FR2881830A1 - PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF PROSTATE CANCER - Google Patents

Abstract

The invention relates to a pharmaceutical composition comprising a compound which negatively regulates the expression of the BCAR-1 protein, in association with at least one pharmaceutically acceptable excipient. Such a pharmaceutical composition makes it possible in particular to treat prostate cancers even when these have become resistant to treatments with antiandrogenic compounds. The invention also relates to methods for detecting and evaluating in vitro the aggressiveness of prostate cancer.

Description

FIELD OF THE INVENTION

  The present invention relates to pharmaceutical compositions for the treatment of prostate cancer and methods for detecting and evaluating the aggressiveness of prostate cancer in vitro.

PRIOR ART

  Currently, prostate cancer is the most common cancer in men over the age of 50 [Cuzin et al. (1998), Grosclaude P. et al. (1997), and IARC Publication (1992)]. Due to the increase in life expectancy, the number of diagnosed prostate cancer is increasing, and is in the order of 30,000 new cases per year in France, and causes the death of 10,000 people per year in average.

  The development of prostate cancer is due to the proliferation of cancer cells and the decrease in the process of androgen-dependent physiological apoptosis. Over time, prostate cancer evolves. Cancer cells proliferation is first observed, then associated with metastatic extension followed progressively by resistance to antiandrogenic compounds.

  This evolution has led to the classification of prostate cancers in three successive stages, for which specific treatments have been developed. We first distinguish the localized stage for which the cancer can be treated by surgical excision or in situ irradiation with or without the addition of antiandrogenic treatment. We then distinguish the advanced metastatic stage for which the cancer is treated by castration and / or antiandrogenic treatment, that is to say a treatment aimed at reducing the biological action of androgens and finally the stage of escape to castration or treatment. anti-androgenic drugs for which cancer is treated with chemotherapy and palliative care.

  Treatment with anti-androgenic compounds or, more broadly, by androgen deprivation is a conventional treatment for treating prostate cancer, aimed at decreasing the biological action of androgens. It is for example to reduce the rate of circulating testosterone by surgical or medical castration; using androgen-competing drugs for the androgen receptor, or drugs that inhibit the enzymes of androgen synthesis. However, although these treatments offer remission and tumor suppression in most cases, they are not devoid of side effects. Indeed, these compounds have in common a liver toxicity requiring close monitoring of patients. In addition, the therapeutic effects of antiandrogens are only temporary. Prostate cancers lose their sensitivity to such treatments over time and their development becomes androgen independent. In other words, cell clones resistant to anti-androgenic treatments appear in tumors and develop over time.

  The molecular mechanisms of escape to antiandrogenic treatments are the subject of numerous studies but are still poorly known. Some changes during cancer development have been highlighted, such as mutations in androgen receptor genes. In addition, certain factors such as IGF or KGF and epithelial growth factors such as EGF have a role in the activation of androgen receptors in the absence of androgens. Finally, intracellular signals involved in the control of proliferation or resistance to apoptosis, such as AKT or MAPK, are generally overexpressed in prostate cancers escaping antiandrogenic treatments.

  Because of the prevalence of prostate cancers, and in particular cancers that evolve towards resistance to antiandrogenic treatments mentioned above, and disadvantages of these existing treatments, there is a need for new therapeutic compounds for treatment prostate cancer. In particular, there is a need for novel therapeutic compounds capable of treating prostate cancers even when they are resistant to antiandrogenic compounds treatments. This need is all the more important as the severity of prostate cancer, that is to say the survival time after diagnosis, is proportional to the importance of the cellular contingent resistant to hormone treatment within the tumor during the diagnosis.

  Prostate cancer is diagnosed on anatomo-pathological criteria usually obtained from prostate biopsies indicated following a rise in blood level of PSA (Prostate Specific Antigen). PSA is a protein normally secreted by the non-cancerous prostate, the rate of which rises when there is a benign or malignant inflammatory prostatic pathology (cancer).

  Since PSA is not specific for prostate cancer, there is also a need for new methods for detecting prostate cancer that can provide an alternative or complement to the PSA detection test.

SUMMARY OF THE INVENTION

  The subject of the invention is a pharmaceutical composition comprising a compound capable of negatively regulating the expression of the BCAR1 protein, in association with at least one pharmaceutically acceptable excipient.

  The subject of the invention is also a method for the in vitro detection of the presence of a prostate cancer in a patient, comprising a step of qualitative or quantitative detection of the concentration of the BCAR-1 protein, in a sample from said patient.

  The invention also relates to a method for in vitro evaluation of the aggressiveness of a prostate cancer in a patient, comprising the following steps: (i) measuring the amount of BCAR-1 protein in a sample previously taken from said patient, and (ii) comparing the amount of BCAR-1 protein measured in step (i) with one or more control values.

  The invention also relates to the use of a compound capable of downregulating or suppressing the expression of BCAR-1 protein for the manufacture of a medicament for treating prostate cancer.

DESCRIPTION OF THE FIGURES:

  Figure 1: histological section of non-cancerous prostate.

  To study the expression of the bcar-1 gene, a histological section of non-cancerous prostate was carried out and the presence of the BCAR1 protein was revealed with a mouse monoclonal anti-BCAR-1 antibody directed against the human protein p130 CAS .

  At the level of the basal gland layer, normal expression of BCAR-1 is found in androgen-independent, non-cancerous prostatic epithelial cells, and at the level of the secretory layer there is a lack of BCAR-1 expression in the cells. non-cancerous, androgen-dependent prostatic epithelial cells of the same glands.

  Figure 2: Histological section of a mild prostate cancer.

  A histological section of cancerous prostate was made and revealed with the mouse monoclonal anti-BCAR-1 antibody described above. There is a low expression of BCAR-1 in mild prostate cancers (less than 10% of tumor cells express the BCAR-1 protein), whose gleason grade is equal to 2 or 3 (well-differentiated cancers) Figure Fig. 3: Histological section of aggressive prostate cancer A histological section of cancerous prostate was performed and revealed with the mouse monoclonal anti-BCAR-1 antibody described above. There is a strong expression of BCAR -1 in aggressive prostate cancers, whose gleason grade is equal to 4 or 5 (poorly differentiated cancers). Figure 4: Histological section of prostate cancer resistant to antiandrogenic treatment.

  A histological section of cancerous prostate, resistant to antiandrogen treatment, was performed and revealed with the mouse monoclonal anti-BCAR-1 antibody described above. There is a strong expression of BCAR-1.

  DETAILED DESCRIPTION OF THE INVENTION

  The invention provides novel pharmaceutical compositions for the treatment of prostate cancer, as well as methods for detecting and evaluating in vitro the aggressiveness of prostate cancer.

  Pharmaceutical Compositions According to the Invention Historically, the BCAR-1 protein, (Breast Cancer Antiestrogen Resistance-1), has been associated with the resistance of breast cancers to antiestrogens. (Dorssers, et al 2001, Dorssers, et al 2004). Surprisingly, the inventors have discovered that this protein is present in samples from prostate cancers, and not present in healthy prostate cell samples. It has thus been discovered according to the invention that the BCAR-1 protein is a marker of prostate cancer.

  It has also been shown according to the invention that the expression of the BCAR1 protein in prostate cancers is surprisingly associated with the resistance of these cancers to antiandrogens, including, for example, estrogens.

  Indeed, the presence of the BCAR-1 protein has been detected by the inventors in histological sections of prostate cancer (FIGS. 2, 3 and 4) whereas this protein is not detected in histological sections of the prostate. non-cancerous prostate cells (Figure 1). The inventors have also shown that the expression of the bcar-1 gene is lower in samples from cancer cells that are not aggressive than in samples from aggressive cancer cells (FIGS. 2, 3, and 4).

