EP1414862A1 - Anti-idiotypic antibodies of hla class i molecules and the use thereof in the preparation of compositions that are designed to inhibit vascular activation - Google Patents

Anti-idiotypic antibodies of hla class i molecules and the use thereof in the preparation of compositions that are designed to inhibit vascular activation

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Publication number
EP1414862A1
EP1414862A1 EP02772487A EP02772487A EP1414862A1 EP 1414862 A1 EP1414862 A1 EP 1414862A1 EP 02772487 A EP02772487 A EP 02772487A EP 02772487 A EP02772487 A EP 02772487A EP 1414862 A1 EP1414862 A1 EP 1414862A1
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EP
European Patent Office
Prior art keywords
hla
antibodies
idiotypic
cells
molecules
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EP02772487A
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German (de)
French (fr)
Inventor
Jean Plouet
Pierre Fons
Marc Trombe
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Abtech
Centre National de la Recherche Scientifique CNRS
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Abtech
Centre National de la Recherche Scientifique CNRS
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    • 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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Abstract

The invention relates to an anti-idiotypic antibody of a HLA class 1 molecule, a fragment or derivative of same and a gene that codes for said antibody. The invention also relates to a pharmaceutical composition that can be used to prevent or inhibit angiogenesis comprising at least one such anti-idiotypic antibody.

Description

ANTICORPS ANTI-IDIOTYPIQUES DE MOLECULES HLA DE CLASSE I ET LEUR UTILISATION POUR LA PREPARATION DE COMPOSITIONS DESTINEES A INHIBER L'ACTIVATION VASCULAIRE . ANTI-IDIOTYPIC ANTIBODIES OF CLASS I HLA MOLECULES AND THEIR USE FOR THE PREPARATION OF COMPOSITIONS FOR INHIBITING VASCULAR ACTIVATION.
La présente invention se rapporte au domaine de l'inhibition de 1 ' angiogénèse impliquée dans des nombreux syndromes prolifératifs pathologiques. L'invention concerne des anticorps anti-idiotypiques de molécules du Complexe Major d'Histocompatibilité (CMH) et tout particulièrement à des anticorps anti-idiotypiques de molécules solubles HLA de classe 1, comme HLA-Gs ou HLA-B7s. L'invention concerne la préparation de ces anticorps et leur utilisation dans des compositions pharmaceutiques utiles pour inhiber 1 ' angiogénèse , et donc destinées au traitement des pathologies angiogéniques impliquant des cellules endothéliales et en particulier pour enrayer leur prolifération tumorale. L'invention vise également l'utilisation de ces anticorps anti-idiotypiques pour vectoriser des substances d' intérêt thérapeutique ou de diagnostic au niveau des cellules endothéliales impliquées dans un processus d' angiogénèse pathologique ou non pathologique.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. 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.
Les cellules endothéliales tapissent l'intérieur de tous les vaisseaux sanguins de l'organisme. Ces cellules ont un phénotype quiescent, elles ne prolifèrent quasiment pas, seulement 0,01% des cellules endothéliales sont engagées dans la division cellulaire à un moment donné. Le rôle physiologique des cellules endothéliales est très vaste, elles participent à de nombreux phénomènes vitaux pour l'organisme : coagulation sanguine, maintien de la pression artérielle, recrutement de cellules circulantes, synthèse de cytokines, construction de nouveaux vaisseaux irriguant les territoires voisins lors d'une hypoxie. Toutes ces fonctions nécessitent ou s'accompagnent d'un changement de phenotype des cellules endothéliales qui deviennent alors «activées», c'est à dire que la paroi vasculaire devient capable de fixer des cellules du sang circulant ou de répondre aux actions chimiotactiques des facteurs de croissance. L'activation endothéliale est donc un état transitoire commun aux cellules endothéliales, préalable à toute réponse fonctionnelle.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.
