WO2004056989A2 - Human variants of kallikrein-2 and kallikrein-3 and uses thereof - Google Patents

Human variants of kallikrein-2 and kallikrein-3 and uses thereof Download PDF

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Publication number
WO2004056989A2
WO2004056989A2 PCT/FR2003/003775 FR0303775W WO2004056989A2 WO 2004056989 A2 WO2004056989 A2 WO 2004056989A2 FR 0303775 W FR0303775 W FR 0303775W WO 2004056989 A2 WO2004056989 A2 WO 2004056989A2
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nucleic acid
psa
polypeptide
seq
klk2
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PCT/FR2003/003775
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French (fr)
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WO2004056989A3 (en
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Laurent Bracco
Brigitta Brinkman
Fanny Coignard
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Exonhit Therapeutics
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Priority to AU2003300655A priority Critical patent/AU2003300655A1/en
Publication of WO2004056989A2 publication Critical patent/WO2004056989A2/en
Publication of WO2004056989A3 publication Critical patent/WO2004056989A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention relates to the field of biology, genetics and medicine. It relates in particular to new nucleotide sequences associated with alternative splicing events of the genes corresponding to the PSA antigen (prostate specific antigen or KLK3) and to kallikrein-2 (KLK2).
  • the invention also relates to methods for detecting the presence or for determining the level of expression of these nucleic acids or the corresponding proteins in biological samples, as well as methods of selecting molecules capable of modulating their activity or their expression.
  • the invention is particularly suitable for screening or monitoring cancers, especially of the prostate and in particular in the differentiation between prostate cancer and benign prostatic hyperplasia (BPH), as well as to the development of new therapeutic approaches. of these diseases.
  • BPH benign prostatic hyperplasia
  • Kallikreins correspond to a group of proteins whose protease activity allows the post-translational modification of protein precursors to biologically active forms.
  • PSA protease activity
  • kallikrein-2 are considered to be the best available markers usable in detection, diagnosis and monitoring of prostate cancer.
  • PSA prostate-specific antigen
  • BPH benign hyperplastic symptom
  • PSA exists in free, uncomplexed form and in complexed form, in particular with alpha-antichymotrypsin. Measuring these different forms and their relationships improves the differentiating factor between Pca and BPH.
  • the alternative splicing constitutes a mechanism of regulation of the expression of the genes making it possible to generate functional diversity from limited genetic information.
  • This highly regulated mechanism can undergo alterations during the development of human pathologies.
  • the deregulation of the splicing machinery in cancers can allow the expression of isoforms or variants specifically expressed in certain human tumors. These isoforms may have a determining functional role in the development or maintenance of the pathological state.
  • the specific expression of such isoforms constitutes an event of choice for a rational and targeted approach to the development of drugs and / or diagnostic methods.
  • a gene expression profiling technology has been recently developed to systematically identify genes and, within these genes, the domains likely to be altered by alternative splicing (WO99 / 46403).
  • the present invention now describes novel genetic events associated with alternative splicing of the PSA and KLK2 genes in prostate tissue.
  • the present invention results in particular from the construction of a directory of splicing alterations associated with prostate tumor tissues, and from the identification of structural alterations in the PSA and KLK2 genes or in the corresponding mRNAs.
  • the present invention thus provides new therapeutic and diagnostic approaches for cancers, in particular prostate cancer.
  • RNA extracted from samples of prostate tissue originating from tumor and non-tumor areas of patients with prostate carcinomas was carried out by qualitative differential screening thanks to the implementation of the DATAS technique (described in application n ° WO99 / 46403) which has unmatched benefits.
  • the application of DATAS technology on RNAs from tumor and non-tumor prostate tissue has made it possible to isolate various cDNA fragments derived from human kalikrein 2 and kalikrein 3 (PSA) mRNAs. These results then made it possible to identify a certain number of cDNAs revealing events linked to alternative splicing.
  • the present invention therefore describes original molecular events which can lead to the specific expression of isoforms or variants of KLK3 (PSA) and KLK2 in prostate tissues and, more specifically, in cancerous tissues or tissues associated with a benign hyperplasia (BPH).
  • PSA KLK3
  • BPH benign hyperplasia
  • the present invention provides molecular data which justify the use of one or more of these variants as new therapeutic and diagnostic targets which can advantageously be used in the diagnosis and treatment of cancers and in particular prostate cancer.
  • a first aspect of the present application therefore relates to variants of human PSA and KLK2, in particular splicing variants.
  • the invention relates to the nucleic acids corresponding to these variants or to the specific alterations which they exhibit, as well as the proteins (or polypeptides or protein domains) encoded.
  • Another aspect of the present application relates to methods and tools for detecting the presence of these variants or alterations in biological samples (blood, plasma, urine, serum, saliva, biopsies or cell cultures, etc.), or for determining their ( s) respective quantity (s) or proportion (s).
  • Such tools include in particular nucleic probes or primers, antibodies or other specific ligands, kits, supports, chips, etc.
  • Detection methods can include hybridization, PCR, chromatography, immunology, etc. These methods are particularly suitable for the detection, characterization, monitoring of progression or the effectiveness of a treatment for cancers, in particular prostate cancer, or in determining a predisposition.
  • Another aspect of the present application relates to tools and methods for the production of compounds active on the variants described, that is to say capable of modulating their expression or their activity.
  • tools and methods include in particular nucleic acids, vectors, recombinant cells (or preparations derived from such cells), binding tests, etc.
  • the invention also relates to the compounds thus identified or produced, to pharmaceutical compositions containing them, as well as to their therapeutic uses.
  • the present invention is thus applicable to the diagnosis and development of therapeutic strategies for cancers, in particular of the prostate.
  • a first aspect of the present application therefore relates to variants of KLK-2 and KLK-3 (PSA), or particular genetic alterations affecting these genes (or the corresponding RNAs or proteins).
  • PSA KLK-2 and KLK-3
  • a more particular subject of the invention relates to nucleic acids corresponding to these variants of human PSA and KLK2 or to the specific alterations which they exhibit, as well as the proteins (or polypeptides or protein domains) encoded.
  • KLK2 The nucleotide numbering refers to the Genbank accession number M18157 unless otherwise specified.
  • the reference protein is KLK2 provided with its signal peptide.
  • KLK2-EHT101 SEQ ID NO: 1
  • This isoform has a deletion of exon 1 associated with a retention of part of intron 1 (deletion of nucleotides 483 to 738).
  • This KLK2-EHT101 isoform deletes the initiation codon used by KLK2 in exon 1.
  • An open reading phase is proposed from two ATG initiation codons in phase at positions 845 and 878 to produce two polypeptides of 77 and 66 amino acids respectively (KLK2-EHT101prota / SEQ ID NO: 8 and KLK2-EHTIOlprotb / SEQ ID NO: 9).
  • KLK2-EHT101protb / SEQ ID NO: 9 presents in its amino-terminal part a signal peptide very homologous to that of KLK2 of wild type.
  • KLK2-EHT101 protb / SEQ ID NO: 9 a secreted polypeptide of 46-50 amino acids (KLK2-EHT101protc / SEQ ID NO: 10).
  • KLK2-EHT102 (SEQ ID NO: 2):
  • This isoform has a deletion in intron 1 between positions 1032 and
  • KLK2-EHT102 isoform puts a peptide of 52 amino acids into phase with the coding phase of secreted KLK2 (from exon 2), thus coding for a protein of 297 amino acids (KLK2-EHT102prota / SEQ ID NO: 11 ).
  • the amino-terminal part of KLK2- EHT102prota / SEQ ID NO: 11 has the same signal peptide as KLK2- EHT101protb / SEQ ID NO: 9.
  • KLK2-EHT102prota / SEQ ID NO: 11 between 16 and 20 amino acids can be cleaved from KLK2-EHT102prota / SEQ ID NO: 11 to form the secreted polypetide KLK2-EHT102protb / SEQ ID NO: 12.
  • the 32 -36 N-terminal amino acids of KLK2-EHT102protb (KLK2-EHT102protc / SEQ ID NO: 13) are new and may have one or more original epitopes.
  • KLK2-EHT103 (SEQ ID NO: 3): This isoform has a deletion in intron 1 between positions 965 and 1774 (junction intron 1 with exon 2). An open reading phase is proposed from two ATG initiation codons in phase at positions 845 and 878 to produce two polypeptides of 91 and 80 amino acids respectively (KLK2- EHT103prota / SEQ ID NO: 14 and KLK2-EHT103protb / SEQ ID NO: 15). Identical to KLK2-EHT101protb, KLK2-EHT103protb / SEQ ID NO: 15 presents in its amino-terminal part a signal peptide very homologous to that of KLK2 of wild type.
  • KLK2-EHT103protb / SEQ ID NO: 15 can be cleaved from KLK2-EHT103protb / SEQ ID NO: 15 to form a secreted polypeptide of 60-64 amino acids (KLK2-EHT103protc / SEQ ID NO: 16).
  • Variants of PSA (or KLK3): The nucleotide numbering refers to the Genbank accession number M27274 unless otherwise specified.
  • the reference protein is PSA provided with its signal peptide.
  • PSA-EHT101 SEQ ID NO: 4
  • This isoform has a deletion of exon 1 associated with a retention of part of intron 1 (deletion of nucleotides 636 to 918).
  • This PSA-EHT101 isoform deletes the initiation codon used by PSA in exon 1.
  • An open reading phase is proposed from an ATG initiation codon at position 1057 to produce a polypeptide of 60 amino acids (PSA -EHT101 prota / SEQ ID NO: 17).
  • PSA-EHT101 prota / SEQ ID NO: 17 presents in its amino-terminal part a signal peptide very homologous to that of PSA of wild type.
  • PSA-EHT101 prota / SEQ ID NO: 17 can be cleaved from PSA-EHT101 prota / SEQ ID NO: 17 to form a secreted polypeptide of 40-44 amino acids (PSA-EHT101 protb / SEQ ID NO: 18) .
  • PSA-EHT102 SEQ ID NO: 5:
  • This isoform has a deletion of exon 1 associated with a retention of part of intron 1 (deletion of nucleotides 636 to 963).
  • PSA-EHT101 deletes
  • this PSA-EHT102 isoform deletes the initiation codon used by PSA in exon 1.
  • An open reading phase is proposed from an ATG initiation codon at position 1057 to produce a polypeptide of 60 amino acids (PSA-EHT101prota / SEQ ID NO: 17).
  • PSA-EHT101 prota / SEQ ID NO: 17 presents in its amino-terminal part a signal peptide very homologous to that of PSA of wild type.
  • PSA-EHT101prota / SEQ ID NO: 17 can be cleaved from PSA-EHT101prota / SEQ ID NO: 17 to form a secreted polypeptide of 40-44 amino acids (PSA-EHT101 protb / SEQ ID NO: 18).
  • PSA-EHT103 SEQ ID NO: 6
  • This isoform has a deletion in intron 1 between positions 1211 and 1958 Gunction intron 1 with exon 2).
  • This PSA-EHT103 isoform puts a peptide of 52 amino acids into phase with the coding phase of secreted PSA (from exon 2), thus coding for a protein of 297 amino acids (PSA- EHT103prota / SEQ ID NO: 19 ).
  • the amino-terminal part of PSA- EHT103prota / SEQ ID NO: 19 has the same signal peptide as PSA- EHT101prota / SEQ ID NO: 17.
  • PSA-EHT103prota / SEQ ID NO: 19 between 16 and 20 amino acids can be cleaved from PSA- EHT103prota / SEQ ID NO: 19 to form the secreted polypeptide PSA-EHT103protb / SEQ ID NO: 20.
  • the 32-36 N-terminal amino acids of PSA-EHT103protb are new and may present one or more original epitopes.
  • PSA-EHT104 (SEQ ID NO: 7): This isoform has a deletion in intron 1 between positions 1144 and 1958 Intron gunction 1 with exon 2). An open reading phase is proposed from an ATG initiation codon at position 1057 to produce a 85 amino acid polypeptide (PSA-EHT104prota / SEQ ID NO: 22). Identical to PSA-EHT101prota, PSA-EHT104prota / SEQ ID NO: 22 has in its amino-terminal part a signal peptide very homologous to that of PSA of wild type.
  • PSA-EHT104prota / SEQ ID NO: 22 can be cleaved from PSA-EHT104prota / SEQ ID NO: 22 to form a secreted polypeptide of 65-69 amino acids (PSA-EHT104protb / SEQ ID NO: 23).
  • a first subject of the invention relates to nucleic acids comprising the sequence of the variants of PSA and KLK-2 described above.
  • nucleic acids specific for the genetic alterations carried by the variants of PSA and KLK-2 described above.
  • Such nucleic acids may in particular be complementary to mutated regions, retained intronic domains or junctions newly created by deletions.
  • Another subject of the invention relates to a nucleic acid comprising all or part of a sequence derived from messenger RNAs (or cDNAs) from KLK2-EHT101 to KLK2-EHT103 and from PSA-EHT101 to PSA-EHT104 or any combination of these. variants and their uses for the implementation of a method for diagnosing, detecting or monitoring cancers, in particular prostate cancer and more particularly of the benign form of the latter, BPH.
  • Another object of the invention resides in any nucleic acid characterized in that it comprises a sequence chosen from: a) the sequences SEQ ID NOs: 1 to 7, b) a variant of the sequences SEQ ID NOs: 1 to 7 resulting degeneracy of the genetic code, c) the complementary strand of the sequences SEQ ID NOs: 1 to 7, and d) a specific fragment of the sequences a) to c).
  • the term “specific” fragment or part designates a fragment characteristic of the variants considered, typically a fragment carrying at least one genetic alteration characteristic of the variants considered.
  • Such specific fragments are therefore distinguished from the wild-type sequence by the presence of their own structural characteristic (eg, mutation, new junction, intron retention, sequence deletion, stop codon, new sequence resulting from a shift in the reading frame, etc.) resulting from an alteration event highlighted by the applicants in patients.
  • This specific structural characteristic is also designated by the expression "target sequence”.
  • Particular target sequences are in particular the sequences of the new junctions contained in the variants of the invention, which result from the deletion events described above.
  • target sequences are therefore in particular the sequences formed by the two codons bordering the junctions created by deletion of the residues 483-738 (SEQ ID NO: 1), 1032-1774 (SEQ ID NO: 2), 965-1774 (SEQ ID NO: 3), 636-918 (SEQ ID NO: 4), 636-963 (SEQ ID NO: 5), 1211-1958 (SEQ ID NO: 6) and 1144-1958 (SEQ ID NO: 7).
  • Specific fragments according to the invention therefore comprise at least one target sequence as defined above.
  • Preferred fragments comprise at least 5 consecutive nucleotides of the sequence considered, preferably at least 8, more preferably at least 12. Fragments can comprise up to 50, 75 or 100 nucleotides, or even more.
  • the nucleic acids may be DNAs, preferably chosen from cDNAs and gDNAs, or RNAs. They can be synthetic or semi-synthetic nucleic acids, PCR fragments, oligonucleotides, double- or single-stranded regions, etc. Nucleic acids can be produced by synthesis, recombinantly, by cloning, by genetic assembly (s), mutagenesis, etc., or a combination of these techniques. The nucleic acids can be used to produce a PSA or KLK-2 variant of the invention in vitro, ex vivo, in vivo or in an acellular transcription system.
  • They can also be used for the manufacture of antisense molecules, capable of reducing the translation of the corresponding mRNAs in a cell. They can also be used for the production of probes, in particular labeled, allowing, by hybridization reactions, to specifically demonstrate the presence of a mutated form of PSA or KLK-2 of the invention in a sample. They can also be used for the production of nucleic primers, useful for the amplification of a PSA or KLK-2 variant (or of a target sequence of such a variant) in a sample, in particular for the purpose of screening. or diagnostic.
  • another object of the invention relates to a nucleic probe characterized in that it allows the detection of a nucleic acid as defined above, typically by selective hybridization from a population of nucleic acids test.
  • the probe comprises the sequence of a nucleic acid as defined above or a (specific) part of the sequence of such a nucleic acid.
  • the specific part is preferably characteristic of a variant as described above, in particular a part containing an alteration associated with prostate cancer.
  • the probe typically comprises from 10 to 1000 nucleotides, preferably from 50 to 800, and is generally single-stranded.
  • a particular example of a probe is represented by an oligonucleotide specific and complementary to a region of at least one nucleic acid as defined above.
  • the oligonucleotide is typically single-stranded, and generally comprises from 10 to 100 bases, preferably from 10 to 60 bases, more preferably from 15 to 50. Specific examples of such oligonucleotides are given in Table 2.
  • the oligonucleotides and / or the nucleic acid probes according to the invention can be labeled, for example by means of radioactive, enzymatic, fluorescent, luminescent markers, etc.
  • a particular object of the invention resides in a single-stranded oligonucleotide, of a length between 10 and 60 bases, complementary and selective of a variant of PSA or KLK2 as defined above, in particular of a junction created by deletion, in the sequence coding for the PSA or KLK antigen, of residues 483- 738 (SEQ ID NO: 1), 1032-1774 (SEQ ID NO: 2), 965-1774 (SEQ ID NO: 3), 636-918 (SEQ ID NO: 4), 636-963 (SEQ ID NO: 5), 1211-1958 (SEQ ID NO: 6) and 1144-1958 (SEQ ID NO: 7).
  • the oligonucleotides are devoid of secondary structure, have a Tm of between 62 and 67 ° C, more preferably around 65 ° C, and have a length of between 20 and 40 bases.
  • the oligonucleotides or probes of the invention may comprise a particular functional group, allowing or facilitating their immobilization on a solid support or their flexibility, for example a terminal group (that is to say 5 ′ or 3 ′, more preferably 5 ') NH 2 -C n , n being an integer between 2 and 10, preferably between 3 and 8, for example 4, 5 or 6.
  • a terminal group that is to say 5 ′ or 3 ′, more preferably 5 '
  • NH 2 -C n n being an integer between 2 and 10, preferably between 3 and 8, for example 4, 5 or 6.
  • Another subject of the invention relates to a nucleic primer, allowing the (selective) amplification of a nucleic acid as defined above or of a (specific) part of such a nucleic acid.
  • the amplified part preferably comprises an alteration characteristic of one of the variants described above, in particular an alteration associated with prostate cancer.
  • a primer according to the invention is typically single-stranded, and advantageously composed of 3 to 50 bases, preferably from 3 to 40 and even more preferably from 3 to 35 bases.
  • a particular primer is complementary to at least one region of the PSA or KLK-2 gene, or of the corresponding RNA.
  • a preferred embodiment resides in a primer consisting of a single-stranded nucleic acid comprising from 3 to 50 nucleotides complementary to at least part of one of the sequences SEQ ID NOs: 1 to 7 or to their complementary strand. Examples of such nucleic primers are provided in the experimental section.
  • the invention also relates to a pair of primers comprising a sense sequence and a reverse sequence, characterized in that the primers of said pair hybridize with a region of a nucleic acid as defined above and allow the amplification. at least a portion of this nucleic acid.
  • Couples of specific primers according to the invention are provided in Table 1.
  • Other specific primers, capable of selectively amplifying a variant of the invention comprise a sequence as described in Table 2.
  • Another object of the present application relates to any vector comprising a nucleic acid as defined above.
  • They can be plasmids, cosmids, episomes, artificial chromosomes, viruses, phages, etc. Mention may be made of various commercial plasmids such as pUC, pcDNA, pBR, etc.