  Conventionally, the aggressiveness of a cancer can be quantified according to the Gleason classification according to the physiological state of prostate cancer. The classification of Gleason is based on the degree of differentiation of the tumor, noted from grade 1 to 5, as shown in Table 1. The Gleason score, between 2 and 10, is the sum of the two highest grades. frequently represented in the tumor analyzed. Gleason's rank and score make it easy to measure the aggressiveness of a cancer.

  By way of example, the inventors have shown that the BCAR-1 protein is strongly expressed in aggressive cancers whose Gleason grade is equal to 4 or 5, and more weakly expressed in non-aggressive cancers whose Gleason grade is equal to 2 or 3. (see the Examples section: Tables 2 and 4) The inventors have therefore shown that there is on the one hand a correlation between the abnormal cellular expression of the protein BCAR-1 and the development of cancer of the prostate, and on the other hand a correlation between the expression of a high level of BCAR-1 protein, or a high number of cells expressing the BCAR-1 protein and the aggressiveness of a cancer of prostate.

  Abnormal cell expression refers to tissue expression, for example in contact with prostatic gland light, or anatomical expression, for example in the lymph nodes or in the blood, where BCAR-1 protein does not usually express itself. in a patient who does not have prostate cancer.

  The subject of the invention is therefore a pharmaceutical composition comprising a compound which negatively regulates the expression of the BCAR-1 protein, in association with at least one pharmaceutically acceptable excipient.

  The BCAR-1 protein according to the invention is defined as the protein encoded by the nucleic acid of sequence SEQ ID No. 1, or by a variant of this nucleic acid.

  By variant of a nucleic acid is meant a nucleic acid which differs from the reference nucleic acid by one or more substitutions, additions or deletions of a nucleotide, relative to the reference nucleic acid. A variant of a nucleic acid according to the invention may be of natural origin, such as an allelic variant which exists naturally. Such a variant nucleic acid may also be an unnatural nucleic acid obtained, for example by mutagenesis techniques.

  In general, the differences between the reference nucleic acid and the variant nucleic acid are reduced so that the reference nucleic acid and the variant nucleic acid have very similar nucleotide sequences and in many identical regions.

  BCAR-1 variants include polypeptides comprising an amino acid sequence having at least 80% amino acid identity with the BCAR-1 protein of sequence SEQ ID No. 3.

  BCAR-1 variants include polypeptides comprising an amino acid sequence of at least 81%, and preferably at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% d amino acid identity with the BCAR-1 protein of sequence SEQ ID N 3.

  The percentage identity between two amino acid sequences, in the sense of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window.

  The portion of the amino acid sequence in the comparison window may thus comprise additions or deletions (for example gaps) with respect to the reference sequence (which does not include these additions or deletions) so as to obtain optimal alignment between the two sequences.

  The percent identity is calculated by determining the number of positions at which an identical amino acid is observed for the two compared sequences, and then dividing the number of positions at which the two amino acids are identified by the total number of positions in the amino acid sequence. the comparison window, then multiplying the result by one hundred to obtain the percentage amino acid identity of the two sequences between them.

  The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms.

  Most preferably, the percentage of sequence identity is determined using the CLUSTAL W software (version 1.82) with the parameters set as follows: (1) CPU MODE = ClustalW mp; (2) ALIGNMENT = full; (3) OUTPUT FORMAT = aln w / numbers; (4) OUTPUT ORDER = aligned; (5) COLOR ALIGNMENT = no; (6) KTUP (word size) = default; (7) WINDOW LENGTH = default; (8) SCORE TYPE = percent; (9) TOPDIAG = default; (10) PAIRGAP = default; (11) PHYLOGENETIC TREE / TREE TYPE = none; (12) MATRIX = default; (13) GAP OPEN = default; (14) END GAPS = default; (15) GAP EXTENSION = default; (16) GAP DISTANCES = default; (17) TREE TYPE = cladogram and (18) TREE GRAP DISTANCES = hide.

  For the purposes of the present invention, the term "expression of the BCAR-1 protein" means the production of an mRNA, an mRNA fragment or the production and / or post-translational processing of the BCAR-1 protein. 1 or a fragment of this protein, encoded by the bcar-1 gene.

  For clarity, the bcar-1 gene is shown in lowercase letters and the BCAR-1 protein is shown in capital letters.

  Negative regulation of the expression of the BCAR-1 protein should be understood as a decrease in the level of the mature native BCAR-1 protein, which can result from the decrease in the transcription rate of the bcar-1 gene, a change in the stability of the transcribed mRNAs, a modification of the translated protein or an increased degradation of the native protein.

  The decrease in the level of the BCAR-1 protein can be measured by performing a qualitative or quantitative detection of this protein on samples taken at regular intervals on a patient to be tested. By way of example, several types of samples may be analyzed, for example one or more prostate cells, or a sample of whole blood or serum of a patient to be tested, or even possibly a urine sample. collected after prostate massage of a patient to be tested. A sample may also consist of a histological section of tissue, as has been done in the examples. The qualitative or quantitative detection of the concentration of the BCAR-1 protein may be carried out using an anti-human antibody. -BCAR-1 as defined below.

  The detection of the concentration of the BCAR-1 protein can also be deduced by quantifying the mRNA coding for the BCAR-1 protein.

  By way of example, such quantification can be carried out by quantitative or semi-quantitative real-time PCR. This technique is based on the same principles as classical PCR, ie denaturation, hybridization, and extension. Unlike conventional PCR, real-time quantitative PCR uses a fluorescent probe that allows quantification and characterization of the amplicon formed in real time. This technique makes it possible to perform a relative quantification of mRNAs between several samples, and for example between samples from patients with or without prostate cancer. It is also possible to perform an absolute quantification, that is to say a determination in number of copies relative to a control.

  Alternatively, the quantification of the mRNAs of interest can be carried out by a conventional competitive PCR technique or by the so-called Northern Blot technique known to those skilled in the art.

  By way of example, such quantification can be carried out by real-time quantitative or semi-quantitative RT-PCR using the primers of sequence SEQ ID No. 4 and SEQ ID No. 5, and a probe, for example a fluorescent probe, of sequence SEQ ID N 6. In particular, the quantification of the RNAs of interest can be carried out according to the protocol described by de Cremoux et al. (2003).

  Preferably, the compound which negatively regulates the expression of the BCAR-1 protein is chosen from an antibody directed against the BCAR-1 protein and an interfering nucleic acid (siNA).

  In particular, the interfering nucleic acid (siNA) comprises a polynucleotide having a length of at least 10 nucleotides which hybridises to the sequence of a nucleic acid of at least 10 nucleotides. sequence SEQ ID N 1. Such a nucleic acid may be called antisense polynucleotide.

  Preferably, the interfering nucleic acid (siNA) comprises a polynucleotide having a nucleotide identity of at least 80% with a sequence complementary to the sequence SEQ ID No. 1, and preferably at least 91%, 92%, 93%. %, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98, 6%, 99%, 99.6% nucleotide identity with a sequence complementary to the sequence SEQ ID N 1.

  The percentage of identity with the sequence complementary to the sequence SEQ ID No. 1, of the polynucleotide of the invention used, will depend on the conditions encountered during the hybridization, and in particular, a polynucleotide of strong identity with the sequence complementary to the sequence SEQ ID N 1 will be used if the conditions are very astringent.

  Preferably, the interfering nucleic acid according to the invention comprises a polynucleotide whose sequence is complementary to the sequence of a nucleic acid of sequence SEQ ID N 1.

  The interferential nucleic acid according to the invention may also comprise a polynucleotide whose sequence is chosen within the sequence SEQ ID No. 2, corresponding to the cDNA of the bcar-1 gene.

  Preferably, the interfering nucleic acid (siNA) of the invention comprises a polynucleotide of a length ranging from 10 nucleotides to 50 nucleotides, and preferably from 15 nucleotides to 30 nucleotides.

