following sequence of two residues: 5'-UG-3'. The construction and production of hammer-action ribozymes is well known in the art and is fully described by Haseloff and Gerlach, Nature 334: 585-591, 1988; and see PCT application WO 89/05852, the contents of which are incorporated herein by reference. Hammerhead ribozyme sequences can be inserted into stable RNA such as transfer RNA (tRNA) to increase the efficiency of in vivo cleavage (Perriman et al., Proc. Natl. Acad. Sci. USA, 92: 6175-79, 1995; Feyter, and Gaudron, Methods in Molecular Biology, Vol.74, Chapter 43, Expressing Ribozymes in Plants, Edited by Turner, P.C., Humana Press Inc., Totowa, N.J.). Expression of chimeric ribozymes with tRNA mediated by RNA polymerase III is particularly well known in the art (see Kawasaki et al., Nature 393: 284-9, 1998; Kuwabara et al., Nature Biotechnol. 16: 961-5, 1998 and Kuwabara et al., Mol. Cell 2: 617-27, 1998; Koseki et al., J Virol 73: 1868-77, 1999; Kuwabara et al., Proc Natl Acad Sci USA 96: 1886-91, 1999; Tanabe et al., Nature 406: 473-4, 2000). Typically, there are several potential cleavage sites for hammer-type ribozymes in the target cDNA sequence. The ribozyme is preferably made so that the cleavage recognition site is located closer to the 5' end of the target mRNA to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts. Moreover, the use of any cleavage recognition site located in the target sequence encoding different C-terminal amino acid domains, e.g., short and long forms of the target, will allow selective production of any form of the target, and thus will achieve a selective effect with respect to the form of the target. gene product.

Ribozymes to genes necessarily contain a hybridization region complementary to two regions, each at least 5 in length, and preferably each of which is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 in length. , 18, 19, or 20 contiguous nucleotides of a target mRNA, such as an mRNA sequence present in any of the human RAP80 proteins. Moreover, ribozymes have a very specific endonuclease activity that autocatalytically cleaves the encoded target mRNA. The present invention extends to ribozymes that hybridize to a coding mRNA encoded by a target gene, such as a target therapeutic drug candidate gene, hence ribozymes that hybridize to and cleave the coding mRNA so that it is no longer capable of being translated to synthesize a functional polypeptide product. .

The ribozymes used in the compositions of the present invention also include RNA endoribonucleases (hereinafter Cech ribozymes) such as those found naturally in Tetrahymena thermophila (known as IVS, or L-19 IVS RNA), which are described in detail by Thomas Cech et al. (Zaug et al., Science 224:574-578, 1984; Zaug et al., Science 231: 470-475, 1986; Zaug et al., Nature 324: 429433, 1986; International Published Application WO88/04300 University Patents Inc.; Been, et al., Cell 47: 207-216, 1986). Cech ribozymes have an eight base pair active site that hybridizes to the target RNA sequence, where the target RNA is later cleaved. The invention includes those Cech ribozymes which have as target sequences an eight base pair active site that is present in the target gene or target nucleotide sequence.

Ribozymes can be created from modified oligonucleotides (eg, to improve stability, orientation, etc.) and must be delivered to a cell expressing the target gene in vivo. The preferred delivery method involves the use of a DNA construct encoding

- 10 040048 ribozyme under the control of a strictly constitutive pol II or pol III promoter such that the transfected cells produce sufficient amounts of the ribozyme to destroy endogenous target messengers and inhibit translation. Because ribozymes are catalytic, unlike other antisense molecules, they require a lower intracellular concentration to be effective.

In some embodiments, the ribozyme may be designed after determining a portion of the sequence sufficient to cause a reduction in efficiency by the mRNA. The same part of the sequence can later be inserted into the ribozyme. In this aspect of the invention, the parts of the ribozyme or mRNA that are directed against the genes are essentially the same sequence in length, at least 5, and preferably 7, 8, 9.10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides of a target nucleic acid, such as any of the human RAP80 sequences. In a long chain of target RNA, a significant number of target sites are not available to ribozymes because they are hidden by secondary and tertiary structures (Birikh et al., Eur J Biochem 245: 1-16, 1997). To overcome the problem of target RNA availability, computer generated secondary structure predictions are typically used to identify targets that are most likely to be single stranded or have an open configuration (see Jaeger et al., Methods Enzymol 183: 281-306, 1989). Other approaches use a systematic secondary structure prediction approach that involves a large number of hybridizable candidate oligonucleotide molecules (see Milner et al., Nat Biotechnol 15: 537-41, 1997; and Patzel and Sczakiel, Mat Biotechnol 16: 64-8, 1998). In addition, US Pat. No. 6,251,588, the contents of which are incorporated herein by reference, describes methods for evaluating oligonucleotide probe sequences to predict the hybridization potential of a target nucleic acid sequence. The method of the invention provides for the use of such methods for selecting preferred segments of a target mRNA sequence that is predicted to be single stranded, and moreover, for the flexible use of it or a substantially identical target mRNA sequence, preferably containing about 10-20 consecutive nucleotides of the target mRNA, in design as mRNA oligonucleotides, and ribozymes according to the invention.

Inhibitory peptides

As used herein, the term inhibitory peptide refers to those peptides that are capable of binding an IL-6-type cytokine and inhibiting its activity as explained above, ie. prevent the ability of the IL-6-type cytokine to induce activation of the JAK-STAT signaling pathway.

An example of an inhibitory peptide is the PEGylated LIF variants described by White et al. (J. Biol. Chem, 2007, Proc. Natl. Acad. Sci. USA, 104: 19357-19362).

Inhibitors of cytokine binding to receptors

As used herein, the expression cytokine receptor binding inhibitors refers to any compound that exhibits affinity for an IL-6-type cytokine and is therefore capable of sequestering a cytokine and preventing it from binding to its physiological receptors. The inhibitory polypeptide is preferably a soluble form of the IL-6-type cytokine receptor (also referred to as a decoy receptor). In the specific case of LIF, a soluble LIF receptor variant or LIF-binding protein (LBP) may be used, a soluble form of the alpha LIF receptor has been found in nature and has been found to be able to effectively prevent the effects of LIF on proteoglycan metabolism in articular cartilage explants (Bell et al ., 1997, J. Rheumatol 24: 2394).

inhibitory antibodies

In the context of the present invention, an inhibitory antibody is understood to be any antibody that is capable of binding an IL-6-type cytokine or receptors for said IL-6-type cytokines, thereby preventing said IL-6-type cytokine from inducing the JAK-STAT signaling pathway. Antibodies can be obtained using any of the methods known to the person skilled in the art. Thus, polyclonal antibodies are obtained by immunizing an animal with an inhibited protein. Monoclonal antibodies are prepared by the methods described by Kohler, Milstein et al. (Nature, 1975, 256:495). Since antibodies with the ability to bind the IL-6-type cytokine or with the ability to bind the receptors of these cytokines have been identified, those that are able to inhibit the activity of this protein will be selected using the inhibitor identification assay described above (Metz, 2007 mentioned above).

Therefore, in a more specific embodiment, the antibodies are inhibitory antibodies specific for said IL-6-type cytokine or antibodies that block IL-6-type cytokine receptors.

Antibodies specific for LIF are described in US 5654157A, Kim et al., (J. Immunol. Meth., 156: 9-17, 1992), Alphonso et al. the Society for Leukocyte Biology, vol.0, no.SP.2 (1991) (NY, N.E., p.49) (Mabs D4.16.9, D25.1.4, and D62.3.2).

In the present invention, the term antibody should be interpreted broadly to include polyclonal, monoclonal multispecific antibodies and fragments thereof (F(ab')2, Fab) and the like. provided that they are capable of specifically recognizing the antigen of interest, which in the context of the present invention is an IL-6-type cytokine or receptors for said IL-6-type cytokines. Examples of antibodies that can be used in the context of the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and the like.

Polyclonal antibodies are initially heterogeneous mixtures of antibody molecules derived from the sera of an animal that has been immunized with an antigen. These include monospecific polyclonal antibodies prepared from heterogeneous mixtures, for example by column chromatography with single epitope peptides of the antigen of interest. A monoclonal antibody is a homogeneous population of antibodies specific for a single epitope of an antigen. These monoclonal antibodies can be obtained by conventional methods already described, for example, Kohler and Milstein [Nature, 1975; 256:495-397] or Harlow and Lane [Using Antibodies. A Laboratory Manual of E. Harlow and D. Lane, Editor: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1998 (ISBN 978-0879695439)].

A chimeric antibody is a monoclonal antibody constructed by cloning or recombining antibodies from different animal species. In a typical, but non-limiting, configuration of the invention, a chimeric antibody comprises a portion of a monoclonal antibody, typically a variable fragment (Fv), including antigen recognition and binding regions, and another portion corresponding to a human antibody, typically a portion including a constant region and a region adjacent to the constant.

