EP1631665A1 - Neue phosphorylierte sequenzen der phosphatase cdc25b, antikörper für die sequenzen und verwendung davon - Google Patents

Neue phosphorylierte sequenzen der phosphatase cdc25b, antikörper für die sequenzen und verwendung davon

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Publication number
EP1631665A1
EP1631665A1 EP04767282A EP04767282A EP1631665A1 EP 1631665 A1 EP1631665 A1 EP 1631665A1 EP 04767282 A EP04767282 A EP 04767282A EP 04767282 A EP04767282 A EP 04767282A EP 1631665 A1 EP1631665 A1 EP 1631665A1
Authority
EP
European Patent Office
Prior art keywords
phosphorylated
peptide sequence
antibody
cdc25b
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04767282A
Other languages
English (en)
French (fr)
Inventor
Bernard Ducommun
Bernard Monsarrat
Claude Prigent
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Universite de Rennes 1
Universite de Toulouse
Original Assignee
Centre National de la Recherche Scientifique CNRS
Universite de Rennes 1
Universite Toulouse III Paul Sabatier
Universite de Toulouse
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Universite de Rennes 1, Universite Toulouse III Paul Sabatier, Universite de Toulouse filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP1631665A1 publication Critical patent/EP1631665A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the subject of the present invention is new phosphorylated sequences of the CDC25B phosphatase as well as polyclonal or monoclonal antibodies directed against these sequences.
  • the present invention also relates to the use of these new phosphorylated sequences in particular for the implementation of a method of in vitro diagnosis of cancers in humans or animals.
  • CDC25B is a regulating phosphatase of the cell cycle essential for the control of the entry into mitosis. It belongs to a family which has three members coded by different genes (CDC25A, B and C) in mammals. The CDC25B protein is expressed and active at the end of the G2 phase of the cell cycle (Baldin et al.,
  • CDC25B can act as a "starter" for early mitotic events (Nllsson et al., 2000). It could play a role in the initial activation of a CDC2 / cyclin B population at the centrosome before its nuclear translocation (Kumagai et al., 1992; Hoffmann et al., 1993). CDC25B activates the CDK / cyclin complexes to allow the architectural and biochemical changes that are necessary to allow the process of cell division. Its activity is regulated by variations in its expression, by its association with regulatory partners and by phosphorylation events.
  • the protein kinase Aurora A also known as STK5
  • STK5 The protein kinase Aurora A
  • This expression is correlated with a high tumor grade (Bischoff et al., 1998; Zhou et al., 1998).
  • This kinase is encoded by the STKl 5 gene localized as locus 20ql 3, an amplicon present in many tumors.
  • This protein is located at the centrosome (Dutertre et al., 2002). . Its function seems important for the separation of centrosomes (Giet et al., 2000), their duplication (Zhou et al., 1998) and the assembly of a bipolar mitotic spindle (Giet et al., 2000).
  • I 1 RNA interference technology leads to the formation of monopolar spindles and its overexpression is responsible for a centrosomal amplification and a polyploidization (Meraldi et al., 2002; Bischoff et al, 1998; Zhou et al., 1998).
  • the CDC25B phosphatase is the first identified substrate which is also co-located at the centrosome level and plays a clear role in controlling the cell cycle and proliferation.
  • the present invention derives from the discovery by the Inventors of the site of in vitro phosphorylation of the phosphatase CDC25B by the kinase Aurora A, and from the identification of the phosphorylated sequence of the splicing variant CDC25B3 of
  • One of the aims of the present invention is to provide new phosphorylated sequences of the different variants of the CDC25B phosphatase.
  • Another object of the invention consists in providing a new antibody directed against a phosphorylated CDC25B phosphatase, said antibody being able to be used within the framework of a medical diagnosis, or for the preparation of screens for the identification of molecules binding said phosphorylated sequence and likely to represent new agents usable in anti-tumor pharmacology.
  • Another object of the invention consists in providing a new tool for the study of molecular mechanisms which lead to polyploidization and cell transformation, as well as a new tool allowing the demonstration of the activity of the Aurora kinase. Has on one of its physiological substrates, and therefore the identification of possible quantitative, temporal and spatial disturbances of this activity.
  • the present invention relates to a peptide sequence characterized in that it comprises or consists of a fragment of at least approximately 10 amino acids derived from the following sequence SEQ ID NO: 1:
  • phosphorylated residue designates an amino acid carrying a phosphate group.
  • the present invention also relates to a peptide sequence as defined above, characterized in that it comprises or consists of the following sequence SEQ ID NO: 2:
  • sequence SEQ ID NO: 2 corresponds to a fragment of the sequence SEQ ID ' NO: 1 mentioned above. More precisely, it corresponds to the fragment of SEQ ID NO: 1 delimited from the amino acid in position 4 to the amino acid in position 17.
  • the present invention also relates to a peptide sequence as defined above, characterized in that it comprises or consists of one of the following sequences:
