MXPA00011919A - Monoclonal antibodies directed against the g3bp protein, and uses - Google Patents

Abstract

The invention concerns monoclonal antibodies directed against the G3BP protein and cell lines producing them. The invention also concerns the use of said antibodies or their derivatives for obtaining medicines and diagnostic reagents.

Description

MONOCLONAL ANTIBODIES DIRECTED AGAINST G3BP PROTEIN, AND ITS USE The present invention concerns monoclonal antibodies directed against the G3BP protein as well as the cell lines that produce them. The invention also concerns the use of these antibodies or their derivatives for obtaining drugs and diagnostic reagents.

The products of ras genes, generally designated p21Ras proteins, play a key role in the control of cell division in all eukaryotic organisms in which they have been investigated. Certain specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic. Thus, a large number of human tumors have been associated with the presence of modified ras genes. Likewise, an over-expression of the p21Ra proteins can lead to a deregulation of cell proliferation. Globally, p21i? As proteins are implicated in 30% of human cancers.

The understanding of the exact role of p21 Ras proteins is therefore one of the targets of the Ref.125319 research in the area of oncology.

The model currently available to explain the functioning of p21i? As proteins is based on the analogies they share with transduction G proteins. There is a balance in the cells between active p21 proteins, linked to GTP and inactive forms, which is fixed to GTP. In an inactive cell, in which Ras proteins are not requested, most of them are in the form of GDP. When the cell is stimulated, the nucleotide exchange factor, GEF, becomes more active and facilitates the evacuation of the GDP and its replacement by GTP. The protein then adopts an active conformation that allows it to recognize and stimulate its effector, the GAP protein "GTPase activating protein". The Ras-GTP-GAP complex actually interacts with one or other proteins that allow the transmission of the signal that implies a biological response of the cell. The association of Ras-GTP with GAP simultaneously initiates the hydrolysis of GTP and the return of the ras protein to the inactive form, GDP.

In the case of oncogenic p21Ra proteins, the mutation they carry prevents the return to the inactive state. The balance is in this last case, displaced towards the active form of Ras.

This complex balance between the active and inactive forms of the p21J? As is controlled both by factors inherent to the biochemical properties of Ras proteins (relative affinity for GDP and GTP, exchange rate of nucleotides ...) and external factors that modulate its activity, particularly the GAP protein.

The GAP protein is a cystolic protein present in all eukaryotic organisms that thus possess the ability to accelerate strongly the hydrolysis of GTP, linked to the normal p21 protein (Trahey and Me Cormick 1987). It has two regions that ensure different functions. Its carboxy terminal end carries the catalytic activity that binds the Ras proteins and increases its GTPase activity. On its other, lower end of the ino-terminal part, there is a juxtaposition of SH2 and SH3 regions that are likely to participate in interactions with other proteins.

The applicant has already identified a supplementary protein, in the Has-dependent signaling cascade. Thus, application 096/16169 describes the identification, cloning, sequence formation and characterization of a protein capable of binding the SH3 region of GAP, designated G3BP ("Protein Link GAP-SH3"). This application shows in particular that G3BP is an effector of GAP, that is to say a companion that intervenes in the lower part in the Ras-dependent signaling path. Thus, the article by Parker and collaborators (Mol Cell. Biol .. 16 (1996) 2561) describes the sequence of this protein, its ability to bind to GAP, etc. This protein constitutes a new major target in the adjustment of anti-cancer therapeutic attempts.

The G3BP protein is a 68 kDa cystosolic protein expressed ubiquitously. The sequence of the G3BP protein and the corresponding geen are presented in SEQ ID NO: 1 and SEQ ID No. 2. This protein of 466 amino acids belongs to the family of hnRNP (heterogeneous nuclear riboproteins) and contains several characteristic regions of the proteins that are linked to the AR ?: - an RRM region (aa 342-385) with the R? P2 regions (aa 342-347) and RNP1 (aa 378 to 385) - a region rich in amino acids arginine, glycine (aa 429 to 461) - an acid auxiliary region (aa 144 to 221) The applicant is currently interested in the mechanism of action of the G3BP protein. In this view, the applicant obtained different antibodies directed against different regions of the G3BP protein in order to antagonize the activity of G3BP or to constitute effectors of G3BP.

The present invention relies on the one hand on the demonstration of an over-expression of the protein G3BP in tumor cells and in human tumors (adenocarcinoma of the colon, and breast tumor) and on the other hand in the surprising discovery that certain antibodies directed against the G3BP protein are capable of inducing apoptosis in human tumor cells. Unexpectedly, it was found that these antibodies induce apoptosis in the tumor cells in which the G3BP protein is overexpressed but are found to be non-toxic in normal human cells, in which G3BP is slightly expressed.

A first object of the invention concerns monoclonal antibodies directed against the G3BP protein and capable of inducing apoptosis in different types of tumor cells.

. More particularly, the invention relates to monoclonal antibodies capable of recognizing an epitope located in the N-terminal part of the G3BP protein. Advantageously, it is an epitope comprised between the amino acids located at positions 1 and 144 of the G3BP protein and preferably it is an epitope comprised between amino acids located at positions 1 to 72 of the G3BP protein. Preferably still, it is an epitope comprised between the amino acids located at positions 22 to 55 of the G3BP protein, and more preferentially still, of an epitope constituted by the amino acids located at positions 22-34.

In a particularly advantageous embodiment, the present invention concerns antibodies designated by the designation Mab 1F1 and secreted by the hybridoma line G3B 1F1 1D1 deposited on June 9, 1998 before the C.N.C.M. with the number 1-2038.

The invention also encompasses the antibodies derived from Iso monoclonal antibodies defined above. In the sense of the present invention, "derived antibodies" is understood to mean any molecule comprising the idiotype of the monoclonal antibodies according to the invention and particularly the chimeric antibodies, the single chain antibodies and the Fab fragments. Such chimeric antibodies can be obtained according to the techniques described by Morrison et al., J. Bacteriol. 159: 870 (1984); Neberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314; 452-454 (1985), incorporated herein by reference. The Fab fragments containing the idiotype of the antibodies according to the invention can be generated by any technique known to the person skilled in the art. For example, such fragments contain, but are not limited to: the F (ab ') 2 fragment, which fragment can be produced by digestion by the antibody pepsin; Fab 'fragments that can be obtained by reduction of the disulfide bridges of the F (ab) 2 fragment, and Fab fragments that can be produced by treatment of the antibodies with papain and a reducing agent.

The invention also relates to single chain ScFv antibodies derived from the monoclonal antibodies defined above. Such single chain antibodies can be obtained according to the techniques described in US Patents 4,949,778, US 5,132,405 and US 5,476,786.

The invention also concerns a sequence of nucleic acids comprising a gene encoding a single-chain antibody derived from the monoclonal antibodies described above. The obtaining of such sequences and their use for the in vivo expression of the antibodies has been described in the application WO 94/29446 incorporated herein by reference.

The invention also relates to viral or plasmid vectors containing a nucleic acid sequence encoding a single chain antibody derived from the monoclonal antibodies described above. More particularly, the vectors of the invention are of viral origin, such as retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, cowpox virus, HSV viruses, etc.