  • viral vectors mention may be made of retroviruses, adenoviruses, AAVs, herpesviruses, etc.
  • the cells can be prokaryotic or eukaryotic.
  • prokaryotic cells mention may in particular be made of bacteria such as E. coli.
  • eukaryotic cells mention may be made of yeast cells or cells of mammals, insects or plants. It can be primary cultures or lines. One can quote the cells COS, CHO, 3T3, HeLa, etc.
  • compositions comprising a nucleic acid as defined above immobilized on a support.
  • the invention relates in particular to compositions comprising a plurality of nucleic acids in admixture, in soluble form or immobilized to a support, the composition comprising at least one nucleic acid as defined above.
  • the support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material.
  • the nucleic acids are preferably immobilized at one end, under conditions leaving the molecule accessible for a hybridization reaction.
  • the nucleic acids can be precisely arranged on the support, and deposited in several copies.
  • Another subject of the invention relates to a (product comprising a) support on which one or more recombinant cells as defined above are immobilized or cultured.
  • the support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material.
  • the cells are, for example, distributed in wells of a microplate or immobilized in a gel or on a suitable support.
  • the invention also relates to the peptides and protein sequences encoded by all or part of the isoforms KLK2-EHT101 to KLK2-EHT103 and PSA-EHT101 to PSA-EHT104, in particular those described among the sequences SEQ ID NO: 8 to 23 as well as their uses for the implementation of a method of diagnosis, detection or monitoring of cancers, in particular prostate cancer and more particularly of the benign form of the latter, BPH.
  • a particular subject of the present application relates to a polypeptide comprising all or a specific part of a sequence chosen from SEQ ID NOs: 8 to 23.
  • Particular polypeptides are composed or comprise a sequence or part of a sequence created by the alteration of the gene or corresponding messenger.
  • the term “part” preferably designates at least 5 contiguous residues, preferably at least 8, more preferably at least 10, even more preferably at least 15.
  • the splicing alterations of the PSA or KLK-2 gene lead to the production of mutated proteins, comprising newly created sequences (target sequences). These can be new sequences (eg, offset translation, insertions), new junctions, etc.
  • Particular peptides of the invention correspond to or comprise these target sequences, coded by the sequences SEQ ID Nos: 1 to 7, for example peptides comprising all or part of the sequences SEQ ID Nos: 10, 13, 16, 18, 20, 21 or 23.
  • Another subject of the invention relates to a (product comprising a) support on which one or more polypeptides as defined above are immobilized.
  • the support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material.
  • the polypeptides are preferably immobilized at one end, under conditions leaving the molecule accessible for an interaction reaction with a specific ligand, such as an antibody.
  • the polypeptides can be precisely arranged on the support, and deposited in several copies.
  • the invention further relates to specific ligands, preferably peptide, in particular antibodies (polyclonal, monoclonal) and their fragments, specific to peptide regions characteristic of the proteins coded by KLK2-EHT101 and KLK2EHT103 and PSA-EHT101-104 (in particular retained intronic domains or specifically created junctions), and their uses for the detection, diagnosis or monitoring of cancers and in particular prostate cancer. In particular, it may involve diagnosing the BPH form and differentiating it from carcinoma of the prostate.
  • another object of the invention relates to any antibody capable of binding, preferably selectively, to a polypeptide as defined above.
  • the antibody can be polyclonal or monoclonal. It can also be fragments and derivatives of antibodies having substantially the same antigenic specificity, in particular antibody fragments (eg, Fab, Fab'2, CDRs), humanized, polyfunctional, single-stranded antibodies (ScFv) , etc.
  • Antibodies can be produced using conventional methods, including immunizing an animal and recovering its serum (polyclonal) or spleen cells (so as to produce hybridomas by fusion with appropriate cell lines).
  • polyclonal antibodies from various species are described in the prior art.
  • the antigen is combined with an adjuvant (eg, Freund's adjuvant) and administered to an animal, typically by subcutaneous injection. Repeated injections can be given. Blood samples are collected and immunoglobulin or serum are separated.
  • Conventional methods of producing monoclonal antibodies include immunizing an animal with an antigen, followed by the recovery of spleen cells which are then fused with immortalized cells, such as myeloma cells. The resulting hybridomas produce monoclonal antibodies and can be selected by limiting dilutions so as to isolate the individual clones.
  • the Fab or F (ab ') 2 fragments can be produced by digestion using a protease according to conventional techniques.
  • the invention also relates to a method for producing antibodies, comprising injecting a polypeptide as defined above or an immunogenic fragment thereof into a non-human animal and recovering the antibody or antibody producing cells.
  • the preferred antibodies are antibodies specific for the isoforms of PSA and KLK-2 described in the present application, and essentially non-specific for wild forms.
  • Particular antibodies are specific for a target sequence as defined above, for example peptides or epitopes comprising all or part of the sequences SEQ ID Nos: 10, 13, 16, 18, 20, 21 or 23.
  • the invention relates to hybridomas producing the monoclonal antibodies described above and their use for producing said antibodies.
  • the antibodies can be coupled to heterologous fragments such as toxins, markers, drugs or any other therapeutic agent, covalently or not, either directly or through coupling agents.
  • the markers can be chosen from radio-markers, enzymes, fluorescent agents, magnetic particles, etc.
  • the antibodies of the invention can be used as screening agents or to detect or quantify the presence or amount of PSA or KLK-2 isoforms in samples collected from a subject, typically a biological fluid from 'a mammal, for example from a human.
  • Another subject of the invention relates to a (product comprising a) support on which one or more antibodies (or fragments or derivatives) as defined above are immobilized.
  • the support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material.
  • the antibodies are preferably immobilized at one end, under conditions leaving the molecule accessible for an interaction reaction with a specific antigen.
  • the antibodies can be precisely arranged on the support, and deposited in several copies. Detection / diagnostic methods
  • the present application also describes new methods for detecting a pathology or a predisposition to a pathology in a subject, comprising determining the presence, in a sample of said subject, of a nucleic acid, or an alteration genetic or a protein or polypeptide as defined above.
  • the determination can be carried out by various techniques, such as sequencing, hybridization and / or selective amplification.
  • Methods that can be used to determine the presence of proteins are based, for example, on immunoenzymatic reactions, such as ELISA, RIA, EIA, etc.
  • Techniques that can be used to determine the presence of altered genes or RNA are, for example, PCR, RT-PCR, ligation chain reaction (LCR), PCE or TMA (“Transcriptional Mediated Amplification”) technique, gel migration, electrophoresis, in particular DGGE (“denaturing gel gradient electrophoresis”), etc.
  • this preferably uses a primer or a pair of primers as defined above.
  • a particular object of the invention relates to the use of complementary and specific nucleic acids of fragments of the genes or messengers of KLK2-EHT101-103 and of PSA-EHT101-104 (eg, retained intronic domains, specifically created junctions, mutations particular, etc.) for the detection of cancers, in particular prostate cancer, and more particularly of its benign form, BPH.
  • This detection may in particular be carried out by means of DNA chips or by the implementation of a PCR from biological fluids such as blood (notably serum), urine, seminal fluid, etc.
  • the invention also resides in the development and use of immunoassays containing one or more antibodies as described above or fragments thereof.
  • a particular method comprises bringing a sample from a subject into contact with a probe as defined above, and demonstrating hybridization.
  • Another particular method comprises bringing a sample from a subject into contact with a primer or a pair of primers as defined above, and demonstrating an amplification product.
  • Another particular method comprises bringing a sample from a subject into contact with an antibody as defined above, and demonstrating an antigen-antibody complex.
  • the method of the invention comprises the determination, in parallel, of the presence of several genetic variants or alterations as described above in a sample of a patient.
  • the methods according to the invention can be implemented using various biological samples, in particular biological fluids (eg, blood, plasma, urine, serum, saliva, etc.), biopsies of tissues or cell cultures, by example and, more generally, from any sample likely to contain nucleic acids or proteins (or polypeptides).
  • the biological sample can be treated before the implementation of the method, to facilitate or allow the accessibility of the polypeptides or nucleic acids which it contains.
  • the sample can also be purified, centrifuged, fixed, etc., possibly frozen or stored before use.
  • the invention relates to a method for detecting the presence of an altered form of KLK-2 or KLK-3 in a subject, comprising bringing into contact, in vitro or ex vivo, of a sample of said subject with a specific probe, primer or ligand as defined above and the determination of the formation of a hybrid, of an amplification product or of a complex, respectively, said formation being indicative the presence of an altered form.
  • kits which can be used for implementing a method as defined above comprising i. a pair of primers or a probe (including an oligonucleotide) or an antibody as defined above, and ii. the reagents necessary for amplification or for a hybridization or immunological reaction.
  • the invention also resides in the development of a method making it possible to detect and / or measure specific partners of one or more of these variants by adding one or more of these variants or their fragments in biological fluids to be tested, such as blood (serum in particular), urine or seminal fluid.
  • KLK2 and KLK3 have been identified and isolated from pathological subjects and therefore represent therapeutic targets which are particularly advantageous for the treatment of cancers and in particular prostate cancer.
  • a particular object of the invention resides in methods of selection, identification, characterization, optimization or production of active compounds, comprising a step of determining the capacity of a test compound to modulate the expression or activity of a polypeptide as defined above.
  • the compounds are more particularly selected on the basis of their capacity to modulate the synthesis of a polypeptide as defined above (that is to say in particular the production or the maturation of the corresponding RNAs, or their translation) or the activity of such a polypeptide (that is to say in particular their maturation, transport, or their interaction with intra- or extracellular targets).
  • the method comprises contacting in vitro or ex vivo a test compound with a polypeptide as defined above or a nucleic acid encoding such a polypeptide (eg, gene, cDNA, RNA), and selecting compounds that bind to said polypeptide or nucleic acid.
  • a polypeptide as defined above or a nucleic acid encoding such a polypeptide (eg, gene, cDNA, RNA)
  • selecting compounds that bind to said polypeptide or nucleic acid eg, gene, cDNA, RNA
  • the binding of the polypeptide to the gene or to the corresponding RNA can be measured by various techniques, such as the displacement of a labeled ligand, migration on gel, electrophoresis, etc. It can be carried out in vitro, for example using the polypeptide or the nucleic acid immobilized on a support.
  • the method comprises contacting in vitro or ex vivo a test compound with a cell expressing a polypeptide as defined above, and the selection or identification of compounds modulating the expression or the activity of said polypeptide.
  • the modulation of expression can be determined by assaying the RNAs or proteins, or by means of a reporter system.
  • the cells used can be any compatible cell, in particular eukaryotic or prokaryotic cells as mentioned above.
  • Such recombinant cells can be prepared by the introduction of a recombinant nucleic acid expressing the polypeptide, or of a vector comprising the latter.
  • Such recombinant cells constitute particular objects of the invention.
  • the method can be implemented to select or identify an activator or an inhibitor of the expression or of the activity of the specific antigen of PSA or of KLK-2.
  • the selection methods can be carried out in different formats, such as for example multi-well plates, by testing multiple candidate compounds in parallel.
  • the compound is an antisense nucleic acid capable of inhibiting the expression of the variants described.
  • the antisense nucleic acid can comprise all or part of specific sequences of the variants described.
  • the antisense can in particular comprise a region complementary to the form of splicing identified (e.g., of a target sequence), and inhibit (or reduce) its translation into protein.
  • the compound is a chemical compound, of natural or synthetic origin, in particular an organic or inorganic molecule, of plant, bacterial, viral, animal, eukaryotic, synthetic or semi-synthetic origin, capable of modulating the expression or activity of one or more of the variants described above.
  • Specific compounds are preferred, that is to say capable of modulating the expression or the activity of the variants, without significantly affecting the expression or the activity of the wild forms.
  • the compounds thus identified can be used for the preparation of a composition intended for the treatment of prostate cancer.
  • Another object of the invention resides in the use of a compound capable of modulating, ie stimulating, inhibiting or reducing, the expression of one or more variants as described above, for the preparation of a composition intended for the treatment of cancers and in particular prostate cancer.
  • treatment designates preventive, curative or palliative treatment, as well as the management of patients (reduction of suffering, improvement of lifespan, slowing down the progression of the disease ), etc.
  • the treatment can also be carried out in combination with other active agents.
  • Another subject of the invention relates to methods of selection, identification, or characterization of active compounds which can be used in the context of the preparation of compositions intended for the treatment of cancerous pathologies, comprising the bringing into contact of one or more test compounds with cellular extracts expressing the proteins described in the present invention, or with said purified proteins.
  • the invention also relates to a method for producing a medicament for the treatment of cancers, in particular prostate cancer, comprising (i) the selection of active compounds according to the above methods and (ii) the conditioning of said compound or a functional analog thereof in the presence of a pharmaceutically acceptable carrier.
  • the functional analog is typically a compound derived from the active compound identified, by chemical modification, in particular with the aim of improving its activity or its pharmacokinetics, or with the aim of reducing its toxicity.
  • the functional analog may be a "pro-drug" of the identified compound.
  • Techniques for preparing functional analogs are well known to those skilled in the art, for example molecular modeling, coupling of NO groups, etc. The method can in this regard comprise an intermediate step of synthesis of the selected compound or of the functional analog thereof.
  • the pharmaceutically acceptable vehicle or excipient can be chosen from buffer solutions, solvents, binders, stabilizers, emulsifiers, etc.
  • Buffer or diluent solutions include dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, starch, powdered sugar and hydroxy propyl methyl cellulose (HPMC) (for delayed release).
  • Binders are for example starch, gelatin and filling solutes such as sucrose, glucose, dextrose, lactose, etc.
  • Natural or synthetic gums can also be used, such as alginate, carboxymethylcellulose, methylcellulose, polyvinyl pyrrolidone, etc.
  • excipients are for example cellulose and magnesium stearate.
  • Stabilizing agents can be incorporated into the formulations, such as for example polysaccharides (acacia, agar, alginic acid, guar gum and tragacanth, chitin or its derivatives and cellulose ethers.
  • Solvents or solutes are for example the Ringer solution, l , distilled water, phosphate buffers, phosphate salt solutions, and other conventional fluids.
  • Another subject of the invention relates to the use of cytotoxic ligands specific for one or more variants as described above located on the surface of cancer cells and in particular prostate cancer cells.
  • Poly A + RNAs are prepared according to techniques known to those skilled in the art. It may in particular be a treatment using chaotropic agents such as guanidium thiocyanate followed by extraction of the total RNAs using solvents (phenol, chloroform for example). Such methods are well known to those skilled in the art (see Maniatis et al., Chomczynsli et al., Anal. Biochem. 162 (1987) 156), and can be easily implemented using commercially available kits . From these total RNAs, the poly A + RNAs are prepared according to conventional methods known to those skilled in the art and described in commercial kits.
  • RNAs serve as a template for reverse transcription reactions using reverse transcriptases.
  • reverse transcriptases lacking RNase H activity are used. They make it possible to obtain complementary DNA strands of sizes larger than those obtained using conventional reverse transcriptases. Such preparations of reverse transcriptases without RNase H activity are commercially available.
  • RNA sequences not paired with complementary DNA are released from these heteroduplexes under the action of RNase H, this enzyme degrading the paired RNA sequences.
  • RNase H this enzyme degrading the paired RNA sequences.
  • These unpaired sequences represent the qualitative differences which exist between RNAs which are otherwise homologous to one another. These qualitative differences can be located anywhere on the RNA sequence, either in 5 ′ or 3 ′ as within the sequence and in particular within the coding sequence. Depending on their location, these sequences can be not only modifications of splicing but also the consequences of translocations or deletions.
  • the RNA sequences representing the qualitative differences are then cloned according to techniques known to those skilled in the art and in particular those described in the patent relating to the DATAS technology.
  • sequences are grouped together within cDNA libraries which constitute qualitative differential banks.
  • One of these banks contains exons and introns specific to the healthy situation; the other banks contain the splicing events characteristic of the pathological conditions.
  • the fragments derived from the human genes of KLK2 and KLK3 come from these banks. Four tumor samples were mixed to form a tumor pool.
  • RNA pool was treated with Dnase using the company's “DNA free” kit
  • RNAs are then reverse transcribed using the reverse transcriptase from the “High capacity cDNA Archive” kit from the company Aplied Biosystems (cat.
  • the cDNA thus produced serves as a template for PCR reactions in order to specifically amplify different regions of messenger RNAs derived from human kallikrein-2 and kallikrein-3 according to the following protocol:
  • Taq polymerase 0.2 ⁇ L
  • oligonucleotides used as PCR primers are as follows
  • KLK2 5 ' AGGCTTTATAGGGCTCCTCA
  • KLK2 intron 1 stop GACTCAGGGTGGGAGGCAG 450 KLK2 intron 1 ATG: GACCCTATCACTGGGCTCT 422: KLK2 exon 2 revl: TGTGCCCATCCATGACTGTA
  • PSA 5 ' AGGTTTTATAGGGCTCCTGG
  • PSA intron 1 stop CATTGCCAGACTGAGGGACC
  • PSA intron 1 ATG GGACCGTATCACTGGTCCAT 416: PSA exon2 revl: GCCCTGCCACGAGAGGC
  • the amplified products are then cloned in the “Topo” system from the company Invitrogen (cat. No. K4600) according to the protocol provided.
  • the ligation products are transformed into competent “Top 10” cells.
  • the colonies are identified on agar / LB medium supplemented with ampicillin.
  • the cDNAs present in these colonies are amplified individually by PCR amplification using primers Sp6 and T7 according to the following protocol:
  • Buffer 10x 4 ⁇ L Taq polymerase 0.2 ⁇ L
  • the amplification products are then purified by P100 to be sequenced using the “Big Dye Terminator” kit from the company Applied Biosystems according to the protocol provided by this supplier. Sequence reactions are analyzed using an Applied Biosystems 3100 sequencer. Table 1 represents the various cDNAs as well as the pairs of priming oligonucleotides used for obtaining them and allowing their amplification in a sample. The complete sequence of variants is provided in the attached sequence list.
  • PSA and KLK2 variants described in this invention were established and validated using a micro-array comprising oligonucleotides capable of specifically hybridizing with these variants.
  • the principle and validation of this approach has already been described for other variants of PSA and KLK2 in application PCT / FR03 / 00833, belonging to the applicant.
  • the PSA and KLK2 splicing variants come from events of different natures, but all presenting deletions of well defined nucleic sequences which thus create new very specific junctions. Discriminating oligonucleotides are drawn to be complementary to (ie, placed on) one or the other of these new junctions.
  • oligonucleotides Given that the probes are shorter than the PCR product probes conventionally used, it is preferable to verify that these probes do not hybridize in an aspecific manner other genes than the one for which they were designed. In addition, it is highly desirable to ensure that the oligonucleotides do not have secondary structures which could interfere with their hybridization capacity.
  • the oligonucleotides are moreover modified in 5 ′ by an NH 2 -C6 group promoting their flexibility and making it possible to establish a covalent bond with the polymer constituting the coating of the glass slide.
  • junction oligonucleotides are ideally centered on the junction, but we have also considered the possibilities of oligonucleotides shifted on the junction.
  • oligonucleotide design software we have selected the Primer Finder software.
  • -% GC 40% to 60% for 24 seas and 30 seas, 30% to 60% for 40 seas.
