Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
  • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
  • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
  • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

• the present invention relates to the field of inhibition of angiogenesis involved in many pathological proliferative syndromes.
• the invention relates to anti-idiotypic antibodies to molecules of the Major Histocompatibility Complex (MHC) and more particularly to anti-idiotypic antibodies to soluble HLA class 1 molecules, such as HLA-Gs or HLA-B7s.
• MHC Major Histocompatibility Complex
• the invention relates to the preparation of these antibodies and their use in pharmaceutical compositions useful for inhibiting angiogenesis, and therefore intended for the treatment of angiogenic pathologies involving endothelial cells and in particular for controlling their tumor proliferation.
• the invention also relates to the use of these anti-idiotypic antibodies to vectorize substances of therapeutic or diagnostic interest at the level of endothelial cells involved in a process of pathological or non-pathological angiogenesis.
• Endothelial cells line the inside of all of the body's blood vessels. These cells have a quiescent phenotype, they hardly proliferate, only 0.01% of endothelial cells are engaged in cell division at any given time.
• the physiological role of endothelial cells is very vast, they participate in many vital phenomena for the organism: blood coagulation, maintenance of blood pressure, recruitment of circulating cells, synthesis of cytokines, construction of new vessels irrigating neighboring territories during hypoxia. All these functions require or are accompanied by a change in phenotype of endothelial cells which then become "activated", that is to say that the vascular wall becomes capable of fixing circulating blood cells or of responding to the chemotactic actions of growth factors. Endothelial activation is therefore a transient state common to endothelial cells, prior to any functional response.
• lymphocytes In adults, the walls of these vessels do not have adhesion molecules to immobilize the lymphocytes of the circulating blood.
• endothelial cells express adhesion molecules on their surface, activated lymphocytes - in particular NK (natural killer) cells - that is, they unwind antennas to immobilize killer lymphocytes.
• NK natural killer cells
• These lymphocytes can cross the layer of endothelial cells or destroy them, then colonize the graft and induce the synthesis of cytokines, which amplifies the immune response and leads to the destruction of the graft.
• Endothelial cells have been shown to express HLA class I molecules on their surface whose role is not clearly established (Majema, 1997; Rebman 1997; Blaschitz 1997). It has been proposed that the overexpression of these class I HLA molecules by endothelial cells could inhibit the transendothelial migration of NK lymphocytes and contribute to attenuating transplant rejection. Furthermore, the dimerization of HLA-I molecules by means of an antibody recognizing all the HLA-I molecules stimulates the expression of FGF2 receptors on the surface of endothelial cells and their proliferation (Harris, 1997). HLA class I molecules have been described as being able to structurally associate with certain membrane receptors, including insulin receptors and EGF receptors.
• HLA class I molecules have also been shown to regulate the function of insulin receptors.
• no description of any non-immunological role of the soluble form of HLA Gl has been reported and there are also no experimental data demonstrating the presence of HLA-I receptors on endothelial cells. No biological effect of HLA-I on these cells or in angiogenesis has been described.
• the inventors have developed expertise in the field of endothelial activation, and have sought to verify whether the dimerization of HLA-I molecules by the antibody W6 / 32 (Barnstable CJ, 1978; Brodsky FM, 1982) stimulated the expression of VEGF receptors. Knowing that certain HLA-I molecules have a significant polymorphism and that they bind to distinct receptors that are still poorly understood, a versatile HLA-I mime has been prepared thanks to the strategy of manufacturing anti-idiotypic antibodies.
• the subject of the invention is an anti-idiotypic antibody of a class 1 HLA molecule.
• the invention contemplates both anti-idiotypic polyclonal or monoclonal antibodies which can be prepared by techniques described in the literature
• HLA class 1 HLA class 1 molecule as reported in the experimental part below. It can also be fragments of such anti-idiotypic antibodies such as Fab'2 or Fab fragments or a combination of such fragments.
• the class 1 HLA molecule is chosen from: HLA-A, B, C, D, F, or G.
• the invention relates to anti-idiotypic antibodies to molecules derived from HLA class 1 molecules, soluble or not, having anti-angiogenic properties that the skilled person is able to test. They may be fragments, in truncated form, sequences modified by deletion, addition or deletion of one or more amino acids.
