WO2012034969A1

WO2012034969A1 - Gt aptamer oligonucleotides and use thereof as antitumor agents

Info

Publication number: WO2012034969A1
Application number: PCT/EP2011/065738
Authority: WO
Inventors: Gabriele Grassi; Bruna Scaggiante
Original assignee: Universita' Degli Studi Di Trieste
Priority date: 2010-09-13
Filing date: 2011-09-12
Publication date: 2012-03-22
Also published as: IT1405761B1; ITPD20100272A1

Abstract

The invention relates to aptamer oligonucleotide having a GT sequence, and derivatives thereof, having a well-defined length, and their use as antitumor agents alone or in association with antitumor agents currently in therapeutic use. The oligonucleotide according to the invention may, in addition to a therapeutic use, have a diagnostic use in that it is capable of detecting proteins of the eEF1A family.

Description

GT aptamer oligonucleotides and use thereof as antitumour agents

Field of the invention

The invention relates to new aptamer oligonucleotides of length 75 nucleotides (nt) with a GT sequence capable of exercising a specific and selective antiproliferative action in human tumour cells and binding/inactivating proteins in the family eukaryotic elongation factor eEF1 A. The invention also relates to the therapeutic use of said oligonucleotides as new antitumour agents with antiproliferative activity in highly aggressive tumours and the diagnostic use thereof as reagents for the specific detection of proteins of the eEF1 A family.

State of the art

Among the components the family of eukaryotic elongation factor eEF1 A, two genes are constitutively transcribed: EEF1 A1 (Chromosome 6 - NC_000006.1 1 ; Gene ID 1915) on chromosome 6q14.1 and EEF1 A2 (Chromosome 20 - NC_000020.10; Gene ID 1917) on chromosome 20q13.3. The proteins eEF1A belong to the superfamily of G proteins; they are among the most abundant proteins in mammalian cells and participate in the process of protein translation, carrying aminoacyl-tRNA (aa-tRNA) to site A of the ribosomes in the form of a ternary complex eEF1 A-GTP-aa-tRNA (Scaggiante B et al., 2008). Both eEF1 A1 proteins (Homo sapiens eukaryotic translation elongation factor 1 alpha 1 : NM_001402.5 NP_001393.1 ) and eEF1 A2 (Homo sapiens eukaryotic translation elongation factor 1 alpha 2: NM_001958.2 NP_001949.1 ) also have non-canonical functions: they are involved in many other essential cellular processes such as cytoskeleton remodelling and cell migration, modulation of kinase activity and phosphorylation, control of the fidelity of translation, modulation of the degradation of ubiquitylated proteins, cell response to stress and thermal shock and modulation activities of the conformation of proteins (Scaggiante B et al., 2008; Koiwai K et al., 2008; Zhong D et al., 2009; Jeganathan S et al., 2008; Leclercq TM et al., 2008; Panasyuk G et al., 2008).