  The antisense polynucleotides according to the invention may be advantageously thioesterified or 2'-methylated or may have a triethylene glycol residue at their 3 'end.

  The most common form of antisense polynucleotides is in the form of antisense polydeoxyribonucleotides. However, the antisense polynucleotides according to the present invention also comprise ribozymes, oligozymes, and catalytic short RNAs or catalytic polynucleotides which hybridize with the target nucleic acid, of sequence SEQ ID No. 1 and modulate its expression.

  The polynucleotides according to the invention may comprise a modified backbone or non-natural internucleoside linkages.

  Specific examples of antisense compounds useful in this invention include polynucleotides containing modified backbone or non-natural internucleoside linkages. Thus, modified framework polynucleotides include those which retain a phosphate atom in their backbone and those which lack it. For the purposes of the present invention, modified polynucleotides lacking a phosphorus atom in their internucleoside linkage may still be regarded as polynucleotides. The backbone of these modified polynucleotides may comprise, for example, phosphorothioate, chiral phosphorothioate, phosphorodithiolate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkylphosphonate groups, including chiral 3'-alkylene phosphonates, 5'-alkylene phosphonates and phosphonates, and phosphinate groups. , Phosphoramidates including 3'amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borophosphates forming normal bonds, 3'-5 ', and their analogues forming 2'5' bonds, as well as those having reversed polarity that is to say comprising at least one internucleoside bond of the type 3'-3 ', 5'-5' or 2'-2 '. The polynucleotide form having an inverted polarity preferably used is that of which the first 3'-3 'internucleoside linkage is of the 3'-3' type. This corresponds to a single inverted nucleoside residue, which can also be abasic, that is to say in which the heterocyclic nitrogen base is missing or replaced by a hydroxyl group. The various forms (saline or free acid) are part of the invention.

  The backbone of the modified polynucleotides devoid of a phosphorus atom is preferably formed of short alkyl or cycloalkyl chains, including their derivatives containing one or more heteroatoms, serving as internucleoside binding. This type of framework may be based on morpholino bonding (consisting in part of the nucleoside sugar), siloxane, formacetyl and thioformacetyl, methylene formacetyl and methylene thioformacetyl, riboacetyl, alkenes, sulfamates and sulfonate. and sulfonamide, methylene imine and methylene hydrazine, amide, and any other group having various nitrogen, sulfur and oxygen atoms or methyl groups.

  For other polynucleotide analogs, sugar and internucleoside binding (i.e., backbone) are both replaced in the nucleotide structure by new groups. The heterocyclic nitrogen base is conserved to provide hybridization with the target nucleic acid. Such compounds, PNA (for Peptide Nucleic Acid), have shown excellent hybridization ability. In these compounds, the backbone of the polynucleotide is replaced by an amide-based backbone, in particular by aminoethyl glycine, grafted directly or indirectly on the nitrogenous bases. Further information on these ANPs can be found in Nielsen et al., Science, 1991, 254, 1497.

  The invention particularly relates to polynucleotides with a phosphorothioate, amide and morpholine backbone, and oligonucleosides with a heteroatom backbone, and more specifically: -CH 2 -NH-O-CH 2 -CH 2 -N (CH 3) -O-CH 2 (referred to as methylene-methylimino skeleton) or MMI-CH2-ON (CH3) -CH2-CH2-N (CH3) -N (CH3) -CH2 -ON (CH3) -CH2-CH2- (in which the phosphodiester bridge is: OPO CH2).

  The modification of the polynucleotides may also relate to sugars: the preferred substitutions are in position 2 '(F; O-, N- or Salcane derivatives, O-, N- or S-alkenes or O-, N- or S-alkynes of the length C 1 to C 1, substituted or unsubstituted) in particular, the preferred derivatives are: O - [(CH 2) n -O] m -CH 3 O- (CH 2) n -O-CH 3 O- (CH 2) n -NH 2 O- (CH 2) n -CH 3 O- (CH 2) n -O-NH 2 O (CH 2) n -O- [(CH 2) n -CH 3] 2 where n and m vary from 1 to 10.

  Other modifications of the 2'-position include the following groups: substituted or unsubstituted aliphatic chains of length CI to C, o, aryl, aryl-alkyl and alkyl-aryl; -SH, -SCH3, -OCN, -Cl, -Br, -CN, -CF3, OCF3, -SO2CH3, -ONO2, -NO2, -N3, -NH2; substituted silyles; reporter group, intercalator group; cleavage group of RNA; grouping to improve the pharmacodynamic capabilities of an oligonucleotide. Preferred modifications include the groups 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3, also known as 2'-O- (2-methoxyethyl) or 2'-MOE) (Martin et al., Helv Chim Acta, 1995). 78f 486-504) 2'dimethylaminooxyethoxy (O (CH2) 2ON (CH3) 2, also called 2'-DMAOE. 2'dimethylaminoethoxyethoxy (2'-O-CH2OCH2-N (CH2) 2, also called 2'dimethylaminoethoxyethyl or 2 '-DMAEOE).

  Another interesting modification leads to the formation of LNAs (Locked Nucleic Acids) in which the hydroxyl at the 2 'position is bonded to the carbon at the 3' or 4 'position of the sugar, thereby forming a sugar with a bicyclic structure. The preferred bridging is through a methyl or ethyl bond between oxygen 2 'and carbon 4'.

  Other preferred 2 'substitutions include: -O-CH 3 (2'-methoxy) -O- (CH 2) 3 -NH 2 (2'-aminopropoxy) -CH 2 -CH = CH 2 (2'-allyl) -O- CH 2 CH = CH 2 (2'-O-allyl) -F (2'fluoro).

  These 2 'modifications can be in ribo (low) or arabino (high) position. The 2'-fluoro substituent is the preferred in the arabino position.

  Similar modifications can be made at other positions, in particular at the 3 'position of the nucleotide sugar at the 3' end or in the 2 '-5' framework oligonucleotides, and at the 5 'position of the 5' end sugar. -terminal. The sugars of the oligonucleotides may also be replaced by analogs (eg, a cyclobutyl may be substituted for a pentofurranyl).

  The oligonucleotides may also include modifications or substitutions at the level of the nucleobases, that is to say, the nitrogenous heterocyclic bases called bases by those skilled in the art. The natural (unmodified) bases are purines (adenine A and guanine G) and pyrimidines (cytosine C, thymine T and uracil U). Among the modified bases are included natural or synthetic molecules such as 5-methylcytosine, 5-hydroxytmethylcytosine, xanthine, hypoxanthine, 2 aminoadenine; 6-methyl, 2-methyl and other alkylated derivatives of purine bases (A and G); 2-thio derivative (C, T and U); 5-halo derivative (U, C); 5-propynyl derivative (U and C) cytosine; 6-azo derivative (U, Tet C); 5-uracil; 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl otheradenines and 8-substituted guanines; 5-halo (in particular 5bromo), 5-trifluoromethyl and other uracils and cytosines substituted at 5-position, 7-methylguanine and 7-methyladenine; 2-fluoroadenine: 2 aminoadenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7deazaadenine; 3-deazaguanine and 3-deazaadenine. In the other modified bases, there are tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido [(-4-b] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5] , 4-b] (1,4] benzothiazin-2 '(3H) -one), substituted phenoxazine cytidine (such as 9-2-aminoethoxy) -H-pyrimido [5,4-b] [1,4] benzoxazin -2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] -indol-2-one).