The invention also concerns the use of nucleic acid sequences coding for these ScFv or of vectors that include them for the preparation of pharmaceutical compositions intended for the surgical or therapeutic treatment of human and animal bodies. It also concerns any pharmaceutical composition comprising vectors, particularly viral or a sequence of nucleic acids such as defined above.

The subject of the invention is also the lines of hybridomas capable of secreting the monoclonal antibodies according to the invention.

The invention relates more particularly to the hybridoma line G3B 1F1 1D1 which secretes the Mab 1F1 antibodies deposited on June 9, 1998 before the "Collection National des Cultures de Microorganism.es" (C.N.C.M.) with the number 1-2038.

The invention also concerns a method of producing monoclonal antibodies capable of inducing apoptosis in different tumor lines comprising (I) the fusion of spleen cells of an animal immunized with the aid of the protein, G3BP or of a fragment comprising minus one part of the N-terminal region (aa 1-144), with myeloma cells under conditions that allow the formation of hybridomas; (2), the detection and isolation of the so-called hybridomas that secrete monoclonal antibodies capable of inducing apoptosis in different tumor lines.

The invention also concerns the use of the antibodies defined above for the production of drugs. The invention relates more particularly to the use of the aforementioned antibodies for the preparation of a medicament for the treatment or prevention of iper-proliferative disorders.

The invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of the antibodies according to the invention, optionally mixed with a pharmaceutically acceptable carrier, the aforesaid amount being therapeutically effective for inducing apoptosis in tumor cells.

The antibodies according to the invention can also be used as diagnostic reagents to identify or dose the G3BP protein. Indeed, it has been found that the G3BP protein is overexpressed in tumor cells and that this overexpression constitutes a marker in the phenomena of cell proliferation. These antibodies are therefore particularly useful for diagnosing hyperproliferative diseases that involve over-expression of the G3BP protein.

Another subject of the invention is therefore the use of the monoclonal antibodies defined above as a diagnostic reagent as well as an immunological diagnostic kit comprising the aforementioned monoclonal antibodies.

Monoclonal antibodies can be coupled to chromogenic, fluorescent, biotinylated, radioactive or other markers ... Antibodies can be used in immunostaining methods in immunohistochemistry, in flow cytometry, for radioimmunological (RIA) or immunoenzymatic (ELISA) assays and in all kinds of known diagnostic equipment.

The antibodies according to the invention have also made it possible to carry out a functional study of the protein G3BP In particular, this functional study has led to the identification of new peptides derived from the G3BP protein and that present an apoptotic activity. In this regard, the invention also relates to novel peptides derived from the G3BP protein and capable of inducing apoptosis. Preferably, the peptides according to the invention comprise the N-terminal fragment, and preferably even these polypeptides comprise at least the region corresponding to the first 14 amino acids of the G3BP protein.

In addition to the foregoing provisions, the present invention also comprises other features and advantages that will be highlighted from the examples that will follow, and which should be considered as illustrative of the invention without limiting the scope.

SUBTITLES OF THE FIGURES Figure 1: Identification by Western Spotting of the different G3BP regions recognized by the Mab 1F1 antibody.

Figure 2 a and 2b: comparison of the expression level of the G3BP protein in the different cell lines.

Figure 3: Effect of the microinjection of Mab 1F1 antibodies on HCT116 cells.

Figure 4: Comparison of sequences between the human proteins G3BP and G3BP2. the comparison of the N-terminal sequences shows several differences evidenced by the umlauts.

MATERIALS AND METHODS Cell lines used HCT116: epithelial cells originating from human colon carcinoma that have a mutated Ki -ras gene and a mutation of the DCC tumor suppressor gene. Required culture medium: DMEM, 10% fetal calf leather, 1% Penicillin / Streptomycin,! % of Glutamine.

Hke-3 and HK2-6: HCT116 cells modified by elimination of the Ki-ras gene and which, from this fact, have lost their tumorigenic power. (ref .: Shirasawa, .Sasazuki. (1993) Science 26Q, 85-88). Required culture medium: DMEM, 10% fetal cow serum, 400} g / ml Geneticin, 1% Penicillin / esterptomycin, 1% Glutamine.

H460: epithelial cells originating from lung carcinoma, not from small human cells that have a mutated ki-ras gene. Required culture medium: DMEM, 10% fetal calf serum, 1% Penicillin / Streptomycin, 1% Glutamine.

H1299: from ATCC. Epithelial cells originating from human lung carcinoma that present a mutated N-ras gene and an elimination of the p53 gene. Required culture medium: DMEM, 10% fetal calf serum, 1% Penicillin / streptomycin, 1% Glutamine.

HeLa: epithelial cells originating from human carcinoma of the cervix that do not have mutations of the ras genes. Required culture medium: DMEM, 10 i fetal calf serum, 1% Penicillin / Streptomycin, 1 % of Glutamine.

HT-29: from ATCC. Epithelial cells originating from carcinoma "of the human colon presenting a mutation of the p53 gene but not mutations in the gens ras. Culture medium required: DMEM, 10% fetal calf serum, 1% Penicillin / streptomycin, 1. Glutamine .

DLD-1: from ATCC. Epithelial cells originating from human colon carcinoma that present a mutation of the ki-ras gene and the p53 gene. Required culture medium: DMEM, 10% fetal calf serum, 1% Penicillin / streptomycin, 1% Glutamine.

T47: from ATCC. Cells isolated from pleural effusion of a patient afflicted with mammary carcinoma. Required culture medium: RPMI, 10% fetal calf serum, 1% Penicillin / Streptomycin, 1% Glutamine.

BT20: cells originating from human mammary carcinoma. Required culture medium: RPMI, 10% fetal calf serum, 1% Penicillin / streptomycin, 1 1 Glutamine.

MCF-7: cells originating from human mammary carcinoma. Required culture medium: RPMI, 10% fetal calf serum, 1% Penicillin / streptomycin, 1% Glutamine.

T24: from ATCC. Cells originating from human carcinoma of the bladder, which have a mutation of the Ha-xas gene. Required culture medium: RPMI, 10% fetal calf serum, 1 I Penicillin / streptomycin, 1% Glutamine.

NHDF: from Boerhinger Ingelheim. Normal human dermal fibroblasts (adults) in primary culture that do not have mutated genes. Medium required by the manufacturer.

