EP1573011A1 - Procede de preparation d'heterocarpine recombinante - Google Patents

Procede de preparation d'heterocarpine recombinante

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Publication number
EP1573011A1
EP1573011A1 EP03815092A EP03815092A EP1573011A1 EP 1573011 A1 EP1573011 A1 EP 1573011A1 EP 03815092 A EP03815092 A EP 03815092A EP 03815092 A EP03815092 A EP 03815092A EP 1573011 A1 EP1573011 A1 EP 1573011A1
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
sequence seq
seq
polypeptide
protein
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Withdrawn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
EP03815092A
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German (de)
English (en)
French (fr)
Inventor
Eric Ferrandis
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.)
Ipsen Pharma SAS
Original Assignee
Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS
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Filing date
Publication date
Application filed by Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS filed Critical Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS
Publication of EP1573011A1 publication Critical patent/EP1573011A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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 invention is a process for the preparation of recombinant heterocarpine.
  • Heterocarpine is a protein with anti-cancer properties described for the first time by the applicant in PCT patent application WO 02/068461.
  • This isolated protein has a molecular mass of approximately 90.9 kDa, contains the fragments of SEQ peptide sequences. ED. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3 (see the part of the description reserved for the sequence list) and is capable of being obtained by extraction of Pilocarpus heterophyllus plant cells cultivated in vitro as described in the abovementioned patent application.
  • the complete sequence of this protein remained unknown to date since the cloning had not been carried out.
  • the present application now describes polynucleotides which can serve as a primer for the cloning of heterocarpine, the DNA coding for heterocaine, the mRNA corresponding to heterocarpine, expression vectors containing said mRNA, cells hosts transformed or transfected with these vectors as well as a process for the preparation of recombinant heterocarpine.
  • the present invention therefore firstly relates to an isolated polynucleotide comprising the polynucleotide sequence SEQ. ED. NO. 8.
  • said isolated polynucleotide consists of the polynucleotide sequence SEQ. ID. NO. 8.
  • an antisense polynucleotide comprising the sequence complementary to that of said isolated polynucleotide comprising the polynucleotide sequence SEQ. ID. NO. 8.
  • said antisense polynucleotide consists of the sequence complementary to the polynucleotide sequence SEQ. ID. NO. 8.
  • the invention also relates to an isolated polynucleotide comprising the polynucleotide sequence SEQ. ID. NO. 8 or one of the fragments thereof, said polynucleotide being such that it codes for a polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and which is associated with the modulation of cell proliferation.
  • said isolated polynucleotide is such that it consists of the polynucleotide sequence SEQ. ID. NO. 8 or a fragment thereof.
  • the invention therefore relates to the isolated polynucleotide of nucleotide sequence SEQ. ED. NO. 9 or the isolated polynucleotide of nucleotide sequence complementary to the nucleotide sequence SEQ. ED. NO. 9.
  • Heterocarpine in other words the protein of sequence SEQ. ID. NO. 10, is encoded by the fragment of the polynucleotide of polynucleotide sequence SEQ. ID. NO. 8 contained between the bases at positions 115 (initiator codon ATG coding for a methionine) and 2437 (stop codon UAA), i.e. by the polynucleotide sequence SEQ. ID. NO. 9.
  • the invention therefore also relates to an expression vector comprising an isolated polynucleotide comprising the polynucleotide sequence SEQ. ED. NO. 8 or one of the fragments of the latter or else the sequence complementary to the polynucleotide sequence SEQ. ID. NO. 8 or one of the fragments thereof, the polypeptide coded by said isolated polynucleotide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation.
  • said expression vector will comprise the polynucleotide sequence SEQ. ID. NO. 9 or the sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9.
  • the invention likewise relates to a host cell transformed or transfected with said expression vector.
  • the invention also relates to an isolated polypeptide comprising a polypeptide of sequence SEQ. ID. NO. 14 or one of its fragments, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation.
  • said isolated polypeptide will consist of a polypeptide of sequence SEQ. ID. NO. 14 or one of its fragments.
  • said polypeptide will have the sequence SEQ. ID. NO. 14.
  • a subject of the invention is also a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds an isolated polypeptide as described above, and in particular a monoclonal antibody, or a binding fragment of it. antigen thereof, which specifically binds the protein of sequence SEQ. ED. NO. 14 but not the protein of sequence SEQ. ID. NO. 10.
  • the invention also relates, as a medicament, to an isolated polynucleotide comprising: the polynucleotide sequence SEQ. ID. NO. 8 or one of its fragments, or - the polynucleotide sequence SEQ. ED. NO. 9 or one of its fragments, said isolated polynucleotide being as it codes for an isolated polypetide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation.
  • the isolated polynucleotide will consist of the polynucleotide of polynucleotide sequence SEQ. ID. NO. 8 or a fragment thereof, or into the polynucleotide of polynucleotide sequence SEQ. ID. NO. 9 or one of its fragments.
  • said isolated polynucleotide will consist of the polynucleotide of polynucleotide sequence SEQ. ED. NO. 8 or into the polynucleotide of polynucleotide sequence SEQ. ED. NO. 9.
  • said isolated polynucleotide used as a medicament will preferably be present in a viral vector, said viral vector being for example selected from the group consisting of an adenovirus, an associated adenovirus, a retrovirus and a pox virus.
  • the invention also relates, as a medicament, to an isolated polypeptide comprising a polypeptide of sequence SEQ. ED. NO. 14 or one of its fragments, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation.
  • said isolated polypeptide will consist of a polypeptide of sequence SEQ. ED. NO. 14 or one of its fragments.
  • said polypeptide will have the sequence SEQ. ID. NO. 14.
  • the invention further relates, as a medicament, to a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds an isolated polypeptide comprising the protein encoded by the polynucleotide sequence SEQ. ID. NO. 9 or one of the fragments of the latter or else by a sequence complementary to the polynucleotide sequence SEQ. ID. NO. 9 or one of the fragments thereof, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation.
  • said monoclonal antibody or said fragment of the antigen thereof specifically binds an isolated polypeptide consisting of the protein encoded by the polynucleotide sequence SEQ.
  • said monoclonal antibody or said fragment of the antigen thereof specifically binds an isolated polypeptide consisting of the protein encoded by the polynucleotide sequence SEQ. ED. NO. 9 or by a sequence complementary to the polynucleotide sequence SEQ. ID. NO. 9 (in other words the protein of sequence SEQ. ID. NO. 10).
  • compositions comprising, as active principle, an isolated polynucleotide comprising: - the polynucleotide sequence SEQ. ID. NO. 8 or one of its fragments, or
  • isolated polynucleotide being such that it codes for an isolated polypetide having at least one immunological and / or biological activity characteristic of a protein binding human GHRH and being associated with the modulation of cell proliferation, with one or more pharmaceutically acceptable excipients.
  • the isolated polynucleotide serving as active ingredient will consist of the polynucleotide of polynucleotide sequence SEQ. ED. NO. 8 or one of its fragments or into the polynucleotide of polynucleotide sequence SEQ. DD. NO. 9 or one of its fragments.