  • oligonucleotides were synthesized and taken up at a concentration of 25 ⁇ M in a 150 mM sodium phosphate buffer. The oligonucleotides were then deposited in quadruplet on glass slides (Codelink, Amersham), then these slides were incubated in a saturated NaCl chamber for
  • the targets are hybridized in the 5X SSC buffer, 0.1% SDS, 0.1 mg / ml DNA of salmon sperm, at a temperature of 55 ° C. for 16 h. They are then washed by increasing the stringency of the baths:
  • the first step is to transfer mRNA in the presence of oligodT using Superscript II.
  • RnaseH will degrade the RNA used as a template and leave primers which will be used by DNA polymerase I for the synthesis of the second strand of cDNA.
  • the synthesized fragments are assembled by DNA ligase.
  • T7 DNA polymerase the T7 DNA polymerase, which will amplify the strand corresponding to the messenger sequence and synthesize cRNA molecules which will hybridize to the probes. of complementary sequence (sense of mRNA).
  • the oligonucleotide KLK2-482-739 corresponding to KLK2-EHT101 produces significant fluorescence signals among the four patients, superior to the oligonucleotide KLK2-482-483 corresponding to the wild type KLK2;
  • the oligonucleotide KLK2-1031 -1775 corresponding to KLK2-EHT102 produces significant fluorescence signals among two of the four patients;
  • the oligonucleotide PSA-635-919 corresponding to PSA-EHT101 produces significant fluorescence signals among the four patients;
  • the oligonucleotide PSA-635-964 corresponding to PSA-EHT102 produces significant fluorescence signals among the four patients. These last two oligonucleotides produce signals superior to those produced by the oligonucleotides PSA-635-636 and PSA-600-630 both corresponding to the wild form of PSA.
  • the oligonucleotide PSA-1210-1959 corresponding to PSA-EHT103 produces significant fluorescence signals among one of the four patients.
  • Fluorescence signals obtained from RNAs from benign and tumor prostate tissue were obtained from RNAs from benign and tumor prostate tissue. Columns B, E, H, K: Signals from healthy tissue. Columns C, F, I, L: Signals from tumor tissue. Columns D, G, J, M: Ratio of benign / tumor signals for the four patients.

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Abstract

The invention concerns the field of biology, genetics and medicine. It concerns in particular novel methods for detecting, characterizing and/or treating cancer pathologies, in particular prostatic cancer. The invention also concerns methods for identifying or screening compounds active in said pathologies. The invention further concerns compounds, genes, cells, plasmids or compositions useful for implementing said methods. The invention concerns in particular the role of human variants of kallikrein-2 and kallikrein-3, also referred to as PSA, in said pathologies and their uses as therapeutic, diagnostic or experimental targets.

Description

Variants de kallikrein-2 et kallikrein-3 humaines et leurs utilisations Variants of human kallikrein-2 and kallikrein-3 and their uses
La présente invention concerne le domaine de la biologie, de la génétique et de la médecine. Elle concerne notamment de nouvelles séquences nucléotidiques associées à des événements d'épissage alternatif des gènes correspondant à l'antigène PSA (antigène spécifique de la prostate ou KLK3) et à la kallikréine- 2 (KLK2). L'invention concerne également des méthodes pour la détection de la présence ou pour la détermination du niveau d'expression de ces acides nucléiques ou des protéines correspondantes dans des échantillons biologiques, ainsi que des méthodes de sélection de molécules capables de moduler leur activité ou leur expression.The present invention relates to the field of biology, genetics and medicine. It relates in particular to new nucleotide sequences associated with alternative splicing events of the genes corresponding to the PSA antigen (prostate specific antigen or KLK3) and to kallikrein-2 (KLK2). The invention also relates to methods for detecting the presence or for determining the level of expression of these nucleic acids or the corresponding proteins in biological samples, as well as methods of selecting molecules capable of modulating their activity or their expression.
L'invention est particulièrement adaptée au dépistage ou au suivi de cancers, notamment de la prostate et notamment dans la différentiation entre le cancer de la prostate et l'hyperplasie bénigne de la prostate (BPH), ainsi qu'au développement de nouvelles approches thérapeutiques de ces maladies.The invention is particularly suitable for screening or monitoring cancers, especially of the prostate and in particular in the differentiation between prostate cancer and benign prostatic hyperplasia (BPH), as well as to the development of new therapeutic approaches. of these diseases.
Les kallikréines correspondent à un groupe de protéines dont l'activité protéasique permet la modification post-traductionnelle de précurseurs protéiques vers des formes biologiquement actives. Certains membres de cette famille, la kallikréine-3, aussi connue sous la dénomination de PSA (« prostate- specific antigen » ou antigène spécifique de la prostate), et plus récemment, la kallikréine-2 sont considérés comme les meilleurs marqueurs disponibles utilisables dans la détection, le diagnostique et le suivi du cancer de la prostate. L'utilisation de tests mesurant la quantité de PSA dans le sang a permis de diagnostiquer un nombre croissant de patients atteints de cancer de la prostate (Pca). Cependant, comme la PSA est également produite par les cellules épithéliales prostatiques non cancéreuses, il est souvent difficile de différencier les patients atteints de cancers de la prostate de ceux présentant un symptôme bénin hyperplasique (BPH). Dans le sérum, la PSA existe sous forme libre, non complexée et sous forme complexée, notamment à l'alpha-antichymotrypsine. La mesure de ces différentes formes et de leurs rapports permet d'améliorer le facteur différentiateur entre Pca et BPH.Kallikreins correspond to a group of proteins whose protease activity allows the post-translational modification of protein precursors to biologically active forms. Some members of this family, kallikrein-3, also known under the name of PSA (“prostate-specific antigen”), and more recently, kallikrein-2 are considered to be the best available markers usable in detection, diagnosis and monitoring of prostate cancer. The use of tests measuring the amount of PSA in the blood has made it possible to diagnose an increasing number of patients with prostate cancer (Pca). However, since PSA is also produced by non-cancerous prostate epithelial cells, it is often difficult to differentiate patients with prostate cancer from those with benign hyperplastic symptom (BPH). In serum, PSA exists in free, uncomplexed form and in complexed form, in particular with alpha-antichymotrypsin. Measuring these different forms and their relationships improves the differentiating factor between Pca and BPH.
L'épissage alternatif constitue un mécanisme de régulation de l'expression des gènes permettant de générer de la diversité fonctionnelle à partir d'une information génétique limitée. Ce mécanisme, fortement régulé, peut subir des altérations au cours du développement de pathologies humaines. Ainsi, la dérégulation de la machinerie d'épissage dans les cancers peut permettre l'expression d'isoformes ou de variants spécifiquement exprimés dans certaines tumeurs humaines. Ces isoformes pourront avoir un rôle fonctionnel déterminant dans le développement ou le maintien de l'état pathologique. L'expression spécifique de telles isoformes constitue un événement de choix pour une approche rationnelle et ciblée de développement de médicaments et/ou de méthodes de diagnostique. Une technologie de profilage de l'expression génétique (DATAS) a été récemment développée pour identifier de façon systématique les gènes et, à l'intérieur de ces gènes, les domaines susceptibles d'être altérés par épissage alternatif (WO99/46403).The alternative splicing constitutes a mechanism of regulation of the expression of the genes making it possible to generate functional diversity from limited genetic information. This highly regulated mechanism can undergo alterations during the development of human pathologies. Thus, the deregulation of the splicing machinery in cancers can allow the expression of isoforms or variants specifically expressed in certain human tumors. These isoforms may have a determining functional role in the development or maintenance of the pathological state. The specific expression of such isoforms constitutes an event of choice for a rational and targeted approach to the development of drugs and / or diagnostic methods. A gene expression profiling technology (DATAS) has been recently developed to systematically identify genes and, within these genes, the domains likely to be altered by alternative splicing (WO99 / 46403).
La présente invention décrit à présent de nouveaux événements génétiques liés à des epissages alternatifs des gènes PSA et KLK2 dans des tissus prostatiques. La présente invention découle notamment de la construction d'un répertoire des altérations d'épissage associées à des tissus tumoraux de la prostate, et de l'identification d'altérations structurales dans les gènes PSA et KLK2 ou dans les ARNm correspondants. La présente invention fournit ainsi de nouvelles approches thérapeutiques et diagnostiques des cancers, notamment du cancer de la prostate.The present invention now describes novel genetic events associated with alternative splicing of the PSA and KLK2 genes in prostate tissue. The present invention results in particular from the construction of a directory of splicing alterations associated with prostate tumor tissues, and from the identification of structural alterations in the PSA and KLK2 genes or in the corresponding mRNAs. The present invention thus provides new therapeutic and diagnostic approaches for cancers, in particular prostate cancer.
Plus particulièrement, une analyse qualitative différentielle a été effectuée à partir d'ARN extraits d'échantillons de tissus prostatiques provenant de zones tumorale et non tumorale de patients atteints de carcinomes de la prostate. Cette analyse a été effectuée par criblage différentiel qualitatif grâce à la mise en œuvre de la technique DATAS (décrite dans la demande n° WO99/46403) qui présente des avantages inégalés. L'application de la technologie DATAS sur des ARNs issus de tissus prostatiques tumoraux et non-tumoraux a permis d'isoler divers fragments d'ADNc dérivés des ARNm de la kalikreine 2 et de la kalikreine 3 (PSA) humaines. Ces résultats ont ensuite permis d'identifier un certain nombre d'ADNc révélateurs d'événements liés à des epissages alternatifs.More particularly, a differential qualitative analysis was carried out using RNA extracted from samples of prostate tissue originating from tumor and non-tumor areas of patients with prostate carcinomas. This analysis was carried out by qualitative differential screening thanks to the implementation of the DATAS technique (described in application n ° WO99 / 46403) which has unmatched benefits. The application of DATAS technology on RNAs from tumor and non-tumor prostate tissue has made it possible to isolate various cDNA fragments derived from human kalikrein 2 and kalikrein 3 (PSA) mRNAs. These results then made it possible to identify a certain number of cDNAs revealing events linked to alternative splicing.
La présente invention décrit donc des événements moléculaires originaux qui peuvent aboutir à l'expression spécifique d'isoformes ou de variants de KLK3 (PSA) et de KLK2 dans les tissus prostatiques et, plus spécifiquement, dans les tissus cancéreux ou les tissus associés à une hyperplasie bénigne (BPH). La présente invention fournit des données moléculaires qui justifient l'utilisation d'un ou de plusieurs de ces variants comme cibles thérapeutiques et diagnostiques nouvelles et utilisables avantageusement dans le diagnostic et le traitement des cancers et notamment du cancer de la prostate.The present invention therefore describes original molecular events which can lead to the specific expression of isoforms or variants of KLK3 (PSA) and KLK2 in prostate tissues and, more specifically, in cancerous tissues or tissues associated with a benign hyperplasia (BPH). The present invention provides molecular data which justify the use of one or more of these variants as new therapeutic and diagnostic targets which can advantageously be used in the diagnosis and treatment of cancers and in particular prostate cancer.
Un premier aspect de la présente demande concerne donc des variants de PSA et KLK2 humains, notamment des variants d'épissage. L'invention concerne les acides nucléiques correspondant à ces variants ou aux altérations spécifiques qu'ils présentent, ainsi que les protéines (ou polypeptides ou domaines protéiques) codés.A first aspect of the present application therefore relates to variants of human PSA and KLK2, in particular splicing variants. The invention relates to the nucleic acids corresponding to these variants or to the specific alterations which they exhibit, as well as the proteins (or polypeptides or protein domains) encoded.
Un autre aspect de la présente demande concerne des méthodes et outils pour détecter la présence de ces variants ou altérations dans des échantillons biologiques (sang, plasma, urine, sérum, salive, biopsies ou cultures cellulaires, etc.), ou pour déterminer leur(s) quantité(s) ou proportion(s) respective(s). De tels outils comprennent notamment des sondes ou amorces nucléiques, des anticorps ou autres ligands spécifiques, des kits, supports, puces, etc. Les méthodes de détection peuvent inclure les méthodes d'hybridation, de PCR, de chromatographie, d'immunologie, etc. Ces méthodes sont particulièrement adaptées à la détection, la caractérisation, le suivi de la progression ou de l'efficacité d'un traitement de cancers, notamment du cancer de la prostate, ou à la détermination d'une prédisposition.Another aspect of the present application relates to methods and tools for detecting the presence of these variants or alterations in biological samples (blood, plasma, urine, serum, saliva, biopsies or cell cultures, etc.), or for determining their ( s) respective quantity (s) or proportion (s). Such tools include in particular nucleic probes or primers, antibodies or other specific ligands, kits, supports, chips, etc. Detection methods can include hybridization, PCR, chromatography, immunology, etc. These methods are particularly suitable for the detection, characterization, monitoring of progression or the effectiveness of a treatment for cancers, in particular prostate cancer, or in determining a predisposition.
Un autre aspect de la présente demande concerne des outils et méthodes pour la production de composés actifs sur les variants décrits, c'est-à-dire capables de moduler leur expression ou leur activité. Ces outils et méthodes incluent notamment des acides nucléiques, des vecteurs, des cellules recombinantes (ou préparations dérivées de telles cellules), des tests de liaison, etc. L'invention vise également les composés ainsi identifiés ou produits, des compositions pharmaceutiques les contenant, ainsi que leurs utilisations thérapeutiques.Another aspect of the present application relates to tools and methods for the production of compounds active on the variants described, that is to say capable of modulating their expression or their activity. These tools and methods include in particular nucleic acids, vectors, recombinant cells (or preparations derived from such cells), binding tests, etc. The invention also relates to the compounds thus identified or produced, to pharmaceutical compositions containing them, as well as to their therapeutic uses.
La présente invention est ainsi applicable au diagnostic et au développement de stratégies thérapeutiques de cancers, notamment de la prostate.The present invention is thus applicable to the diagnosis and development of therapeutic strategies for cancers, in particular of the prostate.
Variants de KLK2 et KLK3Variants of KLK2 and KLK3
Un premier aspect de la présente demande concerne donc des variants de KLK- 2 et KLK-3 (PSA), ou des altérations génétiques particulières affectant ces gènes (ou les ARN ou protéines correspondants). Un objet plus particulier de l'invention concerne des acides nucléiques correspondant à ces variants de PSA et KLK2 humains ou aux altérations spécifiques qu'ils présentent, ainsi que les protéines (ou polypeptides ou domaines protéiques) codées.A first aspect of the present application therefore relates to variants of KLK-2 and KLK-3 (PSA), or particular genetic alterations affecting these genes (or the corresponding RNAs or proteins). A more particular subject of the invention relates to nucleic acids corresponding to these variants of human PSA and KLK2 or to the specific alterations which they exhibit, as well as the proteins (or polypeptides or protein domains) encoded.
Un certain nombre d'isoformes des gènes KLK2 et KLK3 ont été décrites dans l'art antérieur. Ces isoformes correspondent toutes à la rétention totale d'un intron (David et.al (2002), Riegman et.al (1988), Riegman et.al (1991 ), Liu et.al (1999), Heuze et.al (1999), Tanaka et.al (2000)). La présente demande décrit maintenant l'existence de formes différentes des gènes PSA et KLK2, et leur corrélation à des situations pathologiques.A number of isoforms of the KLK2 and KLK3 genes have been described in the prior art. These isoforms all correspond to the total retention of an intron (David et.al (2002), Riegman et.al (1988), Riegman et.al (1991), Liu et.al (1999), Heuze et.al ( 1999), Tanaka et.al (2000)). The present application now describes the existence of different forms of the PSA and KLK2 genes, and their correlation to pathological situations.
Ces isoformes ont été identifiées à partir d'échantillons tumoraux. La description des ADNc et des protéines/polypeptides codés par ces ADNc est indiquée ci- dessous. Les séquences complètes sont fournies dans la Liste de Séquences annexée à la présente demande.These isoforms have been identified from tumor samples. The description of the cDNAs and of the proteins / polypeptides encoded by these cDNAs is given below. below. The complete sequences are provided in the Sequence List annexed to this request.
Variants de KLK2 : La numérotation des nucléotides se réfère au numéro d'accession de Genbank M18157 sauf autre précision. La protéine de référence est la KLK2 munie de son peptide signal.Variants of KLK2: The nucleotide numbering refers to the Genbank accession number M18157 unless otherwise specified. The reference protein is KLK2 provided with its signal peptide.
KLK2-EHT101 (SEQ ID NO : 1) :KLK2-EHT101 (SEQ ID NO: 1):
Cette isoforme présente une délétion de l'exon 1 associée à une rétention d'une partie de l'intron 1 (délétion des nucléotides 483 à 738). Cette isoforme KLK2- EHT101 délète le codon d'initiation utilisé par KLK2 dans l'exon 1. Une phase ouverte de lecture est proposée à partir de deux codons d'initiation ATG en phase aux positions 845 et 878 pour produire deux polypeptides de 77 et 66 acides aminés respectivement (KLK2-EHT101prota / SEQ ID NO : 8 et KLK2- EHTIOlprotb / SEQ ID NO : 9). KLK2-EHT101protb / SEQ ID NO : 9 présente en sa partie amino-terminale un peptide signal très homologue à celui de KLK2 de type sauvage. Selon le site de clivage utilisé, entre 16 et 20 acides aminés peuvent être clivés de KLK2-EHT101 protb / SEQ ID NO : 9 pour former un polypeptide sécrété de 46-50 acides aminés (KLK2-EHT101protc / SEQ ID NO : 10).This isoform has a deletion of exon 1 associated with a retention of part of intron 1 (deletion of nucleotides 483 to 738). This KLK2-EHT101 isoform deletes the initiation codon used by KLK2 in exon 1. An open reading phase is proposed from two ATG initiation codons in phase at positions 845 and 878 to produce two polypeptides of 77 and 66 amino acids respectively (KLK2-EHT101prota / SEQ ID NO: 8 and KLK2-EHTIOlprotb / SEQ ID NO: 9). KLK2-EHT101protb / SEQ ID NO: 9 presents in its amino-terminal part a signal peptide very homologous to that of KLK2 of wild type. Depending on the cleavage site used, between 16 and 20 amino acids can be cleaved from KLK2-EHT101 protb / SEQ ID NO: 9 to form a secreted polypeptide of 46-50 amino acids (KLK2-EHT101protc / SEQ ID NO: 10).
KLK2-EHT102 (SEQ ID NO : 2) :KLK2-EHT102 (SEQ ID NO: 2):
Cette isoforme présente une délétion dans l'intron 1 entre les positions 1032 etThis isoform has a deletion in intron 1 between positions 1032 and
1774 Gonction intron 1 avec exon 2). Cette isoforme KLK2-EHT102 met en phase un peptide de 52 acides aminés avec la phase codante de KLK2 sécrétée (à partir de l'exon 2), codant ainsi pour une protéine de 297 acides aminés (KLK2-EHT102prota / SEQ ID NO : 11 ). La partie amino-terminale de KLK2- EHT102prota / SEQ ID NO : 11 possède le même peptide signal que KLK2- EHT101protb / SEQ ID NO : 9 . Selon le site de clivage utilisé, entre 16 et 20 acides aminés peuvent être clivés de KLK2-EHT102prota / SEQ ID NO : 11 pour former le polypetide sécrété KLK2-EHT102protb / SEQ ID NO : 12. Selon le site de clivage utilisé, les 32-36 acides aminés N-terminaux de KLK2-EHT102protb (KLK2-EHT102protc / SEQ ID NO : 13) sont nouveaux et peuvent présenter un ou plusieurs épitopes originaux.1774 Gunction intron 1 with exon 2). This KLK2-EHT102 isoform puts a peptide of 52 amino acids into phase with the coding phase of secreted KLK2 (from exon 2), thus coding for a protein of 297 amino acids (KLK2-EHT102prota / SEQ ID NO: 11 ). The amino-terminal part of KLK2- EHT102prota / SEQ ID NO: 11 has the same signal peptide as KLK2- EHT101protb / SEQ ID NO: 9. Depending on the cleavage site used, between 16 and 20 amino acids can be cleaved from KLK2-EHT102prota / SEQ ID NO: 11 to form the secreted polypetide KLK2-EHT102protb / SEQ ID NO: 12. Depending on the cleavage site used, the 32 -36 N-terminal amino acids of KLK2-EHT102protb (KLK2-EHT102protc / SEQ ID NO: 13) are new and may have one or more original epitopes.