EXAMPLES Example 1: Obtaining monoclonal antibodies 1.1 - Production of the G3BP protein The SF9 cell cultures were performed according to the protocol described in Parker et al. (Mol.Cell. Biol .. 16 81996) 2561). The cells were lysed in the presence of HNTG buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X100, 10 c glycerol, 1 mM MgCl, 1 mM EGTA, in the presence of inhibitors. of phosphatases (1 mM Na3V04, 10 mM Na4P207, 10 mM NaF) and protease inhibitors (1 μg / ml leupeptin, 1 μg / ml trypsin inhibitor, 1 μg / ml pepstatin A, 2 μg / ml ml of aprotinin, 10 μg / ml of benzamidine, 1 mM of phenylmethanesulfonyl fluoride, 1 μg / ml of antipain, 1 μg / ml of chymostatin.) The lysate is centrifuged (15,000 g, 15 minutes) and diluted. fold in HG wash buffer (50 mM Hepes pH 7.5, 10% glycerol, 1 mM EGTA, in the presence of phosphatase inhibitors and protease inhibitors) The lysate is then incubated 12 hours in the presence of heparin gel / Sepharose gel Fast Flow (Pharmacia LKB) at the rate of 15 mg of protein per milliliter of gel, equilibrated in the same buffer (50 mM Hepes, NaCl 0. 030 M, 0.2% Triton X100, 10% glycerol, 1 mM MgC12, 1 mM EGTA). The complex is transferred to a K26 column of Pharmacia. The column is washed with 10 volumes of equilibration buffer (50 ml / hour), then the proteins are eluted in 100 mM NaCl buffer then in mM NaCl buffer. Fractions containing the binding activity "GAP-SH3 are pooled, diluted 10 'fold in HG buffer and loaded onto an AGPOLY (U) agarose-polyribouridilic acid column (column type 6, 19 cm2 x 1 cm, Pharmacia) at the rate of 1.3 mg of proteins per milliliter of gel The column is pre-equilibrated in the same buffer with a flow of 5 ml / cirrh "1. After washing in HNG buffer (60 M NaCl), the contaminating proteins are eluted with 229 ml of a linear gradient buffer of 0.06 to 0.32 M NaCl, and the G3BP protein is eluted in the presence of 0.7 M? ACl. At the exit of the POLY (U) chromatography, the fractions containing the G3BP protein are combined, diluted 12 times in the presence of phosphate buffer pH 7.5 + protease inhibitors) and loaded onto a 1 ml MonoS HR5 / 5 column (Pharmacia), are pre-equilibrated in the same buffer containing 60 M of α aCl, at a flow of 30 ml h "1. After rinsing the column, the proteins are eluted with 40 ml of a linear gradient buffer de? aCl from 0.06 to 1 M. The G3BP protein is eluted at? aCl between 0.15 M and 0.2 M. 1. 2 Immunization protocol of mice Female BALB / c mice 6 weeks of age received an intraperitoneal injection of 25 μg of G3BP on day 10 of the immunization. A second intraperitoneal injection was made at day 14 of the immunization then a third and fourth injection at days 45 and 80. A fifth injection (10 μg of protein G3BP) was made intravenously at day 165 and the splenocytes were extracted three days afterwards and merged. The splenocytes were fused with murine myeloma cells SP2 / 0-Agl4 on the one hand and X63Ag8 / 653 on the other hand. The splenocyte / myeloma cell ratio is 5: 1. The lymphocyte hybridization is done in the presence of polyethylene glycol PEG pm 1500 (final concentration 40%). Six 96-well plates were prepared in the presence of 10, 000 peritoneal macrophages were Balb / C mouse per well. The amounts of splenocytes used vary between 2.5 104 and 105 cells per well. The medium used contains RPMI 1640 (Biowhitaker and Gibco), 2 mM glutamine, 1 mM sodium pyruvate, HAT, 10 mM hypoxamine, 0.4 μM aminopterin, 16 μM thymidine, 220% decomplemented fetal calf serum, penicillin. 100 U / ml, streptomycin 100 μg / ml.

The first clones appear three days after the fusion, the medium is changed seven days after the fusion, finally 10 days after the fusion, 5000 macrophages are added per well. The wells in which a hybridoma solom grew were identified and the supernatants were sampled for screening.

Hybridoma supernatants originating from the fusion were screened by Western Spotting. Cell extracts (50 μg) of ER22 cells and HCT116 cells, the recombinant G3BP protein as well as a SAM68 control protein were electrophoresed on 7.5% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) then transferred over a polyvinylidene difluoride membrane (PVDF, Millipore Corpo.). the non-specific binding is blocked by 2% skim milk in ECL buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for two hours at room temperature.

The membranes are then incubated with the different supernatants diluted 1 / 50th in the blocking buffer overnight at 4 ° C. After washing with the ECL-0.05% Tween 20 buffer, the bound proteins are detected by incubation with an anti-mouse antibody conjugated to a peroxidase and chemiluminescent ECL reagent (NEN Life Science Product).

Three hybridomas were retained at the exit of this screening. These are the hybridomas G3B 1E1, G3B1F1 1D1 and G3B11H7.

Example 2 - Identification of the epitope recognized by the Mab 1F1 antibody.

Different fragments of the G3BP protein have been produced in an expression system in insect cells (SF9) infected by a recombinant baculovirus according to the technique described in Parker et al. (Mol.

Biol. 16 (1996) 2561). It's about the "1" fragments (aa 72 -. 72 - 350), "2" (aa 72 - 235), "3" (aa 144 - 341), "4" (aa 236 -. 236-350), and "5" (aa 299-466). Experiments of Western staining have been made from these fragments produced in baculorvirus systems. 2. 1 - Detection of proteins by Western Spotting After removal of the culture medium, the proliferating cells were washed twice in phosphate buffer at 4 ° C. Lysis was performed directly on the culture dishes in the presence of 1 ml of HNTG buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1 mM + phosphatase inhibitors (1 mM Na3V04, 10 mM Na4P2? 7, 10 mM NaF) + protease inhibitors (1 μg / ml leupeptin, 1 μg / ml trypsin inhibitor, 1 μg / ml pepstatin A, 10 μg / ml benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 μg / ml analgesic, 1 μg / ml chymostatin.) Proteins were separated on sodium acrylamide dodecyl sulfate (SDS) -polyacrylamide electrophoresis gel ( PAGE) of 7.5% (16 hours, 60 volts) proteins were then transferred onto polyvinylidene difluoride membrane (PVDF) Millipore Corp.) by a semi-liquid transfer method. The transfer was carried out at 4 ° C, for 3 hours, at a voltage of 60 volts, in a transfer buffer of 25 mM Tris base, Glycine 192, SDS 0.15% methanol 20%. The membrane was rinsed in PBS then saturated in PBS supplemented with 0.05 Tween 20 and 2% skim milk for two hours at room temperature. The membrane was incubated for 12 hours at 4 ° C, in an ECL buffer (20 mM TRIS, pH 7.4, 150 mM NaCl) + 0.05% Tween 20 + 2% skim milk, which contained the first antibody. The MAB 1F1 antibody was diluted at 1/10000ava. The membrane was then rinsed 4 or 5 times for 10 minutes, at room temperature in an ECL + 0.05% Tween 20 buffer. The second antibody (anti-mouse antibody) was then added in the presence of ECL + 0.05% Tween 20 during 45 minutes at room temperature. The antibodies were coupled to the peroxidase that allows the revelation of the proteins by the technique of Enhanced Chemiluminescence (ECL, "Enhanced ChemiLuminiscence"). The membrane was rinsed 4 or 5 times in PBS + 0.05% Tween 20 buffer, incubated 1 minute in the ECL reagent. The disclosure was made according to the manufacturer's recommendations (NEN Life Science Product).

The results are presented on Figure 1 and demonstrate that the Mab 1F1 antibodies allow the detection of fragments "1" (aa 72-350) and "2" (aa 72-235). The central fragment "3" (aa 144-341) which contains a zone common to fragment "2" is not recognized. The fragments "4" (aa 236-350) and "5" (aa 299-466) are not recognized either.