  • sequence SEQ ID NO: 3 representing the splicing variant CDC25B1 of the protein of human origin of the phosphatase CDC25B, the serine residue of which at position 339 is phosphorylated
  • sequence SEQ ID NO: 4 representing a variant splicing CDC25B2 of the protein of human origin of the phosphatase CDC25B, the serine residue of which at position 312 is phosphorylated
  • sequence SEQ TD NO: 5 representing a splicing variant CDC25B3 of the protein of human origin of the phosphatase CDC25B, the serine residue of which at position 353 is phosphorylated
  • sequence SEQ TD NO: 6 representing a splicing variant CDC25B4 of the protein of human origin of the phosphatase CDC25B, the serine residue of which at position 374 is phosphorylated
  • 361 is phosphorylated.
  • the present invention also relates to a polyclonal or monoclonal antibody capable of recognizing a peptide sequence as defined above.
  • An advantageous polyclonal antibody of the invention is characterized in that it is capable of recognizing the sequence SEQ ID NO: 2 as defined above.
  • Such an antibody directed against the phosphorylated epitope of sequence SEQ ED NO: 2 is generated by immunizing rabbits with said epitope. More specifically, said epitope is covalently coupled with a carrier protein such as hemocyanin, BSA or Povalbumin. The rabbits are then immunized for 3 months (4 injections in total) and the final bleeding allows the recovery of approximately 50 ml of serum. The serum is then doubly purified by affinity on a column of phosphorylated peptide and then on a column of non-phosphorylated peptide.
  • a carrier protein such as hemocyanin, BSA or Povalbumin
  • the present invention also relates to a process for the preparation of a monoclonal antibody as defined above, directed against the peptide sequence SEQ ED NO: 2 as defined above, characterized in that it comprises the following steps: - immunization of an animal by injection of the peptide sequence
  • the animal used for the immunization step is in particular a mouse.
  • the myébmes used for fusion come in particular from a mouse.
  • the splenocytes used for the fusion come from an animal of the same species as that from which the myelomas originate, namely in particular a mouse.
  • Hybridomas are chosen which secrete the antibodies against the peptide sequence SEQ ED NO: 2 on the basis of the production of antibodies capable of recognizing in an ELISA test the phosphorylated peptide used for immunization but not the nonphosphorylated peptide.
  • the present invention also relates to a pharmaceutical composition characterized in that it contains, as active substance, a peptide sequence as defined above, an antibody as defined above, or an antibody anti-idiotypic as defined above, in combination with a pharmaceutically acceptable carrier.
  • An advantageous pharmaceutical composition according to the invention is characterized in that it contains, as active substance, the peptide sequence represented by the sequence SEQ ID NO: 2.
  • the present invention also relates to the use of a peptide sequence as defined above, of an antibody as defined above, or of an anti-idiotypic antibody as defined above, for the preparation of drugs used to treat cancers, such as breast cancer.
  • the present invention also relates to the use of an antibody as defined above, for the implementation of a method of in vitro diagnosis of cancers in humans or animals, in particular breast cancers.
  • the present invention relates to the use of a polyclonal antibody as defined above, directed against the phosphorylated epitope of sequence SEQ ID NO: 2, for the implementation of a method of
  • the present invention also relates to a method of in vitro diagnosis of cancers, in particular of breast cancers, in humans or animals, characterized in what it includes: - bringing an antibody as defined above into contact with a biological sample taken from an individual, said antibody being optionally fixed on a solid support,
  • a peptide sequence as defined above capable of being present in the biological sample using labeled reagents, in particular labeled antibodies, recognizing either the antibody linked to said peptide sequence, either the peptide sequence linked to said antibody in the complexes formed during the previous step between the antibody and the peptide sequence likely to be present in the biological sample, and this, if necessary, after appropriate rinsing of the solid support.
  • the present invention also relates to a method for in vitro prognosis of cancers, in particular breast cancers, in humans or animals, characterized in that it comprises:
  • an antibody as defined above into contact with a tumor sample taken from an individual, said antibody being optionally fixed on a solid support, the detection of a peptide sequence as defined above, capable of being present in the biological sample using labeled reagents, in particular labeled antibodies, recognizing either the antibody linked to said peptide sequence, or the peptide sequence linked to said antibody in the complexes formed during the previous step between the antibody and the peptide sequence likely to be present in the biological sample, and this, if necessary, after appropriate rinsing of the solid support.
  • the present invention also relates to the use of the abovementioned antibodies of the invention directed against a phosphorylated sequence of CDC25B within the framework of the implementation of a diagnostic test aiming to detect on tumor samples the presence or not of this sequence phosphorylated, this for diagnostic or prognostic purposes.