KLK2-EHT103 (SEQ ID NO : 3) : Cette isoforme présente une délétion dans l'intron 1 entre les positions 965 et 1774 (jonction intron 1 avec exon 2). Une phase ouverte de lecture est proposée à partir de deux codons d'initiation ATG en phase aux positions 845 et 878 pour produire deux polypeptides de 91 et 80 acides aminés respectivement (KLK2- EHT103prota / SEQ ID NO :14 et KLK2-EHT103protb / SEQ ID NO :15). Identique à KLK2-EHT101protb, KLK2-EHT103protb / SEQ ID NO :15 présente en sa partie amino-terminale un peptide signal très homologue à celui de KLK2 de type sauvage. Selon le site de clivage utilisé, entre 16 et 20 acides aminés peuvent être clivés de KLK2-EHT103protb / SEQ ID NO :15 pour former un polypeptide sécrété de 60-64 acides aminés (KLK2-EHT103protc / SEQ ID NO :16 ).KLK2-EHT103 (SEQ ID NO: 3): This isoform has a deletion in intron 1 between positions 965 and 1774 (junction intron 1 with exon 2). An open reading phase is proposed from two ATG initiation codons in phase at positions 845 and 878 to produce two polypeptides of 91 and 80 amino acids respectively (KLK2- EHT103prota / SEQ ID NO: 14 and KLK2-EHT103protb / SEQ ID NO: 15). Identical to KLK2-EHT101protb, KLK2-EHT103protb / SEQ ID NO: 15 presents in its amino-terminal part a signal peptide very homologous to that of KLK2 of wild type. Depending on the cleavage site used, between 16 and 20 amino acids can be cleaved from KLK2-EHT103protb / SEQ ID NO: 15 to form a secreted polypeptide of 60-64 amino acids (KLK2-EHT103protc / SEQ ID NO: 16).
Variants de PSA (ou KLK3) : La numérotation des nucléotides se réfère au numéro d'accession de Genbank M27274 sauf autre précision. La protéine de référence est la PSA munie de son peptide signal.Variants of PSA (or KLK3): The nucleotide numbering refers to the Genbank accession number M27274 unless otherwise specified. The reference protein is PSA provided with its signal peptide.
PSA-EHT101 (SEQ ID NO : 4) :PSA-EHT101 (SEQ ID NO: 4):
Cette isoforme présente une délétion de l'exon 1 associée à une rétention d'une partie de l'intron 1 (délétion des nucléotides 636 à 918). Cette isoforme PSA- EHT101 délète le codon d'initiation utilisé par PSA dans l'exon 1. Une phase ouverte de lecture est proposée à partir d'un codon d'initiation ATG en position 1057 pour produire un polypeptide de 60 acides aminés (PSA-EHT101 prota / SEQ ID NO : 17). PSA-EHT101 prota / SEQ ID NO : 17 présente en sa partie amino-terminale un peptide signal très homologue à celui de PSA de type sauvage. Selon le site de clivage utilisé, entre 16 et 20 acides aminés peuvent être clivés de PSA-EHT101 prota / SEQ ID NO : 17 pour former un polypeptide sécrété de 40-44 acides aminés (PSA-EHT101 protb / SEQ ID NO : 18). PSA-EHT102 (SEQ ID NO :5 ) :This isoform has a deletion of exon 1 associated with a retention of part of intron 1 (deletion of nucleotides 636 to 918). This PSA-EHT101 isoform deletes the initiation codon used by PSA in exon 1. An open reading phase is proposed from an ATG initiation codon at position 1057 to produce a polypeptide of 60 amino acids (PSA -EHT101 prota / SEQ ID NO: 17). PSA-EHT101 prota / SEQ ID NO: 17 presents in its amino-terminal part a signal peptide very homologous to that of PSA of wild type. Depending on the cleavage site used, between 16 and 20 amino acids can be cleaved from PSA-EHT101 prota / SEQ ID NO: 17 to form a secreted polypeptide of 40-44 amino acids (PSA-EHT101 protb / SEQ ID NO: 18) . PSA-EHT102 (SEQ ID NO: 5):
Cette isoforme présente une délétion de l'exon 1 associée à une rétention d'une partie de l'intron 1 (délétion des nucléotides 636 à 963). Comme PSA-EHT101 délète, cette isoforme PSA-EHT102 délète le codon d'initiation utilisé par PSA dans l'exon 1. Une phase ouverte de lecture est proposée à partir d'un codon d'initiation ATG en position 1057 pour produire un polypeptide de 60 acides aminés (PSA-EHT101prota / SEQ ID NO : 17). PSA-EHT101 prota / SEQ ID NO :17 présente en sa partie amino-terminale un peptide signal très homologue à celui de PSA de type sauvage. Selon le site de clivage utilisé, entre 16 et 20 acides aminés peuvent être clivés de PSA-EHT101prota / SEQ ID NO :17 pour former un polypeptide sécrété de 40-44 acides aminés (PSA-EHT101 protb / SEQ ID NO : 18).This isoform has a deletion of exon 1 associated with a retention of part of intron 1 (deletion of nucleotides 636 to 963). As PSA-EHT101 deletes, this PSA-EHT102 isoform deletes the initiation codon used by PSA in exon 1. An open reading phase is proposed from an ATG initiation codon at position 1057 to produce a polypeptide of 60 amino acids (PSA-EHT101prota / SEQ ID NO: 17). PSA-EHT101 prota / SEQ ID NO: 17 presents in its amino-terminal part a signal peptide very homologous to that of PSA of wild type. Depending on the cleavage site used, between 16 and 20 amino acids can be cleaved from PSA-EHT101prota / SEQ ID NO: 17 to form a secreted polypeptide of 40-44 amino acids (PSA-EHT101 protb / SEQ ID NO: 18).
PSA-EHT103 (SEQ ID NO : 6) :PSA-EHT103 (SEQ ID NO: 6):
Cette isoforme présente une délétion dans l'intron 1 entre les positions 1211 et 1958 Gonction intron 1 avec exon 2). Cette isoforme PSA-EHT103 met en phase un peptide de 52 acides aminés avec la phase codante de PSA sécrétée (à partir de l'exon 2), codant ainsi pour une protéine de 297 acides aminés (PSA- EHT103prota / SEQ ID NO : 19). La partie amino-terminale de PSA- EHT103prota / SEQ ID NO : 19 possède le même peptide signal que PSA- EHT101prota / SEQ ID NO : 17. Selon le site de clivage utilisé, entre 16 et 20 acides aminés peuvent être clivés de PSA-EHT103prota / SEQ ID NO : 19 pour former le polypeptide sécrété PSA-EHT103protb / SEQ ID NO : 20. Selon le site de clivage utilisé, les 32-36 acides aminés N-terminaux de PSA-EHT103protb (PSA-EHT103protc / SEQ ID NO : 21 ) sont nouveaux et peuvent présenter un ou plusieurs épitopes originaux.This isoform has a deletion in intron 1 between positions 1211 and 1958 Gunction intron 1 with exon 2). This PSA-EHT103 isoform puts a peptide of 52 amino acids into phase with the coding phase of secreted PSA (from exon 2), thus coding for a protein of 297 amino acids (PSA- EHT103prota / SEQ ID NO: 19 ). The amino-terminal part of PSA- EHT103prota / SEQ ID NO: 19 has the same signal peptide as PSA- EHT101prota / SEQ ID NO: 17. Depending on the cleavage site used, between 16 and 20 amino acids can be cleaved from PSA- EHT103prota / SEQ ID NO: 19 to form the secreted polypeptide PSA-EHT103protb / SEQ ID NO: 20. Depending on the cleavage site used, the 32-36 N-terminal amino acids of PSA-EHT103protb (PSA-EHT103protc / SEQ ID NO : 21) are new and may present one or more original epitopes.
PSA-EHT104 (SEQ ID NO : 7) : Cette isoforme présente une délétion dans l'intron 1 entre les positions 1144 et 1958 Gonction intron 1 avec exon 2). Une phase ouverte de lecture est proposée à partir d'un codon d'initiation ATG en position 1057 pour produire un polypeptide de 85 acides aminés (PSA-EHT104prota / SEQ ID NO :22). Identique à PSA-EHT101prota, PSA-EHT104prota / SEQ ID NO :22 présente en sa partie amino-terminale un peptide signal très homologue à celui de PSA de type sauvage. Selon le site de clivage utilisé, entre 16 et 20 acides aminés peuvent être clivés de PSA-EHT104prota / SEQ ID NO : 22 pour former un polypeptide sécrété de 65-69 acides aminés (PSA-EHT104protb / SEQ ID NO : 23) .PSA-EHT104 (SEQ ID NO: 7): This isoform has a deletion in intron 1 between positions 1144 and 1958 Intron gunction 1 with exon 2). An open reading phase is proposed from an ATG initiation codon at position 1057 to produce a 85 amino acid polypeptide (PSA-EHT104prota / SEQ ID NO: 22). Identical to PSA-EHT101prota, PSA-EHT104prota / SEQ ID NO: 22 has in its amino-terminal part a signal peptide very homologous to that of PSA of wild type. Depending on the cleavage site used, between 16 and 20 amino acids can be cleaved from PSA-EHT104prota / SEQ ID NO: 22 to form a secreted polypeptide of 65-69 amino acids (PSA-EHT104protb / SEQ ID NO: 23).
Un premier objet de l'invention concerne des acides nucléiques comprenant la séquence des variants de PSA et KLK-2 décrits ci-avant.A first subject of the invention relates to nucleic acids comprising the sequence of the variants of PSA and KLK-2 described above.
Un autre objet de l'invention concerne des acides nucléiques spécifiques des altérations génétiques portées par les variants de PSA et KLK-2 décrits ci-avant. De tels acides nucléiques peuvent être notamment complémentaires de régions mutées, de domaines introniques retenus ou de jonctions nouvellement créées par des délétions.Another subject of the invention relates to nucleic acids specific for the genetic alterations carried by the variants of PSA and KLK-2 described above. Such nucleic acids may in particular be complementary to mutated regions, retained intronic domains or junctions newly created by deletions.
Un autre objet de l'invention concerne un acide nucléique comprenant tout ou partie d'une séquence dérivée des ARNs messagers (ou des ADNc) de KLK2- EHT101 à KLK2-EHT103 et de PSA-EHT101 à PSA-EHT104 ou toute combinaison de ces variants ainsi que leurs utilisations pour la mise en œuvre d'une méthode de diagnostic, de détection ou de suivi de cancers, notamment du cancer de la prostate et plus particulièrement de la forme bénigne de ce dernier, BPH.Another subject of the invention relates to a nucleic acid comprising all or part of a sequence derived from messenger RNAs (or cDNAs) from KLK2-EHT101 to KLK2-EHT103 and from PSA-EHT101 to PSA-EHT104 or any combination of these. variants and their uses for the implementation of a method for diagnosing, detecting or monitoring cancers, in particular prostate cancer and more particularly of the benign form of the latter, BPH.
Un autre objet de l'invention réside dans tout acide nucléique caractérisé en ce qu'il comprend une séquence choisie parmi : a) les séquences SEQ ID NOs : 1 à 7, b) un variant des séquences SEQ ID NOs : 1 à 7 résultant de la dégénérescence du code génétique, c) le brin complémentaire des séquences SEQ ID NOs : 1 à 7, et d) un fragment spécifique des séquences a) à c). Le terme fragment ou partie « spécifique » désigne un fragment caractéristique des variants considérés, typiquement un fragment portant au moins une altération génétique caractéristique des variants considérés. De tels fragments spécifiques se distinguent donc de la séquence sauvage par la présence d'une caractéristique structurale propre (e.g., mutation, nouvelle jonction, rétention d'intron, délétion d'une séquence, codon stop, nouvelle séquence résultant d'un décalage du cadre de lecture, etc.) résultant d'un événement d'altération mis en évidence par les demandeurs chez des patients. Cette caractéristique structurale propre est également désignée par l'expression « séquence cible ». Des séquences cibles particulières sont notamment les séquences des nouvelles jonctions contenues dans les variants de l'invention, qui résultent des événements de délétion décrits précédemment. De telles séquences cibles sont donc notamment les séquences formées par les deux codons bordant les jonctions crées par délétion des résidus 483-738 (SEQ ID NO: 1), 1032-1774 (SEQ ID NO: 2), 965-1774 (SEQ ID NO: 3), 636-918 (SEQ ID NO: 4), 636-963 (SEQ ID NO: 5), 1211-1958 (SEQ ID NO: 6) et 1144-1958 (SEQ ID NO: 7). Des fragments spécifiques selon l'invention comprennent donc au moins une séquence cible telle que définie ci-avant. Des fragments préférés comportent au moins 5 nucléotides consécutifs de la séquence considérée, de préférence au moins 8, plus préférentiellement au moins 12. Des fragments peuvent comprendre jusqu'à 50, 75 ou 100 nucléotides, voire plus.Another object of the invention resides in any nucleic acid characterized in that it comprises a sequence chosen from: a) the sequences SEQ ID NOs: 1 to 7, b) a variant of the sequences SEQ ID NOs: 1 to 7 resulting degeneracy of the genetic code, c) the complementary strand of the sequences SEQ ID NOs: 1 to 7, and d) a specific fragment of the sequences a) to c). The term “specific” fragment or part designates a fragment characteristic of the variants considered, typically a fragment carrying at least one genetic alteration characteristic of the variants considered. Such specific fragments are therefore distinguished from the wild-type sequence by the presence of their own structural characteristic (eg, mutation, new junction, intron retention, sequence deletion, stop codon, new sequence resulting from a shift in the reading frame, etc.) resulting from an alteration event highlighted by the applicants in patients. This specific structural characteristic is also designated by the expression "target sequence". Particular target sequences are in particular the sequences of the new junctions contained in the variants of the invention, which result from the deletion events described above. Such target sequences are therefore in particular the sequences formed by the two codons bordering the junctions created by deletion of the residues 483-738 (SEQ ID NO: 1), 1032-1774 (SEQ ID NO: 2), 965-1774 (SEQ ID NO: 3), 636-918 (SEQ ID NO: 4), 636-963 (SEQ ID NO: 5), 1211-1958 (SEQ ID NO: 6) and 1144-1958 (SEQ ID NO: 7). Specific fragments according to the invention therefore comprise at least one target sequence as defined above. Preferred fragments comprise at least 5 consecutive nucleotides of the sequence considered, preferably at least 8, more preferably at least 12. Fragments can comprise up to 50, 75 or 100 nucleotides, or even more.
Au sens de l'invention, les acides nucléiques peuvent être des ADN, choisis de préférence parmi les ADNc et les ADNg, ou des ARN. Il peut s'agir d'acides nucléiques synthétiques ou semi-synthétiques, de fragments PCR, d'oligonucléotides, de régions double- ou simple-brin, etc. Les acides nucléiques peuvent être produits par synthèse, par voie recombinante, par clonage, par assemblage(s) génétique(s), mutagénèse, etc., ou une combinaison de ces techniques. Les acides nucléiques peuvent être utilisés pour produire un variant de PSA ou KLK-2 de l'invention in vitro, ex vivo, in vivo ou dans un système de transcription acellulaire. Ils peuvent également être utilisés pour la fabrication de molécules antisens, capables de réduire la traduction des ARNm correspondants dans une cellule. Ils peuvent aussi servir à la production de sondes, notamment marquées, permettant par des réactions d'hybridation de mettre en évidence de manière spécifique la présence d'une forme mutée de PSA ou KLK-2 de l'invention dans un échantillon. Ils peuvent encore servir à la production d'amorces nucléiques, utiles à l'amplification d'un variant de PSA ou KLK-2 (ou d'une séquence cible d'un tel variant) dans un échantillon, notamment dans un but de dépistage ou de diagnostic.Within the meaning of the invention, the nucleic acids may be DNAs, preferably chosen from cDNAs and gDNAs, or RNAs. They can be synthetic or semi-synthetic nucleic acids, PCR fragments, oligonucleotides, double- or single-stranded regions, etc. Nucleic acids can be produced by synthesis, recombinantly, by cloning, by genetic assembly (s), mutagenesis, etc., or a combination of these techniques. The nucleic acids can be used to produce a PSA or KLK-2 variant of the invention in vitro, ex vivo, in vivo or in an acellular transcription system. They can also be used for the manufacture of antisense molecules, capable of reducing the translation of the corresponding mRNAs in a cell. They can also be used for the production of probes, in particular labeled, allowing, by hybridization reactions, to specifically demonstrate the presence of a mutated form of PSA or KLK-2 of the invention in a sample. They can also be used for the production of nucleic primers, useful for the amplification of a PSA or KLK-2 variant (or of a target sequence of such a variant) in a sample, in particular for the purpose of screening. or diagnostic.
A cet égard, un autre objet de l'invention concerne une sonde nucléique caractérisée en ce qu'elle permet la détection d'un acide nucléique tel que défini ci-avant, typiquement par hybridation sélective à partir d'une population d'acides nucléiques test. Généralement, la sonde comprend la séquence d'un acide nucléique tel que défini ci-avant ou une partie (spécifique) de la séquence d'un tel acide nucléique. La partie spécifique est préférentiellement caractéristique d'un variant tel que décrit ci-avant, notamment une partie contenant une altération associée au cancer de la prostate. La sonde comprend typiquement de 10 à 1000 nucléotides, de préférence de 50 à 800, et est généralement simple-brin. Un exemple particulier de sonde est représenté par un oligonucléotide spécifique et complémentaire d'une région au moins d'un acide nucléique tel que défini ci-avant. L'oligonucléotide est typiquement simple-brin, et comporte généralement de 10 à 100 bases, de préférence de 10 à 60 bases, plus préférentiellement de 15 à 50. Des exemples spécifiques de tels oligonucléotides sont donnés dans le Tableau 2. Les oligonucléotides et/ou les sondes nucléiques selon l'invention peuvent être marqués, par exemple au moyen de marqueurs radioactifs, enzymatiques, fluorescents, luminescents, etc.In this regard, another object of the invention relates to a nucleic probe characterized in that it allows the detection of a nucleic acid as defined above, typically by selective hybridization from a population of nucleic acids test. Generally, the probe comprises the sequence of a nucleic acid as defined above or a (specific) part of the sequence of such a nucleic acid. The specific part is preferably characteristic of a variant as described above, in particular a part containing an alteration associated with prostate cancer. The probe typically comprises from 10 to 1000 nucleotides, preferably from 50 to 800, and is generally single-stranded. A particular example of a probe is represented by an oligonucleotide specific and complementary to a region of at least one nucleic acid as defined above. The oligonucleotide is typically single-stranded, and generally comprises from 10 to 100 bases, preferably from 10 to 60 bases, more preferably from 15 to 50. Specific examples of such oligonucleotides are given in Table 2. The oligonucleotides and / or the nucleic acid probes according to the invention can be labeled, for example by means of radioactive, enzymatic, fluorescent, luminescent markers, etc.