These results also demonstrate that the epitope recognized by the Mab 1F1 antibody is located in the N-terminal region of the G3BP protein, in the area corresponding to the first 144 amino acids and more particularly in the zone containing the first 100 amino acids located in the upper part of the acid region (aa 144-221) of the G3BP protein.

Example 3: Expression levels of the G3BP protein in different cell lines.

The level of expression of the G3BP protein has been studied in human colon rectal tumors and in different tumor lines. 3. 1-Nilevel expression of G3BP in different human cell lines.

For this experience, the cells in culture were harvested by scraping and centrifuging 5 min at 2000 rpm to harvest them in the bottom. Then, in a buffer of 10 mM Hepes pH 7.2, 140 mM KCl, 5 mM MgCl 2, 0.2% NP40 and protease inhibitors were lysed for 45 minutes at 4 ° C. The lysates were centrifuged at 14000 rpm for 10 minutes and the supernatants were sampled. Protein was dosed and the supernatants were denatured in 4X Laemmli buffer and treated for 10 minutes at 95 ° C.

The equivalent of 50 μg of proteins from the different lysates was deposited per well of a minigel of Novex SDS-PAGE, 10% acrylamide, 10 wells and 1.5 mm thickness.

After migration, electroblotting on PVDF membrane, and passivation of the membranes in TTBS buffer [20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 220% 0.2% sodium azide [containing 3% BSA, the samples were subjected to a Western blot.

The membranes were incubated for 2 hours with the Mab 1F1 antibody diluted to 0.2 μg / ml in TTBS buffer containing 3% BSA, then rinsed 4 times with TTBS for 10 minutes, then incubated 1 hour with an anti-body antibody. mouse labeled to peroxidase and again rinsed four times with TTBS for 10 minutes. The membranes were then developed with the ECL (chemiluminescence) NEN-Dupont kit on the Amersham film.

The results are presented in figure 2 a. The obtained film shows the over-expression of the G3BP protein in all the tumor lines examined, in relation to the line of normal fibroblast NHDF. The level of this over-expression is variable from one line to the other but is not dependent on the activation status of the ras genes in these lines; cell lines that have a mutated Ki-ras gene such as HCT116, H460 or DLD-1, express more or less strongly the G3BP protein and cell lines that do not show mutation of ras genes such as HeLa or HT29, have a level of G3BP expression also strong and variable.

It can be confirmed, however, that the Hke-3 and HK2-6 cells in which the Ki -ras gene has been eliminated, will decrease the level of expression of G3BP in relation to the cells from which they are native, HCT116 , without therefore there is an extinction of this expression.

Level of expression of G3BP in human biopsies The samples of healthy tissues were fixed on the slide. Solubilization of the samples was carried out by a deposit of 40 μl of HNTG buffer on each slide at the tissue level. The extracts were centrifuged at 14000 rpm for 10 minutes and the supernatants were sampled. The proteins were dosed and denatured by the 4X laemmli buffer by a heat treatment of 95 ° C for 10 minutes.

Samples of 20 to 30 μg of proteins were subjected to an electrophoresis on a 7.5% sodium dodecylsulfate polyacrylamide gel (SDS_PAGE) then transferred onto a polyvinylidene dilfuoride membrane (PVDF Millipore Corpo.). blocks 2% of skim milk in ECL buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 2 hours, at room temperature.

The membranes are then incubated with the Mab 1F1 antibody diluted in 0.2 μg / ml in the blocking buffer overnight at 4 ° C. After washing with the ECL-0.05% Tween 20 buffer, the bound proteins are detected by incubation with an anti-mouse antibody conjugated with a peroxidase and chemiluminescent ECL reagent (NEN Life Science Product).

The results presented in Figure 2b show that the G3BP protein is overexpressed in moderately well-differentiated adenomas.

Example 4: Effect of the miero-injection of the Mab 1F1 antibody on the feasibility of human tumor cells.

This example illustrates the property of the antibodies according to the invention of inducing apoptosis in different types of tumor cells.

The Mab 1F1 monoclonal antibody was injected according to the protocol described below. The solution to be injected was dialyzed against PBS, passed over Millipore UltraFree filter of 0.45 μm and centrifuged (5 minutes at 14,000 rpm) before being introduced into the microcapillary with the help of a lengthened "micro-charger" tip (Roucaire). The time of the injection was regulated from 0.1 to 0.3 seconds and the injection pressure between 50 hPa and 150 hPa depending on the size of the cells. Approximately 200 cells were injected per condition.

The HCT116, H460, H1299, HeLa or Hke-3 cells were microinjected by 10 mg / ml of Mab 1F1 monoclonal antibody then filmed and recorded by videomicroscope (at 2 images per minute) for 3 days after injection. In the reading of the film, the evolution of the injected cells is observable (division, cell deaths, morphological changes) and quantifiable (number of cells that are divided or killed per hour). Only cells that detach from the support after condensation and fragmentation (observable events of apoptotic cells) were counted.

The results obtained with the injection of the Mab 1F1 antibody in the HCT 116 cells are reported in figure 3. A high percentage of induced cell death is observed from twelve hours after the injection of the Mab 1F1 antibody, with a peak between 24 hours. and 48 hours.

Different doses of Man 1F1 antibodies were tested on HCT116 cells. From 10 mg / ml to 3 mg / ml, the effect of induction of cell death is equivalent, that is to say that the Mab 1F1 antibody induces the cell death of at least 50% of the injected cells. At 1 mg / ml of antibody, the effect is strongly diminished.

If the HCT116 cells are injected by a mouse immunoglobulin at the 10 mg / ml concentration as a control, no death was checked and the injected cells continue to divide normally.

This toxic effect of the Mab 1F1 antibody on HCT116 cells was confirmed by using the "T NEL" method in fluorescence, which allows to specifically detect the DNA cuts of the cells that enter apoptosis: The cells were seeded on gridded glass slides (Cellocates-Roucaire) and placed in 4-well plates. 2 days after sowing, cells were injected onto these lamellae by Mab 1F1 antibodies or with a mouse immunoglobulin, as a control, at 10 mg / ml. After injection, the cells are placed back in the incubator for 40 hours. They are then fixed to formaldehyde at 4% for 15 minutes, permeabilized for 5 minutes to triton X 100 at 0.2% and rinsed several times with phosphate buffer. The double labeling is then carried out for 1 hour at 37 ° C. with: an anti-mouse antibody labeled with Texas Red diluted to 1/400, which will reveal the injected cells, - Boehringer team "Detection of Cell Death In Situ" that will mark specifically in green, the DNA cuts of the apoptotic cells.

The lamellae are then placed on glass sheets in the mounting medium (Vectashield-Biosys).

With the observation in fluorescent light, they were counted, with the help of filters that select the different colors, the cells injected on the one hand and the cells on apoptosis on the other hand.

Thus, in 3 independent experiments, between 9 and 18% of HCT116 cells killed by apoptosis at 40 hours are obtained. Under the same conditions, the death of any HCT116 cell injected with the control immunoglobulin is not observed.

The recording experiences after microinjection of the Mab 1F1 antibody were reproduced with the H460, H1299, HeLa and Hke-3 cells with results similar to the graph presented in Figure 3 showing that at least 50% of the injected cells die from 12 to 60 hours after the injection.