In mammals the protein eEF1 A1 is expressed ubiquitously, whereas the expression of eEF1 A2 protein occurs in specialized tissues such as skeletal muscle, cardiac tissue and the central nervous system (Scaggiante B et al., 2008). The two proteins eEF1 A1 and eEF1 A2 are involved in the transformation and progression of neoplastic cells. In particular, the expression of the gene EEF1 A2 in tissue other than skeletal muscle, cardiac tissue and the central nervous system is associated with the development of tumours and is correlated with their aggressiveness in ovarian tumours, breast tumours, pancreas carcinoma and hepatocellular carcinoma (Scaggiante B et al., 2008; Schlaeger C et al., 2008; Cao H et al., 2009; Sun Y et al., 2008; Lee MH and Surh YJ, 2009). In addition, over- expression of eEF1 A1 and 2 in hepatocellular carcinoma is correlated with an increased rate of proliferation and eEF1 A1 and 2 are expressed most in undifferentiated cells with a more aggressive phenotype (Grassi G et al., 2007). eEF1 A2 has been shown to determine the migration and invasiveness of breast carcinoma cells (Scaggiante B et al., 2008). Furthermore, eEF1 A1 and eEF1 A2 are interactors of the "gene human testis-specific Y-encoded" (TSPY; Chromosome 8 - NC_007306.4; Gene ID 281554) and it has been suggested that the complex TSPY-eEF1 A1/2 promotes and supports the neoplastic transformation of testicular germ cells, but also probably the cells of the prostate or other types of somatic tissue (Kido T and Lau YF, 2008). The over-expression of the gene EEF1A1 has been associated with increased proliferation and transformation of cells (Scaggiante B et al., 2008; Scaggiante B and Manzini G, 2010). The protein eEF1 A1 regulates the half-life of the imRNA of osteopontin (OPN, Accession No. P10451 .1 ), a phosphoprotein whose over-expression is characteristic of advanced-stage metastatic tumours, acting as a transactivating factor and promoting the invasiveness of hepatocellular carcinoma cells (Zhang J et al., 2009). An increase in eEF1 A1 is associated with the resistance of head and neck tumours resistant to cisplatin. An isoform of eEF1 A1 is present in human haematopoietic tumours, but not in normal cells. The deregulation of eEF1 A1 in cells of rodents exposed to chemical and physical carcinogens promotes neoplastic transformation. The reduction of the expression of eEF1 A1 in leukaemic cells of promyelocytes subjected to the action of the differentiating drug "All-trans- retinoic acid" (ATRA) contributes to the survival of malignant cells. The over- expression of eEF1 A1 contributes to the survival of breast cancer cells and is involved in the invasiveness of these cells, whereas in pro-B line murine cells it confers resistance to apoptosis following stress (Scaggiante B et al., 2008; Scaggiante B and Manzini G, 2010). The over-expression of the gene EEF1 A1 is correlated to methotrexate resistance in many solid human tumours and tumours of the haematopoietic system (Selga E et al, 2009). During the alkalinisation of tumour cells, which is a common phenomenon in transformation, an actin-mediated increase in the levels of eEF1 A1 /2 promotes and supports the invasiveness of the neoplastic cells (Kim J et al, 2009).

Phosphodiester or partially phosphorothioated GT aptamer oligodeoxyribonucleotides with lengths between 20 and 60 nucleotides have been shown to be effective in causing a specific, selective and dose-dependent inhibition of the growth of human tumour cells (WO 97/20924; Scaggiante B et al., 1998; Morassutti C et al., 1999a; Morassutti C et al., 1999b). The GT oligonucleotide sequences that act as aptamers are ligands of the eEF1 A proteins with the ability to also recognise a specific isoform produced only by leukaemic tumour cells (Dapas B. et al., 2003). These GT aptamers, in particular one of length of 51 nucleotides, if carried by polycations, are active at nanomolar concentrations in inhibiting the proliferation of haematopoietic tumour cells (Scaggiante B et al., 2005). It has been shown that GT aptamers GT of 23 to 27 nucleotides in length are active as antitumour agents if they do not give rise to particularly stable structures such as G quadruplexes (Scaggiante B et al, 2006). A GT aptamer sequence of length 27 nucleotides is able to increase the therapeutic index of conventional antineoplastic drugs in haematopoietic and solid tumours (Dapas B et al, 2002).

Such oligonucleotides have been shown to be active in many human solid and haematopoietic tumours. However, their activity has never been studied in cells with a high level of malignancy, such as hepatocellular carcinoma. In this type of tumour, it has been shown that proteins of the eEF1 A family are over-expressed and contribute to tumour progression (Grassi G et al., 2007).

On the other hand, as it is known, hepatocellular carcinoma is a type of tumour that has few options for pharmacological treatment, and therefore new tools useful in controlling its expansion/progression are extremely important. There is also a need to find molecules and formulations of molecules that are able to effectively control the growth of undifferentiated tumours and therefore their dissemination in the body and have limited side-effects or have a risk/benefit ratio favourable to the severity of the disease and the possibility of being treated with methods currently in use.

Summary

In seeking a possible therapeutic method of treating highly aggressive tumours, such as hepatocellular carcinoma, and even orphans of adequate therapy, the inventors have identified in the length of these GT aptamer oligonucleotides the essential characteristic of determining their antiproliferative activity. In particular, the inventors found that the oligonucleotide having a length of 75 nucleotides has the ability to inhibit the proliferation of undifferentiated human hepatocellular carcinoma cells in a surprisingly specific and dose-dependent way after a single administration of nanomolar doses.