A fully human antibody is an antibody or antibodies that have been generated in transgenic mice with a human immune system or in vitro by immunization of human immune cells (including both genetic and conventional immunization with or without adjuvants and with purified or crude antigen; or by antigen display method immune system) or from natural/synthetic libraries derived from human immune cells. These antibodies can be obtained and selected from transgenic animals (for example, from mice) in which human immunoglobulin genes have been cloned and which have been immunized with a target antigen (in the present invention, said antigen is an IL-6-type cytokine or IL-6 receptors of said cytokines -type). These antibodies can be produced by selecting human single chain variable fragments (scFv) or antigen binding fragments (Fab) displayed in phage displays, followed by cloning and grafting into a human antibody, or by any other method of production and detection known to the person skilled in the art, of the library obtained cloning of variable fragments of both chains with their subsequent combination/mutation to obtain antibody libraries.

A humanized antibody is a monoclonal antibody that is constructed by cloning and transplanting the hypervariable regions (CDRs) of a mouse monoclonal antibody into a human antibody in place of its own hypervariable regions.

In addition, in the context of the present invention, the term antibody also includes variants with an altered glycosylation pattern, as well as glycosylated and non-glycosylated antibody fragments derived from protein or by recombinant technology, which may consist of (i) antibody variable regions linked to each other. binding peptide (scFv), (ii) variable zone together with heavy chain constant CH1 (Fd) linked to light chain via cysteines or via binding peptides and disulfide bond (scFab), (iii) new variants such as single heavy chains , or (iv) any modification made to antibody fragments to make it more similar, less immunogenic (humanized) or more stable in biological fluids and which, in the context of the present invention, has the ability to prevent IL-6-type cytokines from performing their functions ( activity), i.e. prevent activation of the JAK-STAT signaling pathway.

As will be appreciated by one of skill in the art, antibodies can be produced by conventional genetic engineering and recombinant methods, antibody production methods, extraction and purification methods from biological fluids and tissues, or any other conventional method for producing proteins and antibodies that is widely known to those skilled in the art. this area. Illustrative non-limiting examples of methods for producing antibodies are: methods for immunizing animals, including animals transgenic for human immunoglobulin genes, obtaining monoclonal antibodies using hybridomas, obtaining libraries of antibodies, which can be natural, synthetic or obtained from organisms immunized with an antigen of interest and which can be selected by various differential display methods (phage display, ribosomal display, etc.) and then through genetic engineering techniques can be reshaped and expressed in vectors designed to produce recombinant antibodies of various sizes, compositions and structures. An overview of the main ways to obtain and

- 12 040048 purification of antibodies can be found, for example, in

Handbook of Therapeutic Antibodies, by S. Dubel. Editor: Wiley-VCH, 2007, Vol: I to III (ISBN 9783527314539);

Antibodies: Volume 1: Production and Purification by G. Subramanian Ed., Editor: Springer, 1st Ed,

2004 (ISBN 978-0306482458);

Antibodies: Volume 2: Novel Technologies and Therapeutic Use, by G. Subramanian Ed., Editor: Springer, first edition, 2004 (ISBN 978-0306483158); J Molecular Cloning: a Laboratory manual, by J. Sambrook and D. W. Russel Eds., Publisher: Cold Spring Harbor Laboratory Press, third edition, 2001 (ISBN 9780879695774).

Other compounds that inhibit IL-6-type cytokine activity Other compounds with the ability to inhibit IL-6-type cytokine expression include aptamers and spiegelmers, which are single- or double-stranded D- or L-nucleic acids that specifically bind a protein, resulting in modification its biological activity. Aptamers and Spiegelmers are 15 to 80 nucleotides in length, and preferably 20 to 50 nucleotides in length.

Polypeptides with IL-6-type cytokine inhibitory activity In particular, LIF (IL-6-type cytokine) antagonists which may be useful in the context of the present invention.

LIF variants having mutations at the receptor binding sites that exhibit reduced affinity for it, or that are able to bind to only one of the receptor chains. Examples of these mutants include those described by Hudson et al. (J. Biol. Chem., 1996, 271:11971-11978), LIF variants described in WO 05030803 which have one or more mutations selected from the group Q29A, G124R and N128A and which show reduced affinity for the LIF receptor and for gp130 . A highly effective LIF antagonist variant containing MH35BD/Q29A+G124R is described by Fairlie, W. D. et al. (J. Biol. Chem., 2004, 279: 2125-2134).

The mutant described in WO 9601319 is characterized by one or more substitutions at the receptor binding sites and in particular at positions 25-38, 150-160 or 161-180 according to human LIF numbering.

Soluble variants of the LIF receptor based on the primary structure and capable of binding to LIF and preventing interaction with its natural receptor on the cell surface, such as chimeric proteins containing part of the extracellular region of the LIF receptor and the gp130 ligand-binding domain, as described by Metz S. et al. (J. Biol. Chem., 2008, 283:5985-5995).

As noted at the beginning of the description, the authors have discovered a new therapeutic route for the treatment of diseases associated with unwanted cell proliferation, such as malignancies, especially the treatment of malignancies caused by the activity of the JAK-STAT signaling pathway, using the invention described herein.

In the context of the present invention, a disease associated with unwanted cell proliferation includes the growth, progression and metastasis of malignant neoplasms and tumors.

Examples of diseases associated with unwanted cell proliferation that can be treated in accordance with the methods described in the present invention are malignancies, restenosis, arteriosclerosis, angiogenic diseases, fibrosis, dermatological diseases and inflammatory diseases.

In a specific embodiment of the invention, the disease associated with unwanted cell proliferation is a malignant neoplasm.

The terms malignancy and tumor refer to a physiological condition in mammals that is characterized by deregulated cell growth. The compounds of the present invention are useful in the treatment of tumors of the breast, heart, lung, small intestine, rectum, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis and liver.

Specifically, tumors that can be treated with the compounds of the invention include adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma , sarcoma and teratoma. Specifically, the tumor/malignant tumor is selected from the group of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenocystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma холангиокарциномы, хондросаркомы цистоаденомы, эндодермальной опухоли, гиперплазии эндометрия, эндометриальной стромальной саркомы, эндометриоидной аденокарциномы, эпендимальной саркомы, саркомы Юинга, фокальной нодулярной гиперплазии, гастриномы, герминогенных опухолей, глиобластомы, глюкагономы, гемангиобластомы, гемангиоэндотелиомы, гемангиомы, аденомы печени, аденоматоза печени, гепатоцеллюлярной carcinoma, insulinitis, intraepithelial neoplasia, intraepithelial squamous neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, nerve sheath tumor, medulloblastoma, medulloepithelioma, mesothelioma, mucus-forming squamous cell carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian carcinoma, adenocarcinoma serosa plasmacytoma, pseudosarcoma, lung blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous, vaginal/vulvar carcinomas, VIPomas, Wilms tumors. Even more preferably, the tumors/malignancies to be treated with the compounds of the invention include, brain cancer, head and neck cancer, colorectal carcinoma, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably astrocytoma II , grade III or IV, glioblastoma, preferably glioblastoma multiforme, small cell carcinoma, and non-small cell carcinoma, preferably non-small cell lung cancer, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, and breast cancer, preferably ductal carcinoma, or breast carcinoma.

In a specific embodiment of the invention, the malignant neoplasm or cells that form tumors arising from oncological diseases are characterized by the presence of high levels of the IL-6-type cytokine. In the context of the present invention, by high levels of the IL-6-type cytokine, it should be understood that the cytokine concentrations are greater than in the control sample by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150 % or more. A control sample is understood as a sample in which the levels of the IL-6 type cytokine are useful as a reference for determining the relative levels of said cytokine in the test sample. Reference samples are usually obtained from patients who are clinically well documented and who do not have disease. In these examples, the concentration of the biomarker can be determined, for example, by determining the average concentration in the reference population. When defining a reference population for a particular marker, some characteristics of the sample type, such as age, sex, physical condition, etc., must be taken into account. patient. For example, a reference sample can be derived from identical numbers of a group of at least 2, at least 10, at least 100 to more than 1000 individuals such that the population is statistically significant.

The concentration of the IL-6-type cytokine can be determined intracellularly, in the interstitial space, or in extracts that have both an intracellular protein and a protein found in the interstitial space. Levels of the cytokine IL-6 can be determined by measuring the activity of said cytokine using assays suitable for this purpose, or by measuring the amount of protein using immunological methods or by measuring the mRNA corresponding to the IL-6-type cytokine. In another specific embodiment, the cancer is caused by high activity of the JAK-STAT signaling pathway, which in another even more specific embodiment is a glioma, preferably a grade IV glioma.

As previously mentioned, the inhibitory agent of the invention is capable of inhibiting tumor stem cell proliferation, so that its use is particularly useful in the treatment of diseases that would benefit from inhibition of stem cell proliferation. Thus, in a preferred embodiment, the inhibitory agents act on the self-renewal of tumor stem cells.

The term arteriosclerosis refers to the thickening and hardening of the arterial wall. A particular type of arteriosclerosis is atherosclerosis, which causes most coronary artery diseases, aortic aneurysm, and lower extremity arterial disease, and moreover, contributes to cerebrovascular disease. A normal artery, as a rule, has an internal part (intima) formed by a single layer of endothelial cells. Beneath this layer lies the so-called middle layer, containing only smooth muscle cells. The outer layer, in turn, is the adventitia. With age, the width of the intima continuously increases in part, as a result of the migration and proliferation of smooth muscle cells. A similar increase also occurs in the width of the intima as a result of several traumatic episodes or interventions, such as those that occur when the process of dilatation causes destruction of the vessel wall. The compounds used in the present invention are potentially useful in inhibiting the proliferation of endothelial cells.