The "T NEL" labeling experiments were performed with the HCT116 and H460 cells and gave equivalent results (9 to 18% of the cells enter apoptosis 40 hours after the injection of the antibody).

Example 5: Effect of the microinjection of the Mab 1F1 antibody on the feasibility and on the proliferation of normal human fibroblasts NHDF . 1 - Effect of the microinjection of the Mab 1F1 antibody on the feasibility of normal human fibroblasts NHDF.

The recording experiences of the proliferating NHDF cells microinjected with the MablFl antibody show that no cell death is observed within 60 hours after the injection and that the cells continue to divide normally. . 2 - Effect of the microinjection of MablFl antibody on the proliferation of normal human fibroblasts NHDF.

Normal cells in culture need a contribution of growth factors. If they are deprived of these factors, they enter a phase of "non-proliferation" called quiescence in which they no longer replicate their AD. and can not then incorporate bromodeoxyuridine, an analogue of thymidine added into the culture medium, which only proliferating cells can incorporate into their AD ?.

When injected into inactive HDF cells, the Mab 1F1 antibody at the concentration of 10 mg / ml, and that these cells are put back into a medium containing growth factors and bromodeoxyuridine (BrdU), it is found, after 24 contact hours and then fixation and staining with the help of an anti-BrdU antibody, an inhibition of 33 cell proliferation.

This inhibition is calculated by relating the percentage of cells injected with the Mab 1F1 antibody and that have incorporated the BrdU, to the percentage of cells injected with a mouse immunoglobulin and that have incorporated the BrdU (control).

Another anti-G3BP monoclonal antibody (11H7) has been injected into NHDF cells under the same conditions.

Antibody% incorporation BrdU injection Immunoglobulin of mouse (IgG) 44.7 Mab 1F1 29.7 11H7 30.3 Table 1: Effect of injection of G3BP-specific antibodies on cell growth of HDF cells It is found that immunoglobulin does not prevent cell proliferation when the 11H7 antibody has an effect - similar to that of the Mab 1F1 antibody, namely that they cause an inhibition of proliferation of 32 to 33%.

This inhibition is attributed to the blocking of the endogenous G3BP protein by the Mab 1F1 or 11H7 antibody. In this regard it is interesting to note that this arrest of proliferation is only temporary in normal NHDF cells and does not imply in any case the death of the cells. In effect, the cells again take their normal cycle when the antibody is naturally degraded in the cytoplasm (approximately 40 hours after the injection).

The results presented in Examples 4 and 5 show that the Mab 1F1 antibody induces apoptosis in at least 50% of the human tumor cells examined in which G3BP is overexpressed but nontoxic when injected into normal human cells. , in which G3BP is slightly expressed. This effect on the tumor cells is apparently not related to the activation state of the ras genes. On the other hand, the Mab 1F1 antibody, causes a temporary arrest - or a delay - of the proliferation of normal NHDF cells, when injected into the inactive cells.

Example 6 - Cloning of a DNA sequence encoding an anti-G3BP intracellular antibody from the G3B 1F1 1D1 hybridoma.

This example describes the cloning and expression of a nucleic acid sequence encoding an intracellular antibody that mimics the properties of the monoclonal antibody. 6. I-Preparation of the DNA sequence A DNA sequence encoding an intracellular antibody - (ScFv fragment) is prepared according to the technique described in US Pat. No. 4,946,778. This sequence is then placed under the control of a functional promoter in the mammalian cells.

Poly A RNAs were isolated from a cell culture of the G3B 1F1 1D1 hybridoma, according to the technique described by Chirguin S.H. and collaborators (Biochemistry 18, 5294 (1979)). These RNAs were used for a reverse transcription reaction with the aid of murine primers formed of random hexanucleotides. The AD? C obtained serve as a template for two PCR reactions. - one, designed to amplify the variable fragment of the heavy chain (VH) with specific VH primers. - a second PCR that allows obtaining the VL fragment, using a mixture of 10 primers derived from the murine sequences.

Two fragments of 340 bp are obtained in this way and are combined thanks to a link that allows a correct location of the VH cDNA 5 'of that of VL. PCR ("Recombinant Phage Antibody System Mouse ScFv Module", Pharmacia, 27-9400-01).

The VH-link-VL fused nucleic acid sequence is then inserted into a phagemid which allows expression of the intracellular antibody. This expression easily allows the identification and selection of intracellular antibodies that correctly recognize the antigen. This stage is carried out by selecting the antibodies capable of recognizing G3BP in the ELISA test ("Recombinant Phage Antibody System Expression Module", Pharmacia, 27-9401-01). 6. 2-Functional evaluation of the modified intracellular antibody.

The sequence encoding the anti-G3BP modified intracellular antibody is isolated from the restriction phagemid, then inserted into the SV2 vector (Schweighoffer et al., Science, 256, 825-827 (1992)) or pCDNA3 (In Vitrogen, V790- twenty) . The plasmid thus obtained is called pSV-ScFvG3BP or pCDNA3ScFvG3BP. The functional evaluation can be done through the tests described below. a) by microinjection in the cells of non-immortalized mammals NHDF and in transformed lines (HCT116, H460, Hke-3, H1299, Hela) by examining the apoptosis in the transformed lines according to the techniques described above and measuring the impact of the antibody on the feasibility compared to the activity of the Mab 1F1 antibody. b) by a test of neo-resistance on the transformed lines and normal cells. The plasmid pCDNA3G3BP is transfected in the transformed lines (HCT116, Hke-3, H460, H1299, Hela) as well as in non-transformed human cells. Three days after transfection, the transformed cells are selected in the presence of geneticin. In this test the single chain antibody is compared to fragments of G3BP (whose corresponding nucleic acid sequences are included in the same expression vectors) that inhibit the growth of tumor cells. c) by the inhibition of the formation of foci. The ability of the single chain antibody to inhibit foci formation is performed according to the technique described in W094 / 29446. For tests of decreased tumor capacity, the effect can also be investigated in the casso of transformation by other oncogens different from Ras. d) investigating the impact of the expression of this ScFv on the binding activity in the RNA and / or the RNA endonucleolytic cleavage by G3BP1. indeed, the apoptosis induced by microinjection of the monoclonal antibody Mab 1F1 can be explained either by the dissociation of an interaction with a specific protein partner, or by a modification of the binding activity to the RA? and / or endoribonucleotite fragmentation. The ScFv antibody obtained can also be characterized by its ability to modify these interactions.

Example 7: Identification of another recognition region of the Mab 1F1 antibody on the G3BP1 protein. Sequence comparison between the human proteins G3BP and G3BP2 (or G3BPh).

The G3BP protein is part of a family of proteins in which the short G3BP2 proteins are found (AF 051311, Genbank) that presents 60.9% identity at the nucleotide level, and 58.9% at the protein level with G3BP, and the long G3BP protein ( AB 014560, Genbank). The G2BP2 protein, long form, has an insertion of 99 nucleotides in 790 in the sequence of G3BP2 (short form).

The comparison of the protein sequences between the G3BP and G3BP2 proteins exposes a conservation of the primordial regions of the protein, namely: 1 - . 1 - an N-terminal region (presenting homologies with the NTF2 protein, Nuclear Transport Factor 2), target of the Mab 1F1 monoclonal antibody, 2 - . 2 - an acid region, 3 - . 3 - a region rich in PXXP motifs involved in protein-protein interactions, 4 - a region containing RRM regions of recognition in RNA, and auxiliary motifs, "RGG" groupings (arginine, glycine, glycine).