  • the isolated polynucleotide serving as active principle will consist of the polynucleotide of polynucleotide sequence SEQ. ID. NO. 8 or into the polynucleotide of polynucleotide sequence SEQ. ID. NO. 9.
  • Said isolated polynucleotide incorporated into a pharmaceutical composition according to the invention will preferably be present in a viral vector, said viral vector being for example selected from the group consisting of an adenovirus, an associated adenovirus, a retrovirus and a pox virus.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated polypeptide comprising a polypeptide of sequence SEQ. DD. NO. 14 or one of its fragments, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation.
  • said isolated polypeptide will consist of a polypeptide of sequence SEQ. ED. NO. 14 or one of its fragments.
  • said polypeptide will have the sequence SEQ. ED. NO. 14.
  • a further subject of the invention will be a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds an isolated polypeptide comprising at least one fragment of the protein encoded by the polynucleotide sequence SEQ . DD. NO. 9 or by a sequence complementary to the polynucleotide sequence SEQ. DD. NO. 9, said polypeptide isolated having at least one immunological and / or biological activity characteristic of a protein binding human GHRH and being associated with the modulation of cell proliferation, said composition further comprising one or more pharmaceutically acceptable excipients.
  • said pharmaceutical composition according to the invention will be such that it comprises a monoclonal antibody, or an antigen binding fragment thereof, which specifically fixes an isolated polypeptide consisting of the protein coded by the polynucleotide sequence SEQ . ED. NO. 9 or one of the fragments of the latter or else by a sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9 or one of the fragments thereof.
  • the invention will relate to a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody, or an antigen binding fragment thereof, which specifically fixes the protein coded by the polynucleotide sequence SEQ. ED. NO. 9 or by the sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9 (in other words the protein of sequence SEQ. ED. NO. 10) with one or more pharmaceutically acceptable excipients.
  • Another object of the invention is the use of an isolated polynucleotide comprising:
  • the isolated polynucleotide being such that it codes for an isolated polypetide having at least one immunological and / or biological activity characteristic of a protein binding human GHRH and being associated with the modulation of cell proliferation, to prepare a medicine to treat a proliferative disease.
  • the isolated polynucleotide used will consist of the polynucleotide of polynucleotide sequence SEQ. ED. NO. 8 or one of its fragments or into the polynucleotide of polynucleotide sequence SEQ. ID. NO. 9 or one of its fragments.
  • the isolated polynucleotide used will consist of the polynucleotide of polynucleotide sequence SEQ. ID. NO. 8 or into the polynucleotide of polynucleotide sequence SEQ. ED. NO. 9.
  • Said isolated polynucleotide used is preferably present in a viral vector, said viral vector being for example selected from the group consisting of an adenovirus, an associated adenovirus, a retrovirus and a pox virus.
  • the present invention also relates to the use of an isolated polypeptide comprising a polypeptide of sequence SEQ. ID. NO. 14 or one of its fragments, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and which is associated with the modulation of cell proliferation, for preparing a medicament intended for treating a proliferative disease.
  • said isolated polypeptide will consist of a polypeptide of sequence SEQ. ED. NO. 14 or one of its fragments.
  • said polypeptide will have the sequence SEQ. ED. NO. 14.
  • a monoclonal antibody, or an antigen binding fragment thereof which specifically binds an isolated polypeptide comprising the protein encoded by the polynucleotide sequence SEQ. ED. NO. 9 or one of the fragments of the latter or else by a sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9 or one of the fragments thereof, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation, may be used to prepare a medicament intended to treat a proliferative disease.
  • said monoclonal antibody or said fragment of the antigen thereof will specifically bind a polypeptide consisting of the protein encoded by the polynucleotide sequence SEQ. ED. NO. 9 or one of the fragments of the latter or else by a sequence complementary to the polynucleotide sequence SEQ. DD. NO. 9 or one of the fragments thereof.
  • a monoclonal antibody, or an antigen binding fragment thereof which specifically binds an isolated polypeptide encoded by the polynucleotide sequence SEQ. ED. NO. 9 or by the sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9 (in other words, a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds the protein of sequence SEQ. ED. NO. 10), may be used to prepare a medicament for treating a proliferative disease.
  • the proliferative disease to be treated with the polypeptide or the polynucleotide described above will be cancer.
  • the cancer will be chosen from the group consisting of prostate cancer, breast cancer, lung cancer (and in particular small cell lung cancer) and colorectal cancer. Even more particularly preferred are breast cancer and small cell lung cancer.
  • the invention further provides a method of preparing an isolated polypeptide comprising the protein encoded by the polynucleotide sequence SEQ. ED. NO. 9 or SEQ. ED. NO. 13 or a fragment of the latter or else by a sequence complementary to the polynucleotide sequence SEQ. JD. NO. 9 or one of the fragments thereof, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation, said method of preparation comprising the steps following successive:
  • said preparation method will relate to the preparation of an isolated polypeptide consisting of the protein encoded by the polynucleotide sequence SEQ. JD. NO. 9 or one of the fragments of the latter or else by a sequence complementary to the polynucleotide sequence SEQ. JD. NO. 9 or one of the fragments thereof, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation, said method of preparation comprising the steps following successive:
  • (a) culture under conditions suitable for obtaining the expression of said polypeptide from a host cell transformed or transfected with an expression vector comprising an isolated polynucleotide comprising the polynucleotide sequence SEQ. ED. NO. 9 or SEQ. DD. NO. 13, the sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9 or SEQ. ED. NO. 13 or one of the fragments thereof, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation, and
  • the subject of said method will be the preparation of the isolated polypeptide encoded by the polynucleotide sequence SEQ. JD. NO. 9 or SEQ. JD. NO. 13 or by the sequence complementary to the polynucleotide sequence SEQ. JD. NO. 9 or SEQ. ID. NO. 13 (in other words, the preparation of the protein of sequence SEQ. JD. NO. 10).
  • this will relate to the preparation of the protein of sequence SEQ. JD. NO. 10 and will include the following steps:
  • (a) culture under conditions suitable for obtaining the expression of said polypeptide from a host cell transformed or transfected with an expression vector comprising an isolated polynucleotide comprising the polynucleotide sequence SEQ. JD. NO. 9 or SEQ. JD. NO. 13 or the sequence complementary to the polynucleotide sequence SEQ. JD. NO. 9 or SEQ. ED. NO. 13, said isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation, and
  • the present invention also provides a method for identifying compounds capable of fixing human GHRH and of modulating cell proliferation, which comprises the following successive steps:
  • SEQ. ED. NO. 9 or by a sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9
  • said method for identifying compounds capable of fixing human GHRH and of modulating cell proliferation will comprise, in step (a), bringing each candidate compound into contact, under conditions and during a sufficient time to allow the candidate agent to bind to the polypeptide, with the isolated polypeptide encoded by the polynucleotide sequence SEQ. JD. NO. 9 or by the sequence complementary to the polynucleotide sequence SEQ. JD. NO. 9 (in other words, with the protein of sequence SEQ. ED. NO. 10).