- 14 040048 current, smooth muscle cells and fibroblasts. Accordingly, the labdan-type diterpenoid compounds described herein may also be useful in the treatment of arteriosclerotic conditions. Arteriosclerotic conditions are understood to include classic atherosclerosis, accelerated atherosclerosis, and other arteriosclerotic conditions characterized by unwanted proliferation of endothelial and/or vascular smooth muscle cells, including vascular complications of diabetes and diabetic glomerulosclerosis.

The term restenosis is understood as a disease demonstrating excessive cell proliferation and migration as a result of the release of growth factors, caused by mechanical disruption of the endothelial cells that form the coronary arteries.

Angiogenic disease is understood as a disease or medical condition exhibiting abnormal neovascularization. Such diseases or conditions include diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, and certain malignancies such as hemangioendotheliomas, hemangiomas, and Kaposi's sarcoma. The proliferation of endothelial cells and vascular smooth muscle cells is the main characteristic of neovascularization. The compounds described in the present invention are useful in inhibiting said proliferation and consequently in inhibiting the progression of an angiogenic state which depends in whole or in part on said neovascularization.

The term fibrosis refers to the formation or overdevelopment of fibrous connective tissue in an organ or tissue. Fibrosis includes, for example, endomyocardial fibrosis, idiopathic pulmonary fibrosis, emphysema, pulmonary fibrosis (leading to chronic obstructive pulmonary disease), Peyronie's disease, scleroderma, diffuse parenchymal lung disease, keloids, mediastinal fibrosis, progressive massive fibrosis, proliferative fibrosis, neoplastic fibrosis, interstitial fibrosis of the kidneys, fibrosis of the liver, surgical scars and burns.

The term dermatological diseases is understood as skin diseases exhibiting cell proliferation associated with any proliferative dysfunction. These dysfunctions include, for example, keloids, hypertrophic burn scars, seborrheic keratosis, papillomavirus infection, actinic keratosis, and eczema.

The term inflammatory diseases is understood as diseases caused by inflammation as a result of cell proliferation associated with any proliferative dysfunction. These include, for example, proliferative glomerulonephritis, lupus erythematosus, scleroderma, temporary arthritis, thromboangiitis, and mucocutaneous lymph node syndrome.

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an inhibitory agent of the present invention together with a pharmaceutically acceptable carrier for the treatment of diseases associated with unwanted cell proliferation. Examples of diseases associated with unwanted cell proliferation have been mentioned above in the description.

In the context of the present invention, a therapeutically effective amount is understood as the amount of an agent that inhibits the expression and/or activity of an IL-6-type cytokine that is necessary to achieve the desired effect, which, in this particular case, is the treatment of a disease associated with unwanted cell proliferation. In general, the therapeutically effective amount of the inhibitory agent of the present invention to be administered will depend on the individual being treated, the severity of the disease that the individual is suffering from, the dosage form chosen, and the like, among other factors. For this reason, the doses mentioned in this invention should only be considered as a guide for a person skilled in the art, and the latter should adjust the doses in accordance with the previously mentioned variables. However, the inhibitory agent of the present invention can be administered one or more times a day, for example 1, 2, 3 or 4 times a day, in a typical total daily amount containing from 0.1 to 1000 mg/kg body weight/ day, preferably 10 mg/kg body weight/day.

In the context of this description, the term treatment or treat means the administration of an inhibitory agent of the invention to prevent, alleviate or eliminate a disease or one or more symptoms associated with said disease associated with unwanted cell proliferation. Treatment also includes preventing, attenuating or eliminating the physiological complications of the disease. In the context of the present invention, the term alleviate is understood as any improvement in the situation of the patient being treated - both subjectively (feelings of the patient or about the patient) and objectively (measurable parameters).

The terms carrier, adjuvant and/or carrier refer to the molecular entities or substances with which the active ingredient is administered. Such pharmaceutical carriers, adjuvants or carriers may be sterile liquids such as water or oil, including oils of petroleum, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like, excipients, disintegrating agents, wetting agents or solvents. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin.

- 15 040048

In the context of the present invention, the term pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not generally cause an allergic reaction or similar adverse reaction such as stomach upset, dizziness, and the like. when they are administered to a person. The term pharmaceutically acceptable preferably means approved by a federal and state regulatory agency, or listed in the USP or other widely accepted pharmacopoeia for use in animals, and more particularly in humans. An agent that inhibits the expression and/or activity of an IL-6-type cytokine, as well as pharmaceutical compositions containing it, can be used together with other additional drugs useful in the treatment of diseases associated with unwanted cell proliferation. Said additional drugs may form part of the same pharmaceutical composition or, alternatively, they may be presented as a separate composition for their administration, which may or may not be simultaneous with the administration of a pharmaceutical composition containing said agent that inhibits expression and/or activity of IL-6-type cytokine.

Examples of other additional drugs useful in the treatment of diseases associated with unwanted cell proliferation include, but are not limited to, alkylating agents such as, for example, cyclophosphamide, carmustine, daunorubicin, mechlorethamine, chlorambucil, nimustine, melphalan, and the like; anthracyclines such as, for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, and the like; taxane compounds such as, for example, paclitaxel, docetaxel, and the like; topoisomerase inhibitors such as, for example, etoposide, teniposide, tuliposide, irinotecan, and the like; nucleotide analogs such as, for example, azacitidine, azathioprine, capecitabine, cytarabine, doxyfluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, thioguanine, ftorafur, and the like; platinum-based agents such as, for example, carboplatin, cisplatin, oxaliplatin, and the like; antitumor agents such as, for example, vincristine, leucovorin, lomustine, procarbazine, and the like; hormone modulators such as, for example, tamoxifen, finasteride, 5-α reductase inhibitors, and the like; vinca alkaloids such as, for example, vinblastine, vincristine, vindesine, vinorelbine and the like. Suitable chemotherapeutic agents are described in more detail in the literature, such as the Merck index on CD-ROM, 13th edition.

The pharmaceutical composition of the invention may be administered by any route suitable for administration, for example, orally, parenterally (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, etc.), rectally, etc., usually orally due to chronic the nature of the disease being treated.

Illustrative examples of oral pharmaceutical dosage forms include tablets, capsules, granules, solutions, suspensions, and the like, and may contain conventional excipients such as binders, diluents, disintegrators, lubricants, wetting agents, and the like, and can be prepared by conventional methods. Pharmaceutical compositions may also be suitable for parenteral administration, in the form of, for example, sterile solutions, suspensions or lyophilized products, in a suitable dosage form; in this case, said pharmaceutical compositions will include suitable excipients such as buffers, reagents, and the like. In any case, the excipients are selected in accordance with the selected pharmaceutical dosage form.

An overview of various pharmaceutical dosage forms of drugs and their preparation can be found in Tratado de Farmacia, Galenica, by C. Fauli i Trillo, 10th Edition, 1993, Luzan 5, S.A. de Ediciones.

As one skilled in the art will understand, if an agent that inhibits the expression and/or activity of an IL-6 type cytokine according to the invention contains a nucleotide sequence such as, for example, antisense oligonucleotides, interfering RNA (siRNA), catalytic RNA or specific ribozymes, RNA with decoy activity , etc., the pharmaceutical composition of the invention may be formulated as a composition for use in gene therapy; as a non-limiting illustration, in this case, the pharmaceutical composition of the invention may contain a viral or non-viral vector comprising the specified nucleotide sequence or a gene construct containing the said sequence. By way of non-limiting illustration, these vectors can be viral vectors, such as those based on retroviruses, adenoviruses, and the like, or non-viral, such as DNA-liposome complexes, DNA polymer, DNA polymer-liposome, and the like. [cm. Nonviral Vectors for Gene Therapy, edited by Huang, Hung and Wagner, Academic Press (1999)]. These vectors can be introduced directly into a human or animal body by conventional means, and otherwise they can be used to transform, transfect or infect cells, for example, mammalian cells, including human cells, ex vivo, followed by implantation in a human or animal body. to obtain the desired therapeutic effect. For their introduction into the human or animal body, said cells will be formulated in a suitable medium that does not adversely affect the viability of said cells.

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Screening Methods of the Invention

The discovery made by the authors of the present invention and set forth in the present description, not only relates to the treatment of diseases associated with unwanted cell proliferation or to the diagnosis of these diseases, but also relates to the involvement of LIF in the activation of the JAK/STAT cascade, which can also be used in the development a screening method to identify compounds capable of blocking/inhibiting tumor cell proliferation induced by an IL-6-type cytokine or a functionally equivalent variant thereof.

Therefore, in another aspect, the invention relates to an in vitro method for identifying compounds capable of blocking/inhibiting tumor cell proliferation induced by an IL-6-type cytokine or a functionally equivalent variant thereof, comprising the steps of (i) contacting a cell expressing the IL-cytokine receptor 6-type with an IL-6-type cytokine and a candidate compound, and (ii) identifying compounds that block cell proliferation of said cells.