Un objet particulier de l'invention réside dans un oligonucléotide simple-brin, d'une longueur comprise entre 10 et 60 bases, complémentaire et sélectif d'un variant de PSA ou KLK2 tel que défini ci-avant, notamment d'une jonction créée par délétion, dans la séquence codant l'antigène PSA ou KLK, des résidus 483- 738 (SEQ ID NO: 1), 1032-1774 (SEQ ID NO: 2), 965-1774 (SEQ ID NO: 3), 636-918 (SEQ ID NO: 4), 636-963 (SEQ ID NO: 5), 1211-1958 (SEQ ID NO: 6) et 1144-1958 (SEQ ID NO: 7). De préférence, les oligonucléotides sont dépourvus de structure secondaire, possèdent un Tm compris entre 62 et 67°C, plus préférentiellement de 65°C environ, et présentent une longueur comprise entre 20 et 40 bases.A particular object of the invention resides in a single-stranded oligonucleotide, of a length between 10 and 60 bases, complementary and selective of a variant of PSA or KLK2 as defined above, in particular of a junction created by deletion, in the sequence coding for the PSA or KLK antigen, of residues 483- 738 (SEQ ID NO: 1), 1032-1774 (SEQ ID NO: 2), 965-1774 (SEQ ID NO: 3), 636-918 (SEQ ID NO: 4), 636-963 (SEQ ID NO: 5), 1211-1958 (SEQ ID NO: 6) and 1144-1958 (SEQ ID NO: 7). Preferably, the oligonucleotides are devoid of secondary structure, have a Tm of between 62 and 67 ° C, more preferably around 65 ° C, and have a length of between 20 and 40 bases.
En outre, les oligonucléotides ou sondes de l'invention peuvent comporter un groupe fonctionnel particulier, permettant ou facilitant leur immobilisation sur un support solide ou leur flexibilité, par exemple un groupement terminal (c'est-à- dire 5' ou 3', plus préférentiellement 5') NH2-Cn, n étant un nombre entier compris entre 2 et 10, de préférence entre 3 et 8, par exemple 4, 5 ou 6.In addition, the oligonucleotides or probes of the invention may comprise a particular functional group, allowing or facilitating their immobilization on a solid support or their flexibility, for example a terminal group (that is to say 5 ′ or 3 ′, more preferably 5 ') NH 2 -C n , n being an integer between 2 and 10, preferably between 3 and 8, for example 4, 5 or 6.
Un autre objet de l'invention concerne une amorce nucléique, permettant l'amplification (sélective) d'un acide nucléique tel que défini ci-avant ou d'une partie (spécifique) d'un tel acide nucléique. La partie amplifiée comporte préférentiellement une altération caractéristique d'un des variants décrits ci- avant, notamment d'une altération associée au cancer de la prostate. Une amorce selon l'invention est typiquement simple-brin, et avantageusement composée de 3 à 50 bases, de préférence de 3 à 40 et encore plus préférentiellement de 3 à 35 bases. Une amorce particulière est complémentaire d'une région au moins du gène PSA ou KLK-2, ou de l'ARN correspondant.Another subject of the invention relates to a nucleic primer, allowing the (selective) amplification of a nucleic acid as defined above or of a (specific) part of such a nucleic acid. The amplified part preferably comprises an alteration characteristic of one of the variants described above, in particular an alteration associated with prostate cancer. A primer according to the invention is typically single-stranded, and advantageously composed of 3 to 50 bases, preferably from 3 to 40 and even more preferably from 3 to 35 bases. A particular primer is complementary to at least one region of the PSA or KLK-2 gene, or of the corresponding RNA.
Un mode de réalisation préféré réside dans une amorce constituée d'un acide nucléique simple-brin comprenant de 3 à 50 nucléotides complémentaires d'une partie au moins de l'une des séquences SEQ ID NOs : 1 à 7 ou de leur brin complémentaire. Des exemples de telles amorces nucléiques sont fournies dans la section expérimentale. L'invention concerne également un couple d'amorces comprenant une séquence sens et une séquence inverse, caractérisé en ce que les amorces de ladite paire s'hybrident avec une région d'un acide nucléique tel que défini ci- avant et permettent l'amplification d'au moins une portion de cet acide nucléique.A preferred embodiment resides in a primer consisting of a single-stranded nucleic acid comprising from 3 to 50 nucleotides complementary to at least part of one of the sequences SEQ ID NOs: 1 to 7 or to their complementary strand. Examples of such nucleic primers are provided in the experimental section. The invention also relates to a pair of primers comprising a sense sequence and a reverse sequence, characterized in that the primers of said pair hybridize with a region of a nucleic acid as defined above and allow the amplification. at least a portion of this nucleic acid.
Des couples d'amorces particuliers selon l'invention sont fournis dans le Tableau 1. D'autres amorces particulières, capables d'amplifier sélectivement un variant de l'invention, comprennent une séquence telle que décrite dans le Tableau 2.Couples of specific primers according to the invention are provided in Table 1. Other specific primers, capable of selectively amplifying a variant of the invention, comprise a sequence as described in Table 2.
Un autre objet de la présente demande concerne tout vecteur comprenant un acide nucléique tel que défini ci-avant. Il peut s'agir de plasmides, cosmides, épisomes, chromosomes artificiels, virus, phages, etc. On peut citer divers plasmides commerciaux tels que pUC, pcDNA, pBR, etc. Parmi les vecteurs viraux, on peut citer les rétrovirus, adénovirus, AAV, herpès virus, etc.Another object of the present application relates to any vector comprising a nucleic acid as defined above. They can be plasmids, cosmids, episomes, artificial chromosomes, viruses, phages, etc. Mention may be made of various commercial plasmids such as pUC, pcDNA, pBR, etc. Among the viral vectors, mention may be made of retroviruses, adenoviruses, AAVs, herpesviruses, etc.
Un autre objet de l'invention concerne les cellules recombinantes comprenant un acide nucléique ou un vecteur tels que définis ci-avant. Les cellules peuvent être procaryotes ou eucaryotes. Parmi les cellules procaryotes on peut citer notamment les bactéries telles que E. coli. Parmi les cellules eucaryotes, on peut mentionner les cellules de levure ou les cellules de mammifères, d'insectes ou de plantes. Il peut s'agir de cultures primaires ou de lignées. On peut citer les cellules COS, CHO, 3T3, HeLa, etc.Another subject of the invention relates to recombinant cells comprising a nucleic acid or a vector as defined above. The cells can be prokaryotic or eukaryotic. Among the prokaryotic cells, mention may in particular be made of bacteria such as E. coli. Among the eukaryotic cells, mention may be made of yeast cells or cells of mammals, insects or plants. It can be primary cultures or lines. One can quote the cells COS, CHO, 3T3, HeLa, etc.
Un autre objet de l'invention concerne une composition comprenant un acide nucléique tel que défini ci-avant immobilisé sur un support. L'invention concerne notamment des compositions comprenant une pluralité d'acides nucléiques en mélange, sous forme soluble ou immobilisée à un support, la composition comprenant au moins un acide nucléique tel que défini ci-avant. Un autre objet de l'invention concerne un (produit comprenant un) support sur lequel un ou plusieurs acides nucléiques tels que définis ci-avant sont immobilisés. Le support peut être solide, plan ou non, régulier ou non, comme par exemple du nylon, verre, plastique, métal, fibre, céramique, silice, polymère, etc., ou tout autre matériau compatible. Les acides nucléique sont préférentiellement immobilisés par une extrémité, dans des conditions laissant la molécule accessible pour une réaction d'hybridation. Les acides nucléiques peuvent être arrangés de manière précise sur le support, et déposés en plusieurs exemplaires.Another subject of the invention relates to a composition comprising a nucleic acid as defined above immobilized on a support. The invention relates in particular to compositions comprising a plurality of nucleic acids in admixture, in soluble form or immobilized to a support, the composition comprising at least one nucleic acid as defined above. Another subject of the invention relates to a (product comprising a) support on which one or more nucleic acids as defined above are immobilized. The support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material. The nucleic acids are preferably immobilized at one end, under conditions leaving the molecule accessible for a hybridization reaction. The nucleic acids can be precisely arranged on the support, and deposited in several copies.
Un autre objet de l'invention concerne un (produit comprenant un) support sur lequel une ou plusieurs cellules recombinantes telles que définies ci-avant sont immobilisées ou cultivées. Le support peut être solide, plan ou non, régulier ou non, comme par exemple du nylon, verre, plastique, métal, fibre, céramique, silice, polymère, etc., ou tout autre matériau compatible. Les cellules sont par exemple réparties dans des puits d'une microplaque ou immobilisées dans un gel ou sur un support adapté.Another subject of the invention relates to a (product comprising a) support on which one or more recombinant cells as defined above are immobilized or cultured. The support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material. The cells are, for example, distributed in wells of a microplate or immobilized in a gel or on a suitable support.
L'invention concerne également les peptides et séquences protéiques codés par tout ou partie des isoformes KLK2-EHT101 à KLK2-EHT103 et PSA-EHT101 à PSA-EHT104 , notamment celles décrites parmi les séquences SEQ ID NO : 8 à 23 ainsi que leurs utilisations pour la mise en œuvre d'une méthode de diagnostic, de détection ou de suivi de cancers, notamment du cancer de la prostate et plus particulièrement de la forme bénigne de ce dernier, BPH.The invention also relates to the peptides and protein sequences encoded by all or part of the isoforms KLK2-EHT101 to KLK2-EHT103 and PSA-EHT101 to PSA-EHT104, in particular those described among the sequences SEQ ID NO: 8 to 23 as well as their uses for the implementation of a method of diagnosis, detection or monitoring of cancers, in particular prostate cancer and more particularly of the benign form of the latter, BPH.
Un objet particulier de la présente demande concerne un polypeptide comprenant tout ou une partie spécifique d'une séquence choisie parmi SEQ ID NOs : 8 à 23. Des polypeptides particuliers sont composés ou comprennent une séquence ou une partie de séquence créée par l'altération du gène ou du messager correspondant. Au sens de l'invention, le terme « partie » désigne préférentiellement au moins 5 résidus contigus, de préférence au moins 8, plus préférentiellement au moins 10, encore plus préférentiellement au moins 15. Comme expliqué ci-avant, les altérations d'épissage du gène PSA ou KLK-2 conduisent à la production de protéines mutées, comprenant des séquences nouvellement créées (séquences cibles). Il peut s'agir de séquences nouvelles (e.g., traduction décalée, insertions), de jonctions nouvelles, etc. Des peptides particuliers de l'invention correspondent ou comprennent ces séquences cibles, codées par les séquences SEQ ID Nos :1 à 7, par exemple des peptides comprenant tout ou partie des séquences SEQ ID Nos: 10, 13, 16, 18, 20, 21 ou 23.A particular subject of the present application relates to a polypeptide comprising all or a specific part of a sequence chosen from SEQ ID NOs: 8 to 23. Particular polypeptides are composed or comprise a sequence or part of a sequence created by the alteration of the gene or corresponding messenger. Within the meaning of the invention, the term “part” preferably designates at least 5 contiguous residues, preferably at least 8, more preferably at least 10, even more preferably at least 15. As explained above, the splicing alterations of the PSA or KLK-2 gene lead to the production of mutated proteins, comprising newly created sequences (target sequences). These can be new sequences (eg, offset translation, insertions), new junctions, etc. Particular peptides of the invention correspond to or comprise these target sequences, coded by the sequences SEQ ID Nos: 1 to 7, for example peptides comprising all or part of the sequences SEQ ID Nos: 10, 13, 16, 18, 20, 21 or 23.
Un autre objet de l'invention concerne un (produit comprenant un) support sur lequel un ou plusieurs polypeptides tels que définis ci-avant sont immobilisés. Le support peut être solide, plan ou non, régulier ou non, comme par exemple du nylon, verre, plastique, métal, fibre, céramique, silice, polymère, etc., ou tout autre matériau compatible. Les polypeptides sont préférentiellement immobilisés par une extrémité, dans des conditions laissant la molécule accessible pour une réaction d'interaction avec un ligand spécifique, tel qu'un anticorps. Les polypeptides peuvent être arrangés de manière précise sur le support, et déposés en plusieurs exemplaires.Another subject of the invention relates to a (product comprising a) support on which one or more polypeptides as defined above are immobilized. The support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material. The polypeptides are preferably immobilized at one end, under conditions leaving the molecule accessible for an interaction reaction with a specific ligand, such as an antibody. The polypeptides can be precisely arranged on the support, and deposited in several copies.
Des techniques d'immobilisation de matériels (tels que acides nucléiques, polypeptides, anticorps, etc.) sur des supports ont été décrites dans la littérature, et notamment dans les demandes ou brevets n° EP619 321 , WO91/08307, US4,925,785 et GB2, 197,720, par exemple.Techniques for immobilizing materials (such as nucleic acids, polypeptides, antibodies, etc.) on supports have been described in the literature, and in particular in applications or patents no. EP619,321, WO91 / 08307, US4,925,785 and GB2, 197,720, for example.
Ligands spécifiquesSpecific ligands
L'invention concerne par ailleurs des ligands spécifiques, de préférence peptidiques, en particulier des anticorps (polyclonaux, monoclonaux) et leurs fragments, spécifiques de régions peptidiques caractéristiques des protéines codées par KLK2-EHT101 et KLK2EHT103 et de PSA-EHT101-104 (notamment des domaines introniques retenus ou des jonctions spécifiquement créées), et leurs utilisations pour la détection, le diagnostique ou le suivi de cancers et notamment du cancer de la prostate. En particulier, il peut s'agir de diagnostiquer la forme BPH et de la différencier du carcinome de la prostate.The invention further relates to specific ligands, preferably peptide, in particular antibodies (polyclonal, monoclonal) and their fragments, specific to peptide regions characteristic of the proteins coded by KLK2-EHT101 and KLK2EHT103 and PSA-EHT101-104 (in particular retained intronic domains or specifically created junctions), and their uses for the detection, diagnosis or monitoring of cancers and in particular prostate cancer. In particular, it may involve diagnosing the BPH form and differentiating it from carcinoma of the prostate.
A cet égard, un autre objet de l'invention concerne tout anticorps capable de se lier, de préférence de manière sélective, à un polypeptide tel que défini ci- dessus. L'anticorps peut être polyclonal ou monoclonal. Il peut également s'agir de fragments et dérivés d'anticorps présentant substantiellement la même spécificité antigénique, en particulier de fragments d'anticorps (e.g., Fab, Fab'2, CDRs), d'anticorps humanisés, polyfonctionnels, monocaténaires (ScFv), etc. Les anticorps peuvent être produits à l'aide de méthodes conventionnelles, comprenant l'immunisation d'un animal et la récupération de son sérum (polyclonal) ou de cellules spléniques (de manière à produire des hybridomes par fusion avec des lignées cellulaires appropriées).In this regard, another object of the invention relates to any antibody capable of binding, preferably selectively, to a polypeptide as defined above. The antibody can be polyclonal or monoclonal. It can also be fragments and derivatives of antibodies having substantially the same antigenic specificity, in particular antibody fragments (eg, Fab, Fab'2, CDRs), humanized, polyfunctional, single-stranded antibodies (ScFv) , etc. Antibodies can be produced using conventional methods, including immunizing an animal and recovering its serum (polyclonal) or spleen cells (so as to produce hybridomas by fusion with appropriate cell lines).
Des méthodes de production d'anticorps polyclonaux à partir d'espèces variées sont décrites dans l'art antérieur. Typiquement, l'antigène est combiné avec un adjuvant (e. g., Freund's adjuvant) et administré à un animal, typiquement par injection sous-cutanée. Des injections répétées peuvent être réalisées. Les échantillons sanguins sont collectés et l'immunoglobuline ou le sérum sont séparés. Les méthodes classiques de production d'anticorps monoclonaux comprennent l'immunisation d'un animal avec un antigène, suivie de la récupération des cellules spléniques qui sont ensuite fusionnées avec des cellules immortalisées, telles que des cellules de myélome. Les hybridomes résultant produisent des anticorps monoclonaux et peuvent être sélectionnés par dilutions limites de manière à isoler les clones individuels. Les fragments Fab ou F(ab')2 peuvent être produits par digestion à l'aide d'une protéase selon les techniques conventionnelles.Methods of producing polyclonal antibodies from various species are described in the prior art. Typically, the antigen is combined with an adjuvant (eg, Freund's adjuvant) and administered to an animal, typically by subcutaneous injection. Repeated injections can be given. Blood samples are collected and immunoglobulin or serum are separated. Conventional methods of producing monoclonal antibodies include immunizing an animal with an antigen, followed by the recovery of spleen cells which are then fused with immortalized cells, such as myeloma cells. The resulting hybridomas produce monoclonal antibodies and can be selected by limiting dilutions so as to isolate the individual clones. The Fab or F (ab ') 2 fragments can be produced by digestion using a protease according to conventional techniques.
L'invention concerne également une méthode de production d'anticorps, comprenant l'injection d'un polypeptide tel que défini ci-avant ou d'un fragment immunogène de ce dernier à un animal non humain et la récupération des anticorps ou des cellules productrices d'anticorps. Les anticorps préférés sont des anticorps spécifiques des isoformes de PSA et KLK-2 décrites dans la présente demande, et essentiellement non-spécifique des formes sauvages. Des anticorps particuliers sont spécifiques d'une séquence cible telle que définie ci-avant, par exemple de peptides ou épitopes comprenant tout ou partie des séquences SEQ ID Nos: 10, 13, 16, 18, 20, 21 ou 23.The invention also relates to a method for producing antibodies, comprising injecting a polypeptide as defined above or an immunogenic fragment thereof into a non-human animal and recovering the antibody or antibody producing cells. The preferred antibodies are antibodies specific for the isoforms of PSA and KLK-2 described in the present application, and essentially non-specific for wild forms. Particular antibodies are specific for a target sequence as defined above, for example peptides or epitopes comprising all or part of the sequences SEQ ID Nos: 10, 13, 16, 18, 20, 21 or 23.
L'invention concerne des hybridomes produisant les anticorps monoclonaux décrits ci-dessus et leur utilisation pour produire lesdits anticorps.The invention relates to hybridomas producing the monoclonal antibodies described above and their use for producing said antibodies.
Les anticorps peuvent être couplés à des fragments hétérologues telles que des toxines, des marqueurs, des médicaments ou tout autre agent thérapeutique, de façon covalente ou non, soit directement, soit par l'intermédiaire d'agents de couplage. Les marqueurs peuvent être choisis parmi les radio-marqueurs, des enzymes, des agents fluorescents, des particules magnétiques, etc.The antibodies can be coupled to heterologous fragments such as toxins, markers, drugs or any other therapeutic agent, covalently or not, either directly or through coupling agents. The markers can be chosen from radio-markers, enzymes, fluorescent agents, magnetic particles, etc.
Les anticorps de l'invention peuvent être utilisés comme agents de criblage ou pour détecter ou quantifier la présence ou la quantité d'isoformes de PSA ou KLK-2 dans des échantillons collectés à partir d'un sujet, typiquement, un fluide biologique provenant d'un mammifère, par exemple d'un être humain.The antibodies of the invention can be used as screening agents or to detect or quantify the presence or amount of PSA or KLK-2 isoforms in samples collected from a subject, typically a biological fluid from 'a mammal, for example from a human.