  • An alternative method for identifying compounds capable of fixing human GHRH and of modulating cell proliferation comprises the following successive steps:
  • the isolated polypeptide having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and modulates cell proliferation, and (b) determination of the effect of each candidate compound on the cellular concentration of polypeptide and identification, among the candidate compounds, of compounds capable of fixing human GHRH and of modulating cell proliferation.
  • said alternative method will include, in step (a), bringing each candidate compound into contact, under conditions and for a time sufficient to allow the candidate agent and the cell to interact, with a cell. capable of expressing the isolated polypeptide coded by the polynucleotide sequence SEQ. ID. NO. 9 or by the sequence complementary to the polynucleotide sequence SEQ. JD. NO. 9 (in other words, a cell capable of expressing the protein of sequence SEQ. ID. NO. 10).
  • the candidate compounds will come from libraries of small molecules originating from combinatorial chemistry programs.
  • the pharmacological properties obtained for the polynucleotides and polypeptides according to the invention make the latter suitable for pharmaceutical use.
  • the isolated polypeptides comprising:
  • polypeptides which have at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and which is associated with the modulation of cell proliferation, as well as the polynucleotides coding for said polypeptides, can, according to the invention, be administered to cancer patients in order to slow the progression of their tumors or to cause said tumors to regress.
  • the protein which binds human GHRH and which is associated with the modulation of cell proliferation may in particular be the isolated polypeptide coded by the polynucleotide sequence SEQ. ED. NO. 9 or by the sequence complementary to the polynucleotide sequence SEQ. ED. NO. 9 (in other words, the protein of sequence SEQ. ED. NO. 10).
  • the invention relates to polynucleotides of sequence SEQ. ED. NO. 4, SEQ. ED. NO. 5, SEQ. JD. NO. i l and SEQ. ID. NO. 12, which can in particular be used as a primer in the PCR reactions for the cloning of heterocain.
  • the present invention is generally directed to products and methods for modulating cell growth and treating cancer.
  • the present invention is based, in part, on the identification of "sequences associated with the modulation of cell proliferation" which are polypeptide and polynucleotide sequences associated with the modulation of cell proliferation.
  • sequences associated with the modulation of cell proliferation are polypeptide and polynucleotide sequences associated with the modulation of cell proliferation.
  • Such cDNA molecules can be prepared from RNA or mRNA preparations - li ⁇
  • a protein or polypeptide associated with differentiation comprises the sequence encoded by an mRNA associated with cell differentiation.
  • compositions described herein may include one or more polypeptides, nucleic acid sequences and / or antico ⁇ s.
  • the polypeptides of the present invention comprise at least a portion of the protein or a variant thereof which binds human GHRH and is associated with the modulation of cell proliferation.
  • the nucleic acid sequences of the present invention comprise a DNA or RNA sequence which codes for at least one portion of such a polypeptide or which is complementary to such a coding sequence.
  • Antico ⁇ s are proteins of the immune system or fragments of binding to the antigen thereof, which are capable of binding a portion of the polypeptides described above.
  • the subject of the present invention is in particular the polynucleotides of sequence SEQ. ED. NO. 8, SEQ. ED. NO. 9 or SEQ. ED. NO. 13 as well as the polypeptide or the protein of sequence SEQ. ED. NO. 14.
  • the invention also includes polynucleotides having polynucleotide sequences homologous at least to 75%, preferably at least 85% and even more preferably at least 90% or even 95%, to the sequences of the polynucleotides described above, in particular to the sequences SEQ . ID. NO. 8, SEQ. ED. NO. 9 and SEQ. ED. NO. 13. This also applies mutatis mutandis to the other polynucleotides, polypeptides and proteins forming part of the invention, and in particular to the protein of sequence SEQ. ED. NO. 14.
  • the degree of homology expressed in% is calculated as follows:
  • N 100 - 100 x (N '/ N) with N' representing the number of nucleotides or amino acids modified relative to the sequence SEQ. JD. NO. 8, SEQ. ID. NO. 9, SEQ. JD. NO. 10, SEQ. ID. NO. 13 or SEQ. ED. NO. 14 and N the number of nucleotides of the sequence SEQ. ED. NO. 8, SEQ. ED. NO. 9, SEQ. ED. NO. 10, SEQ. ED. NO. 13 or SEQ. ED. NO. 14.
  • polynucleotide sequences which code for the polypeptides or proteins of the invention, and fragments or fusion proteins of these polypeptides or proteins can be used to generate recombinant DNA molecules that direct the expression of these polypeptides or proteins, or an active portion thereof, in appropriate host cells.
  • polynucleotide sequences which hybridize with portions of the polynucleotide sequences according to the invention can also be used in nucleic acid hybridization tests, Southern blot, Northern blot, etc.
  • DNA sequences encoding substantially for the amino acid sequence of the polypeptides or proteins of the invention can be used for cloning and expression of said polypeptides or proteins.
  • DNA sequences include those capable of hybridizing the polynucleotide sequences of the polynucleotides of the invention under certain stringency conditions which can be adjusted in several ways. For example, during a polymerase chain reaction (PCR), the temperature at which the primers hybridize to the matrix or the concentrations of MgCl 2 in the reaction buffer can be adjusted. When using radio-labeled DNA fragments, or oligonucleotides to probe membranes, the stringency can be adjusted by changing the ionic strengths of the washing solutions or by carefully controlling the washing temperature.
  • PCR polymerase chain reaction
  • such a homologous nucleotide sequence hybridizes specifically with the sequence complementary to the sequence SEQ. JD. NO. 8, SEQ. ID. NO. 9 or SEQ. ID. NO. 13 under stringent conditions (or “strong” stringency conditions).
  • stringent conditions or “strong” stringency conditions.
  • the parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (T m ).
  • T m is defined by the relation:
  • T m 81.5 + 0.41 x (% G + C) + 16.6 x log [cations] - 0.63 x (% formamide) - (600 / number of bases)
  • the stringency conditions will be said to be “strong” when a hybridization temperature of 10 ° C. below T m is used and hybridization buffers containing a solution 6 ⁇ SSC (sodium chloride 0 , 9 M and 0.09 M sodium citrate). Under such conditions, the polynucleotides of aspecific sequences will not hybridize with the polynucleotide of the sequence complementary to the sequence SEQ. JD. NO. 8, SEQ. ID. NO. 9 or SEQ. ID. NO. 13.
  • Altered DNA sequences which can be used in accordance with the present invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence which codes for the same gene product or equivalent function.
  • the gene product may also contain deletions, additions or substitutions of amino acid residues in the sequences of the proteins of the invention, which result in so-called silent changes, thus producing polypeptides and proteins of equivalent function.
  • Such amino acid substitutions can be made based on polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipatic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with polar groups having similar hydrophobicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
  • DNA sequences of the present invention can be modified to alter the sequences of the polynucleotides according to the invention for many reasons including but not limited to alterations which modify the process and expression of the gene product.
  • mutations can be introduced using techniques well known to those skilled in the art, for example site-directed mutagenesis, insertion of new restriction sites, alteration of glycosylations, phosphorylation, etc.