In a first step, the method of the invention comprises contacting a cell expressing an IL-6-type cytokine receptor with an IL-6-type cytokine in the presence of a candidate compound of any purity.

In the context of the present invention, a cell is understood to be any cell expressing an IL-6-type cytokine receptor. Cells in which the receptor for IL-6-type cytokines is expressed and which can be used in the methods of the present invention include cells derived from solid tumors such as breast cancer cells, bladder cancer cells, melanoma cells, ovarian cancer cells, pancreatic cancer cells, prostate cancer cells, colon cancer cells, lung cancer cells and the like, as well as cells derived from liquid tumors such as leukemia and lymphoma cells. In a specific embodiment, the cell expressing the receptor for the IL-6-type cytokine is a glioma cell, preferably a glioma initiator cell (GIC). Examples of receptors for IL-6-type cytokines can be found in Auernhammer and Melmed, 2000 (Endocrine reviews, vol. 21(3): 313-345), such as the LIF receptor (LIFR), the Oncostatin M receptor (OMSR), etc. .P. Thus, the cells of interest include higher eukaryotic cells, preferably mammalian cells. Preferably, the cells to be used in the present invention are those in which receptors for IL-6-type cytokines are constitutively expressed. Alternatively, conventional cell lines can also be used either directly if found to suitably express IL-6-type cytokine receptors, or after prior transfection with DNA constructs that allow the expression of said receptors. Suitable cells for this purpose include CHO, VERO, BHK, HeLa, COS, MDCK 293, 3T3, WI38, and the like. In a preferred embodiment, the cells used in the method of the invention are glioma cells, preferably grade IV glioma cells.

One skilled in the art will appreciate that, depending on the type of receptor that is expressed by the cell used in the screening method of the invention, it will be necessary to use the appropriate cytokine. The cytokine is preferably selected from the group LIF, IL-6, IL-11, oncostatin M, cardiotrophin-1, CNTF and CLC.

Contacting a cell with a candidate compound may be by any method known to one skilled in the art, including direct contact of a cell expressing a receptor for an IL-6-type cytokine, where said cell is in culture, with an IL-6-type cytokine and a candidate compound. a solvent suitable for their interaction, such as DMSO, and the like.

In accordance with the present invention, contacting a cell with a candidate compound includes any possible way of bringing the candidate compound into close proximity to the target cell, either on the surface of the cell or within it. Thus, if the candidate compound is a low molecular weight molecule, it is sufficient to add said molecule to the culture medium. Where the candidate compound is a high molecular weight molecule (eg, a biological polymer such as a nucleic acid or protein, antibodies, or polypeptides), means must be provided to allow that molecule to enter the cell. Where the candidate molecule is a nucleic acid (eg, antisense oligonucleotides, interfering RNA (siRNA), catalytic RNA or specific ribozymes, RNA with decoy activity), conventional transfection techniques can be used to introduce the DNA construct. In the case where the candidate compound is a protein, the cell can contact either directly with the protein or with the nucleic acid encoding it coupled with elements that allow its transcription/translation once it is inside the cell. To this end, any of the previously mentioned methods can be used to allow it to enter the inside of the cell. Otherwise, it is possible for the cell to come into contact with a variant of the protein under study that has been modified with a peptide that is able to promote protein translocation into the cell, for example, a Tat peptide derived from the HIV-1 TAT protein, the third helix of the Antennapedia protein homeodomain from D. melanogaster, VP22 of herpes simplex virus and arginine oligomers (Lindgren A. et al., 2000, Trends Pharmacol. Sci, 21:99-103, Schwarze S. R. et al., 2000, Trends Pharmacol. Sci., 21: 45-48, Lundberg M et al., 2003, Mol. Therapy 8:143-150 and Snyder E L. and Dowdy, S. F., 2004, Pharm. Res. 21: 389-393).

- 17 040048

Preferably, the candidate compound is not isolated, but rather forms part of a more or less complex mixture obtained from a natural source, or forms part of a library of compounds. Examples of compound libraries that can be tested in accordance with the method of the present invention include, but are not limited to, peptide libraries, including both peptides and peptide analogs, containing D-amino acids or peptides containing non-peptide linkages, nucleic acid libraries, including nucleic acids with non-phosphodiester linkages of the phosphorothioate or peptide-nucleic acid type, libraries of antibodies, carbohydrates, or small molecules, preferably organic molecules, peptidomimetics, and the like. If a library of low molecular weight organic compounds is used, the library should be pre-selected so that it contains compounds that can more easily penetrate into the cell. Thus, compounds can be selected based on certain parameters such as size, lipophilicity, hydrophilicity, ability to form hydrogen bonds.

Otherwise, candidate compounds may form part of an extract obtained from a natural source. The natural source can be an animal, plant, sourced from any environment, including, but not limited to, extracts from terrestrial, aerial, marine organisms, and the like.

In the second step, the invention comprises the identification of those compounds which block the cell proliferation of a cell expressing the receptor for the IL-6-type cytokine. Examples of methods suitable for detecting blockage of cell proliferation include, but are not limited to:

determination of telomerase activity

The enzymatic activity of telomerase can be determined by a method known in the art. For example, telomerase activity can be determined by determining the elongation rate of a particular repeat sequence containing 2, 3, or more repeats of a unitary telomeric sequence (Yegorov, E. E. et al., 1997, Mol. Biol, 31:130-136). Cytoplasmic extracts, nuclear extracts, cell lysates or whole cells can be used to measure this activity. An increase in telomerase activity should be understood as an increase in the absolute level of telomerase activity in a particular cell compared to normal cells in the same individual or compared to normal cells in subjects that do not suffer from the condition.

Determination of telomere length

Methods for determining telomere length are well described in the art by Harley C. B., et al. (Nature, 1990, 345:458-460); Levy, M. Z. et al., (J. MoI. Biol., 1992, 225: 951-960); Lindsey J. et al., (Mutat. Res., 1991, 256:45-48) and Allsopp, R. C. et al., (Proc. Natl. Acad. Sci. USA, 1992, 89: 10114-10118), in among others. Restriction endonucleases that do not fragment telomeric DNA are typically used to later separate molecular weight derived fragments and detect telomeres by hybridization with telomere sequence specific probes.

Determination of cell proliferation

Cell proliferation can be determined by methods that are well known to those skilled in the art, including determining cell doubling time as described by Harley et al., (Nature, 1990, 345:458-460). The rate of cell proliferation can be determined by determining the incorporation of tritiated uridine into the cell or by colorimetric assays with BrdU.

In the present invention, a functionally equivalent variant of an IL-6-type cytokine is understood to be a protein whose amino acid sequence (i) is substantially homologous to the amino acid sequence of an IL-6-type cytokine and (ii) performs the same functions as said IL-6-type cytokine. type. The functional similarity of a protein to another specific protein can be determined by interference analyzes of the expression of a gene encoding a specific protein, when a decrease in expression reduces the activity of this protein, and the subsequent restoration of activity is carried out by the expression of a sequence of another protein. These experiments are carried out using interfering RNA sequences specific and complementary to the sequence of a particular protein, and using expression vectors containing a sequence specific for another protein, regulated or unregulated by an inducible promoter.

An amino acid sequence is substantially homologous to a defined amino acid sequence when it has a degree of identity of at least 70/%, preferably at least 75%, typically at least 80%, preferably at least 85%, more preferably at least 90 %, even more preferably at least 95%, 97%, 98%, or 99% relative to said specific amino acid sequence. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, using standard sequence alignment algorithms known in the art, such as, for example, BLAST [Altschul S. F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3):403-10].

One skilled in the art will appreciate that mutations in the nucleotide sequence of a gene that

- 18 040048 generative cytokine IL-6-type, giving conservative mutations of amino acids in positions that are not critical for the functionality of the protein, are evolutionarily neutral mutations that do not affect the overall structure or functionality. These options fall within the scope of the claims of the present invention. These functionally equivalent IL-6-type cytokine variants having insertions, deletions, or modifications of one or more amino acids relative to said IL-6-type cytokine and, moreover, retaining the same functions as said cytokine, are also included within the scope of this inventions.

Therefore, as used herein, the term functionally equivalent variant also includes any functional fragment of an IL-6-type cytokine. The term fragment refers to a peptide containing a portion of a protein. Here, a functional equivalent fragment of an IL-6-type cytokine is a peptide or protein containing a portion of an IL-6-type cytokine and having the same functions as said cytokine.

Diagnostic methods of the invention

Surprisingly, the present inventors found that the IL-6-type cytokine, more specifically LIF, is involved in the activation of the JAK-STAT cascade mediated by TGFe and thus induces cell proliferation and an increase in the number of tumor stem cells (malignant tumor stem cells). cells). This discovery allows the development of diagnostic methods for diagnosing diseases associated with unwanted cell proliferation based on the determination of levels of the IL-6-type cytokine. Thus, in another aspect, the invention relates to an in vitro method for diagnosing diseases associated with unwanted cell proliferation in a subject, or to a method for determining the predisposition of a subject to said disease associated with unwanted cell proliferation, or to a method for determining in a subject the stage or severity of said associated disease. with unwanted cell proliferation, or to a method for monitoring the impact of therapy administered to an object with said disease associated with unwanted cell proliferation, which includes quantifying the expression levels of a gene encoding an IL-6-type cytokine or a protein encoded by said gene, or a functionally equivalent variant the specified protein in a biological sample from the specified object, where an increase in the expression of the gene encoding the cytokine IL-6 type or the protein encoded by the specified gene, or a functionally equivalent variant of the specified protein, relative to the expression of the gene encoding the cytokine an IL-6-type okine or a protein encoded by the specified gene, or a functionally equivalent variant of the specified protein in the control sample, indicates a disease associated with unwanted cell proliferation, or a greater propensity of the specified object for a disease associated with unwanted cell proliferation or non-response therapy administered to the specified object.