Therefore, in a first aspect, the object of the invention is a molecule comprising an aptamer oligonucleotide having the SEQ ID NO.1 TG(TTTG)i₈T (TGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGT TTGTTTGTTTGTTTGTTTGT). This sequence is hereinafter identified as GT75. The invention also extends to derivatives of said aptamer oligonucleotide GT75 having SEQ ID NO.1 having at least the same nucleotide length with different GT oligodeoxynucleotide sequences with orientation 5'-3' or 3'-5', which do not give rise to structures compatible with the G-quadruplexes, and the oligoribonucleotides corresponding to said oligodeoxynucleotide sequence GT75 and derived oligodeoxynucleotide sequences. Also included in the invention are the derivatives of such sequences of oligodeoxynucleotides or oligoribonucleotides with biochemical or chemical modifications intended for stabilisation, compartmentalisation or in-situ localisation. Such derivatives with biochemical or chemical modifications include alkylates, phosphorothioates, and/or conjugates with peptides, polycations or PEG, as well as optical isomers (Spiegelmers).

In a second aspect, the object of the invention is the therapeutic use of the aptamer oligonucleotide having SEQ ID NO.1 TG(TTTG)isT or its derivatives as antitumour agents for the treatment of tumoral pathologies, in particular for highly aggressive and/or undifferentiated tumours. Therefore, pharmaceutical compositions comprising this oligonucleotide with SEQ ID NO.1 or its derivatives for the treatment of highly aggressive and/or undifferentiated tumours are a further object of the invention.

In another aspect, the object of the invention is the diagnostic use of the aptamer oligonucleotide having SEQ ID NO.1 TG(TTTG)isT or its derivatives as reagents for diagnostic kits for the detection of eEF1 A family proteins for the diagnosis and/or prognosis of highly aggressive tumoral pathologies and for monitoring the efficacy of the chemotherapy regimen selected.

In a further aspect, therefore, the object of the invention is the diagnostic kit comprising the aptamer oligonucleotide with SEQ ID NO.1 TG(TTTG)isT or derivatives thereof for the diagnosis and/or prognosis of tumoral pathologies and for monitoring the efficacy of the therapy regimen.

Brief description of the figures

Figure 1. Dose-response effect of GT75 on cellular growth measured with the MTT test as absorbance of the line U2SO after 10 days of culture in comparison with the CT75 oligonucleotide.

Figure 2. Dose-response effect of GT75 on cellular growth measured with the MTT test as absorbance of the line MO59J after 10 days of culture in comparison with the CT75 oligonucleotide.

Figure 3. Dose-response effect of GT75 on cellular growth measured with the MTT test as absorbance of the lines LNCaP, DU-145 and PC-3 after 10 days of culture in comparison with the CT75 oligonucleotide.

Figure 4. Effect of idarubicin at different doses after 10 days of culture of hepatocellular carcinoma cells JHH6 measured with the MTT test as absorbance. Figure 5. Effect of bortezomib (Velcade) at different doses after 10 days of culture of hepatocellular carcinoma cells JHH6 measured with the MTT test as absorbance.

Figure 6. Effect of the association between GT75 and idarubicin (treatment regimen in a single dose 1 : transfection with oligonucleotides day 1 , incubation with drug day 2) at the indicated doses of idarubicin after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance. Figure 7. Effect of the association between GT75 and Velcade (treatment regimen in a single dose 1 as above) at the indicated doses of Velcade after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance.

Figure 8. Effect of the association between GT75 and idarubicin (treatment regimen in a single dose 2: transfection with oligonucleotides day 1 , incubation with drug day 3) at the indicated doses of idarubicin after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance.

Figure 9. Effect of the association between GT75 and Velcade (treatment regimen in a single dose 2 as above) at the indicated doses of Velcade after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance.

Figure 10. Effect of the association between GT75 and idarubicin (treatment regimen in a single dose 3: transfection with oligonucleotides day 1 , incubation with drug day 4) at the indicated doses of idarubicin after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance.

Figure 11. Effect of the association between GT75 and Velcade (treatment regimen in a single dose 3 as above) at the indicated doses of Velcade after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance.

Figure 12. Effect of the association between GT75 and idarubicin (treatment regimen in a single dose 4: transfection with oligonucleotides day 1 , incubation with drug day 8) at the indicated doses of idarubicin after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance.

Figure 13. Effect of the association between GT75 and Velcade (treatment regimen in a single dose 4 as above) at the indicated doses of Velcade after 10 days of culture of hepatocellular carcinoma cells JHH6 treated with GT75 or CT75 or not treated NT and measured with the MTT test as absorbance.