Notably, the MablFl monoclonal antibody is not able to recognize the recombinant G3BP2 protein (short form) in Western blotting experiments. The comparison of the N-terminal sequences shows several differences evidenced by the umlauts in Figure 4.

The use of peptides that cover the different divergent regions has made it possible to specify the region of recognition of the antibody on the G3Bp protein.

In a first time, the comparison of the sequences of the proteins described above has made it possible to synthesize different peptides (A-F): Peptide A: LLNQAPDMLHRFY amino acids 22-34 of the G3BP protein Peptide B: LLNKAPEYLHRFY amino acids 22-34 of the G3BP2 protein Peptide C: HGGLDSNGKPADAV amino acids 42-55 of the G3BP protein Peptide D: HGGVDASGKPQEAV amino acids 42-55 of the G3BP2 protein Peptide E: LLSNNNQALRRFMQ amino acids 97-111 of the G3BP protein Peptide F: HNDIFRYQDEVFG amino acids 127-139 of the G3BP protein These peptides are examined below: (i) for its ability to recognize MablFl antibody in an ELISA test, (ii) for its ability to inhibit the recognition of the G3BP protein by the MablFl antibody in Manch-Western experiments. (i) Tests of the A-F peptides in an ELISA test Peptides in 10 mg / ml solution in carbonate buffer (0.1 M, pH 9.6) were fixed by adsorption overnight at 4 ° C on 96-well plates. After washing in PBS, the wells were saturated for 4 hours at room temperature in PBS / 1 BSA buffer After three washes in PBS / 0.05% Tween, then the different wells were contacted with dilutions of the purified antibody MablFl in PBS / 0.2% BSA (10 ng / ml in 1 pg / ml) for 1 hour at room temperature. After three washes in PBS / 0.05% Tween buffer, the wells were incubated with dilution of anti-mouse antibody coupled to peroxidase (1/1000 dilution in PBS / 0.2% BSA) (Pharmacia) for 1 hour at room temperature. After three washes in PBS / 0.5% Tween buffer, the co-reaction was developed by mixing: 10 ml of H_0, one OPD tablet, 100 ml of methanol, and 25 ml of H20 ^. The reaction was stopped with 50 ml of 4N H2SO4, and the spectrophotometric reading was made at 492 nm (reference filter 620 nm).

The results obtained show that Peptide A is the most reactive in this test and is able to recognize the MablFl antibody in the ELISA test. These results indicate that the epitope recognized by the MablFl antibody is located in the region defined by amino acids 22 to 34 of G3BP. The same experiments performed with the 1E1 antibody led to identical results. (ii) Tests of the A-F peptides for their ability to inhibit the recognition of the G3BP protein by the MablFl antibody in Western blotting experimentson of the proteins by Western blotting was carried out according to the technique described in Example 2 (2.1: Detection of proteins by Western blotting). For the comparisons, the antibody was pre-incubated for two hours at 37 ° C with a dilution of the peptide before being placed in contact with the membrane. For each peptide, two concentrations were tested: molar excess of 10 and 100 relative to the antibody. The development was done as described in example 2.1.

The results obtained show that peptides A and C are capable of displacing the interaction between the G3BP protein and the MablFl antibody (mainly with a 100X molar excess).

The set of these results point to peptide A (amino acids 22-34 of G3BP) and to peptide C (amino acids 42-55 of G3BP) as corresponding to regions of G3BP involved in the recognition with MablFl antibody.

The sequence comparison between G3BP and G3BP2 short form has then allowed the identification of different peptides and the demonstration of their role in the control of apoptosis by G3BP, these results allow to contemplate different applications and particularly the use of these peptides for the molecular modeling, in order to obtain small molecules that mimic the effects of these peptides or polypeptides that incorporate them. On the other hand, these peptides can be used to displace an interaction of the G3BP protein with a cellular pair; this cellular pair can be the same G3BP since it has shown that the protein is able to dimerise, this pair can also be a known pair (RasGKP) or a newly identified pair. For this purpose, these peptides coupled to a resin can be used for cell pair research of G3BP. Finally, a screening of molecules of therapeutic interest can be established on the basis of the inhibition of a protein-protein interaction.

Example 8. Construction of fragments of G3BP1 and apoptotic activity of these fragments.

In view of the results obtained in Example 7, plasmids allowing expression of N-terminal fragments of G3BP were constructed in view of examining the apoptotic activity of these fragments. For this, the fragments were amplified by PCR from the following baits: A - 5 'Oligonucleotide in G3BP (nucleotide 1) cccgtcgacatggtgatggagaagcctagtcccctg B - 5 'Oligonucleotide in G3BP (nucleotide 42) cccgggtcgactttgtgagacagtattacaca C - 3 'Oligonucleotide in G3BP (nucleotide 150) cccgggtgcggccgcctttccatttgaatccaatcc The fragments (1-50) and 42-150) were amplified by PCR (30 cycles at 50 ° C), hydrolyzed by the restriction enzymes Salí and Notl, then inserted into the commercial vector pCMV / cyto (Invitrogen, " pShooter Vector manual II ", version C).

The sequences of the obtained fragments are described below: the sequences that come from the vetor and that will be translated are written in capital letters, the sequences of the G3BP cDNA are indicated in lowercase letters. The underlined regions correspond to the sequences of the vector coding for Tag myc). ^ fragment 1-150) 'ATGGCCCAGGTGCAGCTGCAGGTCatggtgatggagaagcctagtcccctgctggtc gggcggg'aatttgtgagacagtattacacactgctgaaccaggccccagacatgc.gcatagattttatggaaag aaetcttcUatgtccatgggggattgga.tcaaatggaaagGCGGCCGCAGAACAAAAACT CATCTCAG AAG AGGATCTG AATGGGGCCGCATAG_3_' The corresponding polypeptide is presented below. This polypeptide comprises the fragment corresponding to amino acids 1 to 50 of the G3Bp protein whose sequence is presented in the sequence SEQ ID NO: 1, this fragment appears below in bold characters.

MAQVQLQVMVTMEKPSPLLVGREFVRQYYTLLNQAPDM HRFVGKNSSY VHGGLDSNGKAAA EQKLISEEDLNGAA (fragment 42-150) 'ATGGCCCAGGTGCAGCTGCAGGTCtttgtgagacagtattacacactgctgaaccagg ccccagacatgctgcataga't ^ atggaaagaactcttcttatgtccatgggggattggattcaaatggaaagGC GGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGC CGCATAG_3_' The corresponding gone polypeptide is presented below. This polypeptide comprises the fragment Constructs containing fragments 1-150 and 42-150 of the nucleotide sequence of G3BP were transfected into transformed lines NIH3T3Ras and NIH3T3Raf. These lines were obtained after the transformation of NIH3T3 cells by the oncogene Ki-Ras Vall2, or by the viral oncogenic form of v-Raf.

For the detection of apoptosis, 2 mg of plasmid was transfected by lipofectamine (Gibco-BRL), and apoptosis was measured 48 hours after transfection by an analysis to FACS, Cells in apoptosis correspond to cells in subGl phase .