  Modified bases include compounds whose purine or pyrimidine heterocycle is replaced by another heterocycle, for example 7-deazaadenine 7-deazaguanosine, 2-aminopyridine or 2-pyridone (The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859 Kroschwitz). J.1, ed., John Wiley & Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, YS Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B. ed., CRC Press, 1993). Some of these modified bases may be of great interest for increasing the affinity of the polynucleotides of the invention, such as 5-substituted pyrimidines, azapyrimidines, N- and O-substituted purines (such as 2-aminopropyladenine). 5-propynyl uracil, 5-propynyl cytosine). The substituted 5-methylcytosines have a positive effect on the stability of the nucleic acid duplex-oligomers (Sanghvif YS, Crooke ST, and Lebleu, Breds.f Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278) and are the preferred substitution, especially in combination with 2'-methoxyethyl modifications of sugars.

  Preferably, these polynucleotides are in the single-stranded form. Preferably, the polynucleotides according to the present invention are DNAs.

  As will be detailed below, when the pharmaceutical composition comprises polynucleotides as described above, it will preferably be formulated as injectable compositions intravenously.

  RNAi According to the Invention Alternatively, the interfering nucleic acid (siNA) is a ribonucleic acid composed of a single strand comprising a polynucleotide sequence (a) which hybridises to the sequence SEQ ID No. 1 or to a portion of that and a polynucleotide sequence (b) which hybridizes to a sequence complementary to the sequence SEQ ID No. 1 or to a portion thereof, the sequences (a) and (b) being separated by one or more nucleotides .

  The sequences (a) and (b) are separated by one or more nucleotides so that the sequences (a) and (b) hybridize with each other, and form a so-called hairpin structure.

  Such nucleic acids are called RNAi (interferential ribonucleic acid). These RNAi hybridize specifically to the sequence SEQ ID No. 1 The nucleotide modifications described for antisense nucleic acids are also possible for those used in the composition of RNAi.

  RNAi, according to the invention can be synthesized by chemical synthesis, when it is synthetic RNAi or expressed in situ using vectors expressing such polynucleotides. RNAi can be obtained from different suppliers or can be synthesized from kits marketed by different companies such as DHARMACON, AMBION, and PROLIGO.

  The use of RNAi within the pharmaceutical composition according to the invention is to be understood in a broad sense. Therefore, the RNAi described above can be integrated into vectors for their expression.

  The subject of the invention is therefore a pharmaceutical composition comprising a vector allowing the expression of an RNAi hybridizing specifically to the sequence SEQ ID No. 1, in association with at least one pharmaceutically acceptable excipient.

  Antibody According to the Invention Alternatively, the pharmaceutical composition according to the invention comprises an antibody capable of downregulating or suppressing the expression of the BCAR-1 protein. Such an antibody will be referred to hereinafter as anti-BCAR-1 antibody.

  The anti-BCAR-1 antibodies according to the invention comprise the antibodies which are specific for the mature form of the BCAR-1 protein, that is to say of the form of the BCAR-1 protein, several or all the posttranslational modifications that this protein undergoes in the eukaryotic cell in which it is produced.

  An anti-BCAR-1 antibody may consist of a polyclonal antibody that can be obtained by different biosynthetic pathways known to those skilled in the art.

  By way of example, polyclonal anti-BCAR-1 antibodies can be obtained according to the following steps: (i) administering an immunologically effective amount of purified BCAR-1 protein to an animal, preferably in combination with an adjuvant of the immunity such as Freund's complete adjuvant, (ii) collect the whole blood of the animal thus immunized and (iii) purify the polyclonal anti-BCAR-1 antibodies obtained, for example using an immunoaffinity chromatography substrate on which the purified BCAR-1 protein has been immobilized.

  Alternatively, the anti-BCAR-1 antibody can be prepared from hybridomas obtained after fusion of B cells of animals immunized against the purified human BCAR-1 protein with myeloma cells, according to well known techniques of the invention. skilled in the art, described by KOHLER and MILSTEIN in 1975.

  The anti-BCAR-1 antibody may also be an antibody that has been produced by the trioma cell technique or by the human B-cell hybridoma technique described by KOZBOR et al. in 1983.

  The anti-BCAR-1 antibody may also consist of an antibody fragment, and in particular an Fv chain fragment, obtained from phage display libraries or from an antibody library. humanized.

  Most preferably, the anti-BCAR-1 antibody is a monoclonal antibody which is obtained according to the following steps: (i) preparing a purified recombinant BCAR-1 protein batch, (ii) immunizing a mouse, for example a BALB / c mice with an effective amount of purified BCAR-1 protein obtained in step (i), for example by successive injection of the purified protein over a period of one month, (iii) preparing lines of hybridoma cells by fusion with 10 B cells of the mouse immunized in step (ii) for example using a cloning kit such as the clona-cell-hybridoma cloning kit marketed by (Stemcell Technologies Inc. VANCOUVER BC Canada ).

  (iv) culturing hybridoma clones as prepared in step (ii) and selecting the clone which secretes a monoclonal antibody directed against the BCAR-1 protein, and (v) purifying the monoclonal antibody produced by the hybridoma which was selected in step (iv).

  To prepare a batch of recombinant protein BCAR-1 purified according to step 1 above, the skilled person will use common biotechnology techniques.

  Most preferably an antibody suitable for the detection of BCAR-1 protein in a sample from a patient with prostate cancer is the mouse monoclonal antibody directed against the human protein p130CAS, clone CAS14 The antibodies described above are suitable for the detection of BCAR-1 protein in a sample, but they can also be administered to a patient directly by passive immunotherapy.

  Galenic Formulations of the Pharmaceutical Compositions of the Invention The term pharmaceutically acceptable excipient includes, for example, solvents, dispersion media, antibacterial agents, antifungal agents or dilution agents for achieving isotonicity.

  Among these excipients according to the invention, there may be mentioned in particular water, phosphate buffers, dextrose, glycerol, ethanol and combinations thereof.

  It is often preferable to add to the composition isotonic agents such as sugars, polyalcohols for example mannitol or sorbitol or sodium chloride. The composition may further comprise auxiliary substances, such as emulsifiers, preservatives or buffers which will increase the effectiveness or duration of use of the compositions according to the invention.

  The pharmaceutical compositions according to the invention may have several forms, and may for example be liquid or in the form of solid or semi-solid injectable doses intended to be infused before injection. Other forms are conceivable, such as, for example, suspensions, dispersions, tablets, powders, liposomes or suppositories.

  The pharmaceutical compositions of the invention must be sterile and stable under the usual manufacturing and storage conditions. The compositions according to the invention may be solutions, microemulsions, dispersions, liposomes or other structures. The preparation of sterile injectable compositions can be done by incorporating the active principle, that is to say the compound which negatively regulates the expression of the BCAR-1 protein, in an appropriate amount of solvent and other excipients, as described. above, then perform sterilization by filtration.

  The pharmaceutical compositions of the invention may include an effective pharmaceutical amount of a compound that negatively regulates the expression of BCAR-1 protein. By effective pharmaceutical amount is meant an effective amount, in dosages and for a sufficient time to achieve the desired therapeutic result.

  This amount varies depending on such factors as the medical condition, and in particular the stage of prostate cancer, sex, weight, age of the patient to be treated, and the ability of the selected compound to induce an effect. in the patient. Such an amount is the one that causes side effects or toxicities lower than the benefits that the treatment can bring.

  The dosage must be adapted to obtain an optimum response. For example, the pharmaceutical composition can be administered in a single dose or in several doses at regular time intervals. The pharmaceutical composition according to the invention is intended to treat prostate cancer, which can therefore be injected directly into the area affected by the cancer.

  When the pharmaceutical composition of the invention comprises antisense polynucleotides as described above, it will preferably be formulated as an injectable composition, for example intravenously. Indeed, the administration of such polynucleotides has already been successfully performed in vivo by various authors using simple intravenous injection protocols.