As used herein, diagnosis refers to an assessment of the likelihood that a subject is suffering from a disease. As will be understood by those skilled in the art, such an estimate is usually not correct for 100% of the objects that are diagnosed, although it is preferred. However, the term implies the ability to identify a statistically significant proportion of subjects suffering from a disease or having a predisposition to it. One skilled in the art can determine whether a portion is statistically significant by simply using several well-known statistical evaluation tools, for example, confidence region determination, p-value determination, Student's t-test, Mann-Whitney's test, and the like. See Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 for details. Preferred confidence regions are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values are preferably 0.2, 0.1, 0.05.

As used herein, predisposition means that the subject has not developed the disease or any symptom of the disease mentioned above, or other diagnostic criteria, but, however, will develop the disease in the future with a certain probability. This probability will be significantly different from the statistical probability of occurrence of a disease associated with unwanted cell proliferation. Preferably, at diagnosis, the probability of developing a disease associated with unwanted cell proliferation is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% , at least 90 or 100% predisposition. The diagnosis of predisposition may sometimes be referred to as prognosis or prediction of the subject's likelihood of developing a disease.

In the context of the present invention, a control sample is understood as a reference sample, which is used to determine the variability in the expression levels of genes and proteins used in the present invention. In an embodiment, the reference value is obtained from the available signal using a tissue sample obtained from a healthy individual. Preferably, if the samples are taken from the same tissue of several healthy individuals and combined, so that the amount of mRNA or polypeptides in the sample reflects the average value of these molecules in the population.

Quantitative assessment of the expression levels of the gene encoding the IL-6-type cytokine can be

- 19 040048 carried out on RNA resulting from the transcription of the specified gene (mRNA), or otherwise, the transcription of complementary DNA (cDNA) of the specified gene. In addition, the method of the invention may include carrying out an extraction step to obtain total RNA, which can be carried out using conventional methods (Chomczynski et al., Anal. Biochem., 1987, 162:156; Chomczynski P.,

Biotechniques, 1993, 15:532).

Almost any conventional method can be used to detect and quantify levels of mRNA encoded by a gene encoding an IL-6-type cytokine or its corresponding cDNA within the context of the invention. As a non-limiting illustration, the levels of mRNA encoded by said gene can be quantified using conventional methods, such as methods involving amplifying the mRNA and quantifying the amplification product of said mRNA, such as electrophoresis and staining, or otherwise using northern -blotting and probes specific for the mRNA of the gene of interest or its corresponding cDNA, S1 nuclease mapping, RT-LCR, hybridization, microarrays, and the like. preferably can be quantified by quantitative real-time PCR using suitable sets of probes and primers. Similarly, cDNA levels corresponding to said mRNA encoded by a gene encoding an IL-6-type cytokine can also be quantified using conventional methods; in this case, the method of the invention includes the step of synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNAs, followed by amplification and quantification of the amplification product of said cDNA. Conventional methods for quantifying expression levels can be found, for example, in Sambrook et al., 2001. Molecular cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, N. E., Vol. 1-3.

Thus, in a specific embodiment of the method of the invention, quantifying expression levels of a gene encoding an IL-6-type cytokine comprises quantifying messenger RNA (mRNA) of said gene, a fragment of said mRNA, complementary DNA (cDNA) of said gene, a fragment of said cDNA, or their mixtures.

In another specific embodiment, quantitative analysis of the expression levels of the gene encoding the cytokine IL-6-type, carried out by quantitative polymerase chain reaction.

In addition, when the method of the invention is practiced, the expression levels of the protein encoded by said gene encoding the IL-6-type cytokine, i. a gene encoding IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-I), ciliary neurotrophic factor (CNTF), or cardiotrophin-like cytokine (CLC) may also be quantified. As one of skill in the art will appreciate, the level of protein expression can be quantified using any conventional method. As a non-limiting illustration, protein levels can be quantified, for example, by using antibodies with the ability to bind said proteins (or antigenic determinant-containing fragments thereof) and then quantifying the complexes formed. The antibodies used in these assays may be labeled or unlabeled. Illustrative examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and the like. There are many known types of assays useful in the present invention that use unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these methods include Western blot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive enzyme immunoassay, two-antibody sandwich ELISA, immunocytochemical and immunohistochemical methods, methods based on the use of protein biochips or microerrevs, which include specific antibodies or analyzes based on colloidal precipitation in formats similar to indicator strips. Other ways to detect and quantify a protein include affinity chromatography methods, ligand binding assays, and the like. In another specific embodiment, protein levels are quantified by Western blotting, immunohistochemistry, or ELISA.

In another preferred embodiment, the determination of expression levels of the IL-6-type cytokine can be carried out by determining the activity of said protein, since high levels of expression result in a higher specific activity of said protein in the sample. Assays for determining the activity of IL-6-type cytokines have previously been described in the context of the therapeutic methods of the invention.

IL-6-type cytokines, levels of which can be used as markers of unwanted cell proliferation, have previously been described herein and are applicable to the methods of the invention. Similarly, the diagnostic method of the invention can be applied to any disease associated with unwanted cell proliferation as defined above. In a preferred embodiment, the disease associated with unwanted cell proliferation is a malignancy, preferably a malignancy having high levels of the IL-6-type cytokine or high activity of the JAK-STAT signaling pathway.

The implementation of the method of the invention in practice involves obtaining a biological sample from

- 20 040048 object being treated. Illustrative, non-limiting examples of these samples include various types of body fluids such as blood, serum, plasma, cerebrospinal fluid, peritoneal fluid, feces, urine and saliva, as well as tissue samples. Samples of biological fluids can be obtained by any conventional method suitable for tissue samples; for illustration, these tissue samples may be biopsy samples obtained by surgical resection.

In another aspect, the invention relates to the use of a kit containing reagents for quantifying expression levels of a gene encoding an IL-6 cytokine or a protein encoded by said gene or a functionally equivalent variant of said protein for diagnosing malignancy in a subject or for determining a subject's propensity to the specified malignant neoplasm, or to determine the stage or severity of the specified malignant neoplasm in the object, or to monitor the impact of therapy administered to the object with the specified malignant neoplasm, in which the reagents detect an increase in the expression of the specified gene or the specified protein or its functionally equivalent variant, relative to the control sample, where the specified object may suffer from a disease associated with unwanted cell proliferation, or he has a greater predisposition to the specified disease associated with unwanted cell proliferation her, or he has a greater severity of the indicated disease, or the therapy administered is not effective.

All terms and expressions used to define the use of the kit have previously been described and explained for other original aspects and certain embodiments of the present invention, and are also suitable for use of the kit described in this document.

Methods for designing specialized therapies and selecting patients who may benefit from therapy based on IL-6 inhibitors

The present inventors have shown that inhibitors of cytokines belonging to the IL-6 family, and more particularly LIF, cause inhibition of tumor cell proliferation. The authors also noted that there are tumors that have very high levels of these cytokines, so the authors suggest that therapy based on the use of IL-6 inhibitors may be particularly useful in the treatment of patients with high levels of IL-6-type cytokines.

Thus, in another aspect, the invention relates to an in vitro method for developing a specialized therapy for a patient suffering from a disease associated with unwanted cell proliferation, comprising:

(a) quantifying expression levels of the IL-6-type cytokine in said patient and (b) comparing said expression levels with control levels, where if the expression levels of the IL-6-type cytokine in said patient are higher than the control values, then said patient administering an agent that inhibits the IL-6 type cytokine.

In another aspect, the invention relates to an in vitro method for selecting patients suffering from a disease associated with unwanted cell proliferation to receive an IL-6-type cytokine inhibitory agent, comprising:

a) quantifying expression levels of the IL-6-type cytokine in said patient, and

b) comparing said expression levels with control levels, where if the expression levels of the IL-6-type cytokine in said patient are higher than the control values, then said patient is selected to receive treatment with an IL-6-type cytokine inhibitory agent.

In both aspects, a preferred embodiment is one in which the unwanted cell proliferation disease is associated with unwanted stem cell proliferation.

In a preferred embodiment, the IL-6 cytokine inhibitory agent is selected from the group consisting of siRNAs, antisense oligonucleotides, specific ribozymes, antibodies and polypeptides. The inhibitory agents are preferably antibodies, and more preferably inhibitory antibodies specific for said IL-6 type cytokine or antibodies blocking IL-6 type cytokine receptors.