  • the host cell can over-glycosylate the gene product.
  • modified polynucleotide sequences linked to heterologous sequences to encode a fusion protein which can for example be the protein of sequence SEQ. JD. NO. 14
  • the fusion protein can be modified to contain a cleavage site located between the sequence of the protein according to the invention (for example the sequence SEQ. ID NO. 10) and the sequence of the heterologous protein, so that the protein sequence according to the invention can be cleaved from the heterologous part.
  • polynucleotide which codes for a polypeptide or a portion or variant thereof as described herein which fixes human GHRH and which is associated with the modulation of cell proliferation is covered by the present invention.
  • Such polynucleotides can be single strand (coding or antisense) or double strand and can be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • Polynucleotides encoding polypeptides that bind human GHRH and are associated with the modulation of cell proliferation can be prepared using any technique available to those of skill in the art.
  • a polynucleotide can be amplified via a polymerase chain reaction (PCR) from cDNA prepared from cells.
  • PCR polymerase chain reaction
  • specific primers can be drawn and ordered or synthesized; these primers are based on the sequence of said polynucleotide.
  • An amplified portion can then be used to isolate the complete gene from a genomic DNA library or from a cDNA library from any cell or tissue, using well-established techniques. known to those skilled in the art and briefly recalled below.
  • a complete gene can be constructed from several PCR fragments.
  • the cDNA molecules coding for a protein which binds human GHRH and which is associated with the modulation of cell proliferation, or a portion thereof, can also be prepared by screening a cDNA library obtained for example from Cell or tissue mRNA. Such libraries may be commercially available or may be prepared using conventional techniques (cf. Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989).
  • a cDNA molecule coding for a polypeptide which binds human GHRH and which is associated with the modulation of cell proliferation can be sequenced using standard techniques using enzymes such as the Klenow fragment of DNA polymerase I, Sequenase X ( US Biochemical Co ⁇ ., Cleveland, OH, USA), Taq polymerase (Perkin Elmer, Foster City, CA, USA), T7 thermostable polymerase (Amersham, Chicago, JL, USA) or a combination of Recombinant polymerases and exonucleases with replay activity such as the Elongase amplification system (Gibco BRL, Gaithersburg, MD, United States).
  • a system of Automatic sequencing can be used using instruments available from commercial suppliers like Perkin Elmer and Pharmacia.
  • the partial sequence of a cDNA can be used to identify a polynucleotide sequence which codes for the complete protein associated with the modulation of cell proliferation using standard techniques well known to those skilled in the art. Among these techniques, a cDNA library is screened using one or more polynucleotide probes using the recombinant properties of RecA (ClonCapture cDNA Selection Kit, Clontech Laboratories, USA).
  • a partial sequence can be radiolabeled (for example by cut translation or by marking the ends using 32 P or 33 P) using conventional techniques.
  • a library of bacteria or bacteriophages is then screened by hybridization on filters containing the denatured bacterial colonies (or the imprints containing the phage plates) with the labeled probe (cf. Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). The positive colonies or plaques are then selected and amplified and the DNA is isolated for future analyzes.
  • the complete sequence can then be determined using standard techniques.
  • the overlapping sequences are then assembled into a single continuous sequence.
  • a complete cDNA molecule can be generated by ligation of the fragments of interest using standard techniques.
  • the amplification is generally carried out via PCR. All of the commercially available kits can be used for the amplification steps.
  • the primers can be drawn using, for example, software well known in the art.
  • the nucleotide primers are preferably molecules of 20 to 30 nucleotides having a guanine and cytosine content of at least 50% and which hybridize with the target sequence at temperatures between 50 and 72 ° C.
  • the amplified region can be sequenced as described above and the overlapping sequences assembled into a continuous sequence.
  • sequences adjacent to the partial sequence can be found by amplification with a primer of the binding sequence and a primer specific for a known region.
  • the amplified sequences are then subjected to a second amplification cycle. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. (1991), 1, 111-19) and progressive PCR (Parker et al., Nucl. Acids. Res. (1991), 19, 3055-60). Other methods using amplification can also be used to obtain a complete cDNA sequence.
  • ESTs Expressed Sequence Tags
  • polynucleotide sequences described above are also included in the scope of the present invention.
  • the polynucleotide variants can contain one or more substitutions, deletions or insertions (cf. also above in the section entitled "Polynucleotides, polypeptides and proteins according to the invention").
  • a portion of the sequence complementary to the coding sequence can also be used as a probe or as a modulator of gene expression.
  • the cDNA constructs which can be transcribed into antisense RNA can be introduced into cells or into tissues to facilitate the production of antisense RNA.
  • An antisense polynucleotide can be used, as described herein, to inhibit the expression of a gene associated with the modulation of cell proliferation.
  • Anti-sense technology can be used to control gene expression by forming a triple helix, which compromises the ability of the double helix to open enough for binding of polymerases, transcription factors or regulatory molecules (see Gee et al in Huber and Carr, Molecular and Immunologie Approaches (1994), Futura Publishing Co., Mt. Kisco, NY).
  • an antisense molecule can be used to hybridize with a gene control region (e.g., a promoter or transcription initiation site) and block gene transcription, or block translation by inhibiting fixation of ribosomes to the transcript.
  • the polynucleotides can then be modified to increase their stability in vivo. Possible modifications include (but are not limited to): addition of sequences at the 5 'and / or 3'ends; the use of phosphorothioate or 2 'O-methyl rather than phosphodiesterase bonds in the backbone; and / or the introduction of bases such as inosine, quesosine and wybutosine as well as acetyladenine, methylthioadenine and other modified forms of adenine, cytidine, guanine, thymine and uridine.
  • polynucleotides of the present invention have moreover already been described previously in the section entitled “Polynucleotides, polypeptides and proteins according to the invention”.
  • nucleotide sequences as described in the present invention can be joined to other nucleotide sequences using established recombinant DNA techniques.
  • a polynucleotide can be cloned into a wide range of expression vectors, including plasmids, phagemids, phage lambda derivatives and cosmids.
  • Vectors of particular interest include expression vectors, replication vectors and sequencing vectors.
  • a vector contains an origin of functional replication in at least one organism, suitable endonuclease restriction sites and one or more selection markers. The presence of other elements will depend on the specific use desired by a person skilled in the art who will select the characteristics of the expression vector according to his needs and the techniques available.
  • Polynucleotides can be formulated to enter the cell and express the corresponding polypeptide. Such formulations are particularly useful in therapy as described below.
  • a polynucleotide in a target cell, and that any suitable technique can be employed.
  • a polynucleotide can be incorporated into a viral vector such as an adenovirus or a retrovirus (but also in others).
  • a retroviral vector can transfer or incorporate a gene for a selection marker and / or a targeting entity such as a gene coding for the ligand of a receptor specific for a target cell, in order to make the vector target-specific.
  • colloidal dispersion systems such as macromolecular complexes, nano-capsules, microspheres, beads, and systems based on the use of lipids including oil / water emulsions, micelles, mixed micelles and liposomes.