  Alternatively, when the pharmaceutical composition of the invention comprises anti-BCAR-1 antibodies as described above, it may be formulated in the form of liposomes, albumin-based microspheres, microemulsions, or else microcapsules, comprising the antibodies. Examples of microcapsules include microcapsules made from gelatin, hydroxypropylcellulose, hydroxymethylcellulose or their derivatives.

  The pharmaceutical composition of the invention comprising anti-BCAR-1 antibodies as described above, may also be formulated in the form of a sustained-release preparation, for example in the form of semipermeable matrices, based on polymers. hydrophobes comprising the anti-BCAR-1 antibody. Such matrices include, for example, hydrogels and polyester-based compositions, and in particular poly (2-hydroxyethyl-methyl acrylate), or polyvinyl alcohols, polylactides, or copolymers of L-glutamic acid and ethyl-L-glutamate. Such formulations allow the release of anti-BCAR-1 antibodies over long periods. Nevertheless, body moisture and heat can denature the antibodies and make them less active with respect to the decreased activity of the BCAR1 protein. It may therefore be necessary to chemically modify the antibodies to stabilize them, for example by transforming the disulfide bonds into disulfide bonds, or by using appropriate additives.

  It can also be envisaged that the pharmaceutical composition of the invention is associated with a second active ingredient also intended to treat prostate cancer.

  The inventors have discovered that the level of expression of the BCAR1 protein is low in the samples from non-aggressive cancers, and more important in the samples from aggressive cancers (Table 2). The inventors have also discovered that the expression of the BCAR-1 protein is very important in prostate cancers resistant to antiandrogen treatments, compared to non-aggressive cancers (Table 4).

  Without wishing to be bound to a theory, the inventors believe that increasing the level of the BCAR-1 protein may cause an increase in cancer resistance to antiandrogenic treatments.

  The pharmaceutical composition according to the invention may also comprise at least one active ingredient intended for the treatment of prostate cancer, and in particular, the pharmaceutical composition according to the invention may comprise an antiandrogen.

  An antiandrogen is defined as an antagonist of cells whose proliferation or apoptosis is controlled by androgens. An antagonist refers to a substance capable of blocking the action of androgens on the proliferative or metastatic potential of prostate cancer cells. In particular, antiandrogens can compete with androgens for binding to androgen receptors.

  The term antiandrogen is to be understood in its broadest sense, so that an antiandrogenic treatment includes in particular a medical castration using analogs or LHRH antagonists (Luteinizing hormone-releasing hormone), but also steroids such as estrogen. or progestins or non-steroidal antiandrogen compounds such as diethylstylbestrol, leuprolide acetates, bicalutamides, flutamides, nilutamides, and 5-a reductase inhibitors such as finasteride or glutasteride.

  Therefore, advantageously, said active principle is an antiandrogen chosen from estrogenic derivatives such as diethylstilbestrol, progestational derivatives such as cyproterone acetate, non-steroidal antiandrogens such as bicalutamide, flutamide, nilutamide, the analogs and LHRH antagonists or leuprolide acetates and 5-a reductase inhibitors.

  Alternatively, said additional active ingredient for the treatment of prostate cancer may be a chemotherapeutic agent selected from mitoxantrone, prednisone, paclitaxel, docetaxel, estramustine, adriamycin, estramustine phosphate and mitoxantrone. .

  In a preferred embodiment of the invention, the pharmaceutical composition is in the form of a kit, said kit comprising: (i) a formulation comprising a compound that negatively regulates the expression of the BCAR-1 protein, and (ii) a formulation comprising an active ingredient for the treatment of prostate cancer.

  Thus, both formulations can be administered simultaneously or separately over time, so that the active ingredient for the treatment of prostate cancer can be administered before, at the same time, or after the compound that negatively regulates the expression. of the BCAR-1 protein.

  Methods of detecting prostate cancer according to the invention

  The inventors have shown that the BCAR-1 protein is detected in histological sections of prostate cancer (Figures 2, 3 and 4) while the protein is not detected on histological sections of non-cancerous prostate (Figure 1). The BCAR-1 protein can therefore be considered a marker of prostate cancer.

  Therefore, the subject of the invention is a method for detecting in vitro the presence of a prostate cancer in a patient comprising a step of qualitative or quantitative detection of the BCAR-1 protein in a sample from said patient.

  The qualitative or quantitative detection of the BCAR-1 protein can be carried out using an anti-BCAR-1 antibody as defined above. A sample may also consist of a histological section of tissue, as was done in the examples.

  The invention also relates to a method for the in vitro detection of the presence of a prostate cancer in a patient, comprising a step of qualitative or quantitative detection of the mRNA encoding the BCAR-1 protein, in a sample from said patient.

  By sample, we mean one or more cells of the prostate or a sample of whole blood or serum of a patient to be tested or even a sample of urine possibly collected after massage of the prostate of a patient to be tested.

  The quantification of the mRNA coding for the BCAR-1 protein can be carried out by quantitative or semi-quantitative real-time PCR. This technique is based on the same principles as classical PCR, ie denaturation, hybridization, and extension. Unlike conventional PCR, real-time quantitative PCR uses a fluorescent probe that allows quantification and characterization of the amplicon formed in real time. This technique makes it possible to perform a relative quantification of mRNAs between several samples, and for example between samples from patients with or without prostate cancer. It is also possible to perform an absolute quantification, that is to say a determination in number of copies relative to a control.

  Alternatively, the quantification of the RNAs of interest can be performed by a conventional competitive PCR technique or by the so-called Northern Blot technique known to those skilled in the art.

  By way of example, such a quantification can be carried out by quantitative RT-PCR in real time using the primers of sequence SEQ ID N 4 and SEQ ID N 5, and a probe, for example a fluorescent probe, of sequence SEQ ID In particular, the quantification of the RNAs of interest can be carried out according to the protocol described by de Cremoux et al. (2003).

  The methods for detecting the presence of prostate cancer above may comprise a second step which is a function of the method under consideration, to: compare the amount of the BCAR-1 protein measured in the first step above above, to the amount measured in a control sample or at a mean value, or - compare the amount of mRNA encoding the BCAR-1 protein measured in the first step above, to the amount of mRNA measured in a sample sample or at a mean value.

  A control sample may be derived from an individual with prostate cancer, and a control value may be derived from several samples from individuals with and without prostate cancer.

  The detection methods described in the present application may also comprise an additional step of measuring the PSA (prostate specific antigen) level of said sample.

  A method, including detection of BCAR-1 protein, or corresponding mRNA, associated with measurement of PSA antigen level, is particularly advantageous. Indeed, as recalled above, although the PSA antigen is widely used as a diagnostic agent, the test using the assay of this antigen has a limit of sensitivity and ability to detect early cancers, and generally, estimates that about 20 to 30% of tumors may be missed when the PSA assay is performed alone. The detection of the BCAR-1 protein, or the corresponding mRNA associated with the measurement of the PSA level, can therefore make it possible to detect a larger number of early cancers.

  The inventors have also shown that the BCAR-1 protein is expressed in the non-cancerous androgen-independent epithelial cells of the basal layer of the prostatic glands, and absent in the non-cancerous androgen-dependent prostatic epithelial cells of the secretory layer of the prostatic glands (FIG. 1).

  However, during cancer development, the inventors have observed that the BCAR-1 protein is expressed in contact with the light of the prostatic glands, or in the lymph nodes or in the blood, that is to say in sites tissue and anatomic in which the protein is not usually expressed.

  Therefore, the subject of the invention is also a process for the in vitro detection of the presence of a prostate cancer in a patient comprising a step of identifying the anatomical location of the cells expressing the BCAR-1 protein, in a sample from said patient.

  In general, the identification of the anatomical localization of the cells expressing the BCAR-1 protein can be carried out using an anti-BCAR-1 antibody as defined above, on a histological section of tissue, such as this has been done in the examples.