• the invention also relates to a nucleic acid molecule comprising or consisting of a polynucleotide sequence coding for an anti-idiotypic antibody of any class I HLA molecule, soluble or not, a fragment or derivative thereof.
• a nucleic acid molecule is useful for the preparation of the anti-idiotypic antibodies according to the invention by genetic engineering techniques.
• the invention contemplates, as a nucleic acid molecule comprising a polynucleotide sequence encoding an anti-idiotypic antibody of a soluble HLA class 1 molecules, a nucleic acid molecule comprising said sequence placed under the control of regulatory sequences of his expression.
• a nucleic acid molecule is a vector, such as for example a plasmid, a recombinant virus, a cell transfected with a plasmid or viral expression vector.
• the anti-idiotypic antibodies according to the invention are remarkable in that they are capable of binding to endothelial cells and of inhibiting endothelial activation leading to angiogenesis.
• the invention therefore also relates to a pharmaceutical composition useful for preventing or inhibiting angiogenesis comprising at least one anti-idiotypic antibody or a nucleic acid molecule encoding such an antibody as defined above.
• nucleic acid molecule is useful in gene therapy or cell therapy protocols of administering said nucleic acid molecule or cells transformed by said nucleic acid molecule to an individual so as to express the anti-idiotypic antibodies and thus inhibit
• these anti-idiotypic antibodies and the corresponding nucleic acid molecules are particularly suitable for the preparation of medicaments intended to prevent or to treat angiogenesis and therefore the proliferation of endothelial cells.
• endothelial activation we can cite the rejection of allografts and xenografts, acrocyanosis, scleroderma, cancer, diabetic retinopathy, rheumatoid arthritis, angiomas, angiocarcinomas, in particular Castelman's disease and Kaposi's sarcoma.
• the pharmaceutical compositions according to the invention are also useful for the preparation of grafts between the removal and the transplantation.
• the invention therefore relates to a process for the preparation of grafts between harvesting and transplantation consisting in bringing said graft into contact with anti-idiotypic antibodies according to the invention.
• concentration of anti-idiotypic antibodies for this application would be, for example, of the order of 100 micrograms to 100 mg per kilogram of body weight.
• HLA class 1 molecules to a receptor on the surface of endothelial cells
• anti-idiotypic antibodies of these molecules are useful either for stimulating or for inhibiting endothelial activation and thus having new active agents for treating pathologies.
• pathologies requiring stimulating endothelial activation and therefore angiogenesis mention may be made of scarring, maturation of the corpus luteum of the ovary, the perfusion of the ischemic territories during vascular thromboses such as in arteritis of the lower limbs and myocardial infarction.
• monoclonal or polyclonal antibodies directed against HLA class 1 molecules such as the antibody 6/32.
• a method of identifying receptors for class 1 HLA molecules on the surface of endothelial cells according to the invention comprises the following steps:
• a method for identifying ligands for receptors for class 1 HLA molecules on the surface of endothelial cells according to the invention comprises similar steps, except that the matrix of affinity chromatography will present the previously purified receptor or a fragment.
• the receptor can be replaced by a recombinant receptor obtained by genetic engineering techniques.
• the invention also relates to the assay of HLA class I molecules or of anti-idiotypic antibodies, as markers of angiogenic pathologies.
• the W6 / 32 antibody or any other anti-HLA class I molecule antibody is immobilized on a support.
• a fixed quantity of the anti-idiotypic antibody labeled with biotin or a radioactive element such as iodine 125 is brought into contact with a known volume of the biological fluid containing the molecule to be tested which therefore competes with the binding of the anti-idiotypic antibody labeled on the anti-HLA class I antibody.
• the decrease in the binding of the anti-idiotypic labeled antibody is proportional to the quantity of anti-idiotypic antibody or of HLA class molecules. I in the biological environment.
• the subject of the invention is a method for assaying HLA class I molecules or anti-idiotypic antibodies for these molecules possibly present in a biological sample comprising: bringing said molecules or antibodies from the sample into contact with a determined quantity of anti-idiotypic antibodies of the invention, advantageously labeled and attached to HLA class I antimolecule antibodies,
• this method is carried out by immobilizing the anti-HLA class I molecule antibodies on a support.
• the anti-idiotypic antibodies according to the invention are remarkable in that they can bind to the surface of endothelial cells thanks to the receptors for HLA class 1 molecules.