Detailed description of the invention The invention relates to an aptamer oligonucleotide having the SEQ ID NO.1 TG(TTTG)i₈T (hereinafter referred to as GT75) and its derivatives having different GT sequences with orientation 5'-3' or 3'-5', which do not result in structures compatible with the G-quadruplexes. The invention also extends to derivatives of these oligodeoxynucleotides, including those with different GT sequences, consisting of the oligoribonucleotides corresponding to the oligodeoxynucleotide sequences. The invention also includes the derivatives of these oligonucleotide sequences, being oligodeoxynucleotides or oligoribonucleotides, phosphodiesters or chemically and/or biochemically modified as known to the person skilled in the art to increase stability, and cell penetration in situ (see review by Grassi M et al 2010), for example phosphorothioates, alkylates, methylates, etc., and/or conjugates with peptides, polycations or PEG, and optical isomers.

In order to assess if the length of the oligonucleotide, together with the sequence, was the determining factor for the antiproliferative activity and for the specificity on highly aggressive and undifferentiated tumour cells, the oligonucleotide GT75 was compared with GT oligonucleotides with similar sequences but shorter lengths, that is oligonucleotides with SEQ ID NO. 2 TG(TTTG)eT (hereinafter referred to as GT27) and SEQ ID NO. 3 TG(TTTG)i₂T (hereinafter referred to as GT51 ), and with oligonucleotides of sequence CT of the same sequence and length SEQ ID NO. 4 TC(TTTC)₁₈T (hereinafter referred to as CT75) or of lesser length of SEQ ID NO. 5 TC(TTTC)₆T (hereinafter referred to as CT27) and SEQ ID NO. 6 TC(TTTC)i₂T (hereinafter referred to as CT51 ).

The extended sequences of GT and CT oligonucleotide aptamers are listed below: GT75 (SEQ ID NO. 1 )

TGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTT

TGTTTGTTTGTTTGTTTGT;

GT27 (SEQ ID NO. 2)

TGTTTGTTTGTTTGTTTGTTTGTTTGT ;

GT51 (SEQ ID NO. 3)

TGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGT ;

CT75 (SEQ ID NO. 4)

TCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTT CTTTCTTTCTTTCTTTCT ;

CT27 (SEQ ID NO. 5)

TCTTTCTTTCTTTCTTTCTTTCTTTCT ;

CT51 (SEQ ID NO. 6)

TCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCT.

The comparison has shown that GT75 has demonstrated the ability to inhibit the proliferation of undifferentiated human hepatocellular carcinoma cells in a surprisingly specific and dose-dependent way after a single administration of nanomolar doses. In contrast, GT oligonucleotides with a length shorter than 75 nt, and in particular GT27 and GT51 , have not demonstrated any significant activity of inhibiting the growth of cells of undifferentiated hepatocellular carcinoma cells. GT75 has also been shown to be active on other lines of human solid tumours that do not have any effective treatment, such as osteosarcoma and glioblastoma. In a model of prostate adenocarcinoma cells with different degrees of aggressiveness, GT75 has been shown to be very active in the most undifferentiated cells, which corroborates the result of its remarkable antiproliferative action on highly aggressive human tumours.

Based on the results obtained, the oligonucleotide with SEQ ID NO. 1 and its derivatives can therefore be used as antiproliferative agents for the treatment of highly aggressive undifferentiated tumours and, in particular, for the treatment of tumours such as from hepatocellular carcinoma, osteosarcoma, glioblastoma, prostate adenocarcinoma, melanoma, colonadenocarcinoma, leukaemias/lymphomas and pulmonary carcinoma.

In addition, GT75 has also been shown to significantly increase the therapeutic index of conventional antineoplastics, with the result of inhibiting the growth of hepatocellular carcinoma at subtoxic doses and with different administration regimens of the active molecules. This potentiating effect is particularly interesting because it allows GT75 to be used in combined action with conventional antineoplastic drugs at doses that minimise the risk of side-effects in many types of tumoral pathologies for which the conventional antineoplastic drug is considered the therapy of choice.

The present invention therefore also relates to a therapeutic method comprising the administration to a patient with highly aggressive and/or undifferentiated tumoral pathologies of the aptamer oligonucleotide of SEQ ID NO. 1 and derivatives thereof according to the invention, alone or in combination with other antineoplastic drugs of the same.

Therefore, the present invention extends to pharmaceutical compositions in which the active ingredients according to the invention are combined with suitable excipients and/or diluents or with other active ingredients for systemic or local administration. The active ingredients according to the invention are also preferably administered through drug-delivery systems, such as liposomes, lipid nanoparticles and/or biodelivery systems and/or systems of molecular targeting mediated by protein sequences (for example "RGD" domains) at their site of action, i.e. the tumour is either primary or secondary.