Under these conditions, the construct expressing the 1-150 fragment of the G3BP sequence (which codes for the G3BP fragment comprising amino acids 1 to 50) is capable of inducing apoptosis (factor 4), while the construct expressing the 42-150 fragment of the G3BP sequence (coding for the G3BP fragment comprising amino acids 15 to 50) does not have this property.

These results indicate the region corresponding to amino acids 1 to 14 of G3BP as a region involved in the induction of apoptosis. The identification of this region of G3BP and the demonstration of the role of the polypeptides comprising the region corresponding to amino acids 1 to 14 of G3BP in the control of apoptosis allows contemplating the use of these polypeptides for the prevention, improvement and / or the treatment of pathologies that involve cellular hyperproliferation.

Example 9. Construction of fusion proteins with huG3BPl ragments.

Constructs were made that allow the expression of the peptides identified in Example 7. The sequences corresponding to the peptides were fused to the GST protein (Glutathione S-Transferase) in order to stabilize them.

Sequences of the oligonucleotides: Each oligonucleotide in the sense is limited to 5 'of 4 nucleotides to reconstitute an Ncol site, and 3' of a stop codon (TAA) and of a nucleotide to form a BamHl site. sq22s: tatgctgctgaaccaggccccagacatgctgcatagattttattaag sq22as • gatccttaataaaatctatgcagcatgtctggggcctggttcagcagca (sequence 66-105) sq 44gl s: tatgggattggattcaaatggaaagccagcagatgcagtctaag sq44glas: gatccttagactgcatctgctggctttccatttgaatccaatccca (sequence 130-166) sq44g2s: tatgggagtagatgctagtggaaagccccaggaagctgtttaag sq44g2as: gatccttaaacagcttcctggggctttccactagcatctactccca (sequence 130-166) sq64s: tatgcagaaagaaatccacaggaaagtgatgtcacaaaacttctaag sq64as: gatcct.agaagttttgtgacatcactttcctgtggatttctttctgca (Sequence 172-211) sq65s: tatgaaagtgatgtcacaaaacttcaccaactgctaag sq65as: gatccttagcagttggtgaagttttgtgacatcactttca (sequence 190-220] Oligonucleotides were hybridized in pairs by cooling to 80 ° C at room temperature, phosphorylated by T4 polynucleotide kinases (Boehringer), and were inserted into the "fusion pBCooIIGCT" vector at the Ncol and BamHl sites. This vector allows the expression in eukaryotic cells of a GST fusion protein from the SV40 promoter (Genbank, X78316). The plasmids were then transfected into the human tumor lines, Hela and H1299 (described in the material and methods); The induced apoptosis was determined to the FACS as described in example 8.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property in the following: LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: RHONE-POULEN RORER, S.A. (B) STREET: 20, Raymond Aron Avenue (C) CITY: ANTHONY (E) COUNTRY: FRANCE (F) POSTAL CODE: 92165 (II) TITLE OF THE INVENTION: PEPTIDO CAPABLE OF JOINING ION SH3 OF GAP PROTEIN (iii) SEQUENCE NUMBER: 2 (iv) READING COMPUTER: (A) TYPE OF SUPPORT: TAPE (B) TYPE: amino acid (C) OPERATING SYSTEM: PC-DOS / MS. TWO (D) LOGICAL SYSTEM: Patentin Relay # 1.0, version # (OEB) (2) INFORMATION FOR SEQ ID NO: 1. (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 466 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 Met Val Met Glu Lys Pro Ser Pro Leu Leu Val Gly Arg Glu Phe Val 1 5 10 15 Arg Gln Tyr Tyr Thr Leu Leu Asn Gln Wing Pro Asp Met Leu His Arg 20 25 30 Phe Tyr Gly Lys Asn Ser Ser Tyr Val His Gly Gly Leu Asp Ser Asn 35 40 45 Gly Lys Pro Wing Asp Wing Val Tyr Gly Gln Lys Glu He His Arg Lys 50 55 60 Val Met Ser Gln A? .n Phe Thr Asn Cys His Thr Lys He Arg His Val 65 70 75 80 Asp Ala His Ala Thr Leu Asn Asp Gly Val Val Val Gln Val Met Gly 85 90 95 Leu Leu Ser Asn Asn Asn Gln Wing Leu Arg Arg Phe Met Gln Thr Phe 100 105 110 Val Leu Wing Pro Glu Gly Ser Val Wing Asn Lys Phe Tyr Val His Asn 115 120 125 Asp He Arg Tyr Gln Asp Glu Val Phe Gly Gly Phe Val Thr Glu Pro 130 135 140 Gln Glu Glu Ser Glu Glu Glu Val Glu Glu Pro Glu Glu Arg Gln Gln '145 150 155 160 'Thr Pro Glu Val Val Pro Asp Asp Ser Gly Thr Phe Tyr Asp Gln Wing 165 170 175 Val Val Ser Asn Asp Met Glu Glu His Leu Glu Glu Pro Val Wing Glu 180 185 190 Pro Glu Pro Asp Pro Glu Pro Glu Pro Glu Gln Glu Pro Val Ser Glu 195 200 205 He Gln Glu Glu Lys Pro Glu Pro Val Leu Glu Glu Thr Ala Pro Glu 210 215 220 Asp Wing Gln Lys Ser Ser Ser Pro Pro Wing Pro Wing Asp He Wing Gln Thr 225 230 235 240 Val Gln Glu Asp Leu Arg Thr Phe Ser Trp Wing Ser Val Thr Ser Lys 245 250 255 Asn Leu Pro Pro Ser Gly Ala Val Pro Val Thr Gly He Pro Pro His 260 265 270 Val Val Lys Val Pro Wing Ser Gln Pro Arg Pro Glu Ser Lys Pro Glu 275 280 285 Ser Gln He Pro Pro Gln Arg Pro Gln Arg Asp Gln Arg Val Arg Glu 290 295 300 Gln Arg He Asn He Pro Pro Gln Arg Gly Pro Arg Pro He Arg Glu 305 310 315 320 Wing Gly Glu Gln Gly Asp He Glu Pro Arg Arg Met Val Arg His Pro 325 330 - 335 Asp Ser His Gln Leu Phe He Gly Asn Leu Pro His Glu Val Asp Lys 340 345 350 Ser Glu Leu Lys Asp Phe Phe Gln Ser Tyr Gly Asn Val Val Glu Leu 355 360 365 Arg He Asn Ser Gly Gly Lys Leu Pro Asn Phe Gly Phe Val Val Phe 370 375 380 Asp Asp Ser Glu Pro Val Gln Lys Val Leu Ser Asn Arg Pro He Met 385 390 395 400 Phe Arg Gly Glu Val Arg Leu Asn Val Glu Glu Lys Lys Thr Arg Ala 405 410 415 Wing Arg Glu Gly Asp Arg Arg Asp Asn Arg Leu Arg Gly Pro Gly Gly 420 425 430 Pro Arg Gly Gly Lely Gly Gly Met Arg Gly Pro Pro Arg Gly Gly 435 440 445 Met Val Gln Lys Pro Gly Phe Gly Val Gly Arg Gly Leu Ala Pro Arg 450 455 460 Gln Glx 465 (2) INFORMATION FOR SEQ ID NO: 2 I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2129 PAIRS OF BASES (B) TYPE: nucleotide (C) NUMBER OF FILAMENTS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO 2 GCTTGCCTGT CAGGTCGACT CTAGAGCCCG GGTACCGAGC TCGAATTCGG CGGGGTTTGT 60 ACTATCCTCG GTGCTGTGGT GCAGAGCTAG TTCCTCTCCA GCTCAGCCGC GTAGGTTTGG 120 ACATATTTAC TCTTTTCCCC CCAGGTTGAA TTGACCAAAG CAATGGTGAT GGAGAAGCCT 180 AGTCCCCTGC TGGTCGGGCG GGAATTTGTG AGACAGTATT ACACACTGCT GAACCAGGCC 240 CCAGACATGC TGCATAGATT TTATGGAAAG AACTCTTCTT ATGTCCATGG GGGATTGGAT 300 TCAAATGGAA AGCCAGCAGA TGCAGTCTAC GGACAGAAAG AAATCCACAG GAAAGTGATG. 360 TCACAAAACT TCACCAACTG CCACACCAAG ATTCGCCATG TTGATGCTCA TGCCACGCTA 420 AATGATGGTG TGGTAGTCCA GGTGATGGGG CTTCTCTCTA ACAACAACCA GGCTTTGAGG 480 AGATTCATGC AAACGTTTGT CCTTGCTCCT GAGGGGTCTG TTGCAAATAA ATTCTATGTT 540 CACAATGATA TCTTCAGATA CCAAGATGAG GTCTTTGGTG GGTTTGTCAC TGAGCCTCAG 600 GAGGAGTCTG AAGAAGAAGT AGAGGAACCT GAAGAAAGCA GCAAACACCT GAGGTGGTAC 660 CTGATGATTC TGGAACTTTC TATGATCAGG CAGTTGTCAG TAATGACATG GAAGAACATT 720 TAGAGGAGCC TGTTGCTGAA CCAGAGCCTG ATCCTGAACC AGAACCAGAA CAAGAACCTG 780 TATCTGAAAT CCAAGAGGAA AAGCCTGAGC CAGTATTAGA AGAAACTGCC CCTGAGGATG 840 CTCAGAAGAG TTCTTCTCCA GCACCTGCAG ACATAGCTCA GACAGTACAG GAAGACTTGA 900 GGACATTTTC TTGGGCATCT GTGACCAGTA AGAATCTTCC ACCCAGTGGA GCTGTTCCAG 960 TTACTGGGAT ACCACCTCAT GTTGTTAAAG TACCAGCTTC ACAGCCCCGT CCAGAGTCTA 1020 AGCCTGAATC TCAGATTCCA CCACAAAGAC CTCAGCGGGA TCAAAGAGTG CGAGAACAAC 1080 GAATAAATAT TCCTCCCCAA AGGGGACCCA GACCAATCCG TGAGGCTGGT GAGCAAGGTG 1140 ACATTGAACC CCGAAGAATG GTGAGACACC CTGACAGTCA CCAACTCTTC ATTGGCAACC 1200 TGCCTCATGA AGTGGACAAA TCAGAGCTTA AAGATTTCTT TCAAAGTTAT GGAAACGTGG 1260 TGGAGTTGCG CATTAACAGT GGTGGGAAAT TACCCAATTT TGGTTTTGTT GTGTTTGATG 1320 ATTCTGAGCC TGTTCAGAAA GTCCTTAGCA ACAGGCCCAT CATGTTCAGA GGTGAGGTCC 1380 GTCTGAATGT CGAAGAGAAG AAGACTCGAG CTGCCAGGGA AGGCGACCGA CGAGATAATC 1440 GCCTTCGGGG ACCTGGAGGC CCTCGAGGTG GGCTGGGTGG TGGAATGAGA GGCCCTCCCC 1500 GTGGAGGCAT GGTGCAGAAA CCAGGATTTG GAGTGGGAAG GGGGCTCTGCG CCACGGCAGT 1560 AATCTTCATG GATCTTCATG CAGCCATACA AACCCTGGTT CCAACAGAAT GGTGAATTTT 1620 CGACAGCCTT TGGTATCTTG GAGTATGACC CCAGTCTGTT ATAAACTGCT TAAGTTTGTA 1680 TAATTTTACT TTTTTTTGTGT GTTAATGGTG TGTGCTCCCT CTCCCTCTCT TCCCTTTCCT 1740 GACCTTTAGT CTTTCACTTC CAATTTTGTG GAATGATATT TTAGGAATAA CGGACTTTTA 1800 CCCGAATTCG TAATCATGGT CATAGCTGTT TCCGTGTGAA ATTGTTATCC GCTCACAATT 1860 CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC 1920 TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC 1980 CAGCGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCCAGG 2040 GTGGTTTTCT TTTCACCAGT GAGACGGGCA ACAGCTGATT GCCCTTCACC GCTGGCCCTG 2100 AGAGAGTTGC AGCAAGCGGT CCACGCTGG 2129