• the said anti-idiotypic antibodies are useful for vectorizing substances of interest in endothelial activation foci pathological or not, like angiogenic sites.
• the subject of the invention is therefore the use of an anti-idiotypic antibody of a class 1 HLA molecule alone or associated with a substance of interest for the preparation of a composition intended to inhibit angiogenesis or to detect angiogenic sites.
• the substance of interest can be a chemical or biological substance active like a drug.
• drugs that may be mentioned include: an animal or plant toxin, a radioactive compound, a cytostatic or cytolytic drug, a suicide gene or an antisense.
• the substance of interest can also be a diagnostic substance such as a radioactive tracer or a contrast agent for magnetic resonance imaging making it possible to mark the angiogenic sites.
• the endothelial activation foci can thus be viewed in any medical imaging system.
• the invention therefore relates to a compound consisting of an anti-idiotypic antibody to a class 1 HLA molecule, or a derivative thereof, coupled to a substance of interest as defined above, said compound being able to attach to endothelial cells via a receptor.
• the antibodies of the invention therefore constitute a vectorization system for substances of interest in the endothelial cells involved in angiogenic phenomena which are particularly useful for treating or detecting the pathologies indicated above.
• the coupling of the anti-idiotypic antibody with the substance of interest can be carried out by any cleavable or non-cleavable binding means in a biological medium such as for example:
• These compounds of the invention can also be proteins or fusion genes.
• FIG. 1 shows the titer of mice on Fv cells and shows that Igld / HLA-I recognizes receptors on endothelial cells.
• FIG. 2 shows that the chemotactic effect of 6/32 is not inhibited by VEGFRls or VEGFR2s while the chemotactic effect of VEGF is abolished.
• FIG. 3 shows the inhibition of the chemotactic action of VEGF by Igld / HLA-I.
• FIG. 5 shows the action of the anti-idiotypic HLA class 1 monoclonal antibodies on the proliferation of endothelial cells.
• VEGF 165 amino acid isoform
• a recombinant baculovirus containing the corresponding cDNA and FGF2 is produced in Escherichia Coli (Plou ⁇ t et al., 1997).
• the antibody 6/32 is produced by injection of the hybridoma into the peritoneum of Balb / c mice. Ascites are collected 10-15 days after inoculation and IgG purified by precipitation with ammonium sulfate followed by protein A sepharose chromatography. The Fab fragments are prepared by cleavage with papain.
• Soluble VEGF receptors are produced in the conditioned medium of CHO cells transfected with the corresponding construct.
• the extracellular domains of the VEGFR1 and VEGFR2 receptors are synthesized by PCR, then sequenced to verify that there has been no mutation.
• the inserts are then inserted into the vector pcDNA-3 / myc / His (Invitrogen).
• the plasmids are transfected into pgsA-745 CHO cells and the secretory clones selected by their resistance to geneticin.
• the conditioned medium (1 liter) is then precipitated with 50% (weight / weight) of ammonium sulphate, dialysis against 0.01 M Tris buffer, 0.05 M NaCl and then chromatography on a Sp Sepharose column.
• the active fractions are collected by a discontinuous gradient of NaCl (0.2-0.5 M) and then chromatographed on a nickel-sepharose column.
• the purity of the purified VEGFR1 and VEGFR2 proteins is then certified by polyacrylamide-SDS gel (> 90%) and stored at -80 ° C until use. 1.2 - Cells used.
• the culture media and the sera come from the company Life Technologies TM.
• the endothelial cells of human umbilical cord veins (HUVEC) are cultured on plastic coated with gelatin diluted to 0.2% in PBS.
• the cells are cultured in endothelial medium SFM supplemented with 50 units / ml of penicillin, 50 ⁇ g / ml of streptomycin, and 20% of fetal calf serum and kept in an oven at 10% C02. These cultures are maintained with VEGF at 2 ng / ml added to the medium every two days.
• Fetal bovine aorta endothelial cells are cultured on plastic coated with gelatin diluted to 0.2% in PBS.
• the cells are cultured in DMEM-glutamax medium supplemented with 50 units / ml of penicillin, 50 ⁇ g / ml of streptomycin, and 10% of newborn calf serum and kept in an oven at 10% C02.