Moreover, given the ability of these aptamers to recognise eEF1 A family proteins, further use of GT75 is in the diagnostics field through the use of oligomers of this type marked with fluorescent substances or dyes for histological examinations or for aptamer DNA-based assays.

The diagnostic method may also be prognostic in the evaluation of tumour progression. In fact, when the histological examination shows over-expression of the eEF1 A proteins, this may be correlated with an positive/negative prognosis together with the scores already accredited and in use for the typing of the tumour stages. For diagnostic purposes, in particular, the aptamer oligonucleotide GT75, conjugated with a fluorochrome or colorimetric detection system, can be anchored to a support where proteins extracted from biopsy specimens are brought into contact with the aptamer GT 75 or a derivative thereof in a highly stringent solution that ensures the specificity of the reaction. If the specific proteins are present, they bind to the aptamer and are measured using the appropriate detection signal of the established bond. Alternatively, the aptamer conjugated with a fluorophore or a chromophore can be used as an antibody (aptabody) on paraffinised histological samples or fresh histological samples for histochemical analysis for diagnostic and prognostic purposes. Additionally, the aptamer appropriately conjugated at both ends with a fluorophore and a quencer can be used to quantify very precisely the eEF1 A proteins present in a tumour sample using techniques such as FRET. Typically, the diagnostic method can include at least the following phases:

a. provision of a biological sample by biopsy;

b. bringing the biological sample into contact with the oligonucleotide with SEQ ID NO. 1 or its derivative, optionally labelled; c. detection of the reaction between the oligonucleotide with SEQ ID

NO. 1 or its derivative and the proteins of the eEF1 A family which are present in the biological sample;

d. measuring the signal for detection of the occurred reaction. The diagnostic kit for determining the expression of the eEF1A proteins using the GT75 aptamer oligonucleotide or a derivative thereof may comprise at least: a vial containing the GT75 aptamer oligonucleotide of SEQ ID NO.1 or a derivative thereof, optionally labelled (preferably with fluorophores and/or chromophores), a vial containing a sample of the eEF1 A proteins to be used as competitive ligands, optionally labelled, and an illustrative leaflet with instructions.

There follow some examples of embodiments of the invention and evaluations of the benefits obtained from the GT75 aptamer oligonucleotide according to the invention, given by way of example and not limiting the invention itself.

EXAMPLES

Example 1: synthesis of the oligonucleotides (GT)_n and (CT)_n (n= 75, 51, 27) The deoxyribonucleotide sequences of the phosphodiester oligonucleotides GT75 and CT75 were synthesised by the known chemical method of phosphoramidites (Mag M and Engels J, 1988) and purchased from the company Eurofins MWG/operon. Before being used in the cells for the biological activity assessment, the oligonucleotides were deprotected, desalinised and purified of all toxic products by HPSF and their purity was checked by the MALDI-TOF MS.

The oligonucleotides thus prepared were assayed on different human cell lines: undifferentiated human hepatocellular carcinoma, androgen-dependent prostate adenocarcinoma, androgen-independent prostate adenocarcinoma, osteosarcoma and glioblastoma according to the experimental methods described below.

Methods of culturing of the cell lines and administration of the oligonucleotides All of the human cell lines were cultured in a complete medium containing 10% FBS supplemented with 10 U/ml penicillin, 10 pg/iml streptomycin and 2 imM L- glutamine. The media, supplements and FBS were purchased from Invitrogen. The cell lines used and the respective media are listed below:

JHH6 undifferentiated hepatocellular carcinoma, RPMI medium;

LNCaP androgen-dependent prostate adenocarcinoma and DU-145 and PC-3 androgen-independent prostate adenocarcinoma

(aggressiveness of tumour cells: PC-3>DU-145>LNCaP), RPMI medium;

U2OS osteosarcoma, DMEM medium;

MO59J glioblastoma, DMEM medium.

The cells, unless otherwise indicated, were seeded in 200 μΙ of the appropriate complete medium in following densities in 96 microtitre wells:

the cells of the lines JHH6, DU-145, PC-3, U2OS at the density of 2.5 x10³ and 10³;

the cells of the line MO59J at the density of 2.5x10³;

- the cells of the line LNCaP at the density of 4x10³.