  • the preferred colloidal system for product delivery use in vitro and in vivo is the liposome (ie an artificial membrane vesicle).
  • polypeptides of the present invention comprise at least a portion of the protein associated with the modulation of cell proliferation or a variant thereof, said portion being immunologically and / or biologically active.
  • polypeptides can be any length, including the complete protein, an oligopeptide (i.e. consisting of a relatively limited number of amino acids, such as 8-10 residues, joined by peptide bonds) or a peptide of intermediate size.
  • a polypeptide can also include additional sequences.
  • polypeptide is "biologically active" if it has one or more structural, regulatory and / or biochemical functions from the native protein associated with the binding of human GHRH and being associated with the modulation of cell proliferation.
  • a polypeptide will be considered as “having at least one immunological and / or biological activity characteristic of a protein which binds human GHRH and being associated with the modulation of cell proliferation” therefore that its inhibitory concentration CI 5 o measured under the conditions described in Example 6 of the present application will be less than or equal to 10 nM (and preferably less than or equal to 1 nM).
  • sequence comparison studies may indicate a particular biological activity of the protein.
  • the tests tending to evaluate said activity can then be carried out on the basis of tests already known in the art.
  • Certain portions and other variants of such proteins should also show this property according to an in vitro or in vivo test.
  • the polypeptides according to the present invention can comprise one or more portions of a variant of the endogenous protein where the portion is immunologically and / or biologically active (ie the portion exhibits one or more antigenic, immunogenic and / or biological characteristics. of complete protein). Preferably, such a portion is at least as active as the total protein during tests allowing the detection of such properties.
  • a "variant" polypeptide is a polypeptide which differs from the native protein by substitutions, insertions, deletions and / or modifications to amino acids. Some variants contain conservative substitutions.
  • a conservative substitution is a substitution in which an amino acid is substituted by another amino acid having the same properties, as those determined by a person skilled in the art who does not expect any change in the secondary structure, as well as in nature hydropathic of the polypeptide.
  • Amino acid substitutions can generally be made based on similarity of polarity, charge, solubility, hydrophobicity, hydrophylicity, and / or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and polar uncharged amino acids having similar hydrophobicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine, threonine, phenylalanine and tyrosine.
  • Other groups of amino acids that may represent conservative changes include: (1) Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr (3) Nal, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; and (5) Phe, Tyr, T ⁇ , His.
  • a variant may also, or alternatively, contain non-conservative changes.
  • Variants forming part of this invention also include polypeptides in which the primary structure of the native protein is modified by formation of covalent conjugates or not with other polypeptides or chemical structures such as lipid groups or glycosyl groups, or acetyl phosphate .
  • the present invention also includes polypeptides with or without glycosylation patterns.
  • the polypeptides expressed in yeast or mammalian cell expression systems may be, in terms of molecular weight and glycosylation pattern, similar to or slightly different from the native molecule depending on the expression system used.
  • N-glycosylation sites of eukaryotic proteins are characterized by the amino acid triplet Asn-Al-Z where Al is any amino acid except Pro, and Z is a serine or threonine.
  • polypeptides and proteins of the present invention have moreover already been described previously in the section entitled “Polypeptides and polynucleotides according to the invention”.
  • any expression vector known to those of skill in the art can be used to express recombinant polypeptides of this invention.
  • Expression can be obtained in any suitable host cell which has been transformed or transfected with an expression vector containing a DNA sequence which codes for the recombinant polypeptide.
  • suitable host cells include prokaryotic, higher eukaryotic or yeast cells.
  • the host cells used are E. Coli, yeast cells or mammalian cells such as COS, CHO, HEK-293, MCF7 (human tumor cells isolated from a mammary carcinoma) or DU 145 (tumor cells isolated from prostate cancer).
  • portions and other variants can also be generated by synthetic means using techniques well known to those skilled in the art. For example, portions and other variants having less than 500 amino acids, preferably less than 100 amino acids and more preferably less than 50 amino acids can be synthesized chemically.
  • Polypeptides can be synthesized using commercially available solid phase synthesis techniques, such as the Merrifield resin synthesis method where amino acids are sequentially added to a chain of amino acids being synthesized (see Merrifield, J Am. Chem. Soc. (1963), 85, 2149-2146). Many other solid phase synthesis techniques are also available (for example the method of Roberge et al., Science (1995), 269, 202-204).
  • Equipment for the automatic synthesis of polypeptides is commercially available from suppliers such as Applied Biosystems, Inc. (Foster City, CA, USA); the synthesis of the polypeptides can then be carried out by following the manufacturer's recommendations.
  • polypeptides and polynucleotides described in the present invention are isolated.
  • An "isolated" polypeptide or polynucleotide is a polynucleotide or peptide removed from its original environment.
  • a natural protein is isolated if it is separated from the biological material with which it coexists in the natural system.
  • a polynucleotide is considered isolated if, for example, it is cloned into a vector that is not part of the natural environment.
  • the present invention provides binding agents, such as antico ⁇ s which specifically bind the protein associated with the binding of human GHRH and being associated with the modulation of cell proliferation.
  • binding agents such as antico ⁇ s which specifically bind the protein associated with the binding of human GHRH and being associated with the modulation of cell proliferation.
  • Such an agent is said to "specifically bind" to the cell proliferation modulation protein if it reacts at a detectable level (for example by an ELISA assay) with a protein associated with cell proliferation modulation or a portion or a variant thereof and does not detectably react with other proteins.
  • "Fixation” refers to a non-covalent association between 2 separate molecules so that a complex is formed.
  • the binding capacity can be assessed, for example, by determining the binding constant for the formation of the complex.
  • the binding constant is the value obtained when the value of the concentration of the complex is divided by the product of the values of the concentration of the components. In general, 2 products are said to be “fixed” when the fixing constant reaches 103
  • Any agent capable of meeting the above criteria can be considered a fixing agent.
  • a fixing agent is preferably an antico ⁇ s or a fragment thereof.
  • Antico ⁇ s can be prepared by any technique available to a person skilled in the art (cf. Harlow and Lane, Antibodies. A laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques including the generation of monoclonal antibodies or via transfections of antibody genes in host cells of bacteria or mammals in order to produce recombinant antibodies.
  • an immunogen containing the polypeptide is injected into a group of mammals (eg mice, rats, rabbits, sheep or goats).
  • the polypeptides of the present invention can serve as immunogens without modification.
  • a higher immune response can be induced if the polypeptide is joined to a transport protein such as bovine serum albumin or keyhole limpet hemocyanin.
  • the immunogen is injected into the host animal, preferably according to a predetermined schedule, and the animals are bled periodically.
  • Polyclonal antico ⁇ s specific for the polypeptide can thus be purified from such antisera, for example by affinity chromatography using the peptide coupled to an adequate solid support.
  • the protein of sequence A is injected into the host animal, preferably according to a predetermined scheme, and the animals are bled periodically.
  • Polyclonal antico ⁇ s specific for the polypeptide of sequence A can thus be purified from such antisera, for example by affinity chromatography using the protein of sequence B coupled to an adequate solid support.