  The identification of the anatomical localization of the cells expressing the BCAR-1 protein can also be carried out by using a kit for detecting the mRNA of the BCAR-1 protein by Northern blot or else RNA dot blot, the implementation of such kits being conventional for those skilled in the art, these methods will not be described in detail.

  In particular, the identification of the anatomical localization of the cells expressing the BCAR-1 protein can be carried out using a probe consisting of a labeled nucleic acid, at least 10 nucleotides long and whose sequence is identical to all or part of the sequence SEQ ID N 1.

  In vitro evaluation method of the aggressiveness of a cancer of a prostate cancer according to the invention.

  The inventors have shown that the expression of the BCAR-1 protein is more important in samples from aggressive prostate cancers (whose gleason grade is equal to 4 or 5), than in non-aggressive cancers (the gleason grade is equal to 2 or 3). (See Tables 2 and 4) Therefore, the inventors have also developed a method of in vitro evaluation of the aggressiveness of prostate cancer in a patient comprising the steps of: (i) measuring the amount of BCAR-1 protein in a sample previously taken from said patient, (ii) comparing the amount of BCAR-1 protein measured in step (i) with one or more control values calculated from the measured amount of BCAR-1 protein. in a sample from an individual with prostate cancer.

  The invention also relates to a method for in vitro evaluation of the aggressiveness of a prostate cancer in a patient, comprising the steps of: (i) measuring the amount of mRNA encoding the BCAR-1 protein in a sample previously taken from said patient, (ii) comparing the amount of mRNA measured in step (i) with one or more control values calculated from the amount of mRNA encoding the BCAR-1 protein, measured in a sample from an individual with prostate cancer.

  The methods described above may include an additional step to positively conclude on the aggressiveness of a cancer if the amount of BCAR-1 protein or mRNA measured in step (i) is greater than the amount of protein. BCAR-1 or mRNA measured in a sample from an individual with non-aggressive prostate cancer, for which the Gleason grade is less than or equal to 3, and the Gleason score is less than or equal to 6 .

  Alternatively, and in a completely symmetrical manner, the method described above may comprise an additional step also aimed at concluding positively on the aggressiveness of a cancer if the amount of BCAR-1 protein or mRNA measured at the same time. step (i) is less than the amount of BCAR-1 protein or mRNA measured in a sample from an individual with aggressive prostate cancer, for which the Gleason grade is greater than or equal to 4, and the Gleason score is greater than or equal to 7.

  The subject of the invention is also a method for evaluating in vitro the aggressiveness of a prostate cancer in a patient, comprising the following steps: (i) identifying the anatomical location of the cells expressing the BCAR-1 protein originating from of a sample previously taken from said patient, (ii) comparing the anatomical location of the cells in step (i) with the anatomical location of the cells expressing the BCAR-1 protein from a sample previously taken from an individual suffering from 'prostate cancer.

  The invention also relates to a method for evaluating in vitro the resistance of a patient to treatment with an antiandrogenic active principle comprising the following steps: (i) measuring the amount of BCAR-1 protein in a sample previously taken from said patient, and (ii) comparing the amount of BCAR-1 protein measured in step (i) with one or more control values calculated from the amount of BCAR-1 protein measured in a sample from an individual with prostate cancer resistant to antiandrogenic treatment.

  The invention also relates to a method of in vitro evaluation of the resistance of a patient to a treatment with an antiandrogenic active principle comprising the following steps: (i) measuring the amount of mRNA coding for the BCAR-1 protein in a sample previously taken from said patient, (ii) comparing the amount of mRNA measured in step (i) with one or more control values calculated from the amount of mRNA coding for the BCAR-1 protein, measured in a sample from an individual with prostate cancer resistant to antiandrogenic treatment.

  The invention also relates to a method for in vitro evaluation of a patient's resistance to antiandrogenic drug treatment comprising the steps of: (i) identifying the anatomical location of cells expressing BCAR-1 protein from a sample previously taken from said patient, (ii) comparing the anatomical location of the cells in step (i) with the anatomical location of the cells expressing the BCAR-1 protein from a sample previously taken from an individual suffering from a prostate cancer resistant to antiandrogenic treatment.

  In general, the identification of the anatomical localization of the cells expressing the BCAR-1 protein can be carried out using an anti-BCAR-1 antibody as defined above, on a histological section of tissue, such as this has been done in the examples.

  The method described above may comprise an additional step to positively conclude on the resistance of a cancer to an antiandrogenic compound if the amount of BCAR-1 protein or mRNA measured in step (i) is greater than or equal to equal to the amount of BCAR-1 protein or mRNA measured in a sample from an individual with anti-androgen-resistant cancer.

  The invention finally relates to a method for determining in vitro the treatment to be administered to an individual suffering from a prostate cancer comprising the following steps: measuring the BCAR-1 protein in a sample previously taken from said individual, - recommend the use of a chemotherapeutic active ingredient or the use of an antiandrogenic active ingredient as a function of the amount of BCAR-1 protein measured.

  The subject of the invention is also the use of a compound which negatively regulates the expression of the BCAR-1 protein for the manufacture of a medicament for treating prostate cancer.

  Preferably, the compound which negatively regulates the expression of the BCAR-1 protein is selected from an antibody directed against the BCAR-1 protein, and an interfering nucleic acid (siNA).

  Preferably, the antibody directed against the BCAR-1 protein, and / or the interfering nucleic acid (siNA) are as described above with regard to the pharmaceutical compositions according to the invention.

  Advantageously, the prostate cancer treated is an aggressive cancer, for which the Gleason grade is greater than or equal to 4 and the gleason score is greater than or equal to 7.

  The prostate cancer treated can also be a cancer resistant to treatment with an antiandrogenic active ingredient. In general, the treated prostate cancer may be of the adenocarcinoma type.

  Preferably, the pharmaceutical composition further comprises an active ingredient for the treatment of prostate cancer.

  The above compound, which negatively regulates the expression of the BCAR-1 protein, can be included in a pharmaceutical composition that is distinct from the active principle intended for the treatment of prostate cancer, and can be presented, for example in the form of a kit, so that the compound that negatively regulates the expression of the BCAR-1 protein can be administered before, at the same time or after the active ingredient for the treatment of prostate cancer.

  Other compounds and compositions according to the invention, neutralizing the activity of the BCAR-1 protein.

  BCAR-1 protein in immunogenic form The inventors have shown that the BCAR-1 protein is strongly expressed in aggressive cancers whose Gleason grade is equal to 4 or 5, more weakly expressed in non-aggressive cancers whose Gleason is equal to 2 or 3, and finally non-expressed non-cancerous cells.

  The inventors have therefore shown that there is a correlation between the expression of the BCAR-1 protein and the development of prostate cancer.

  The invention therefore also relates to a BCAR-1 protein in an immunogenic form.

  According to the invention, the term "protein BCAR-1" in an immunogenic form is intended to mean a protein capable of inducing an immune response in humans, it being understood that the corresponding native protein, which is a self-protein, is not immunogenic to humans. makes the immune system immune to self proteins.

  For example, BCAR-1 protein in immunogenic form (form different from native form) induces an antibody-producing immune response that recognizes both the immunized BCAR-1 protein administered and the native BCAR-1 protein produced in the human body.

  More particularly, it is considered that an immunogenic BCAR-1 protein is capable of inducing an immune response based on neutralizing antibodies that inhibit the biological activity of the BCAR-1 protein. The immunogenic BCAR-1 protein can be obtained from a native BCAR-1 protein, in particular by mutation, chemical, physical or immunological treatment.

  Suitable physical treatment may be heat treatment or irradiation while suitable chemical treatment may be treatment with carboxymethylation, or treatment with an aldehyde such as formaldehyde or polyaldehyde.

  Preferably, the BCAR-1 protein in an immunogenic form is rendered immunogenic by mutation or chemical treatment.