IL-6-type cytokines, which can be used as a marker for patient selection or for the development of specialized therapies, have been detailed above and are selected from LIF, IL-6, IL-11, oncostatin M, cardiotrophin-1, CNTF, and CLC. . Diseases caused by unwanted cell proliferation are those described above. In a preferred embodiment, said disease caused by unwanted cell proliferation is a malignant neoplasm. Even more preferably, said cancer is caused by high activity of the JAK-STAT signaling pathway. In a preferred embodiment, said cancer is any of glioma, pre-B lymphoblastic leukemia, acute myeloid leukemia, colorectal carcinoma, bladder cancer, ductal breast cancer, or breast carcinoma. Even more preferably, said glioma is grade IV glioma.

- 21 040048

Predictive Methods of the Invention

In another aspect, the invention relates to an in vitro predictive method for predicting life expectancy in patients suffering from a disease associated with unwanted cell proliferation. This method is based on the observation that, for example, in the case of glioma, life expectancy is reduced in patients showing higher levels of LIF expression compared to control patients (FIG. 12).

The method is based on

a) quantifying expression levels of the IL-6-type cytokine in said patient, and

b) comparing said expression levels with control levels, where if the expression levels of the IL-6-type cytokine in said patient are greater than values in control patients with the same disease, then said patient is likely to have a lower life expectancy than in control group.

In a more specific aspect, the concentration of the IL-6-type cytokine can be measured for prognostic purposes, namely to predict the average life expectancy of an individual suffering from said disease. For this purpose, the concentration of an IL-6-type cytokine in a tumor patient is compared with a reference concentration of the same IL-6-type cytokine. The reference patient group typically consists of patients who are well documented and who suffer from the same disease. For example, a reference sample can be obtained from identical numbers of a group of at least 2, at least 10, at least 100 to more than 1000 individuals, such that the population of patients suffering from said disease is statistically significant. A reference group may consist of one or more of the following:

a) all patients suffering from said disease,

b) all patients suffering from said disease in which no significantly upregulated levels of the IL-6-type cytokine are shown

c) all patients suffering from said disease who exhibit significantly down-regulated levels of the IL-6-type cytokine.

The concentration of the IL-6-type cytokine can be determined intracellularly, in the interstitial space, or in extracts containing both an intracellular protein and a protein found in the interstitial space. Levels of the cytokine IL-6 can be determined by measuring the activity of said cytokine using assays suitable for this purpose, or by measuring the amount of protein by immunological methods or by measuring the mRNA corresponding to the IL-6-type cytokine.

In this aspect, the preferred embodiment is a disease associated with unwanted cell proliferation. In a more specific embodiment, the disease associated with unwanted cell proliferation is a malignant neoplasm. Even more preferably, the type of cancer is associated with abnormally high levels of the IL-6 type cytokine in a subgroup of patients with said cancer. In a more specific embodiment, the cancer is one of the following: leukemia, glioma, colorectal carcinoma, bladder cancer, breast cancer. In a more specific embodiment, the leukemia is pre-B cell acute lymphoblastic leukemia or acute myeloid leukemia and the breast cancer is ductal breast cancer or breast carcinoma.

IL-6-type cytokines, which can be used as a marker to screen patients for prognostic purposes, have been detailed above and are selected from LIF, IL-6, IL-11, oncostatin M, cardiotrophin-1, CNTF, and CLC.

Statistical methods will predict the value of the average life expectancy of patients, based on the levels of the cytokine IL-6-type.

Information confirming the possibility of carrying out the invention

The invention is described below by way of examples, which are to be considered as merely illustrative and non-limiting.

1. Materials and methods

1.1. Cell lines and primary cell cultures.

U373MG and A172 cells were a generous gift from J. Rich and D. Bigner and were cultured in DMEM with 10% fetal bovine serum (PBS). Primary culture of tumor cells (PCTC) and GBM neurospheres were formed as described (Bruna et al., (2007) Cancer Cell 11, 147-160; Gunther et al., (2008) Oncogene. May 1; 27 (20): 2897-909). Briefly, tumor samples were processed within 30 min after surgical resection. Pulverized pieces of human glioma samples were digested with 200 U/ml collagenase I (Sigma) and 500 U. /ml DNase I (Sigma) in PBS for 2 h at 37°C with constant vigorous stirring. The individual cell suspension was filtered through a 70 μm cell filter (BD Falcon) and washed with PBS. Finally, cells were resuspended and then cultured in DMEM with 10% FBS for PCTC culture, or in neurosphere medium for GBM neurospheres. Neurospheres of normal human neuroprogenitors were obtained from

- 22 040048 human fetal cortex tissue (12-16 weeks after conception) collected after voluntary abortions. Samples were processed and cultured as described (Poltavtseva et al. (2002) Brain Res Dev

Brain Res 134, 149-154). Neurosphere medium consists of neurobasal medium (Gibco) supplemented with

B27 (Gibco), glutamine (Gibco), penicillin/streptomycin, and growth factors (20 ng/ml EGF and 20 ng/ml FGF-2 (Peprotech)).

Samples of human gliomas and human fetal tissues were obtained from Vall d'Hebron Hospital. The clinical protocols were approved by the ethics committee (CEIC) of Vail d'Hebron with informed consent obtained from all subjects.

1.2. Plasmids and reagents

U373MG genomic DNA was used to amplify the -634/+32 region of the human LIF promoter, which was cloned into the pGL2-basic luciferase vector. Deletion constructs (267/+32) and (-73/+32) were generated by digestion of the construct (-634/+32). Two point mutations were introduced at positions -83 bp. and -184 b.p. into the construct (-267/+32) to disrupt the Smad binding element (SBE) and obtain the construct (-267/4-32) mutSBE. TGFe1, TGFe2, TGFe3 (R&D Systems), TeRI Inhibitor (SB431542, Tocris), LIF (Chemicon), Anti-LIF Neutralizing Antibodies (R&D), JAK Inhibitor (Tetracyclic Pyridone 6 (P6) Calbiochem), Smad2 siRNA SMART System, Smad3 and Smad4 (Dharmacon) and control siGlo siRNA (Dharmacon) were used at the indicated concentrations. Antibodies specific for p-Smad2, Smad2, p-STAT3 (p-Tyr705) and total STAT3 (Cell Signaling) and for Smad2, Smad3 and Smad4 (Hata et al., (2000) Cell 200, 229-240) were used to Western blotting.

1.3. Immunohistochemistry, ELISA and chromatin immunoprecipitation

Immunohistochemistry of neurospheres and differentiated neurospheres was performed as described (Geshwind et al., 2001) using the following antibodies: anti-nestin antibody (Chemicon), anti-GFAP antibody (Dako), anti-TuJ 1 antibody (Chemicon), anti-04 antibody (Chemicon) , anti-Sox2 antibody (Chemicon), anti-αtubulin antibody (Sigma). The nuclei were counterstained with 4',6-diamino-2-phenylindole (DAPI).

To quantify the levels of LIF protein secreted into the medium, a human LIF ELISA kit (R&D Systems) was used, following the manufacturer's descriptions. The supernatant of U373MG cells and GBM neurospheres, previously serum-free, was collected 48 hours after said treatment. Floating cells were discarded and 5 ml of supernatants were concentrated using Amicon Ultra-4 PLCC Ultracel-PL 5 kDa membranes (Millipore) to a final volume of 200 μl.

Chromatin immunoprecipitation was performed as described (Bruna et al., mentioned above). The proximal and distal primer series of the LIF promoter covered the (-410/-165) and (-4534/-4293) regions, respectively.

1.4. Self-updating analyzes

The self-renewal of the neurospheres was assessed by seeding the same number of very low density cells into the wells of a 96-well plate. Cells were treated, in the absence of growth factors, with these compounds and the total number of new neurospheres formed after 7 days in culture was counted (Lee et al. (2008) Cancer Cell 13, 69-80; Reynolds and Weiss, (1996) Dev Biol 175, 1-13; Seaberg and van der Kooy, (2002) J Neurosci 22, 1784-1793).

1.5. Quantitative real-time PCR

Quantitative RT-PCR was performed using Taqman probes from Applied Biosystems, according to the manufacturer's recommendations. Reactions were performed using an ABI 7000 sequence detector (Perkin Elmer) and the results were expressed as the fold change calculated by the DDCt method from the control sample or the first sample counted. The 18S ribosomal unit or β-actin were used as internal controls for normalization.

1.6. Luciferase assays

A172 cells were transiently transfected with Lipofectamine 2000 (Invitrogen) with various LIF promoter indicator constructs and luciferase plasmid pRL-TK from Renilla (Promega) as a normalization control.

1.7. Analysis of intracranial tumors

The indicated numbers of cells were stereotaxically inoculated into the striatum of the right hemisphere of the brain (1 mm anteriorly, 1.8 mm lateral to the bregma, and 3.0 mm intraparenchymal) of seven-week-old female Balbc nu/nu mice (Charles River Laboratories). Mice were sacrificed when they showed neutral symptoms and significant weight loss. The studies were performed using magnetic resonance imaging (MRI) with a 9.4 T vertical magnet in interface with the AVANCE 400 system (Bruker). Under xylazine/ketamine anesthesia, mice were intravenously injected with the MRI contrast agent, gadolinium-diethylenetriamine-pentaacetic acid, at a dose of mmol Gd/kg body weight and placed in a 18 RF coil (inner diameter, 35 mm). Whole-brain images of a mouse were acquired after image capture by the localizer along three orthogonal axes.