After 12 h of incubation at 37°C, with 5% CO₂ to allow their adherence, the cells were transfected with the oligonucleotides synthesised as described in example 1 . The oligonucleotides were administered at the concentrations indicated in the examples below, using the method of transfection with cationic liposomes and in particular with Lipofectamine as described by Yu JY et al., 2002. The Lipofectamine 2000 (Invitrogen) was hydrated in 100 μΙ of Optimem (Invitrogen), then mixed with the oligonucleotides in the ratio 1 :1 (w/w) and the mixture was incubated for 20 min at room temperature. The liposome/oligonucleotide mixture was then diluted with Optimem to obtain the desired concentration of oligonucleotides/100 μΙ of medium. 100 μΙ of medium containing the oligonucleotides and the transfectant were added to the cells seeded in the 96-well microtitre in substitution of their complete medium. After 3 hours of incubation at 37 °C, the medium with the oligonucleotides and transfectant was replaced with 200 μΙ of fresh complete medium. The medium of the cells was replaced with fresh medium every 72 h of culture for the first two intervals and then after 48 h, with the following schedule:

day 0 seeding; day 1 transfection and change of medium after 3 h;

day 4 medium change;

day 7 medium change;

day 9 medium change.

The cell growth was monitored with the MTT test (Scudiero DA et al., 1988) at the specified times (day 3, day 6 and day 10 after administration of the selected oligonucleotide). 20 μΙ of MTT (4 mg/ml) was added to the cells in each well. The cells were incubated for 4 hours at 37 °C and 5% CO₂, then the medium was removed and the cells solubilised in 200 μΙ DMSO. The colour development of the tetrazolium salts was read at 540-690 nm in a microtitre plate reader.

Example 2: effect on antiproliferative activity in cells of undifferentiated human hepatocellular carcinoma JHH6 - comparison between (GT)_n and (CT)_n (n= 75, 51, 27)

The comparison between GT75 and GT sequences of shorter length was performed on undifferentiated human hepatocellular carcinoma cells JHH6 according to the methods of cultivation of the cell cultures and administration of the oligomers described above, by measuring the effect under different conditions of concentration of oligomers, cell density and observation times after administration of the oligomers.

A first assay was performed to evaluate the cell growth measured by the MTT test after a single administration of 250nM of oligomers to 2.5 x10³ cells in a 6-well microtitre after 3 and 6 days of culture. The GT aptamer sequences shorter in length than 75-mer (27-mer and 51 -mer) were compared with respect to GT75 as mean ± SD of the absorbance and as % of cell growth compared to the control sequences CT75, CT51 and CT27 or the untreated samples (NT). The cell growth was evaluated with the MTT test as previously indicated, and therefore the measurements performed are measurements of absorbance.

The results obtained are shown in Table 1 below.

Table 1. Comparative effect of a single dose of 250nM of oligonucleotides 3 and 6 days after transfection of 2.5 x10³ cells of JHH6

SD ±0.05 ±0.02 ±0.06 ±0.07 ±0.03 ±0.09 ±0.08

% CT 95 95 16

% NT 58 60 22 24 4 24

6 0.787 0.839 1 .203 1 .029 0.688 1 .122 1 .122

SD ±0.12 ±0.09 ±0.09 ±0.05 ±0.06 ±0.09 ±0.09

% CT 94 1 17 61

% NT 47 51 73 62 41 68

As can be seen from the table, only GT75 is able to exert a significant inhibitory activity against proliferation of JHH6 tumour cells. In addition, it is noted that the inhibition of the growth comes from a specific biological effect, as can be deduced from the comparison with the control sequence CT75. It is also noted that in the control CT75, the lower cell proliferation comes from the non-specific toxicity due to the treatment of transfection.

In a second test the cell viability at the dose of 125 nM of cells with a density of 10³ in a 96-well microtitre and with observation at 3, 6 and 10 days after transfection was measured.

Table 2 shows the comparative data, measured as absorbance, obtained between GT75 and shorter GT sequences and the related CT control sequences. It is noted that, 10 days after the start of a single administration, only GT75 shows a significant antiproliferative effect compared to the control CT75.