Chez l'adulte, les parois de ces vaisseaux ne présentent pas de molécules d'adhésion pour immobiliser les lymphocytes du sang circulant. Lors d'une inflammation, les cellules endothéliales expriment à leur surface des molécules d'adhésion les lymphocytes activés -en particulier les cellules NK (natural killer) - c'est à dire qu'elles déroulent des antennes pour immobiliser les lymphocytes tueurs. Lors des greffes par exemple le maintien au froid du greffon suffit à activer les cellules endothéliales du greffon qui alors deviennent capables d'immobiliser les lymphocytes. Ces lymphocytes pourront traverser la couche des cellules endothéliales ou les détruire puis coloniser le greffon et induire la synthèse de cytokines, ce qui amplifie la réaction immunitaire et aboutit à la destruction du greffon.In adults, the walls of these vessels do not have adhesion molecules to immobilize the lymphocytes of the circulating blood. During inflammation, 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. During transplants, for example, keeping the graft cold is enough to activate the endothelial cells of the graft which then become capable of immobilizing the lymphocytes. 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.
Lorsqu'un territoire requiert un apport accru en oxygène et en nutriments, il réagit en relarguant des facteurs solubles qui convertissent le phenotype des cellules endothéliales en un phenotype activé, et des facteurs mitogènes pour les cellules endothéliales de phenotype activé. Ces cellules endothéliales acquièrent enfin un phenotype angiogenique et deviennent capables d'effectuer le programme d ' angiogénèse, c'est-à-dire le développement de nouveaux vaisseaux sanguins à partir du réseau préexistant. Ce phénomène est normal et contrôlé lors d'une blessure ou dans la maturation du corps jaune de l'ovaire; en revanche, il devient dangereux dans des situations pathologiques où 1 ' angiogénèse n'est plus contrôlée, par exemple dans la rétinopathie diabétique, la polyarthrite et le développement tumoral. L'entrée d'une cellule dans un programme d' angiogénèse nécessite son activation préalable (Ortéga, 1997 ; Ortéga, 1999) .When a territory requires an increased supply of oxygen and nutrients, it reacts by releasing soluble factors which convert the phenotype of endothelial cells into an activated phenotype, and mitogenic factors for endothelial cells of activated phenotype. These endothelial cells finally acquire an angiogenic phenotype and become capable of carrying out the angiogenesis program, that is to say the development of new blood vessels from the preexisting network. This phenomenon is normal and controlled during an injury or in the maturation of the yellow body of the ovary; on the other hand, it becomes dangerous in pathological situations where angiogenesis is no longer controlled, for example in diabetic retinopathy, polyarthritis and tumor development. The entry of a cell into an angiogenesis program requires its prior activation (Ortéga, 1997; Ortéga, 1999).
Il a été montré que les cellules endothéliales expriment des molécules HLA de classe I à leur surface dont le rôle n'est pas clairement établi (Majema, 1997 ; Rebman 1997 ; Blaschitz 1997) . Il a été proposé que la surexpression de ces molécules HLA de classe I par les cellules endothéliales pourrait inhiber la migration transendothéliale des lymphocytes NK et contribuer à atténuer le rejet de greffe. Par ailleurs la dimérisation de molécules HLA-I par l'intermédiaire d'un anticorps reconnaissant toutes les molécules HLA-I stimule l'expression de récepteurs du FGF2 à la surface des cellules endothéliales et leur prolifération (Harris, 1997) . Les molécules HLA de classe I ont été décrites comme pouvant s'associer structurellement à certains récepteurs membranaires , y compris des récepteurs à l'insuline et récepteurs à EGF. Il a aussi été démontré que des peptides dérivés de molécules HLA de classe I pouvaient réguler la fonction des récepteurs à l'insuline. Cependant, à ce jour aucune description d'un quelconque rôle non immunologique de la forme soluble de HLA Gl n'a été rapporté et il n'existe non plus de données expérimentales démontrant la présence de récepteurs au HLA-I sur les cellules endothéliales. Aucun effet biologique des HLA-I sur ces cellules ou dans 1 ' angiogénèse n'a été décrit.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. Peptides derived from HLA class I molecules have also been shown to regulate the function of insulin receptors. However, to date 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.