  • the corresponding eluate contains the antico ⁇ s specifically fixing a protein of sequence A but not a protein of sequence B.
  • Any fusion gene can be produced by a person skilled in the art to analyze the sub-cellular localization of a protein according to the invention, in particular the sub-cellular localization of the protein of sequence SEQ. ED. NO. 10.
  • Numerous plasmid constructs are commercially available such as the Glutathione S Transferase protein (GST) or fluorescent proteins such as the Green Fluorescent Protein (GFP) or alternatively and not exclusively a poly-Histidine labeling.
  • GST Glutathione S Transferase protein
  • GFP Green Fluorescent Protein
  • Human eukaryotic host cells for example HEK-293 are subcultured for 24 h before the transfection protocol allowing normal cell metabolism and better transfection efficiency.
  • Increasing concentrations (1, 5 and 10 ⁇ g) of vector alone containing the revealing protein (GFP, GST or Tag Histidine) or of vector containing the polynucleotide of sequence SEQ. JD. NO. 8 or the polynucleotide of sequence SEQ. ED. NO. 9 fused with the revealing protein were performed using the Effectene reagent according to the manufacturer's recommendations (Qiagen).
  • the cells are then analyzed by confocal microscopy, for example, to detect the location of the protein. If the protein is suspected of being secreted, for example, the supernatants are recovered, lyophilized, deposited on acrylamide gel and analyzed by the Western blot technique using antico cors directed against the revealing protein.
  • products such as polypeptides, antico ⁇ s and / or nucleic acids can be incorporated into pharmaceutical compositions or vaccines.
  • the pharmaceutical compositions comprise one or more of these products and one or more pharmaceutically acceptable excipients (transporters).
  • Certain pharmaceutical compositions which can be used as vaccines may include one or more polypeptides and an activator of the immune response, such as an adjuvant or a liposome (in which the product is inco ⁇ orated).
  • Pharmaceutical compositions and vaccines may further contain a delivery system, such as biodegradable microspheres (and for example microspheres composed of copolymers of lactic acid and glycolic acid or PLGA).
  • Pharmaceutical compositions and vaccines within the scope of the disclosure of the present invention may also contain other products which may be biologically active or inactive.
  • a pharmaceutical composition or vaccine may contain DNA encoding one or more polypeptides as described above, so that the polypeptide is generated in situ.
  • DNA can be present in any form of administration known to those skilled in the art, including nucleic acid, bacterial or viral acid expression systems. Appropriate nucleic acid expression systems contain the DNA sequences necessary for expression in the patient.
  • Administration systems based on a bacterium involve the administration of bacterium (such as Bacillus-Calmette-Guerrin) which expresses an immunogenic portion of the polypeptide on its surface.
  • bacterium such as Bacillus-Calmette-Guerrin
  • the DNA can be introduced using a viral expression system (for example a pox virus, a retrovirus or an adenovirus) involving the use of non-pathogenic agents (defective).
  • compositions of the present invention may be formulated for each appropriate mode of administration, including, for example, the topical, nasal, intravenous, intra-cranial, intra-peritoneal, subcutaneous and intramuscular routes.
  • the transporter preferably contains water, salt, alcohol, fat, paraffin or a tampon.
  • any transporter mentioned above or a solid transporter such as mannitol, lactose, starch, magnesium stearate, talc, cellulose, glucose, sucrose and magnesium carbonate can be used.
  • Biodegradable microspheres can also be used as transporters for the pharmaceutical compositions of this invention.
  • formulations such as creams or lotions are preferred.
  • Such compositions can also include buffers (e.g. neutral buffered saline or phosphate), carbohydrates (e.g.
  • compositions of the present invention can be in the form of a lyophilisate. Products can also be encapsulated in liposomes using conventional technologies.
  • each of the varieties of adjuvants can be used in vaccines to induce the immune response.
  • Most adjuvants contain a substance protecting the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses such as lipid A, proteins derived from Bordetella Pertussis or Mycobacteium tuberculosis.
  • Adequate adjuvants are commercially available such as, for example: Freund's adjuvant and the complete adjuvant (Difco Laboratories, Detroit, MJY, USA; Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ, USA) United)), biodegradable microspheres; monophosphoryl lipid A; and cytokines such as GM-CSF or interleukin-2, -7 or -12.
  • compositions described above can also be administered in the form of depot formulations (i.e. a formulation such as a capsule or sponge which triggers the slow release of the product after administration).
  • depot formulations i.e. a formulation such as a capsule or sponge which triggers the slow release of the product after administration.
  • Such formulations can generally be prepared using technologies well known to those skilled in the art and administered, for example, orally, rectally or by subcutaneous implantation or by implantation on the desired target site.
  • the delay formulations may contain a polypeptide, a polynucleotide or an antico ⁇ s dispersed in a transport matrix and / or contained in a reservoir protected by a diffusion membrane.
  • the carriers for the use of such formulations are biocompatible and must also be biodegradable; preferably the formulation provides a relatively constant level of release of the active component.
  • the amount of active product contained in the delay formulation depends on the implantation site.
  • the products described can be used in anticancer therapy.
  • the polynucleotides and polypeptides associated with the binding of human GHRH and being associated with the modulation of proliferation cells can be used to inhibit growth and induce modulation of cell proliferation in specific breast, prostate or lung cancer tumors.
  • polypeptides or polynucleotides can also be used for the therapy of numerous carcinomas including melanomas, multiple forms of glioblastomas, carcinomas of the lung as well as colorectal cancers. Agents which activate the expression of such polypeptides or polynucleotides can also be used in the context of these therapies.
  • the products (which may be polypeptides or nucleic acids) are preferably incorporated in pharmaceutical compositions as described above.
  • Patients suitable for therapy are all warm-blooded animals, preferably humans.
  • a patient eligible for therapy according to the invention may or may not be diagnosed as being affected by cancer.
  • the pharmaceutical compositions described above can thus be used to inhibit the development of cancer at different stages of the disease (to prevent the appearance of cancer or to treat a patient affected by cancer).
  • compositions of the present invention will be administered appropriately for each specific cancer to be treated.
  • the route, duration and frequency of administration will be determined based on the patient's condition, the type and severity of the disease, and the method of administration.
  • the routes and frequencies of administration may vary from one individual to another.
  • the pharmaceutical compositions and the vaccines can be administered by injection (for example by the intra-cutaneous, intramuscular, intravenous or subcutaneous route), by intra-nasal route (for example by inhalation) or by oral route.
  • Alternative protocols may be appropriate for each patient individually.
  • an appropriate dosage and treatment regimen will contain the active product in sufficient quantity to provide therapeutic and / or prophylactic benefit.
  • Such a response can be followed by the establishment of an improved clinical outcome (for example, more frequent remissions, survival in the absence of complete, partial or longer disease) in treated patients compared to patients not treated or treated with lower doses.
  • a polypeptide can be administered at doses ranging from 100 ⁇ g to 5 mg.
  • the DNA molecules encoding such polypeptides can generally be administered in an amount sufficient to generate comparable levels of polypeptides.