Claims (12)

1. A monoclonal antibody that is characterized in that it is directed against the G3BP protein and is capable of inducing apoptosis in different types of tumor cells.

2. Monoclonal antibody that is characterized in that it is able to recognize an epitope comprised between the amino acids located at positions 1 and 144 of the G3BP protein.

3. Monoclonal antibody according to claim 1 or 2 which is characterized in that it is capable of recognizing an epitope comprised between the amino acids located at positions 1 to 72 of the G3BP protein, preferably an epitope comprised between the amino acids located at positions 22 to 55 of the G3BP protein, and preferably even an epitope consisting of the amino acids located at positions 22 to 34.

4. Antibody according to claims 1 to 3 which is characterized in that it is the antibody Mab 1F1, secreted by the hybridoma line G3B 1F1 1D1 deposited on June 9, 1998 before the C. N. C. M., with the number I-2038.

5. Use of an antibody according to any of claims 1 to 4 for the preparation of a medicament.

6. Use of an antibody according to any of claims 1 to 4 for the preparation of a medicament for the treatment or prevention of hyperproliferative disorders.

7. A pharmaceutical composition which is characterized in that it comprises a therapeutically effective amount of the monoclonal antibodies according to any one of claims 1 to 4, optionally mixed with an acceptable support, the aforesaid amount being therapeutically efficient for inducing apoptosis in tumor cells.

8. Hybridoma line which is characterized in that it is capable of secreting monoclonal antibodies according to any of claims 1 to 4.

9. Hybridoma line G3B 1F1 1D1 deposited on June 9, 1998 before the C.N.C.M. with the deposit number I-2038.

10. Procedure for the production of monoclonal antibodies capable of inducing apoptosis in different tumor lines which is characterized in that it comprises (1) the fusion of splenic cells of an animal immunized with the help of the G3BP protein or of a fragment comprising at least a part of the N-terminal region (aa 1-144), with myeloma cells under conditions that allow the formation of hybridomas; (2), the detection and isolation of the aforementioned hybridomas that secrete monoclonal antibodies capable of inducing apoptosis in different tumor lines.

11. Use of an antibody according to any one of claims 1 to 4, as a diagnostic reagent.

12. Diagnostic equipment which is characterized in that it comprises an antibody according to any one of claims 1 to 4.