Un autre objet de l'invention concerne un (produit comprenant un) support sur lequel un ou plusieurs anticorps (ou fragments ou dérivés) tels que définis ci- avant sont immobilisés. Le support peut être solide, plan ou non, régulier ou non, comme par exemple du nylon, verre, plastique, métal, fibre, céramique, silice, polymère, etc., ou tout autre matériau compatible. Les anticorps sont préférentiellement immobilisés par une extrémité, dans des conditions laissant la molécule accessible pour une réaction d'interaction avec un antigène spécifique. Les anticorps peuvent être arrangés de manière précise sur le support, et déposés en plusieurs exemplaires. Méthodes de détection/diagnosticAnother subject of the invention relates to a (product comprising a) support on which one or more antibodies (or fragments or derivatives) as defined above are immobilized. The support can be solid, flat or not, regular or not, such as for example nylon, glass, plastic, metal, fiber, ceramic, silica, polymer, etc., or any other compatible material. The antibodies are preferably immobilized at one end, under conditions leaving the molecule accessible for an interaction reaction with a specific antigen. The antibodies can be precisely arranged on the support, and deposited in several copies. Detection / diagnostic methods
La présente demande décrit également de nouveaux procédés de détection d'une pathologie ou d'une prédisposition à une pathologie chez un sujet, comprenant la détermination de la présence, dans un échantillon dudit sujet, d'un acide nucléique, ou d'une altération génétique ou d'une protéine ou polypeptide tels que définis ci-avant.The present application also describes new methods for detecting a pathology or a predisposition to a pathology in a subject, comprising determining the presence, in a sample of said subject, of a nucleic acid, or an alteration genetic or a protein or polypeptide as defined above.
La détermination peut être réalisée par différentes techniques, telles que séquençage, hybridation et/ou amplification sélectives. Des méthodes utilisables pour déterminer la présence de protéines sont basées par exemple sur des réactions immuno-enzymatiques, telles que ELISA, RIA, EIA, etc. Des techniques utilisables pour déterminer la présence de gènes ou d'ARN altérés sont par exemple la PCR, la RT-PCR, la réaction de ligation en chaîne (LCR), la technique de PCE ou TMA (« Transcriptional Mediated Amplification »), la migration sur gel, l'électrophorèse, notamment la DGGE (« denaturing gel gradient electrophoresis »), etc.The determination can be carried out by various techniques, such as sequencing, hybridization and / or selective amplification. Methods that can be used to determine the presence of proteins are based, for example, on immunoenzymatic reactions, such as ELISA, RIA, EIA, etc. Techniques that can be used to determine the presence of altered genes or RNA are, for example, PCR, RT-PCR, ligation chain reaction (LCR), PCE or TMA (“Transcriptional Mediated Amplification”) technique, gel migration, electrophoresis, in particular DGGE (“denaturing gel gradient electrophoresis”), etc.
Dans le cas où une étape d'amplification est réalisée, celle-ci met préférentiellement en oeuvre une amorce ou un couple d'amorces tel que défini ci-avant.In the case where an amplification step is carried out, this preferably uses a primer or a pair of primers as defined above.
Un objet particulier de l'invention concerne l'utilisation d'acides nucléiques complémentaires et spécifiques de fragments des gènes ou des messagers de KLK2-EHT101-103 et de PSA-EHT101-104 (e.g., domaines introniques retenus, jonctions spécifiquement crées, mutations particulières, etc.) pour la détection de cancers, notamment du cancer de la prostate, et plus particulièrement de sa forme bénigne, BPH. Cette détection pourra en particulier s'effectuer grâce à des puces à ADN ou à la mise en œuvre d'une PCR à partir de fluides biologiques tels que du sang (le sérum notamment), des urines, du fluide séminal, etc. L'invention réside également dans la mise au point et l'utilisation de tests immunologiques contenant un ou plusieurs anticorps tels que décrits ci-dessus ou fragments de ces derniers. Ces tests permettent de détecter et/ou mesurer individuellement un variant à l'aide d'un anticorps spécifique, plusieurs variants, en parallèle, grâce aux anticorps spécifiques appropriés, ou un ou plusieurs rapports entre les isoformes telles que décrites ci-dessus ou entre lesdites isoformes et d'autres formes décrites pour kallikreine-2 et PSA.A particular object of the invention relates to the use of complementary and specific nucleic acids of fragments of the genes or messengers of KLK2-EHT101-103 and of PSA-EHT101-104 (eg, retained intronic domains, specifically created junctions, mutations particular, etc.) for the detection of cancers, in particular prostate cancer, and more particularly of its benign form, BPH. This detection may in particular be carried out by means of DNA chips or by the implementation of a PCR from biological fluids such as blood (notably serum), urine, seminal fluid, etc. The invention also resides in the development and use of immunoassays containing one or more antibodies as described above or fragments thereof. These tests make it possible to individually detect and / or measure a variant using a specific antibody, several variants, in parallel, using the appropriate specific antibodies, or one or more relationships between the isoforms as described above or between said isoforms and other forms described for kallikrein-2 and PSA.
Une méthode particulière comprend la mise en contact d'un échantillon provenant d'un sujet avec une sonde telle que définie ci-avant, et la mise en évidence d'une hybridation.A particular method comprises bringing a sample from a subject into contact with a probe as defined above, and demonstrating hybridization.
Une autre méthode particulière comprend la mise en contact d'un échantillon provenant d'un sujet avec une amorce ou une paire d'amorces telles que définies ci-avant, et la mise en évidence d'un produit d'amplification.Another particular method comprises bringing a sample from a subject into contact with a primer or a pair of primers as defined above, and demonstrating an amplification product.
Une autre méthode particulière comprend la mise en contact d'un échantillon provenant d'un sujet avec un anticorps tel que défini ci-avant, et la mise en évidence d'un complexe antigène-anticorps.Another particular method comprises bringing a sample from a subject into contact with an antibody as defined above, and demonstrating an antigen-antibody complex.
Typiquement, plusieurs tests sont réalisés en parallèle, à partir de plusieurs échantillons et/ou en utilisant plusieurs sondes, amorces et/ou anticorps. Ainsi, dans un mode particulier, le procédé de l'invention comprend la détermination, en parallèle, de la présence de plusieurs variants ou altérations génétiques tels que décrits ci-avant dans un échantillon d'un patient. Les procédés selon l'invention peuvent être mis en œuvre à partir d'échantillons biologiques variés, notamment de fluides biologiques (e.g., sang, plasma, urine, sérum, salive, etc.), de biopsies de tissus ou de cultures cellulaires, par exemple et, plus généralement, à partir de tout échantillon susceptible de contenir des acides nucléiques ou des protéines (ou polypeptides). L'échantillon biologique peut être traité avant la mise en œuvre du procédé, pour faciliter ou permettre l'accessibilité des polypeptides ou acides nucléiques qu'il contient. L'échantillon peut également être purifié, centrifugé, fixé, etc., éventuellement congelé ou conservé avant usage.Typically, several tests are carried out in parallel, using several samples and / or using several probes, primers and / or antibodies. Thus, in a particular embodiment, the method of the invention comprises the determination, in parallel, of the presence of several genetic variants or alterations as described above in a sample of a patient. The methods according to the invention can be implemented using various biological samples, in particular biological fluids (eg, blood, plasma, urine, serum, saliva, etc.), biopsies of tissues or cell cultures, by example and, more generally, from any sample likely to contain nucleic acids or proteins (or polypeptides). The biological sample can be treated before the implementation of the method, to facilitate or allow the accessibility of the polypeptides or nucleic acids which it contains. The sample can also be purified, centrifuged, fixed, etc., possibly frozen or stored before use.
Dans un mode de réalisation particulier, l'invention concerne un procédé de détection de la présence d'une forme altérée de KLK-2 ou KLK-3 chez un sujet, comprenant la mise en contact, in vitro ou ex vivo, d'un échantillon dudit sujet avec une sonde, une amorce ou un ligand spécifique tels que définis ci-avant et la détermination de la formation d'un hybride, d'un produit d'amplification ou d'un complexe, respectivement, la dite formation étant indicative de la présence d'une forme altérée.In a particular embodiment, the invention relates to a method for detecting the presence of an altered form of KLK-2 or KLK-3 in a subject, comprising bringing into contact, in vitro or ex vivo, of a sample of said subject with a specific probe, primer or ligand as defined above and the determination of the formation of a hybrid, of an amplification product or of a complex, respectively, said formation being indicative the presence of an altered form.
Un autre objet de l'invention réside dans un kit utilisable pour la mise en œuvre d'un procédé tel que défini ci-avant comprenant i. un couple d'amorces ou une sonde (y compris un oligonucléotide) ou un anticorps tels que définis ci-avant, et ii. les réactifs nécessaires à l'amplification ou à une réaction d'hybridation ou immunologique.Another object of the invention resides in a kit which can be used for implementing a method as defined above comprising i. a pair of primers or a probe (including an oligonucleotide) or an antibody as defined above, and ii. the reagents necessary for amplification or for a hybridization or immunological reaction.
L'invention réside également dans la mise au point d'une méthode permettant de détecter et/ou de mesurer des partenaires spécifiques d'un ou de plusieurs de ces variants par ajout de l'un ou de plusieurs de ces variants ou de leurs fragments dans des fluides biologiques à tester, tels que du sang (sérum notamment), des urines ou du fluide séminal.The invention also resides in the development of a method making it possible to detect and / or measure specific partners of one or more of these variants by adding one or more of these variants or their fragments in biological fluids to be tested, such as blood (serum in particular), urine or seminal fluid.
Criblage de composés actifsScreening of active compounds
Les variants spécifiques de KLK2 et de KLK3 selon l'invention ont été identifiés et isolés à partir de sujets pathologiques et représentent donc des cibles thérapeutiques particulièrement intéressantes pour le traitement des cancers et notamment du cancer de la prostate. A cet égard, un objet particulier de l'invention réside dans des méthodes de sélection, d'identification, de caractérisation, d'optimisation ou de production de composés actifs, comprenant une étape de détermination de la capacité d'un composé test à moduler l'expression ou l'activité d'un polypeptide tel que défini ci-avant.The specific variants of KLK2 and KLK3 according to the invention have been identified and isolated from pathological subjects and therefore represent therapeutic targets which are particularly advantageous for the treatment of cancers and in particular prostate cancer. In this regard, a particular object of the invention resides in methods of selection, identification, characterization, optimization or production of active compounds, comprising a step of determining the capacity of a test compound to modulate the expression or activity of a polypeptide as defined above.
Les composés sont plus particulièrement sélectionnés sur la base de leur capacité à moduler la synthèse d'un polypeptide tel que défini ci-avant (c'est-à- dire notamment la production ou la maturation des ARNs correspondants, ou leur traduction) ou l'activité d'un tel polypeptide (c'est-à-dire notamment leur maturation, transport, ou leur interaction avec des cibles intra- ou extracellulaires).The compounds are more particularly selected on the basis of their capacity to modulate the synthesis of a polypeptide as defined above (that is to say in particular the production or the maturation of the corresponding RNAs, or their translation) or the activity of such a polypeptide (that is to say in particular their maturation, transport, or their interaction with intra- or extracellular targets).
Dans une variante de réalisation, la méthode comprend la mise en contact in vitro ou ex vivo d'un composé test avec un polypeptide tel que défini ci-avant ou un acide nucléique codant un tel polypeptide (e.g., gène, ADNc, ARN), et la sélection des composés se liant audit polypeptide ou acide nucléique. La liaison au polypeptide au gène ou à l'ARN correspondant peut être mesurée par différentes techniques, telles que le déplacement d'un ligand marqué, la migration sur gel, l'électrophorèse, etc. Elle peut être réalisée in vitro, par exemple en utilisant le polypeptide ou l'acide nucléique immobilisé sur un support.In an alternative embodiment, the method comprises contacting in vitro or ex vivo a test compound with a polypeptide as defined above or a nucleic acid encoding such a polypeptide (eg, gene, cDNA, RNA), and selecting compounds that bind to said polypeptide or nucleic acid. The binding of the polypeptide to the gene or to the corresponding RNA can be measured by various techniques, such as the displacement of a labeled ligand, migration on gel, electrophoresis, etc. It can be carried out in vitro, for example using the polypeptide or the nucleic acid immobilized on a support.
Dans une autre variante de réalisation, la méthode comprend la mise en contact in vitro ou ex vivo d'un composé test avec une cellule exprimant un polypeptide tel que défini ci-avant, et la sélection ou l'identification de composés modulant l'expression ou l'activité dudit polypeptide. La modulation de l'expression peut être déterminée par dosage des ARN ou des protéines, ou au moyen d'un système rapporteur.In another alternative embodiment, the method comprises contacting in vitro or ex vivo a test compound with a cell expressing a polypeptide as defined above, and the selection or identification of compounds modulating the expression or the activity of said polypeptide. The modulation of expression can be determined by assaying the RNAs or proteins, or by means of a reporter system.
Les cellules utilisées peuvent être toute cellule compatible, notamment des cellules eucaryotes ou procaryotes telles que mentionnées plus haut. On utilise typiquement une cellule modifiée pour exprimer ladite molécule, notamment des cellules recombinantes. De telles cellules recombinantes peuvent être préparées par introduction d'un acide nucléique recombinant exprimant le polypeptide, ou d'un vecteur comprenant celui-ci. De telles cellules recombinantes constituent des objets particuliers de l'invention.The cells used can be any compatible cell, in particular eukaryotic or prokaryotic cells as mentioned above. We use typically a cell modified to express said molecule, in particular recombinant cells. Such recombinant cells can be prepared by the introduction of a recombinant nucleic acid expressing the polypeptide, or of a vector comprising the latter. Such recombinant cells constitute particular objects of the invention.
Le procédé peut être mis en œuvre pour sélectionner ou identifier un activateur ou un inhibiteur de l'expression ou de l'activité de l'antigène spécifique de la PSA ou de KLK-2. Les procédés de sélection peuvent être réalisés en différents formats, comme par exemple en plaques multi-puits, en testant en parallèle de multiples composés candidats.The method can be implemented to select or identify an activator or an inhibitor of the expression or of the activity of the specific antigen of PSA or of KLK-2. The selection methods can be carried out in different formats, such as for example multi-well plates, by testing multiple candidate compounds in parallel.
Dans un mode de réalisation particulier, le composé est un acide nucléique antisens, capable d'inhiber l'expression des variants décrits. L'acide nucléique anti- sens peut comprendre tout ou partie de séquences spécifiques des variants décrits. L'anti-sens peut notamment comprendre une région complémentaire de la forme d'épissage identifiée (e.g., d'une séquence cible), et inhiber (ou réduire) sa traduction en protéine.In a particular embodiment, the compound is an antisense nucleic acid capable of inhibiting the expression of the variants described. The antisense nucleic acid can comprise all or part of specific sequences of the variants described. The antisense can in particular comprise a region complementary to the form of splicing identified (e.g., of a target sequence), and inhibit (or reduce) its translation into protein.
Selon un autre mode de réalisation, le composé est un composé chimique, d'origine naturelle ou synthétique, notamment une molécule organique ou inorganique, d'origine végétale, bactérienne, virale, animale, eucaryote, synthétique ou semi-synthétique, capable de moduler l'expression ou l'activité de l'un ou de plusieurs des variants décrits ci-dessus.According to another embodiment, the compound is a chemical compound, of natural or synthetic origin, in particular an organic or inorganic molecule, of plant, bacterial, viral, animal, eukaryotic, synthetic or semi-synthetic origin, capable of modulating the expression or activity of one or more of the variants described above.
On préfère des composés spécifiques, c'est-à-dire capables de moduler l'expression ou l'activité des variants, sans affecter significativement l'expression ou l'activité des formes sauvages.Specific compounds are preferred, that is to say capable of modulating the expression or the activity of the variants, without significantly affecting the expression or the activity of the wild forms.
Les composés ainsi identifiés sont utilisables pour la préparation d'une composition destinée au traitement du cancer de la prostate. Un autre objet de l'invention réside dans l'utilisation d'un composé capable de moduler, i.e. de stimuler, d'inhiber ou de réduire, l'expression d'un ou de plusieurs variants tels que décrits ci-dessus, pour la préparation d'une composition destinée au traitement des cancers et notamment du cancer de la prostate.The compounds thus identified can be used for the preparation of a composition intended for the treatment of prostate cancer. Another object of the invention resides in the use of a compound capable of modulating, ie stimulating, inhibiting or reducing, the expression of one or more variants as described above, for the preparation of a composition intended for the treatment of cancers and in particular prostate cancer.
Dans le contexte de l'invention, le terme « traitement » désigne le traitement préventif, curatif ou palliatif, ainsi que la prise en charge des patients (réduction de la souffrance, amélioration de la durée de vie, ralentissement de la progression de la maladie), etc. Le traitement peut en outre être réalisé en combinaison avec d'autres agents actifs.In the context of the invention, the term “treatment” designates preventive, curative or palliative treatment, as well as the management of patients (reduction of suffering, improvement of lifespan, slowing down the progression of the disease ), etc. The treatment can also be carried out in combination with other active agents.
Un autre objet de l'invention concernent des méthodes de sélection, d'identification, ou de caractérisation de composés actifs utilisables dans le cadre de la préparation de compositions destinées au traitement de pathologies cancéreuses, comprenant la mise en contact d'un ou de plusieurs composés tests avec des extraits cellulaires exprimant les protéines décrites dans la présente invention, ou avec lesdites protéines purifiées.Another subject of the invention relates to methods of selection, identification, or characterization of active compounds which can be used in the context of the preparation of compositions intended for the treatment of cancerous pathologies, comprising the bringing into contact of one or more test compounds with cellular extracts expressing the proteins described in the present invention, or with said purified proteins.