  • Appropriate dosages can generally be determined using experimental models and / or clinical trials. In general, the use of the minimum dose sufficient to provide effective therapy is preferred. Patients can generally be followed with regard to the effectiveness of therapy using tests suitable for the conditions of treatment or prevention which will appear familiar to those skilled in the art.
  • RNAs are retro-transcribed according to two different operating modes in order to dissociate and promote reverse transcriptions of the 5 'and 3' parts of the RNAs using the SMART TM RACE cDNA Amplification Kit (Clontech). 1-3) Design__et . summary ⁇
  • the amplification of the 2 specific sequences of the heterocain cDNA was carried out by polymerase chain reaction (PCR) on the products of reverse transcription using the primer Rev1 for the 5 'specific cDNA products and the primer Fwdl for the 3 'specific cDNA products of respective sequences SEQ. JD. NO. 4 and SEQ. ID. NO. 5.
  • PCR polymerase chain reaction
  • SEQ sequences. ED. NO. 4 and SEQ. ED. NO. 5 are as follows:
  • Reaction conditions include 0.2 ⁇ M Fwdl for 3 'specific cDNA products and 0.2 ⁇ M Revl for 5' specific cDNA products, 200 ⁇ M dNTPs, 40 niM Tricin-KOH (pH 8, 7), 15 mM KOAc, 3.5 mM Mg (OAc) 2 , 3.75 ⁇ g / ml
  • Perkin-Elmer 9700 using the following thermal cycle parameters: 5 cycles including denaturation at 94 ° C for 5 s, hybridization of primers at 72 ° C,
  • the products obtained by the PCR are separated on 1% agarose gel and visualized using an ethidium bromide staining.
  • nucleic acid sequences of the 5 'specific and 3' specific cDNA PCR products are determined using an automatic sequencer. These are respectively the SEQ sequences. ED. NO. 6 and SEQ. ED. NO. 7 reproduced below:
  • gagttcaatt cctccatgtg caactcaaag cttattggag ctagatattt cgataagggt
  • gagttcaatt cctccatgtg caactcaaag cttattggag ctagatattt cgataagggt
  • sequence SEQ. ED. NO. 8 an open reading phase is observed with the presence of an initiating codon (ATG) coding for an initiating methionine at position 115 and a stop codon (UAA) at position 2437.
  • ATG initiating codon
  • UAA stop codon
  • polynucleotide thus translated corresponds to a protein of sequence SEQ. ED. NO.10, composed of 774 amino acids and reproduced below:
  • the cells in culture are stored at -80 ° C. before the steps of extraction of the total RNAs.
  • the extraction of total RNAs is based on a technique described in the scientific literature (Chomczynski and Sacchi, Anal. Biochem. (1987), 162, 156) using the reagent Trizol (Gibco / BRL).
  • the quality of the RNAs thus extracted is analyzed on 1% agarose gel in the presence of ethidium bromide. 2..2) Transcription ⁇ Merse_from__RNA _
  • RNAs are reverse transcribed with Oligo primers (dT) using Superscript ® reverse transcriptase as suggested in the manufacturer's manual (Gibco / BRL).
  • Amplification of the heterocaine cDNA was carried out by chain polymerization reaction (PCR) on the products of reverse transcription using the primer Fwd2 and Rev2 of respective sequences SEQ. ED. NO. 11 and SEQ. ED. NO. 12.
  • SEQ sequences. ED. NO. 11 and SEQ. ED. NO. 12 are as follows: - SEQ. BD. NO. he :
  • the primer Fwd2 corresponds to nucleotides 118 to 140 of SEQ. ED. NO. 8 and includes a BamHI site (underlined) and three additional nucleotides in part 5 ′ to facilitate cloning.
  • the primer Rev2 corresponds to the complementary sequence of the region containing nucleotides 2405 to 2436 of SEQ. ED. NO. 8 and includes an Xhol site (underlined) and three additional nucleotides in part 5 ′ to facilitate cloning.
  • the reaction conditions include 50 ng of the cDNAs produced from the reverse transcription reaction described above, 0.2 ⁇ M of Fwd2 (SEQ. ED. NO. 11) and of Rev2 (SEQ. ED. NO. 12), 200 ⁇ M dNTPs, 40 mM Tricin-KOH (pH 8.7), 15 mM KOAc, 3.5 mM Mg (Oac) 2 , 3.75 ⁇ g / ml BSA, 0.005% Tween-20, 0.005% Nonidet-P40, and 0.5 U Taq DNA polymerase in a final volume of 50 ⁇ l.
  • the PCR reactions are carried out on a Perkin-Elmer 9700 thermocycler using the following thermal cycle parameters: 5 cycles comprising denaturation at 94 ° C for 5 s, hybridization of primers at 72 ° C, 5 cycles comprising denaturation at 94 ° C for 5 s, a hybridization of the primers at 70 ° C for 10 s, and a polymerization extension at 72 ° C for 3 minutes and finally 25 cycles comprising denaturation at 94 ° C for 5 s, a hybridization of the primers at 68 ° C for 10 s, and a polymerization extension at 72 ° C for 3 minutes.
  • the products obtained by the PCR are separated on 1% agarose gel and visualized using an ethidium bromide staining. A band of about 2.3 kb is obtained.
  • the nucleic acid sequence of the PCR product is checked using an automatic sequencer and corresponds to the sequence SEQ. ID. NO. 9 having artificially undergone a deletion of the initiator ATG codon in order to allow the expression of the recombinant heterocain from the vector pQE-TriSystem (Qiagen) in phase with the BamHI site as well as a deletion of the stop codon to preserve the translation into phase of the protein and thus allow the synthesis of an 8xHis sequence in the C-terminal region of the heterocaine.
  • This sequence corresponds to the sequence SEQ. ED. NO. 13 reproduced below:
  • the part of the cDNA coding for heterocaine is inserted at the BamHI / Xhol sites of the expression vector pQE-TriSystem (Qiagen) and is expressed using the E. coli Ml 5 bacteria as host bacteria.
  • 20 ml of LB medium containing 100 ⁇ g / ml of ampicillin and 25 ⁇ g / ml of kanamycin are inoculated and the bacteria put at 37 ° C. with stirring for 12 h. From this culture, 1 liter of LB medium containing 100 ⁇ g / ml of ampicillin and 25 ⁇ g / ml of kanamycin is inoculated and the bacteria are stirred at 37 ° C. until an optical density is obtained at 600 nm of 0.6.
  • the expression of the recombinant heterocain is achieved by the addition of EPTG at the final concentration of 1 mM for 4 to 5 h.
  • the bacteria are then recovered by centrifugation at 4000xg for 20 min and then frozen in liquid nitrogen.
  • the pellet is then thawed in ice for 15 min and suspended in a lysis buffer pH 8.0 composed of 50 mM NaH 2 PO 4 , 300 mM NaCl and 10 mM imidazole in the presence of 1 mg / ml of lysozyme for 30 min in ice.
  • the lysis is finally completed by a sonication step and the debris removed by centrifugation.
  • the lysate (4 ml) thus clarified is mixed with 1 ml of a suspension of nickel matrix and stirred at 4 ° C for 60 min.