  The carboxymethylation reaction makes it possible to modify the thiol groups optionally present at the level of the cysteine residues of the amino acid sequence of the BCAR-1 protein. These groups react with iodoacetic acid or iodoacetamide by an S-carboxymethylation or S-carboxyamidomethylation reaction respectively.

  The carboxymethylation reaction may also be carried out with other chemical agents such as performic acid, 3-bromopropionic acid, ethyleneimine, (2-bromoacetyl) trimethylammonium bromide, 2-bromoethane sulfonate or propane sulfone.

  When an aldehyde treatment is carried out, it is possible to treat, for example, 100 μg to 10 mg of BCAR-1 protein / ml in the presence of formaldehyde having a concentration of 10 to 100 mM, at a temperature of 10 to 60 ° C. for 2 minutes. at 9 days.

  The subject of the invention is also a pharmaceutical composition or an immunogenic composition which comprises at least the immunogenic BCAR-1 protein mentioned above, as active principle.

  The invention also relates to a vaccine composition characterized in that it comprises, as active principle, a BCAR-1 protein in an immunogenic form as defined above, in combination with one or more physiologically compatible excipients.

  Preferably, the physiologically compatible excipient or excipients comprise a systemic or mucosal immunity adjuvant.

  Among the systemic adjuvants, mention may be made of adjuvants of IFA type (Incomplete Freund's Adjuvant), calcium phosphate or alumina hydroxide.

  Among the mucosal adjuvants, adjuvants such as chloro toxin B (CTB) or a mutant of LT toxin (LTp) are preferably used. Such a vaccine composition may also comprise one or more immunostimulatory agents, in combination with an adjuvant of the immunity, such as for example the immunostimulatory agent CpG well known in the state of the art.

  The above pharmaceutical compositions may be formulated in a manner suitable for any conventional route used in the field of vaccines, and in particular to the subcutaneous route, the intramuscular route, the intravenous route or the oral route. .

  Alternatively, the pharmaceutical compositions described above comprise a nucleic acid of sequence SEQ ID No. 1 or a part thereof.

  Immunogenic cellular compositions Dendritic cells are capable of priming the antigens and then presenting the peptides from the antigen-priming process to naive T cells. Dendritic cells activate T cells more efficiently than any other antigen presenting cell. The use of dendritic cells is therefore of particular interest in the context of cancer immunotherapy.

  The invention therefore also relates to a process for producing an immunogenic cellular composition characterized in that it comprises the following steps: a) cultivating in vitro a population of cells enriched in human or animal dendritic cells in an appropriate culture medium; b) adding to the cells cultured in step a), an antigen of interest selected from the BCAR-1 protein, or a part thereof, or a nucleic acid of sequence SEQ ID No. 1 or a part thereof. this.

  In an adjuvant cell composition according to the invention, the dendritic cells are preferably suspended in a saline liquid medium necessary for their survival for a few hours, preferably at least three hours.

  Most preferably, the dendritic cells are suspended in a suitable culture medium comprising all the nutrients for their long-term survival, for example for several days, preferably for at least 2 days.

  The population of cells enriched in human or animal dendritic cells used in step a) of the method can be obtained from a bone marrow sample or a sample of human or animal blood, according to techniques well known to the patient. those skilled in the art, such as the technique described by Mayordomo et al. (1995).

  Most preferably, the cells are obtained from a bone marrow sample or blood from an individual with prostate cancer.

  To purify and cultivate dendritic cells, one skilled in the art can also refer to the technique described by Steinam and Young (1991).

  The invention also relates to a method for the treatment of prostate cancer comprising a step of administering to a patient a compound that negatively regulates expression of the BCAR-1 protein.

  In particular, the treatment method according to the invention may comprise the intravenous administration of a composition comprising an antisense polynucleotide, or an antibody as defined above, which negatively regulate the expression of the BCAR protein. -1.

  The above method of treatment may be associated with a physical treatment of prostate cancer, for example by surgery or radiotherapy.

  Preferably, the compound that negatively regulates the expression of BCAR-1 protein is administered in combination with a compound for treating prostate cancer.

  The compound which negatively regulates the expression of the BCAR-1 protein can be included in a pharmaceutical composition distinct from the active principle for the treatment of prostate cancer, so that the compound which negatively regulates the expression of the BCAR-1 protein 1 may be administered before, at the same time or after the active ingredient for the treatment of prostate cancer.

  In particular said active ingredient may be an antiandrogen selected from diethylstilbestrol, leuprolide acetates, bicalutamides, flutamides, and nilutamides.

  Alternatively, said active ingredient is a chemotherapeutic agent selected from mitoxantrone, prednisone, paclitaxel, docetaxel, estramustine, adriamycin, estramustine phosphate and mitoxantrone.

  The invention also relates to a method of treating prostate cancer comprising a step of administering a composition comprising (i) a BCAR-1 protein in immunogenic form, and (ii) at least one pharmaceutically acceptable excipient.

  By way of example, such a pharmaceutically acceptable adjuvant may be, in the case of a vaccine composition, an adjuvant of the immunity and / or an immunostimulant as previously described in the present description.

  The invention also relates to a method for the treatment of prostate cancer comprising a step of administering to a patient a nucleic acid of sequence SEQ ID No. 1 or a portion thereof.

  Advantageously, the treatment methods according to the invention will be preceded or followed by an in vitro evaluation step of the aggressiveness of the prostate cancer to be treated, comprising the following steps: measuring the amount of BCAR-1 protein in a sample previously taken from said patient, - comparing the amount of BCAR-1 protein measured in step (i) with one or more control values.

  The invention also relates to a method for the treatment of prostate cancer comprising a step of administering to a patient an immunogenic cellular composition as defined above.

  In Vivo Diagnostic Methods The invention also relates to a method for detecting the presence of prostate cancer in vivo, comprising a step of identifying the anatomical location of the cells expressing the BCAR-1 protein.

  In particular, the anatomical location of the cells expressing the BCAR-1 protein can be carried out by using a compound capable of detecting the qualitative or quantitative presence of the BCAR-1 protein in said cells, for example by immunoscintigraphy, or more generally any method using the antibodies and the nucleic acid interference described in the invention.

  The inventors have shown that the expression of the BCAR-1 protein is more important in samples from aggressive prostate cancers (whose gleason grade is equal to 4 or 5), than in non-aggressive cancers (the gleason grade is equal to 2 or 3). (See Tables 2 and 4) Accordingly, the invention also relates to a method for in vivo detection of the presence of prostate cancer, comprising the steps of: (i) identifying the anatomical location of the cells expressing the protein BCAR-1 and (ii) measuring the amount of BCAR-1 protein in a sample taken from said patient.

  The invention also relates to a method for in vivo evaluation of the resistance of a patient to treatment with an antiandrogenic active principle comprising the following steps: (i) identifying the anatomical location of the cells expressing the BCAR-1 protein and ( ii) measuring the amount of BCAR-1 protein in a sample taken from said patient.

  In general, the identification of the anatomical localization of the cells expressing the BCAR-1 protein can be carried out using an anti-BCAR-1 antibody as defined above, on a histological section of tissue, such as this has been done in the examples.

  The present invention is further illustrated, without being limited, by the following examples.

EXAMPLES

  MATERIALS AND METHODS To study the expression of the bcar-1 gene, and detect the presence or absence of the BCAR-1 protein, histological sections of the prostate were made and revealed with an anti-mouse monoclonal antibody BCAR-1. directed against the human p130CAS protein, clone CAS14, marketed by Chemicon International (Temecula, CA) with a 1/500 dilution according to a usual procedure.

  Briefly, the sections were incubated for 30 minutes at room temperature on the Ventana type system marketed by Ventana Medical Systems, SA (Illkirch, France).