• DMEM-glutamax medium supplemented with 50 units / ml of penicillin, 50 ⁇ g / ml of streptomycin, and 10% of newborn calf serum and kept in an oven at 10% C02.
• mice receive subcutaneously 10 ⁇ g of 6/32 mixed volume to volume with complete Freund's adjuvant in a final volume of 100 ⁇ l.
• Four booster shots are given every two weeks by intraperitoneal injection of the mixture, except that the complete adjuvant is replaced by incomplete adjuvant.
• mice The day before the fusion, that is on D 67, five non-immunized Balbc mice are sacrificed to obtain the macrophages serving as feeder cells for the hybridomas.
• the macrophages are recovered by injection of 8 ml of ISCOVE culture medium into the peritoneum of the mice. After centrifugation, the macrophages are taken up in ISCOVE medium containing 20% fetal calf serum (Biochrom, lot 5612). These cells are then distributed in 20 96-well plates at a rate of 100 ⁇ l per well
• mice having the highest titers of anti-Id antibodies are sacrificed. Their rats are removed and diluted in ISCOVE medium to release the splenocytes. The connective tissue is exposed and the splenocytes are centrifuged and counted.
• the mouse myeloma line Ag8X63 was cultured for 10 days in ISCOVE medium containing 20% Myoclone serum. These cells are washed and scanned.
• the two cell types, splenocytes and myelomas, are mixed so as to obtain a ratio of 1 Ag8X63 myeloma cell for 6 splenocytes.
• the fusion is carried out by adding 20 times 50 ⁇ l of polyethylene glycol (PEG) at 30 second intervals. 4 ml of ISCOVE medium preheated to 37 ° C. are then added dropwise to the cell suspension, then after an incubation period of 4 minutes at 37 ° C., 4 ml are added.
• PEG polyethylene glycol
• the suspension is centrifuged then the cell pellet is then taken up in 100 ml of ISCOVE medium supplemented with 20% fetal calf serum and HAT IX (50X: Hypmanthanthine 5mM, Aminopterin 20 ⁇ M and Thymidine 0.8mM) and distributed at the rate of 100 ⁇ l per well on macrophages. After 5 days, 100 ⁇ l of HAT medium is added, and between 8 and 14 days the conditioned medium of each hybridoma is removed to measure the quantity of antibodies directed against the Fab fragments of the antibody 6/32
• the hybridomas selected for their capacity to secrete anti-Id antibodies are then cloned, that is to say that the cells are seeded under limiting dilution condition (5 cells / ml) in a volume of 0.1 ml per well. .
• the medium is changed after 10 days. After 15 days, some wells contain cell foci which have multiplied from the cell seeded at the start, so all these cells are identical and come from the same clone.
• the surface occupied by the cells represents at least half of the total surface of the well, the medium is removed and analyzed as before. At this stage, the clones producing anti-Id antibodies can be selected and their specificity known.
• a third cloning is then carried out to ensure that the clones are indeed monoclonal.
• the plastic boxes are lined with Fab fragments of the W6 / 32 antibody.
• the wells are incubated with the immunoglobulins to be tested (2 h, 37 ° C). After rinsing, the fixed immunoglobulins are revealed using peroxidase-conjugated goat antibodies directed against the Fc fragments of mouse immunoglobulins.
• an antibody binds to the Fab fragments of 6/32, it is either directed against the isotype or against the idiot.
• the subsequent use of a second ELISA in which the boxes are lined with Fab fragments of an antibody of the same isotype will make it possible to eliminate false positives and to keep only the anti-idiotypic antibodies, subsequently called Igld / HLA-I .
• the anti-idiotypic nature of the hybridoma supernatants was evaluated by ELISA on plates lined with 6/32 Fab fragments.
• the Fv cells are incubated at 5000 cells per well of multiplaque 96. After the confluence, the cells are rinsed and fixed by a treatment not inducing permeabilization (0.25% of glutaraldehyde), saturated with a 1M glycine solution ⁇ pH8 then with a rabbit IgG solution. The cells are then rinsed thoroughly and the antibodies incubated for 2 h at room temperature. After rinsing, the fixed immunoglobulins are revealed using peroxidase-conjugated goat antibodies directed against mouse immunoglobulins.