  • the entire reaction medium is placed in a column, washed in the presence of 50 mM NaH 2 PO, 300 mM NaCl and 20 mM imidazole.
  • the heteroca ⁇ ine is finally eluted with 4x 0.5 ml of buffer consisting of 50 mM NaH 2 PO 4 , 300 mM NaCl and 250 mM imidazole.
  • Example 4 Production of Recombinant Heterocarpine by Baculovirus Infected Insect Cells:
  • the part of the cDNA coding for Heterocaine is inserted at the BamHI / Xhol sites of the expression vector pQE-TriSystem (Qiagen).
  • the vector pQE-TriSystem contains the viral sequences of Autographa california nuclear polyhedrosis virus (AcNPV) allowing homologous recombination.
  • the recombinant baculovirus containing the heterocaine sequence is prepared by co-transfection of the vector pQE-TriSystem with the linearized genomic DNA of the baculovirus in insect cells sf9 or sf21 established from ovarian tissues of the larva Spodoptera frugiperda.
  • the transfected cells are washed with phosphate buffer and collected by centrifugation at 10000 xg for 5 min.
  • the pellet is suspended in a lysis buffer pH 8.0 composed of 50 mM NaH 2 PO 4 , 300 mM NaCl and 10 mM imidazole.
  • the lysis is finally completed by a sonication step and the debris removed by centrifugation.
  • the lysate (4 ml) thus clarified is mixed with 200 ⁇ l of a nickel matrix suspension and stirred at 4 ° C for 60 min.
  • the entire reaction medium is placed in a column, washed in the presence of 50 mM NaH PO 4 , 300 mM NaCl and 20 mM imidazole.
  • the heteroca ⁇ ine is finally eluted with 4x 0.5 ml of buffer consisting of 50 mM NaH 2 PO 4 , 300 mM NaCl and 250 mM imidazole.
  • the part of the cDNA coding for heterocaine is inserted at the BamHI / Xhol sites of the expression vector pQE-TriSystem (Qiagen).
  • the vector pQE-TriSystem contains the activating sequences of the cytomegalovirus (CMN) fused to the chicken beta-actin promoter allowing a very important heterologous expression.
  • CNN cytomegalovirus
  • Human embryonic kidney cells (HEK-293) are cultured in DMEM medium (Eagle medium modified by Dulbeco) containing 100 U / ml of penicillin and 100 ⁇ g / ml of streptomycin sulfate, supplemented with fetal calf serum 10%.
  • the cells are subcultured 24 hours before the transfection protocol allowing normal cell metabolism and better transfection efficiency.
  • Transfecting 1 ug of pQE-TriSystem containing the cDNA encoding the hcieroca ⁇ ine was performed using the Effectene reagent ® following the manufacturer's recommendations (Qiagen).
  • the transfected cells are washed with phosphate buffer and collected by centrifugation at 10000 xg for 5 min.
  • the pellet is suspended in lysis buffer pH 8.0 consisting of 50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM imidazole in the presence of 0.05% Tween ® 20.
  • the lysis is finally completed by a sonication step and debris removed by centrifugation.
  • the lysate (4 ml) thus clarified is mixed with 200 ⁇ l of a nickel matrix suspension and stirred at 4 ° C for 60 min.
  • the entire reaction medium is placed in a column, washed in the presence of 50 mM NaH 2 PO 4 , 300 mM NaCl and 20 mM imidazole.
  • the heteroca ⁇ ine is finally eluted with 4x 0.5 ml of buffer consisting of 50 mM NaH 2 PO, 300 mM NaCl and 250 mM imidazole.
  • HEK-293 Human kidney embryonic cells, HEK-293, (a cell line developed by Dr. Stuart Sealfon, Mount Sinai Medical School, New York, New York) stably expressing the human GHRH receptor were obtained from Dr. Kelly Mayo (Northwestern University, Chicago, JL).
  • the HEK-293 cells stably transfected with the human GHRH receptor described above are cultured in DMEM (Eagle medium modified by Dulbecco, high glucose content; supplied by Life technologies) supplemented with 0.4 mg / ml of G418 (Life technologies) in the presence of 10% fetal calf serum and 4 mM L-glutamine (Life technologies).
  • DMEM Stemcell medium modified by Dulbecco, high glucose content; supplied by Life technologies
  • the cells are homogenized in buffer A containing 50 mM HEPES (pH 7.4), 5 mM magnesium chloride (MgCl 2 ), 2 mM ethylene glycol-bis (2-amino-ethyl) -N, N, N ', N'-tetraacetic (EGTA) and 50 ⁇ g / ml of bacitracin then are subjected to sonication in the same buffer A.
  • the cells thus homogenized are centrifuged at 4 ° C at 39,000 g for 10 minutes, suspended in the buffer A and re-centrifuged at 4 ° C at 40,000 g for 10 minutes. Total membrane proteins are quantified by the Bradford technique.
  • the diaphragm membranes are thus stored at -80 ° C for later use.
  • the membranes of the HEK-293 cells transfected stably with the human GHRH receptor are diluted to the concentration of 100 ⁇ g / ml in the reaction buffer containing 50 mM HEPES (pH 7.4), 5 mM MgCl 2 , 2 mM EGTA, 50 ⁇ g / ml bacitracin and 0.5% bovine serum albumin (BSA).
  • the membranes are incubated with 0.05 nM of [ 125 fjGHRH (l-44 amide) (Amersham) in a final volume of 200 ⁇ l in the presence of increasing concentrations of heterocaine for 2 hours at 23 ° C.
  • the reaction is stopped by rapid filtration on 96-well GF / C filters preloaded with 0.1% polyethyleneimine.
  • the filters are then washed three times at 4 ° C with washing buffer containing 50 mM Tris (pH 7.4) using a 96-well Packard filtration station.
  • the filters thus dried are submerged with 20 ⁇ l of scintillating cocktail (Microscint O, Packard) and are subject to counting on the Topcount (Packard).
  • the non-specific activity is determined in the presence of 100 nM of hGHRH.
  • a dose-response curve is generated for hGHRH (0.001 nM-100 nM) and makes it possible to determine the inhibitory concentration IC 5 0 of protein / polypeptide at which 50% of human GHRH is not fixed on the human GHRH receptor.

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FR2821359B1 (fr) * 2001-02-27 2003-05-09 Sod Conseils Rech Applic L'heterocarpine, une proteine fixant le ghrh humain

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004063376A1 *

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CA2509422A1 (fr) 2004-07-29
FR2848221A1 (fr) 2004-06-11
AU2003296782A1 (en) 2004-08-10
US20100279949A1 (en) 2010-11-04
JP2007528192A (ja) 2007-10-11
US7655438B2 (en) 2010-02-02
WO2004063376A1 (fr) 2004-07-29
FR2848221B1 (fr) 2007-09-28
PL377523A1 (pl) 2006-02-06
US20060228773A1 (en) 2006-10-12
RU2359033C2 (ru) 2009-06-20
RU2005121536A (ru) 2006-01-20
MXPA05006070A (es) 2005-08-16

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