Table 2. Comparative effect of a single dose of 125nM of oligonucleotides 3, 6 and

10 days after transfection of 10³ cells of JHH6

SD ±0.199 ±0.127 ±0.125 ±0.067 ±0.144 ±0.189 ±0.022

% CT 1 10 108 27

% NT 120 109 108 99 21 80

Example 3: effect on antiproliferative activity in cells of undifferentiated human hepatocellular carcinoma JHH6 at the density of 10^s cells at different doses of oligonucleotides (GT)_n and (CT)_n (n= 75, 51, 27)

The comparison assay was performed as in example 2, using in this case the cell density of 10³ and progressive doses of oligonucleotides 125, 250 and 500nM. Tables 3-5 show the comparative dose-response effects between GT75 and GT of shorter length on JHH6 seeded at the density of 10³ cells in a 96-well microtitre and monitored at different times from the only administration compared to the CT control oligomers and the non-treated samples (NT). The determinations are also from absorbance in this case.

Table 3. Comparative effect of the dose of 125nM of oligonucleotides 3, 6 and 10 days after transfection of 10³ cells of JHH6

Table 4. Comparative effect of the dose of 250nM of oligonucleotides 3, 6 and 10 days after transfection of 10³ cells of JHH6 Day GT27 CT27 GT51 CT51 GT75 CT75 NT

3 0.153 0.145 0.021 0.019 -0.006 0.01 0.86

SD ±0.03 ±0.03 ±0.01 ±0.01 ±0.01 ±0.005 ±0.07

% CT 105 1 10 -0.5

% NT 18 17 2.4 2.2 -0.7 1 .5

6 0.696 0.659 0.1 0.14 -0.001 0.06 2.02

SD ±0.06 ±0.07 ±0.003 ±0.009 ±0.01 ±0.03 ±0.1 1

% CT 106 71 -0.2

% NT 34 33 5 7 -0.05 3

10 2.35 2.17 0.743 0.789 0.05 0.581 2.52

SD ±0.13 ±0.14 ±0.15 ±0.29 ±0.06 ±0.28 ±0.10

% CT 108 94 8.6

% NT 93 86 29 31 2 23

Table 5. Comparative effect of the dose of 500nM of oligonucleotides 3, 6 and 10 days after transfection of 10³ cells of JHH6

It can be noted that only GT75 is able to exercise a specific dose-dependent inhibition effect of the growth of JHH6. Example 4: study of the antiproliferative effect of GT75 and CT75 in other tumour cell lines

The antiproliferative activity of GT75 and CT75 was monitored after 10 days of cell culture. In addition, for each tumour line, the transfection efficiency was evaluated, and only the lines with the incorporation of at least 90% of the oligomers were considered.

As shown in figure 1 , GT75 exercises a significant and specific dose-dependent antiproliferative effect on the osteosarcoma line compared to the control CT75. Figure 2 shows the dose-response effect of GT75 on a line of glioblastoma compared to the control CT75. Note the specific antiproliferative effect starting from the dose from 125 nM.

Figure 3 shows the antiproliferative effect of a panel of prostate tumour lines with different degrees of aggressiveness (PC-3>DU-145>LNCaP). Note that GT75 exercises a significant and specific antiproliferative effect only in the line of more aggressive prostate adenocarcinoma.

These results indicate that GT75 is particularly active in undifferentiated human tumour lines and with a higher level of aggressiveness than hormone-independent lines (PC-3).

Example 5: effect of GT75 and CT75 in association with conventional antineoplastics in hepatocellular carcinoma cells

The antiproliferative effect of the combination of GT75 and two conventional antineoplastic drugs (idarubicin and bortezomib) was studied in undifferentiated hepatocellular carcinoma lines JHH6. Idarubicin is an anthracycline whose mechanism of action, like its derivative daunomycin, occurs via its insertion into DNA, inhibiting its transcription. Bortezomib (Velcade) is a dipeptidyl derivative of boronic acid that inhibits the activity of the proteasome 26S that degrades ubiquitylated proteins. The resulting effect is inhibition of a whole cascade of molecular events that lead to apoptosis.

GT75 and the conventional antineoplastics were administered according to different therapeutic regimens in subtoxic doses that alone did not significantly alter the growth of the cells to see if the combined treatment is able to have an antiproliferative activity on cell growth. The effect was studied on the growth of 10³ JHH6 cultivated in a 96-well microtitre according to the following therapeutic regimens:

- treatment regimen in single dose 1 : transfection with oligonucleotides day 1 , incubation with drug day 2;

- treatment regimen in single dose 2: transfection with oligonucleotides day 1 , incubation with drug day 3;

- treatment regimen in single dose 3: transfection with oligonucleotides day 1 , incubation with drug day 4;

- treatment regimen in single dose 4: transfection with oligonucleotides day 1 , incubation with drug day 8.