  Results The expression of the bcar-1 gene was measured on 92 prostate cancers by immunohistochemistry. Selected tissues were selected for H / E staining (hematoxylin and eosin) and examined for histological characteristics.

  86 prostate cancers were classified as aggressive or non-aggressive cancers based on their Gleason score, and 38 were characterized for their status with respect to the loss of chromosome 16 heterozygosity (q2223). Six cancers were classified as resistant to castration and antiandrogens.

  As seen in FIG. 1, at the level of the basal layer of the prostatic glands, a normal expression of BCAR-1 is found in androgen-independent and non-cancerous prostatic epithelial cells, and at the level of the secretory layer there is an absence of of BCAR-1 expression in androgen-dependent non-cancerous prostate epithelial cells of the same glands.

  There is low expression of BCAR-1 protein on a histological section of mild prostate cancer with a Gleason grade of 2 or 3 (well-differentiated cancers) (Figure 2). In contrast, a strong expression of BCAR-1 is observed in aggressive prostate cancers, whose Gleason grade is equal to 4 or 5 (poorly differentiated cancers) (Figure 3). Finally, a histological section of cancerous prostate, resistant to antiandrogenic treatment was performed and revealed with the anti-mouse monoclonal antibody BCAR-1 described above. There is a strong expression of BCAR-1 in this type of cancer (Figure 4).

  The results have been grouped in Tables 2, 3 and 4 below. Table 2 represents the distribution of the expression of the BCAR-1 protein according to the type of cancer (aggressive, or non-aggressive).

TABLE 2

  P ^ 0.003 Protein BCAR1 Protein BCAR1 undetected detected Non-aggressive cancers 9 47 (Gleason grade 3 or less) Aggressive cancers 13 15 (Gleason grade 4 or higher) Table 2 shows BCAR protein -1 is mainly expressed in aggressive cancers.

  Table 3 represents the distribution of the expression of the BCAR-1 protein according to the karyotype of the cancer cells (allelic loss).

TABLE 3

  P ^ 0.0001 Protein BCAR1 Protein BCAR1 undetected detected Allele loss 16q 0 14 No allelic loss 16q 13 11 Table 3 shows that BCAR-1 protein is not expressed in prostate cancers with allele loss. long arm of chromosome 16 (q22-23).

  Table 4 represents the distribution of the expression of the BCAR-1 protein according to the type of cancer (non-aggressive, or resistant to antiandrogens).

TABLE 4

  P ^ 0.001 Protein BCAR1 Undetected BCAR1 Protein Detected Non-Aggressive Cancers 9 47 (Gleason Grade 3 or less) Anti-Androgen Resistant Cancers Table 4 shows that BCAR-1 is predominantly found in cancer from prostate to antiandrogens treatment compared to non-aggressive cancers before treatment with antiandrogens.

  Therefore, BCAR-1 protein is present in androgen-independent basal epithelial cells and absent from normal prostate cells in non-tumor prostate samples.

  Expression of the BCRA-1 gene is important in aggressive cancer cells (high Gleason grade, poorly differentiated tumors) and low-level, low-aggressive cancer cells (low Gleason grade, differentiated tumors) in prostate cancer samples. Expression of the BCRA-1 gene is important in 16q22-23 allele-free prostate cancer and low in 16q22-23 allelic cancer. Finally, the expression of the BCRA-1 gene is very important in prostate cancers resistant to antiandrogen treatments.

TABLE 1

  Grade Glands Tumor Histological Aspects / Epithelium 1 Monotonic Proliferation Nodules rounded to single glands, well-rounded, tightly curved edges 2 Simple glands, Vague, rounded masses, more rounded, with poorly defined margins _ Glands simple, size Irregular masses at 3A medium, shaped, jagged edges size and irregular spacing 3B Simple glands, very irregular Small size masses, shape, jagged edges size and irregular spacing 3C Epithelial Masses Irregular areas cribriform or consisting of papillary cylinders, with regular rounded borders and massifs 4A Massive epithelial massifs and cords fused irregular glands of fused glands 4B Same appearance as 4A, Massifs and cords with presence of irregulars, clear cell aspects of hyper-nephroma 5A Rounded masses, Cylinders and papillary masses or rounded cribriforms The following sequence listing is available: The sequence SEQ ID No. 1 is the sequence of a nucleic acid encoding the BCAR-1 protein.

  The sequence SEQ ID No. 2 is the sequence of the cDNA of the bcar-1 gene.

  The sequence SEQ ID No. 3 is the sequence of the BCAR-1 protein.

  The sequence SEQ ID No. 4 and the sequence SEQ ID No. 5 are primers allowing quantification of the mRNA encoding the BCAR-E protein. The sequence SEQ ID No. 6 is a fluorescent probe.

BIBLIOGRAPHIC REFERENCES

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Claims (10)

  A method for detecting in vitro the presence of prostate cancer in a patient, comprising a step of qualitatively or quantitatively detecting BCAR-1 protein in a sample from said patient.   2. A method for detecting in vitro the presence of a prostate cancer in a patient, comprising a qualitative or quantitative detection step of the mRNA encoding the BCAR-1 protein, in a sample from said patient.   3. A method for in vitro detection of the presence of prostate cancer in a patient, comprising a step of identifying the anatomical location of the cells expressing the BCAR-1 protein, from a sample from said patient .   4. A method for in vitro evaluation of the aggressiveness of prostate cancer in a patient, comprising the steps of: (i) measuring the amount of BCAR-1 protein in a sample previously taken from said patient, ( ii) comparing the amount of BCAR-1 protein measured in step (i) with one or more control values calculated from the amount of BCAR-1 protein measured in a sample from an individual with cancer of the prostate.   5. A method for in vitro evaluation of the aggressiveness of a prostate cancer in a patient, comprising the following steps: (i) measuring the amount of mRNA coding for the BCAR-1 protein in a sample previously taken from said patient, (ii) comparing the amount of mRNA measured in step (i) with one or more control values calculated from the amount of mRNA encoding the BCAR-1 protein, measured in a sample from an individual with prostate cancer.   6. A method for in vitro evaluation of the aggressiveness of prostate cancer in a patient, comprising the steps of: (i) identifying the anatomical location of cells expressing BCAR-1 protein from a sample beforehand taken from said patient, (ii) comparing the anatomical location of the cells in step (i) with the anatomical location of the cells expressing the BCAR-1 protein from a sample previously taken from an individual suffering from cancer of the prostate.   7. A method of assessing cancer resistance to antiandrogenic drug treatment in a patient comprising the steps of: (i) measuring the amount of BCAR-1 protein in a sample previously taken from said patient (ii) comparing the amount of BCAR-1 protein measured in step (i) with one or more control values calculated from the amount of BCAR-1 protein measured in a sample from an individual with a prostate cancer resistant to antiandrogenic treatment.   A method for in vitro evaluation of cancer resistance to antiandrogenic drug treatment in a patient, comprising the steps of: (i) measuring the amount of mRNA encoding the BCAR-1 protein in a sample previously taken from said patient, (ii) comparing the amount of mRNA measured in step (i) with one or more control values calculated from the amount of mRNA coding for the BCAR-1 protein, measured in a sample from an individual with prostate cancer resistant to antiandrogenic treatment.   9. A method for in vitro evaluation of the resistance of a cancer to treatment with an antiandrogenic active principle in a patient, comprising the following steps: (i) identifying the anatomical location of the cells expressing the BCAR-1 protein originating from a sample previously taken from said patient, (ii) comparing the anatomical location of the cells in step (i) with the anatomical location of the cells expressing the BCAR-1 protein from a sample previously taken from an individual suffering from a prostate cancer resistant to antiandrogenic treatment.   10. The method of claim 6 or 9, characterized in that said sample is a histological section of prostatic tissue.