• an antibody binds to non-permeable endothelial cells when the preimmune immunoglobulins do not, it means that it recognizes a specific receptor on these cells.
• the 27 monoclonal antibodies were then subjected to an ELISA test on F / V cells.
• Igld / HLA-I antibodies recognize receptors on endothelial cells. III.2 - Cell migration.
• the FV cells are seeded at high density in 12-well plates in DMEM medium supplemented with 10% newborn calf serum and 1 ng / ml of VEGF (50,000 cells per well). Once the confluence is reached, the cells are transferred to a medium without serum and without VEGF. The next day the cells are removed by scraping using a foam tip and rinsed 3 times. The modulators are added to the medium and 24 hours later, the cells are fixed and stained with May Grunwald Giemsa then the cells that have migrated are counted in at least 8 fields.
• the HUVEC cells are seeded at low density (2000 cells / cm 2) in boxes of 12 wells, the surface of which has been previously coated with gelatin.
• the modulators are added after the cells have adhered to the culture plastic, then after 2 days. Each condition is studied in three different wells. The cells are trypsinized and counted on the fifth day. The percentage of inhibition is calculated as the ratio:
• the anti-idiotypic nature of the purified immunoglobulins was evaluated by ELISA on plates lined with Fab fragments of 6/32. Pre-immune immunoglobulins do not bind to Fabs and Igld / HLA-I does.
• Immunoglobulins were then assessed for their ability to recognize activated endothelial cells. As shown in Figure 1, pre-immune immunoglobulins do not bind to endothelial cells and Igld / HLA-I does. Figure 1 therefore shows that Igld / HLA-I recognizes receptors on endothelial cells.
• HLA class I transmembrane induced by ligation using the antibody 6/32 stimulated the proliferation of HUVEC cells. This effect is thought to be due to the overexpression of FGF2 receptors (Harris, 1997). To determine whether this ligation also involved the VEGF system, the cells were incubated with 10 ⁇ g / ml of 6/32 and 1 ⁇ g / ml of VEGFRls or VEGFR2s.
• the FV cells are seeded at high density (50,000 cells / well) in 12-well dishes in the presence of 2 ng / ml of VEGF. 2 days after confluence, cell proliferation is stopped by transfer to a medium containing neither serum nor VEGF for 24 hours. An injury is then practiced in the monohull by scraping using a foam tip, the cells rinsed thoroughly then 10 ⁇ g / ml of 6/32 or 50 ng / ml of VEGF are added in the presence or absence of 1 ⁇ g / ml of VEGFRls or
• VEGFR2s Each condition is studied in two different wells. 24 hours after the cells are rinsed with DMEM medium, stained with May Grunwald-Giemsa and the cells which have migrated are counted in 8 fields by condition.
• Figure 2 shows that the chemotactic effect of W6 / 32 is not inhibited by VEGFRls or VEGFR2s while the chemotactic effect of VEGF is abolished. Consequently, the signaling of 6/32 is not linked to that of VEGF.
• FIG. 3 shows the inhibition of the chemotactic action of VEGF by Igld / HLA-I.
• the FV cells are seeded at high density (50,000 cells / well) in 12-well dishes in the presence of 2 ng / ml of VEGF. 2 days after confluence, cell proliferation is stopped by transfer to a medium containing neither serum nor VEGF for 24 hours. A wound is then practiced in the monolayer by scraping using a foam tip, the cells rinsed thoroughly then 50 ng / ml of VEGF are added in the presence or absence of 5-50 ⁇ g / ml of Igld / HLA-I . Each condition is studied in two different wells. 24 hours after the cells are rinsed with DMEM medium, stained with May Grunwald-Giemsa and migrated cells are counted in 8 fields by condition.
• Igld / HLA-I has no effect on the spontaneous migration of FV cells. On the other hand, a 100-fold molar excess is sufficient to almost completely inhibit the chemotactic activity of VEGF. Consequently, the binding of Igld / HLA-I to its endothelial receptors induces an effect inhibiting the activity of VEGF.
• Igld / HLA-I has no effect on the basal proliferation of HUVEC cells ( Figure 4). However, 2 classes of Igld / HLA-I antibodies can be distinguished:
• Anti-HLA class I antibodies transduce signais in endothelial cells resulting in FGF receptor translocation, down-regulation of ICAM-1 and cell proliferation.

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