Les Inventeurs ont développé une expertise dans le domaine de l' activation endothéliale, et ont cherché à vérifier si la dimérisation de molécules HLA-I par l'anticorps W6/32 (Barnstable CJ, 1978 ; Brodsky FM, 1982) stimulait l'expression des récepteurs du VEGF. Sachant que certaines molécules HLA-I possèdent un polymorphisme important et qu'elles se lient à des récepteurs distincts encore mal connus, un mime polyvalent des HLA-I a été préparé grâce à la stratégie de fabrication d'anticorps anti - idiotypiques .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.
En conséquence, l'invention a pour objet un anticorps anti-idiotypique d'une molécule HLA de classe 1. L'invention envisage tant des anticorps anti- idiotypiques polyclonaux ou monoclonaux qui peuvent être préparés par des techniques décrites dans la littératureConsequently, 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
(Kohler G, 1976) à partir de molécule HLA de classe 1 comme rapportées dans la partie expérimentale ci-après. Il peut aussi s'agir de fragments de tels anticorps anti- idiotypique comme des fragments Fab'2 ou Fab ou d'une combinaison de tels fragments.(Kohler G, 1976) from 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.
Il s'agit de préférence d'anticorps anti- idiotypique de toute molécule HLA de classe I soluble ou non. Avantageusement, la molécule HLA de classe 1 est choisie parmi : HLA-A, B, C, D, F, ou G.They are preferably anti-idiotypic antibodies of any HLA class I molecule, soluble or not. Advantageously, the class 1 HLA molecule is chosen from: HLA-A, B, C, D, F, or G.
Ainsi, l'invention concerne des anticorps anti- idiotypiques de molécules dérivés des molécules HLA de classe 1, solubles ou non, présentant des propriétés anti- angiogéniques que l'homme du métier est à même de tester. Il peut s'agir de fragments, de forme tronquée, de séquences modifiées par délétion, addition ou suppression d'un ou plusieurs acides aminés.Thus, 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.
L'invention concerne aussi une molécule d'acide nucléique comprenant ou constituée par une séquence polynucléotidique codant pour un anticorps anti-idiotypique de toute molécule HLA de classe I, soluble ou non, un fragment ou dérivé de celui-ci. Une telle molécule d'acide nucléique est utile pour la préparation des anticorps anti-idiotypiques selon l'invention par des techniques de génie génétique.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. Such a nucleic acid molecule is useful for the preparation of the anti-idiotypic antibodies according to the invention by genetic engineering techniques.
L'invention envisage à titre de molécule d'acide nucléique comprenant une séquence polynucléotidique codant un anticorps anti-idiotypique d'une molécules soluble HLA de classe 1, une molécule d'acide nucléique comprenant ladite séquence placée sous le contrôle de séquences de régulation de son expression. Une telle molécule d'acide nucléique est un vecteur, comme par exemple un plasmide, un virus recombinant, une cellule transfectée par un vecteur d'expression plasmidique ou viral.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. Such 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.
Les anticorps anti-idiotypiques selon l'invention sont remarquables en ce qu' ils sont capables de se fixer sur les cellules endothéliales et d'inhiber l' activation endothéliale menant à 1 ' angiogénèse .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.
L'invention a donc aussi pour objet une composition pharmaceutique utile pour prévenir ou inhiber 1 ' angiogénèse comprenant au moins un anticorps anti-idiotypique ou une molécule d'acide nucléique codant un tel anticorps comme défini précédemment.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.
Une telle molécule d'acide nucléique est utile dans des protocoles de thérapie génique ou de thérapie cellulaire consistant à administrer la dite molécule d'acide nucléique ou des cellules transformées par la dite molécule d'acide nucléique a un individu de façon à exprimer l'anticorps anti-idiotypique et ainsi inhiberSuch a 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
1 ' angiogénèse .1 angiogenesis.