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1.8. Statistical analysis

The Spearman correlation test was used to analyze the interactions between LIF and TGFe2, Musashi1, Sox2 and Nestin. The data in the graph are presented as mean ± standard deviation.

Example 1

TGFe Induces Self-Renewal of Patient-Derived GICs To investigate the effect of TGFe on the self-renewal capacity of GICs (glioma initiating cells), cells were obtained from surgically removed GBM (glioblastoma multiforme) samples. From one tumor sample, on the one hand, primary cultures of tumor cells (PCTC, primary cultures of tumor cells) were obtained in the presence of serum, and in parallel, tumor cells from the same sample were cultured in a serum-free medium in the presence of EGF and FGF. Cells cultured in serum-free medium supplemented with EGF and FGF rapidly formed non-adherent multicellular spheres (neurospheres) as described (Galli et al. (2004) Cancer Res 64, 7011-7021; Gunther et al., cited above; Lee et al., (2006) Cancer Cell 9, 391-403 Singh et al.(2003) Cancer Res 63, 5821-5828) (Fig. 1A). Neurospheres derived from tumor samples expressed high levels of neuroprogenitor cell markers, Musashi-1, Sox2, and Nestine (Fig. 1B), and as they progressed along the multilinear differentiation pathway, they acquired the expression of GFAP (an astrocyte marker), Tuj-1 (a neuronal marker) and 04 (oligodendrocyte marker), when cultured in the presence of serum (FIG. 2). Moreover, tumor-derived neurospheres were highly oncogenic compared to PCTS. Neurosphere cells and PCTS were orthotopically implanted in the brains of immunosuppressed mice. Tumor growth was assessed by magnetic resonance imaging (MRI), the weight of mice was checked. Neurosphere cells formed tumors 30-60 days after inoculation, which caused intense weight loss, while PCTC did not grow in the tumor during the same time interval in all cases (Fig. 1C). Thus, neurospheres obtained from human GBM samples expressed markers of neuroprogenitor cells, demonstrating the potential for multilinear differentiation, and were highly oncogenic. All of these characteristics indicate that the neurospheres obtained from GBM patients were enriched in GIC.

It was decided to evaluate the effect of TGFe on the self-renewal ability of the GIC by the following well-described protocol based on the ability of the GIC to form neurospheres (Reynolds and Weiss, 1996, Dev Biol. 175:1-13; Seaberg and van der Kooy, 2002, J Neurosci 22:1784 -1793). Neurospheres obtained from patients were dissociated into individual cells, treated with TGFe or left untreated for 7 days in the absence of growth factors, after which the newly formed neurospheres and the total number of cells were counted. Following this protocol, the effect of TGFe on the self-renewal capacity of GICs obtained from three different patients was evaluated. Treatment with TGFe significantly increased the number of neurospheres and the total number of cells (Fig. 1D, 1E, 1F). These effects were blocked when a TGFeI receptor inhibitor (TeRI) was added concurrently with TGFe. Separately, the TeRI inhibitor had no significant effect (Fig. 1D, 1E, 1F). These results suggest that the TGFe pathway enhances GIC self-renewal.

Example 2 TGFe Induces LIF Expression in Human GBM Cells

It was decided to investigate the molecular mechanisms responsible for the effect of TGFe on the GIC. Gene responses to TGFe were studied in GBM cells, which may be involved in the regulation of GIC self-renewal. In previous work (Bruna et al., 2007), transcriptome analyzes were performed in the U373MG glioma cell line treated with TGFe and/or TeRI inhibitor. In U373MG, LIF was among 63 gene responses to TGFe dependent on TeRI activity. The LIF-LIFR/gp130JAK-STAT signaling pathway is involved in stem cell self-renewal like embryonic stem cells (Niwa et al., 1998, Genes Dev, 12:2048-2060; Williams et al., 1988, Nature, 336:684-687 ) and neuroprogenitor cells (Bauer and Paterson, 2006, J Neurosci, 26:12089-12099; Molne et al., 2000, J Neurosci Res, 59:301-311; Wright et al., 2003, J. Neurochem, 86 :179-195), leading to the suggestion that LIF may mediate the effect of TGFe on the GIC. For the first time, TGFe-mediated induction of the LIF transcript has been shown to occur in patient-derived tumor cells. A PCTC panel prepared from 11 different human GBMs was treated with TGFe for 3 h and LIF mRNA levels were determined. TGFe induces LIF in all PCTCs analyzed (FIG. 3A). These results indicate that LIF induction by TGFe is a common phenomenon that occurs in most human GBMs. Moreover, TGFe was able to induce the LIF transcript in patient-derived neurospheres (FIG. 3B), and this effect depended on TeRI activity, since LIF induction by TGFe was blocked in the presence of the TeRI inhibitor (FIG. 3C). Three members of the TGFe family (TGFe1, TGFe2, and TGFe 3) were able to induce LIF in the neurospheres obtained from their patients (Fig. 3D) and, as expected, induction of the LIF transcript by TGFe resulted in increased secretion of the LIF protein, as measured by ELISA in conditioned medium for neurospheres (Fig. 3E).

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Example 3 TGFe induces LIF expression through an activated Smad complex that binds to the LIF promoter

To study the transcriptional regulation of LIF by TGFe, the human LIF promoter was cloned into the pGL2-basic reporter construct. In U373MG cells, TGFe was able to transactivate report constructs that contain the -634/+32 and -276/+32 regions of the LIF promoter. The -73/+32 fragment of the LIF promoter lost the transcriptional response to TGFe, indicating that the TGFe response element was included in the -276/-73 region (FIGS. 4A, 4B). This region contains a single Smad binding element (SBE, 5'GTCT-3') near the SP1 binding site (FIG. 4A). The SBEs were mutated and abolition of the response to TGFe was observed (FIG. 4B), indicating that the activated Smad complex binds the proximal SBE in the LIF promoter to induce transcription. Chromatin immunoprecipitation (ChIP) assays were performed and it was observed that endogenous Smad2 binds the proximal region of the LIF promoter and does not bind the distal 4 kb region. upstream of the transcription initiation site in a TGFe-treated cell (Fig. 4C). In order to definitively demonstrate that Smad is involved in the induction of LIF expression by TGFe, Smad2, Smad3, Smad2 and 3 and Smad4 were silenced by RNA interference. LIF induction by TGFe was reduced with decreasing levels of Smad2 and Smad3, or Smad4, indicating that an activated Smad complex is required for the transcriptional response of LIF to TGFe (Fig. 4D). Smad2 and Smad3 are redundant in this process because silencing of each Smad alone does not significantly affect TGFe-induced LIF levels (Fig. 4D). As expected, LIF induction by TGFe in patient-derived neurospheres was also dependent on Smad. Silencing Smad4 in human GBM abolished the LIF response to TGFe (Fig. 4E).

Example 4 TGFe Induces the JAK-STAT Pathway Through LIF Induction in Patient-Derived Neurospheres

In order to determine if the LIF signaling pathway is functional in GBM neurospheres, the neurospheres were treated with recombinant LIF and the levels of phosphorylation of the downstream substrate of the LIF receptor complex, STAT3, were determined. Recombinant LIF induces rapid STAT3 phosphorylation, suggesting that patient-derived neurospheres express a functional LIF receptor complex (FIG. 5A). Moreover, p-STAT3 induction was abolished in the presence of the pharmacological JAK-specific tetracyclic pyridone 6 (P6) (Pedranzini et al., 2006, Cancer Res, 66: 9714-9721; Thompson et al., 2002, Bioorg Med Chem Lett, 12 :1219-1223) (Fig. 5B). Interestingly, TGFe induces STAT3 phosphorylation in GBM neurospheres, and the TeRI inhibitor interferes with this effect (Fig. 5C). The decision was made to evaluate whether LIF mediated p-STAT3 induction by TGFe. For this purpose, a neutralizing anti-LIF antibody was used to specifically block the effects of secreted LIF on cells treated with TGFe. The presence of a neutralizing antibody to LIF reduces the induction of p-STAT3 by TGFe. Moreover, p-STAT3 levels in TGFe treated cells were repressed by P6 treatment (FIG. 5D). These data indicate that TGFe is able to activate the JAK-STAT pathway in patient-derived neurospheres by inducing the secretion of LIF, which acts through an autocrine/paracrine loop.

Example 5 LIF mediates GIC self-renewal induction by TGFe

The decision was made to evaluate whether LIF and JAK-STAT signaling mediate increased GIC self-renewal via TGFe. For this purpose, a neutralizing antibody against LIF and P6 was used to specifically block the effect of secreted LIF on cells treated with TGFe. The neurospheres were dissociated into individual cells and treated with TGFeα, recombinant LIF, anti-LIF and/or P6 antibodies. Newly formed neurospheres and the total number of cells were counted. Recombinant LIF increased the number of newly formed neurospheres as well as the total number of cells, indicating that LIF induces GIC self-renewal (FIGS. 6A, 6B, 6C). Treatment with a neutralizing antibody to LIF reduces the induction of GIC self-renewal by TGFe. Moreover, P6 represses the effect of TGFe on GIC self-renewal, indicating that the effect of TGFe on self-renewal depends on JAK activity (FIGS. 6A, 6B, 6C). Overall, these data suggest that TGFe induces the self-renewing ability of patient-derived GICs via the LIF-JAK-STAT pathway.