L'invention porte également sur un procédé de production d'un médicament pour le traitement de cancers, notamment du cancer de la prostate, comprenant (i) la sélection de composés actifs selon les procédés ci-dessus et (ii) le conditionnement dudit composé ou d'un analogue fonctionnel de celui-ci en présence d'un véhicule acceptable sur le plan pharmaceutique. L'analogue fonctionnel est typiquement un composé dérivé du composé actif identifié, par modification chimique, notamment dans le but d'améliorer son activité ou sa pharmaco-cinétique, ou dans le but de réduire sa toxicité. L'analogue fonctionnel peut être une « pro-drogue » du composé identifié. Des techniques de préparation d'analogues fonctionnels sont bien connues de l'homme du métier, par exemple la modélisation moléculaire, le couplage de groupes NO, etc. Le procédé peut à cet égard comprendre une étape intermédiaire de synthèse du composé sélectionné ou de l'analogue fonctionnel de celui-ci. Le véhicule ou excipient acceptable sur le plan pharmaceutique peut être choisi parmi des solutés tampons, solvants, liants, stabilisants, émulsifiants, etc. Des solutés tampons ou diluant sont notamment le dicalcium phosphate, sulfate de calcium, lactose, cellulose, kaolin, mannitol, chlorure de sodium, amidon, sucre en poudre et hydroxy propyl methyl cellulose (HPMC) (pour libération retard). Des liants sont par exemple l'amidon, la gélatine et des solutés de remplissage comme le sucrose, glucose, dextrose, lactose, etc. Des gommes naturelles ou synthétiques peuvent aussi être utilisées, comme notamment l'alginate, la carboxymethylcellulose, la méthylcellulose, la polyvinyl pyrrolidone, etc. D'autres excipients sont par exemple la cellulose et du stéarate de magnésium. Des agents stabilisants peuvent être incorporés aux formulations, comme par exemple des polysaccharides (acacia, agar, acide alginique, gomme guar et tragacanth, la chitine ou ses dérivés et des éthers de cellulose. Des solvants ou solutés sont par exemple la solution Ringer, l'eau, l'eau distillée, des tampons phosphates, des solutions salines phosphatées, et autres fluides conventionnels.The invention also relates to a method for producing a medicament for the treatment of cancers, in particular prostate cancer, comprising (i) the selection of active compounds according to the above methods and (ii) the conditioning of said compound or a functional analog thereof in the presence of a pharmaceutically acceptable carrier. The functional analog is typically a compound derived from the active compound identified, by chemical modification, in particular with the aim of improving its activity or its pharmacokinetics, or with the aim of reducing its toxicity. The functional analog may be a "pro-drug" of the identified compound. Techniques for preparing functional analogs are well known to those skilled in the art, for example molecular modeling, coupling of NO groups, etc. The method can in this regard comprise an intermediate step of synthesis of the selected compound or of the functional analog thereof. The pharmaceutically acceptable vehicle or excipient can be chosen from buffer solutions, solvents, binders, stabilizers, emulsifiers, etc. Buffer or diluent solutions include dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, starch, powdered sugar and hydroxy propyl methyl cellulose (HPMC) (for delayed release). Binders are for example starch, gelatin and filling solutes such as sucrose, glucose, dextrose, lactose, etc. Natural or synthetic gums can also be used, such as alginate, carboxymethylcellulose, methylcellulose, polyvinyl pyrrolidone, etc. Other excipients are for example cellulose and magnesium stearate. Stabilizing agents can be incorporated into the formulations, such as for example polysaccharides (acacia, agar, alginic acid, guar gum and tragacanth, chitin or its derivatives and cellulose ethers. Solvents or solutes are for example the Ringer solution, l , distilled water, phosphate buffers, phosphate salt solutions, and other conventional fluids.
Un autre objet de l'invention concerne l'utilisation de ligands cytotoxiques spécifiques d'un ou de plusieurs variants tels que décrits ci-dessus localisés à la surface des cellules cancéreuses et en particulier des cellules cancéreuses prostatiques.Another subject of the invention relates to the use of cytotoxic ligands specific for one or more variants as described above located on the surface of cancer cells and in particular prostate cancer cells.
D'autres aspects et avantages de la présente invention apparaîtront à la lecture des exemples qui suivent, qui doivent être considérés comme illustratifs et non limitatifs.Other aspects and advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.
A - IDENTIFICATION DE VARIANTS SPECIFIQUESA - IDENTIFICATION OF SPECIFIC VARIANTS
Une analyse qualitative différentielle a été effectuée à partir d'ARN poly adénylés (poly A+) extraits d'échantillons tumoraux et normaux prostatiques. Les ARN poly A+ sont préparés selon des techniques connues de l'homme de métier. Il peut s'agir en particulier d'un traitement au moyen d'agents chaotropiques tels que le thiocyanate de guanidium suivi d'une extraction des ARN totaux au moyen de solvants (phénol, chloroforme par exemple). De telles méthodes sont bien connues de l'homme du métier (voir Maniatis et al., Chomczynsli et al., Anal. Biochem. 162 (1987) 156), et peuvent être aisément mises en œuvre en utilisant des kits disponibles dans le commerce. A partir de ces ARN totaux, les ARN poly A+ sont préparés selon des méthodes classiques connues de l'homme de métier et décrites dans les kits commerciaux. Ces ARN poly A+ servent de matrice à des réactions de transcription inverse à l'aide de transcriptases inverses. Des transcriptases inverses dépourvues d'activité RNase H sont avantageusement utilisées. Elles permettent d'obtenir des brins d'ADN complémentaire de tailles supérieures à ceux obtenus à l'aide de transcriptases inverses classiques. De telles préparations de transcriptases inverses sans activité RNase H sont disponibles commercialement.A differential qualitative analysis was carried out using poly adenylated RNA (poly A +) extracted from tumor and normal prostate samples. Poly A + RNAs are prepared according to techniques known to those skilled in the art. It may in particular be a treatment using chaotropic agents such as guanidium thiocyanate followed by extraction of the total RNAs using solvents (phenol, chloroform for example). Such methods are well known to those skilled in the art (see Maniatis et al., Chomczynsli et al., Anal. Biochem. 162 (1987) 156), and can be easily implemented using commercially available kits . From these total RNAs, the poly A + RNAs are prepared according to conventional methods known to those skilled in the art and described in commercial kits. These poly A + RNAs serve as a template for reverse transcription reactions using reverse transcriptases. Advantageously, reverse transcriptases lacking RNase H activity are used. They make it possible to obtain complementary DNA strands of sizes larger than those obtained using conventional reverse transcriptases. Such preparations of reverse transcriptases without RNase H activity are commercially available.
Conformément à la technique DATAS, pour chaque point de la cinétique des hybridations d'ARNm (C) avec des ADNc (T) et des hybridations réciproques d'ARNm (T) avec des ADNc (C) sont réalisées. Ces hétéroduplexes ARNm/ADNc sont ensuite purifiés selon les protocoles prévus par la technique DATAS.In accordance with the DATAS technique, for each point of the kinetics hybridizations of mRNA (C) with cDNAs (T) and reciprocal hybridizations of mRNA (T) with cDNAs (C) are carried out. These mRNA / cDNA heteroduplexes are then purified according to the protocols provided by the DATAS technique.
Les séquences d'ARN non appariées avec un ADN complémentaire sont libérées de ces hétéroduplex sous l'action de la RNase H, cette enzyme dégradant les séquences d'ARN appariées. Ces séquences non appariées représentent les différences qualitatives qui existent entre des ARN par ailleurs homologues entre eux. Ces différences qualitatives peuvent être localisées n'importe où sur la séquence des ARN, aussi bien en 5' ou en 3' qu'à l'intérieur de la séquence et notamment à l'intérieur de la séquence codante. Selon leur localisation, ces séquences peuvent être non seulement des modifications d'épissage mais également des conséquences de translocations ou de délétions. Les séquences d'ARN représentant les différences qualitatives sont ensuite clonées selon les techniques connues de l'homme de métier et notamment celles décrites dans le brevet relatif à la technologie DATAS.RNA sequences not paired with complementary DNA are released from these heteroduplexes under the action of RNase H, this enzyme degrading the paired RNA sequences. These unpaired sequences represent the qualitative differences which exist between RNAs which are otherwise homologous to one another. These qualitative differences can be located anywhere on the RNA sequence, either in 5 ′ or 3 ′ as within the sequence and in particular within the coding sequence. Depending on their location, these sequences can be not only modifications of splicing but also the consequences of translocations or deletions. The RNA sequences representing the qualitative differences are then cloned according to techniques known to those skilled in the art and in particular those described in the patent relating to the DATAS technology.
Ces séquences sont regroupées au sein de banques de ADNc qui constituent des banques qualitatives différentielles. Une de ces banques contient les exons et les introns spécifiques de la situation saine ; les autres banques contiennent les événements d'épissage caractéristiques des conditions pathologiques.These sequences are grouped together within cDNA libraries which constitute qualitative differential banks. One of these banks contains exons and introns specific to the healthy situation; the other banks contain the splicing events characteristic of the pathological conditions.
Les fragments issus des gènes humains de KLK2 et de KLK3 proviennent de ces banques. Quatre échantillons tumoraux ont été mélangés pour former un « pool » tumoral.The fragments derived from the human genes of KLK2 and KLK3 come from these banks. Four tumor samples were mixed to form a tumor pool.
Ce pool d'ARN a été traité à la Dnase à l'aide du kit « DNA free » de la sociétéThis RNA pool was treated with Dnase using the company's “DNA free” kit
Ambion (n° cat. 1906). Un pool de lignées cellulaires tumorales de prostate a également été constitué.Ambion (cat. No. 1906). A pool of prostate tumor cell lines has also been established.
Ces ARNs sont ensuite reverse-transcrits à l'aide de la transcriptase inverse du kit « High capacity cDNA Archive » de la société Aplied Biosystems (n° cat.These RNAs are then reverse transcribed using the reverse transcriptase from the “High capacity cDNA Archive” kit from the company Aplied Biosystems (cat.
4322171 ).4322171).
L'ADNc ainsi produit sert de matrice pour des réactions de PCR afin d'amplifier spécifiquement différentes régions d'ARNs messagers dérivés de la kallikreine-2 et de la kallikreine-3 humaines selon le protocole suivant :The cDNA thus produced serves as a template for PCR reactions in order to specifically amplify different regions of messenger RNAs derived from human kallikrein-2 and kallikrein-3 according to the following protocol:
Tampon Invitrogen 10X : 2μlInvitrogen 10X buffer: 2μl
DNTPs 2mM: 2μLDNTPs 2mM: 2μL
MgCI2 50mM: 0.6μLMgCI2 50mM: 0.6μL
Primer amont 10μM: 0.4μLUpstream primer 10μM: 0.4μL
Primer aval 10μM: 0.4μL10μM downstream primer: 0.4μL
Taq polymerase : 0.2μLTaq polymerase: 0.2μL
H20: 13.4μL cDNA 1 μLH20: 13.4μL cDNA 1 μL
Volume final : 19μLFinal volume: 19μL
Selon le programme de cycles suivant: 94°C 3min 94°C 30sec )According to the following cycle program: 94 ° C 3min 94 ° C 30sec)
55°C 1 min ) 35 cycles55 ° C 1 min) 35 cycles
72°C 3min )72 ° C 3min)
72°C 6min72 ° C 6min
Les oligonucléotides utilisés comme amorces de PCR sont les suivantsThe oligonucleotides used as PCR primers are as follows
Pour KLK2 :For KLK2:
419: KLK2 5': AGGCTTTATAGGGCTCCTCA419: KLK2 5 ': AGGCTTTATAGGGCTCCTCA
421 : KLK2 intron 1 stop : GACTCAGGGTGGGAGGCAG 450 KLK2 intron 1 ATG: GACCCTATCACTGGGCTGC 422: KLK2 exon 2 revl : TGTGCCCATCCATGACTGTA421: KLK2 intron 1 stop: GACTCAGGGTGGGAGGCAG 450 KLK2 intron 1 ATG: GACCCTATCACTGGGCTCT 422: KLK2 exon 2 revl: TGTGCCCATCCATGACTGTA
Pour PSA :For PSA:
413 : PSA 5' : AGGTTTTATAGGGCTCCTGG413: PSA 5 ': AGGTTTTATAGGGCTCCTGG
415 : PSA intron 1 stop : CATTGCCAGACTGAGGGACC415: PSA intron 1 stop: CATTGCCAGACTGAGGGACC
449: PSA intron 1 ATG : GGACCGTATCACTGGTCCAT 416: PSA exon2 revl : GCCCTGCCACGAGAGGC449: PSA intron 1 ATG: GGACCGTATCACTGGTCCAT 416: PSA exon2 revl: GCCCTGCCACGAGAGGC
Les produits amplifiés sont ensuite clones dans le système « Topo » de la société Invitrogen (n° cat. K4600) selon le protocole fourni. Les produits de ligation sont transformés dans des cellules « Top 10 » compétentes. Les colonies sont identifiées sur milieu agar/LB supplémenté d'ampicilline.The amplified products are then cloned in the “Topo” system from the company Invitrogen (cat. No. K4600) according to the protocol provided. The ligation products are transformed into competent “Top 10” cells. The colonies are identified on agar / LB medium supplemented with ampicillin.
Les ADNc présents dans ces colonies sont amplifiés de façon individuelle par amplification PCR à l'aide d'amorces Sp6 et T7 selon le protocole suivant :The cDNAs present in these colonies are amplified individually by PCR amplification using primers Sp6 and T7 according to the following protocol:
Primer T7 10μ M : 2μL Primer Sp6 10μM 2μLPrimer T7 10μ M: 2μL Primer Sp6 10μM 2μL
MgCI2 50mM: 1.2μLMgCI2 50mM: 1.2μL
DNTPs 2mM: 4μLDNTPs 2mM: 4μL
Tampon 10x 4μL Taq polymerase: 0.2μLBuffer 10x 4μL Taq polymerase: 0.2μL
H20: 25.6μLH20: 25.6μL
Colonie : 1 μLColony: 1 μL
vol final 40μLfinal flight 40μL
selon le programme de cycles suivant:according to the following cycle program:
94°C 5min94 ° C 5min
94°C 30sec)94 ° C 30sec)
55°C 30sec) 30cycles55 ° C 30sec) 30cycles
72°C 1min)72 ° C 1min)
72°C 5min72 ° C 5min
Les produits d'amplification sont ensuite purifiés par P100 pour être séquences à l'aide du kit « Big Dye Terminator » de la société Applied Biosystems selon le protocole fourni par ce fournisseur. Les réactions de séquence sont analysées à l'aide d'un séquenceur 3100 d'Applied Biosystems. Le tableau 1 représente les différents ADNc ainsi que les couples d'oligonucleotides amorces utilisés pour leur obtention et permettant leur amplification dans un échantillon. La séquence complète des variants est fournie dans la liste de séquence jointe.The amplification products are then purified by P100 to be sequenced using the “Big Dye Terminator” kit from the company Applied Biosystems according to the protocol provided by this supplier. Sequence reactions are analyzed using an Applied Biosystems 3100 sequencer. Table 1 represents the various cDNAs as well as the pairs of priming oligonucleotides used for obtaining them and allowing their amplification in a sample. The complete sequence of variants is provided in the attached sequence list.
B - UTILISATION D'UN MICRO-ARRAY COMPORTANT DES OLIGONUCLEOTIDES DE JONCTIONB - USE OF A MICRO-ARRAY COMPRISING JUNCTION OLIGONUCLEOTIDES
L'expression de variants de PSA et de KLK2 décrits dans cette invention a été établie et validée à l'aide d'un micro-array comportant des oligonucléotides capables de s'hybrider de façon spécifique avec ces variants. Le principe et la validation de cette approche a déjà été décrit pour d'autres variants de PSA et de KLK2 dans la demande PCT/FR03/00833, appartenant au demandeur. Sur la base de leurs séquences, les variants d'épissage de PSA et de KLK2 proviennent d'événements de natures différentes, mais présentant tous des délétions de séquences nucléiques bien définies qui créent ainsi de nouvelles jonctions bien spécifiques. Des oligonucléotides discriminants sont dessinés pour être complémentaires de (i.e., placés sur) l'une ou l'autre de ces nouvelles jonctions.The expression of PSA and KLK2 variants described in this invention was established and validated using a micro-array comprising oligonucleotides capable of specifically hybridizing with these variants. The principle and validation of this approach has already been described for other variants of PSA and KLK2 in application PCT / FR03 / 00833, belonging to the applicant. Based on their sequences, the PSA and KLK2 splicing variants come from events of different natures, but all presenting deletions of well defined nucleic sequences which thus create new very specific junctions. Discriminating oligonucleotides are drawn to be complementary to (ie, placed on) one or the other of these new junctions.
B1 - Description du micro-arrav à oligonucléotides de jonction.B1 - Description of the micro-arrav with junction oligonucleotides.
Cette étude a consisté à générer plusieurs oligonucléotides de 24 à 25 mers. La séquence de ces oligonucléotides est décrite dans le Tableau 2. Les oligonucléotides « discriminants » sont complémentaires des jonctions spécifiques créées par les variants décrits. Des oligonucléotides correspondant aux formes sauvages sont également inclus.This study consisted in generating several oligonucleotides from 24 to 25 seas. The sequence of these oligonucleotides is described in Table 2. The "discriminating" oligonucleotides are complementary to the specific junctions created by the variants described. Oligonucleotides corresponding to wild forms are also included.
Concernant le design des oligonucléotides, étant donné que les sondes sont plus courtes que les sondes produits PCR classiquement utilisées, il est préférable de vérifier que ces sondes n'hybrident pas de manière aspécifique d'autres gènes que celui pour lequel elles ont été dessinées. De plus, il est hautement souhaitable de s'assurer que les oligonucléotides ne présentent pas de structures secondaires qui pourraient interférer avec leur capacité d'hybridation.Regarding the design of oligonucleotides, given that the probes are shorter than the PCR product probes conventionally used, it is preferable to verify that these probes do not hybridize in an aspecific manner other genes than the one for which they were designed. In addition, it is highly desirable to ensure that the oligonucleotides do not have secondary structures which could interfere with their hybridization capacity.
D'un manière générale, il est préférable d'avoir sur la puce un profil thermodynamique homogène pour l'ensemble des oligos générés, à savoir le Tm (65°C) et la longueur (24-25 mers). Au cours de leur synthèse, les oligonucléotides sont par ailleurs modifiés en 5' par un groupement NH2-C6 favorisant leur flexibilité et permettant d'établir une liaison covalente avec le polymère constituant le revêtement de la lame de verre.In general, it is preferable to have a homogeneous thermodynamic profile on the chip for all of the oligos generated, namely the Tm (65 ° C) and the length (24-25 seas). During their synthesis, the oligonucleotides are moreover modified in 5 ′ by an NH 2 -C6 group promoting their flexibility and making it possible to establish a covalent bond with the polymer constituting the coating of the glass slide.
Concernant plus précisément les oligonucléotides de jonction, ils sont idéalement centrés sur les jonctions, mais nous avons considéré également les possibilités d'oligonucleotides décalés sur la jonction. En terme de logiciel de design d'oligonucleotides, nous avons sélectionné le logiciel Primer Finder.Concerning more precisely the junction oligonucleotides, they are ideally centered on the junction, but we have also considered the possibilities of oligonucleotides shifted on the junction. In terms of oligonucleotide design software, we have selected the Primer Finder software.
Les critères que nous avons sélectionnés sont les suivants :The criteria we have selected are as follows:
- %GC : 40% à 60% pour les 24-mers et les 30-mers, 30% à 60% pour les 40 mers.-% GC: 40% to 60% for 24 seas and 30 seas, 30% to 60% for 40 seas.
- Concentrations en oligonucléotides : 50 nM- Concentrations of oligonucleotides: 50 nM
- Concentrations en sels : 50 mM- Salt concentrations: 50 mM
- Ignorer les oligonucléotides ayant des tendances à former des structures secondaires en « épingles à cheveux » ou ayant des possibilités de s'autodimériser.- Ignore oligonucleotides which tend to form secondary structures in "hairpins" or which have the possibility of self-dimerization.
Ces oligonucléotides ont été synthétisés et repris à une concentration de 25 μM dans un tampon Phosphate de Sodium 150 mM. Les oligonucléotides ont ensuite été déposés en quadruplet sur lames de verre (Codelink , Amersham), puis ces lames ont été incubées dans une chambre saturante en NaCI pendantThese oligonucleotides were synthesized and taken up at a concentration of 25 μM in a 150 mM sodium phosphate buffer. The oligonucleotides were then deposited in quadruplet on glass slides (Codelink, Amersham), then these slides were incubated in a saturated NaCl chamber for
16h. Les sites réactifs non utilisés sont ensuite bloqués en utilisant une solution d'éthanolamine 50 mM, Tris 0.1 M, SDS 0.1% à pH 9. Les lames sont ensuite lavées dans une solution à 4xSSC/0.1%SDS.16h. The unused reactive sites are then blocked using a 50 mM ethanolamine solution, 0.1 M Tris, 0.1% SDS at pH 9. The slides are then washed in a 4xSSC / 0.1% SDS solution.