As can be seen from Figures 4 and 5, idarubicin does not significantly alter cell growth measured by the MTT test up to 5 ng/ml, and for Velcade up to at least 15 nM.

As the standard subtoxic dose of GT75, 125 nM was chosen which, under these experimental conditions, causes no more than 20% inhibition of cell growth. The subtoxic doses of idarubicin and Velcade and that of GT75 are therefore used to study the effect of combined treatment, since these alone are not able to produce a significant antiproliferative effect.

For the specificity of the action of GT75, experiments were also conducted in parallel with the control sequence CT75 at the same nanomolar dose. The cell growth was monitored with the MTT test after 10 days of culture.

Figure 6 shows the effects of the combination of GT75 and different subtoxic doses of idarubicin up to 5 ng/ml, while Figure 7 shows the effects of the combination of GT75 and subtoxic doses of Velcade up to 15 nM, in both cases with treatment regimen 1 .

Figure 8 shows the effects of the combination of GT75 and different subtoxic doses of idarubicin up to 5 ng/ml, while Figure 9 shows the effects of the combination of GT75 and subtoxic doses of Velcade up to 15 nM, in both cases with treatment regimen 2.

Figure 10 shows the effects of the combination of GT75 and different subtoxic doses of idarubicin up to 5 ng/ml, while Figure 1 1 shows the effects of the combination of GT75 and subtoxic doses of Velcade up to 15 nM, in both cases with treatment regimen 3.

Figure 12 shows the effects of the combination of GT75 and different subtoxic doses of idarubicin up to 5 ng/ml, while Figure 13 shows the effects of the combination of GT75 and subtoxic doses of Velcade up to 15 nM, in both cases with treatment regimen 4.

From the results obtained, it can be concluded that GT75 is able to produce a significant and specific increase in the therapeutic index of the two drugs in all of the different treatment schedules used.

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Claims

1 . A molecule comprising an aptamer oligonucleotide having SEQ ID NO.1 and derivatives thereof.

2. A molecule according to claim 1 , wherein the aptamer oligonucleotide derivatives having SEQ ID NO.1 are oligonucleotides having different GT sequences with orientation 5'-3' or 3'-5' which do not give rise to compatible structures with G quadruples.

3. A molecule according to claims 1 and 2, wherein said derivatives are oligoribonucleotide derivatives thereof.

4. A molecule according to claims 1 -3, wherein said derivatives are phosphorodiesters, alkyl derivatives, phosphorothioates and/or conjugated with peptides, polycations or PEGs, and optical isomers (spiegelmer).

5. A molecule according to claims 1 -4 for use as a medicament.

6. A molecule according to claims 1 -4 for use for the treatment of cancer diseases.

7. A molecule according to claim 6, wherein the cancer diseases undifferentiated tumors.

8. A molecule according to claim 6, wherein the cancer diseases are hepatocarcinoma, osteosarcoma, glioblastoma, prostate adenocarcinoma, melanoma, colon adenocarcinoma, leukemias, lymphomas and lung carcinoma.

9. Pharmaceutical compositions comprising as active ingredient a molecule according to claims 1 -4, in combination with suitable excipients and/or diluents and/or carrier systems for the same.

10. Pharmaceutical compositions according to claim 9 for systemic and/or local administration.

1 1 . A molecule according to claims 1 -4 for use as reagent for diagnosis and/or prognosis of cancer diseases.

12. A molecule according to claims 1 -4 for use as reagent for monitoring the efficacy of the therapeutic treatment in cancer diseases.

13. A diagnostic method for diagnosis of cancer diseases by using a molecule according to claims 1 -4 for detecting the eEF1 A protein expression.

14. The method according to claim 13, comprising at least the steps of:

a. providing a biopsy-type biological sample;

b. contacting the biological sample with an optionally labeled molecule according to claims 1 -4;

c. detecting the reaction between the selected molecule and the proteins from eEF1 A family which are present in the biological sample;

d. measuring the signal for detection of the occurred reaction.

15. A diagnostic kit comprising at least one vial containing a molecule according to claims 1 -4, optionally labeled, and one vial containing a sample of optionally labeled eEF1 A proteins, and a leaflet for use in diagnosis and/or prognosis and/or monitoring the efficacy of the therapeutic treatment in cancer diseases.