Example 6 TGFe Prevents GIC Differentiation by LIF

GBM-derived neurospheres grown in the absence of growth factors and scattered in polylysine-coated dishes tend to differentiate with loss of expression of neuroprogenitor markers Musashi-1, Sox2 and Nestin and attach to the culture dish. The decision was made to evaluate the impact of TGFe and LIF on this differentiation process. Neurospheres were cultured in the presence of TGFe or LIF without EGF or FGF for 7 days and then processed for immunohistochemical staining and quantitative RT-PCR to determine the levels of neuroprogenitor markers Musashi-1, Sox-2 and Nestin. Neurospheres treated with TGFe or LIF differed morphologically from control cells in that they were less attached to the culture medium.

- 25 040048 cup, keeping the spherical shape. Moreover, TGFe or LIF-treated cells maintained Musashi-1, Sox2 and Nestin expression as detected by immunohistochemical analyzes (FIG. 7A) and as determined by qPCR-RT (FIG. 7B). This indicates that TGFe and LIF are factors that not only regulate GIC self-renewal, but are also involved in preventing GIC differentiation.

Example 7 Effects of TGFe and LIF on Normal Human Neuroprogenitors

These data indicate that TGFe and LIF regulated GIC self-renewal and differentiation. The decision was made to assess whether this effect was specific to tumor cells or if it is also present in normal neuroprogenitor cells. To answer this question, neuroprogenitor cells were obtained from human fetal cortex samples (12 to 16 weeks after fertilization). Previously described (Carpenter et al., 1999, Exp Neurol, 158: 265-278; Poltavtseva et al., 2002, Brain Res Dev Brain Res, 134: 149-154; Wright et al., 2003, J Neurochem, 86 : 179195) that neurospheres derived from human neuroprogenitors, when grown in serum-free medium supplemented with EGF and FGF, express Musashi-1, Sox-2, Nestin like GBM neurospheres (Fig. 8A). First, LIF induction by TGFe was determined in normal human neuroprogenitors. Normal neurospheres did not induce LIF in response to TGFe 1 , TGFe2 or TGFe3 compared to GBM neurospheres (FIG. 8B). Moreover, TGFe does not induce LIF in mouse neuroprogenitors derived from mouse embryos or from the subventricular region of adult mice (data not shown). This indicates that LIF induction by TGFe is specific to GBM neurospheres. As expected, since TGFe did not induce LIF, TGFe did not increase the self-renewal capacity of normal neuroprogenitors, and the number and size of neuroprogenitor neurospheres were not increased by TGFe treatment. In fact, TGFe treated neurospheres were smaller and the total number of cells decreased due to the presence of TGFe (FIGS. 8C, 8D). On the other hand, LIF increased the number and size of newly formed neurospheres, as well as the total number of cells (Fig. 8C, 8D) in accordance with previous work (Bauer and Patterson, 2006; Wright et al., 2003). Thus, LIF has the same effect on the self-renewal of GBM and normal neurospheres. Conversely, there is a difference in the effect of TGFe on the self-renewal capacity of normal and tumor neurospheres due to the inability of TGFe to induce LIF in normal neuroprogenitors.

Example 8 LIF expression in human gliomas correlates with TGFe2 and neuroprogenitor markers

To assess LIF expression in human gliomas, LIF levels were analyzed in a panel of 39 gliomas. It was noted that LIF was expressed in 17, and was highly expressed in 4-6 of 39 gliomas (FIG. 9A), indicating that the majority of human gliomas express LIF. Since LIF is induced by TGFe and previous work has shown that TGFe2 is responsible for the high TGFe activity observed in gliomas (Bruna et al., 2007, cited above), it was decided to evaluate the involvement of TGFe2 in LIF expression. That LIF levels correlated with TGFe2 in a panel of gliomas, further supporting the fact that TGFe2 is responsible for LIF induction in human gliomas (Fig. 9A, B). If LIF promotes GIC self-renewal, then tumors that express high levels of LIF should be enriched for a group of cells of this type. To test this hypothesis, LIF levels were compared with the expression of GIC/neuroprogenitor markers. LIF levels correlate with Musashi-1 and Nestin expression, but not Sox2 (Fig. 9A,B), indicating that LIF activates GIC self-renewal and increases the GIC group present in the tumor mass.

Example 9 Patients with glioma or glioblastoma have a shorter overall life expectancy

In a subset of all patients with glioma, LIF levels are positively regulated >2-fold. Over a period of time, these patients have a significantly reduced likelihood of survival compared to control patients. For example, the probability of survival after 1000 days is reduced by about 50% compared to all glioma patients, and to about 35% compared to glioma patients with LIF levels that are positively unregulated >2-fold (FIG. 10). Data obtained from the National Cancer Institute's REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) program

In the subset of all patients with glioblastoma, LIF levels are up-regulated >9-fold. Over a period of time, these patients have a significantly reduced survival rate compared to control patients. For example, the probability of survival after 500 days is reduced by about 50% compared to all patients with glioblastoma (Fig. 11). Data obtained from the REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) program of the National Cancer Institute.

Example 10 LIF mRNA levels are abnormally high in various types of tumors

Some patients with certain types of tumors have aberrantly high levels of LIF,

- 26 040048 indicating aberrantly high levels of LIF mRNA (Fig. 12). Data obtained from the GeneSapiens bioinformatics team (www.genesapiens.org).

This indicates that LIF may provide a selective advantage in the progression of different tumor types. Tumor types from >10 patients tested with LIF mRNA levels above the error margin: pre-B cell acute lymphocytic leukemia (B-ALL), acute myeloid leukemia (AML), glioma, adenocarcinoma of the lung, colorectal carcinoma, bladder cancer, ductal breast cancer and breast carcinoma.

In these patients with tumors expressing high levels of LIF, LIF may act as an oncogenic factor through the regulation of malignant tumor stem cells. Thus, blockade of LIF may be useful in this group of tumors and LIF may also be used as a diagnostic and/or prognostic factor in these patients.

Claims (17)

CLAIM 1. A method of treating glioma characterized by high LIF levels, comprising administering a LIF inhibitory agent selected from an anti-LIF antibody or an anti-LIF siRNA. 2. The method of claim 1, wherein the antibody acts through inhibition of self-renewal of tumor stem cells. 3. The method of claim 1, wherein the glioma is caused by high activity of the JAK-STAT signaling pathway. 4. The method according to claims 1 to 3, wherein said glioma is a grade IV glioma. 5. A pharmaceutical composition comprising a therapeutically effective amount of an LIF inhibitory agent, together with a pharmaceutically acceptable carrier, for use in the method of any one of claims 1 to 4, wherein the inhibitory agent is selected from an antibody and an siRNA. 6. Method for the treatment of glioma, including: (a) quantifying the expression level of LIF in a patient having glioma, (b) comparing the expression level to a control level, and (c) administering an LIF inhibitory agent to a patient having a LIF expression level greater than the control level, wherein the inhibitory agent is selected from anti-LIF antibodies; and anti-LIF miRNAs. 7. An in vitro method for selecting patients suffering from glioma to be treated with a LIF inhibitory agent, comprising: (a) quantifying the level of LIF expression in a sample obtained from said patient, and (b) comparing said expression level with a control level, where if the LIF expression level in said sample is higher than the control value, then said patient is selected for treatment with an agent, an inhibitory LIF, wherein the inhibitory agent is selected from an anti-LIF antibody and an anti-LIF siRNA. 8. The method according to any one of claims 6 or 7, wherein said agent acts through inhibition of self-renewal of tumor stem cells. 9. The method according to any one of claims 6 or 7, wherein the LIF-specific antibody blocks LIF receptors. 10. The method according to any one of claims 6 to 9, wherein said glioma is caused by high activity of the JAK-STAT signaling pathway. 11. The method according to any one of claims 6 to 10, wherein said glioma is a grade IV glioma. 12. In vitro method for predicting the life expectancy of patients with glioma, including quantifying the level of expression of the gene encoding LIF or the protein encoded by the specified gene in a biological sample obtained from the specified patient, where an increase in the expression of the gene encoding LIF or the protein encoded the specified gene, relative to the expression of the gene encoding LIF, or the protein encoded by the specified gene in the control sample, indicates a decrease in life expectancy. 13. The method of claim 12 wherein the glioma is glioblastoma. 14. The method according to any one of claims 12, 13, in which quantitative analysis of the level of expression of a gene encoding LIF includes quantitative analysis of messenger RNA (mRNA) of said gene, a fragment of said mRNA, complementary DNA (cDNA) of said gene, a fragment of said cDNA, or their combinations. 15. The method of claim 14, wherein the quantitative analysis of expression levels of the gene encoding LIF is carried out by quantitative polymerase chain reaction (PNR). 16. The method according to any one of claims 12, 13, wherein the quantitative analysis of protein levels is carried out by Western blotting, immunohistochemistry or ELISA. 17. The method according to any one of claims 12, 13, in which the quantitative analysis of protein levels is carried out by determining the activity of said protein.