  • the present invention also relates to a method for screening a molecule capable of binding to a peptide sequence as defined above, said molecule being capable of being used as an anti-tumor agent or an anti-proliferative agent both on cells in culture only in a living organism or against infectious agents (parasites, pathogenic fungi), characterized in that it comprises:
  • the bond between said molecule and the phosphorylated peptide sequence can be detected according to the following method: the phosphorylated sequence (phosphorylated substrate) is linked to a solid support; incubation with the above-mentioned antibody in solution then allows its fixation which is revealed by the use of a secondary antibody carrying a chromophore or by the direct labeling of the primary antibody (antibody of the invention directed against the phosphorylated sequence). Simultaneous incubation with a compound capable of binding said phosphorylated sequence results in its fixation and the masking of the site recognized by the antibody. The visualization of this interaction could therefore be carried out and quantified by the drop in binding of the antibody. DESCRIPTION OF THE FIGURES
  • Figure 1 shows a mass spectrum of the monophosphorylated peptide, 353- S (P) VTPPEEQQEAEEPK-367.
  • the abscissa axis corresponds to the m / z ratio and the ordinate axis corresponds to the percentage of relative abundance.
  • Figures 2A, 2B and 2C show the results of western blot analyzes with the monoclonal antibody SE96 (Figure 2A), with the anti- ⁇ MBP antibody (New England Biolabs) ( Figure 2B) and with the anti- ⁇ Aurora antibody A (see French patent application 02/07212) ( Figure 2C).
  • the first column corresponds to the protein kinase Aurora A
  • the second column to a recombinant protein MBP-CDC25B
  • the third column corresponds to the Aurora A protein kinase and to the recombinant protein MBP-CDC25B
  • the fourth column corresponds to MBP alone.
  • FIGS. 3a to 3h represent images of indirect immunofluorescence produced on HeLa cells with the antibody SE96.
  • the HeLa cells of FIGS. 3 c and 3d were put in competition with the phosphorylated peptide which served for the immunization (SEQ ID NO: 2); the HeLa cells of Figures 3e and 3f were competed with the non-phosphorylated peptide (QNECRRRSVTPPEEQ); and the HeLa cells of Figures 3g and 3h were competed with a phosphorylated peptide unrelated to serine 353
  • the recombinant protein CDC25B3 is phosphorylated in vitro by the recombinant kinase Aurora A.
  • the product of the phosphorylation reaction was analyzed by mass spectrometry after excision of the electrophoresis gel and tryptic digestion.
  • the MSMS spectrum of the monophosphorylated peptide, 353-SVTPPEEQQEAEEPK-367 is presented in Figure 1. Its analysis indicates that it is the serine 353 which is phosphorylated by the kinase.
  • the recombinant Aurora A kinase phosphorylates
  • the peptide of sequence Q] SIE-RRRS (P) VTPPEEQ was used for the immunization of rabbits. After sacrifice of the animals, the serum was purified by chromatography in two stages: the first on a column of phosphorylated peptide to retain the specific antibodies, then the second on a column of the same non-phosphorylated peptide of sequence QNKRRRSVTPPEEQ, so as to purify in
  • the SE96 antibody recognizes CDC25B phosphorylated by Aurora A
  • HeLa cells were fixed and used to perform immunofluorescence analysis with SE96 antibodies. The cells were also stained with 4'-6 diamino-2-phenylindole (DAPI) to locate the nucleus.
  • DAPI 4'-6 diamino-2-phenylindole
  • the images shown in Figure 3 are representative of observations on a large number of cells. They indicate that the protein CDC25B phosphorylated on serine 353 is localized at the level of the centrosomes of the cells in mitosis. This marking is abolished when a competition is carried out with the phosphorylated peptide used for immunization (SEQ ID NO: 2), but not with the non-phosphorylated peptide
  • Tumor amplified kinase STK15 / BTAK induces centrosome amplification, aneuploidy and transformation, Nat Genêt, 20, 189-93.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP04767282A 2003-06-12 2004-06-08 Neue phosphorylierte sequenzen der phosphatase cdc25b, antikörper für die sequenzen und verwendung davon Withdrawn EP1631665A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR0307095A FR2856068B1 (fr) 2003-06-12 2003-06-12 Nouvelles sequences phosphorylees de la phosphatase cd25b, anticorps diriges contre ces sequences ainsi que leur utilisation
PCT/FR2004/001416 WO2004111215A1 (fr) 2003-06-12 2004-06-08 Nouvelles sequences phosphorylees de la phosphatase cdc25b, anticorps diriges contre ces sequences ainsi que leur utilisation

Publications (1)

Publication Number Publication Date
EP1631665A1 true EP1631665A1 (de) 2006-03-08

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EP04767282A Withdrawn EP1631665A1 (de) 2003-06-12 2004-06-08 Neue phosphorylierte sequenzen der phosphatase cdc25b, antikörper für die sequenzen und verwendung davon

Country Status (5)

Country Link
US (1) US20080008712A1 (de)
EP (1) EP1631665A1 (de)
CA (1) CA2528844A1 (de)
FR (1) FR2856068B1 (de)
WO (1) WO2004111215A1 (de)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040151713A1 (en) * 2001-06-04 2004-08-05 Makoto Nakanishi Cell cycle regulatory factor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004111215A1 *

Also Published As

Publication number Publication date
WO2004111215A1 (fr) 2004-12-23
FR2856068B1 (fr) 2005-08-19
FR2856068A1 (fr) 2004-12-17
CA2528844A1 (fr) 2004-12-23
US20080008712A1 (en) 2008-01-10

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