Les cibles (acides nucléiques provenant d'un échantillon à tester) sont hybridées dans le tampon 5X SSC, 0.1% SDS, 0.1 mg/ml ADN de sperme de saumon, à une température de 55°C pendant 16h. Elles sont ensuite lavées en augmentant la stringence des bains :The targets (nucleic acids coming from a test sample) are hybridized in the 5X SSC buffer, 0.1% SDS, 0.1 mg / ml DNA of salmon sperm, at a temperature of 55 ° C. for 16 h. They are then washed by increasing the stringency of the baths:
4X SSC pour éliminer le cover slip4X SSC to eliminate cover slip
2X SSC / 0.1% SDS pendant 5min à 55°C 0.2X SSC pendant 5 minutes à température ambiante2X SSC / 0.1% SDS for 5 min at 55 ° C 0.2X SSC for 5 minutes at room temperature
0.1X SSC pendant 5 minutes à température ambiante0.1X SSC for 5 minutes at room temperature
C2 - Etudes d'échantillons tumoraux et sains de patients Des ARNs provenant de tissus bénins et tumoraux prostatiques de patients atteints de cancer de la prostate (Genomics Collaborative, USA) ont servi de matrice aux expériences d'hybridations suivantes.C2 - Studies of tumor and healthy samples from patients RNAs from benign and prostate tumor tissue from patients with prostate cancer (Genomics Collaborative, USA) served as a template for the following hybridization experiments.
Puisque la quantité de matériel biologique dont nous disposons le plus souvent est faible, nous avons eu recours à une amplification d'ARN . La première étape consiste à reverser de l'ARNm en présence d'oligodT grâce à la Superscript II.Since the amount of biological material most often available to us is small, we have used RNA amplification. The first step is to transfer mRNA in the presence of oligodT using Superscript II.
La RnaseH va dégrader l'ARN ayant servi de matrice et laisser des amorces qui seront utilisées par la DNA polymerase I pour la synthèse du second brin d'ADNc. Les fragments synthétisés sont assemblés par la DNA ligase. A l'issue de cette étape , on se retrouve avec une structure d'ADN double brin, reconnue par la T7 DNA polymerase, qui va amplifier le brin correspondant à la séquence du messager et synthétiser des molécules de cRNA qui s'hybrideront aux sondes de séquence complémentaire (sens du mRNA).RnaseH will degrade the RNA used as a template and leave primers which will be used by DNA polymerase I for the synthesis of the second strand of cDNA. The synthesized fragments are assembled by DNA ligase. At the end of this step, we are left with a double stranded DNA structure, recognized by the T7 DNA polymerase, which will amplify the strand corresponding to the messenger sequence and synthesize cRNA molecules which will hybridize to the probes. of complementary sequence (sense of mRNA).
Pour chaque patient, 8 μg de cibles correspondant aux échantillons tumoraux et sains et marqués à l'aide de 2 fluorochromes différents ont été cohybrides sur une même lame. Les intensités de fluorescence ont été mesurées dans les 2 canaux et normalisées selon la méthode de l'intensité globale dans un logiciel d'analyse de fluorescence sur lame de verre (GeneTraffic).For each patient, 8 μg of targets corresponding to the tumor and healthy samples and marked with 2 different fluorochromes were cohybrid on the same slide. The fluorescence intensities were measured in the 2 channels and normalized according to the global intensity method in a fluorescence analysis software on a glass slide (GeneTraffic).
Pour les 4 patients analysés nous pouvons faire les remarques suivantes :For the 4 patients analyzed we can make the following remarks:
- La qualité du signal de fluorescence est bonne, étant donné que le bruit de fonds était de l'ordre de 150-200 unités de fluorescence (Tableau 3);- The quality of the fluorescence signal is good, given that the background noise was of the order of 150-200 fluorescence units (Table 3);
- L'oligonucléotide KLK2-482-739 correspondant à KLK2-EHT101 produit des signaux de fluorescence significatifs parmi les quatre patients, supérieurs à l'oligonucléotide KLK2-482-483 correspondant à la forme KLK2 de type sauvage;- The oligonucleotide KLK2-482-739 corresponding to KLK2-EHT101 produces significant fluorescence signals among the four patients, superior to the oligonucleotide KLK2-482-483 corresponding to the wild type KLK2;
- L'oligonucléotide KLK2-1031 -1775 correspondant à KLK2-EHT102 produit des signaux de fluorescence significatifs parmi deux des quatre patients;- The oligonucleotide KLK2-1031 -1775 corresponding to KLK2-EHT102 produces significant fluorescence signals among two of the four patients;
- L'oligonucléotide PSA-635-919 correspondant à PSA-EHT101 produit des signaux de fluorescence significatifs parmi les quatre patients;- The oligonucleotide PSA-635-919 corresponding to PSA-EHT101 produces significant fluorescence signals among the four patients;
- L'oligonucléotide PSA-635-964 correspondant à PSA-EHT102 produit des signaux de fluorescence significatifs parmi les quatre patients. Ces deux derniers oligonucléotides produisent des signaux supérieurs à ceux produits par les oligonucléotides PSA-635-636 et PSA-600-630 correspondant tous deux à la forme sauvage de PSA. L'oligonucléotide PSA-1210-1959 correspondant à PSA-EHT103 produit des signaux de fluorescence significatifs parmi un des quatres patients.- The oligonucleotide PSA-635-964 corresponding to PSA-EHT102 produces significant fluorescence signals among the four patients. These last two oligonucleotides produce signals superior to those produced by the oligonucleotides PSA-635-636 and PSA-600-630 both corresponding to the wild form of PSA. The oligonucleotide PSA-1210-1959 corresponding to PSA-EHT103 produces significant fluorescence signals among one of the four patients.
Ces résultats démontrent donc l'expression effective d'isoformes de l'invention dans des tissus prostatiques. Ils démontrent également une expression différentielle entre les tissus bénins et tumoraux, confirmant la pertinence biologique de ces variants pour le suivi de pathologies cancéreuses, notamment du cancer de la prostate. These results therefore demonstrate the effective expression of isoforms of the invention in prostate tissue. They also demonstrate a differential expression between benign and tumor tissues, confirming the biological relevance of these variants for monitoring cancer pathologies, in particular prostate cancer.
REFERENCESREFERENCES
David et.al, (2002) J. Biol. ChemDavid et.al, (2002) J. Biol. Chem
Riegman et al (1988) Biochem. Biophys. Res. Commun. 155, 181-188.Riegman et al (1988) Biochem. Biophys. Res. Common. 155, 181-188.
Riegman et.al (1991) Mol. Cell. Endicronol. 76, 181-190.Riegman et.al (1991) Mol. Cell. Endicronol. 76, 181-190.
Liu et.al (1999) Biochem. Biophys. Res. Commun. 264, 833-839Liu et.al (1999) Biochem. Biophys. Res. Common. 264, 833-839
Heuze et.al (1999) Cancer Res. 59, 2820-2824.Heuze et.al (1999) Cancer Res. 59, 2820-2824.
Tanaka et.al (2000) Cancer Res. 60, 56-59. Tanaka et.al (2000) Cancer Res. 60, 56-59.
Tableau 1Table 1
Figure imgf000034_0001
Figure imgf000034_0001
Tableau 2Table 2
Figure imgf000034_0002
Tableau 3
Figure imgf000035_0001
Figure imgf000034_0002
Table 3
Figure imgf000035_0001
Figure imgf000035_0002
Figure imgf000035_0002
Signaux de fluorescence obtenus à partir d'ARNS issus de tissus prostatiques bénins et tumoraux. Colonnes B, E, H,K : Signaux à partir des tissus sains. Colonnes C,F,I,L : Signaux à partir des tissus tumoraux. Colonnes D,G,J,M : Rapport des signaux bénins / tumoraux pour les quatre patients. Fluorescence signals obtained from RNAs from benign and tumor prostate tissue. Columns B, E, H, K: Signals from healthy tissue. Columns C, F, I, L: Signals from tumor tissue. Columns D, G, J, M: Ratio of benign / tumor signals for the four patients.

Claims

REVENDICATIONS
1. Acide nucléique caractérisé en ce qu'il comprend une séquence choisie parmi : a) les séquences SEQ ID NOs : 1 à 7, b) un variant des séquences SEQ ID NOs : 1 à 7 résultant de la dégénérescence du code génétique, c) le brin complémentaire des séquences SEQ ID NOs : 1 à 7, et d) un fragment spécifique des séquences a) à c) comportant au moins 5 nucléotides consécutifs, le fragment portant au moins une altération génétique (par exemple une mutation, une nouvelle jonction ou une rétention d'intron) caractéristique de ladite séquence.1. Nucleic acid characterized in that it comprises a sequence chosen from: a) the sequences SEQ ID NOs: 1 to 7, b) a variant of the sequences SEQ ID NOs: 1 to 7 resulting from the degeneration of the genetic code, c ) the complementary strand of the sequences SEQ ID NOs: 1 to 7, and d) a specific fragment of the sequences a) to c) comprising at least 5 consecutive nucleotides, the fragment carrying at least one genetic alteration (for example a mutation, a new junction or intron retention) characteristic of said sequence.
2. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il s'agit d'un ADN, choisi de préférence parmi les ADNc et les ADNg, ou d'un ARN.2. Nucleic acid according to claim 1, characterized in that it is a DNA, preferably chosen from cDNAs and gDNAs, or an RNA.
3. Polypeptide codé par un acide nucléique selon l'une quelconque des revendications précédentes, de préférence choisi parmi un polypeptide comprenant tout ou une partie spécifique d'une séquence choisie parmi SEQ ID Nos : 8 à 23.3. A polypeptide encoded by a nucleic acid according to any one of the preceding claims, preferably chosen from a polypeptide comprising all or a specific part of a sequence chosen from SEQ ID Nos: 8 to 23.
4. Protéine choisie parmi les variants KLK2-EHT101 à KLK2-EHT103 et PSA-EHT101 à PSA-EHT104 de séquence SEQ ID Nos : 8 à 23, respectivement.4. Protein chosen from variants KLK2-EHT101 to KLK2-EHT103 and PSA-EHT101 to PSA-EHT104 of sequence SEQ ID Nos: 8 to 23, respectively.
5. Sonde nucléique caractérisée en ce qu'elle permet la détection par hybridation sélective d'un acide nucléique selon l'une des revendications 1 à 2. 5. Nucleic probe characterized in that it allows the detection by selective hybridization of a nucleic acid according to one of claims 1 to 2.
6. Sonde selon la revendication 5, caractérisée en ce qu'elle comprend la séquence d'un acide nucléique selon l'une des revendications 1 à 2.6. A probe according to claim 5, characterized in that it comprises the sequence of a nucleic acid according to one of claims 1 to 2.
7. Sonde selon la revendication 6, caractérisée en ce qu'elle comprend de 10 à 1000 nucléotides, de préférence de 50 à 800.7. A probe according to claim 6, characterized in that it comprises from 10 to 1000 nucleotides, preferably from 50 to 800.
8. Amorce, caractérisée en ce qu'elle permet l'amplification sélective d'un acide nucléique selon l'une des revendications 1 à 2.8. Primer, characterized in that it allows the selective amplification of a nucleic acid according to one of claims 1 to 2.
9. Amorce selon la revendication 8, caractérisée en ce qu'elle est composée de 3 à 50 bases, de préférence de 3 à 40 et encore plus préférentiellement de 3 à 35 bases.9. A primer according to claim 8, characterized in that it is composed of 3 to 50 bases, preferably from 3 to 40 and even more preferably from 3 to 35 bases.
10. Amorce selon l'une des revendications 8 ou 9, caractérisée en ce qu'elle est complémentaire d'une région au moins du gène codant pour l'antigène spécifique de PSA ou de celui codant pour KLK-2 portant une mutation impliquée dans un cancer.10. Primer according to one of claims 8 or 9, characterized in that it is complementary to at least one region of the gene coding for the specific PSA antigen or of that coding for KLK-2 carrying a mutation involved in cancer.
11. Amorce selon la revendication 10, caractérisée en ce qu'elle est constituée d'un acide nucléique simple-brin comprenant de 3 à 50 nucléotides complémentaires d'une partie au moins de l'une des séquences SEQ ID NO : 1 à 7 ou de leur brin complémentaire.11. Primer according to claim 10, characterized in that it consists of a single-stranded nucleic acid comprising from 3 to 50 nucleotides complementary to at least part of one of the sequences SEQ ID NO: 1 to 7 or their complementary strand.
12. Couple d'amorces comprenant une séquence sens et une séquence inverse, caractérisé en ce que les amorces de ladite paire s'hybrident avec une région d'un acide nucléique selon l'une des revendications 1 à 2 et permettent l'amplification d'au moins une portion de cet acide nucléique.12. A pair of primers comprising a sense sequence and a reverse sequence, characterized in that the primers of said pair hybridize with a region of a nucleic acid according to one of claims 1 to 2 and allow the amplification of 'at least a portion of this nucleic acid.
13. Anticorps, caractérisé en ce qu'il est spécifique d'une protéine ou d'un polypeptide selon les revendications 3 ou 4. 13. Antibody, characterized in that it is specific for a protein or a polypeptide according to claims 3 or 4.
14. Anticorps selon la revendication 13, caractérisé en ce qu'il s'agit d'un polyclonal, d'un monoclonal ou d'un dérivé de ceux-ci.14. Antibody according to claim 13, characterized in that it is a polyclonal, a monoclonal or a derivative thereof.
15. Procédé de détection d'une pathologie ou d'une prédisposition à une pathologie chez un sujet, comprenant la détermination de la présence, dans un échantillon dudit sujet, d'un acide nucléique selon l'une des revendications 1 à 2 ou d'une protéine ou polypeptide selon les revendications 3 et 4.15. Method for detecting a pathology or a predisposition to a pathology in a subject, comprising determining the presence, in a sample of said subject, of a nucleic acid according to one of claims 1 to 2 or d 'a protein or polypeptide according to claims 3 and 4.
16. Procédé selon la revendication 15, caractérisé en ce que la détermination est réalisée par séquençage, hybridation et/ou amplification sélectives.16. Method according to claim 15, characterized in that the determination is carried out by sequencing, hybridization and / or selective amplification.
17. Procédé selon la revendication 16, caractérisé en ce que l'amplification est réalisée en utilisant un couple d'amorces selon la revendication 12.17. Method according to claim 16, characterized in that the amplification is carried out using a pair of primers according to claim 12.
18. Kit utilisable pour la mise en œuvre d'un procédé tel que défini dans les revendications 15 à 17 comprenant18. Kit usable for the implementation of a method as defined in claims 15 to 17 comprising
- un couple d'amorces selon la revendication 12 ou une sonde selon l'une des revendications 5 à 7 ou un anticorps selon l'une des revendications 13 et 14, et les réactifs nécessaires à l'amplification ou à une réaction d'hybridation ou immunologique.- a pair of primers according to claim 12 or a probe according to one of claims 5 to 7 or an antibody according to one of claims 13 and 14, and the reagents necessary for the amplification or for a hybridization reaction or immunological.
19. Méthode de sélection ou d'identification de composés actifs, comprenant la mise en contact in vitro ou ex vivo d'un composé test avec une cellule exprimant un polypeptide selon la revendication 3, et la sélection ou l'identification de composés modulant l'expression ou l'activité dudit polypeptide. 19. A method of selection or identification of active compounds, comprising contacting in vitro or ex vivo a test compound with a cell expressing a polypeptide according to claim 3, and the selection or identification of compounds modulating l expression or activity of said polypeptide.
20. Méthode selon la revendication 19, caractérisée en ce qu'elle comprend la sélection des composés se liant audit polypeptide.20. Method according to claim 19, characterized in that it comprises the selection of the compounds binding to said polypeptide.
21. Méthode selon la revendication 19, caractérisée en ce qu'elle comprend la sélection des composés modulant l'expression du dit polypeptide.21. Method according to claim 19, characterized in that it comprises the selection of the compounds modulating the expression of said polypeptide.
22. Vecteur comprenant un acide nucléique selon la revendication 1 ou 2.22. Vector comprising a nucleic acid according to claim 1 or 2.
23. Cellule recombinant comprenant un vecteur selon la revendication 22.23. Recombinant cell comprising a vector according to claim 22.
24. Produit comprenant un acide nucléique selon l'une des revendications 1 , 2, 5, 6 et 7, un vecteur selon la revendication 22, un polypeptide selon la revendication 3 ou 4 ou un anticorps selon la revendication 13 ou 14 immobilisé sur un support. 24. Product comprising a nucleic acid according to one of claims 1, 2, 5, 6 and 7, a vector according to claim 22, a polypeptide according to claim 3 or 4 or an antibody according to claim 13 or 14 immobilized on a support.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046403A1 (en) * 1998-03-11 1999-09-16 Exonhit Therapeutics S.A. Qualitative differential screening
WO2000011015A1 (en) * 1998-08-24 2000-03-02 Alphagene, Inc. Secreted proteins and polynucleotides encoding them
WO2000049158A2 (en) * 1999-02-18 2000-08-24 Compugen Ltd. Psa and klk-2 splicing variants
WO2003076610A2 (en) * 2002-03-14 2003-09-18 Exonhit Therapeutics Sa Variants of human kallikrein-2 and kallikrein-3 and uses thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3395900A (en) * 1999-03-12 2000-10-04 Human Genome Sciences, Inc. Human lung cancer associated gene sequences and polypeptides
EP1368475A4 (en) * 2001-03-15 2004-10-20 Nuvelo Inc Novel nucleic acids and polypeptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046403A1 (en) * 1998-03-11 1999-09-16 Exonhit Therapeutics S.A. Qualitative differential screening
WO2000011015A1 (en) * 1998-08-24 2000-03-02 Alphagene, Inc. Secreted proteins and polynucleotides encoding them
WO2000049158A2 (en) * 1999-02-18 2000-08-24 Compugen Ltd. Psa and klk-2 splicing variants
WO2003076610A2 (en) * 2002-03-14 2003-09-18 Exonhit Therapeutics Sa Variants of human kallikrein-2 and kallikrein-3 and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DAVID ANAT ET AL: "Unusual alternative splicing within the human kallikrein genes KLK2 and KLK3 gives rise to novel prostate-specific proteins." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 20, 17 mai 2002 (2002-05-17), pages 18084-18090, XP002241550 May 17, 2002 ISSN: 0021-9258 cité dans la demande *
EXONHIT THERAPEUTICS: "Exonhit Therapeutics and IDEC Pharmaceuticals announce collaborative agreement in target discovery" PRESS RELEASE, 1 juillet 2002 (2002-07-01), XP002222065 *
SHARIAT SHAHROKH F ET AL: "Preoperative blood reverse transcriptase-PCR assays for prostate-specific antigen and human glandular kallikrein for prediction of prostate cancer progression after radical prostatectomy" CANCER RESEARCH, vol. 62, no. 20, 15 octobre 2002 (2002-10-15), pages 5974-5979, XP002290010 ISSN: 0008-5472 *
YOUSEF GEORGE M ET AL: "Human tissue kallikreins: A new enzymatic cascade pathway?" BIOLOGICAL CHEMISTRY, vol. 383, no. 7-8, juillet 2002 (2002-07), pages 1045-1057, XP008033390 ISSN: 1431-6730 *

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