Ainsi, ces anticorps anti-idiotypiques et les molécules d'acide nucléique correspondantes sont particulièrement adaptées pour la préparation de médicaments destinés à prévenir ou à traiter 1 ' angiogénèse et donc la prolifération des cellules endothéliales. Parmi les pathologies dont le traitement nécessite l'inhibition de l' activation endothéliale, on peut citer le rejet d' allogreffes et de xénogreffes, les acrocyanoses , les sclérodermies, les cancers, les rétinopathies diabétiques, la polyarthrite rhumatoïde, les angiomes, les angiocarcinomes, en particulier la maladie de Castelman et le sarcome de Kaposi. Les compositions pharmaceutiques selon l'invention sont aussi utiles pour la préparation de greffons entre le prélèvement et la transplantation. On sait en effet, que le froid suffit à induire la translocation d' intégrines permettant l'adhésion de lymphocytes activés sur les cellules endothéliales, et donc à activer les cellules endothéliales. La désactivation endothéliale réalisée par la mise en oeuvre des anticorps anti-idiotypiques et des compositions les contenant selon l'invention permettrait donc de s'opposer à la réaction de rejet. L'invention concerne donc un procédé de préparation de greffons entre le prélèvement et la transplantation consistant à mettre en contact le dit greffon avec des anticorps anti-idiotypiques selon l'invention. La concentration en anticorps anti-idiotypiques pour cette application serait par exemple de l'ordre de 100 microgrammes à 100 mg par kilo de poids corporel.Thus, 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. Among pathologies whose treatment requires the inhibition of 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. It is known in fact that the cold is sufficient to induce the translocation of integrins allowing the adhesion of activated lymphocytes to the endothelial cells, and therefore to activate the endothelial cells. The endothelial deactivation carried out by the use of anti-idiotypic antibodies and of the compositions containing them according to the invention would therefore make it possible to oppose the rejection reaction. 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. The 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.
La mise en évidence dans le cadre de l'invention de la fixation des molécules HLA de classe 1 sur un récepteur à la surface des cellules endothéliales permettent également d'utiliser les anticorps anti-idiotypiques de ces molécules pour identifier plus précisément ces récepteurs et identifier des ligands. De tels ligands sont utiles soit pour stimuler soit pour inhiber l' activation endothéliale et ainsi disposer de nouveaux agents actifs pour traiter les pathologies. Parmi les pathologies nécessitant de stimuler l' activation endothéliale et donc 1 ' angiogénèse on peut citer la cicatrisation, la maturation du corps jaune de l'ovaire, la perfusion des territoires ischémies lors de thromboses vasculaires comme dans l'artérite des membres inférieurs et l'infarctus du myocarde. A titre de substance de référence capable de stimuler 1 ' angiogénèse on peut citer des anticorps monoclonaux ou polyclonaux dirigés contre les molécules HLA de classe 1, comme l'anticorps 6/32.The demonstration in the context of the invention of the attachment of HLA class 1 molecules to a receptor on the surface of endothelial cells also makes it possible to use the anti-idiotypic antibodies of these molecules to more precisely identify these receptors and identify ligands. Such ligands are useful either for stimulating or for inhibiting endothelial activation and thus having new active agents for treating pathologies. Among the 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. As a reference substance capable of stimulating angiogenesis, mention may be made of monoclonal or polyclonal antibodies directed against HLA class 1 molecules, such as the antibody 6/32.
Une méthode d' identification des récepteurs des molécules HLA de classe 1 à la surface des cellules endothéliales selon l'invention comprend les étapes suivantes :A method of identifying receptors for class 1 HLA molecules on the surface of endothelial cells according to the invention comprises the following steps:
- Purification de l'anticorps monoclonal selon les techniques connues de l'homme du métier.- Purification of the monoclonal antibody according to techniques known to those skilled in the art.
Préparation d'une matrice de chromatographie d'affinité dans laquelle l'anticorps monoclonal purifié est conjugué à la phase solide.Preparation of an affinity chromatography matrix in which the purified monoclonal antibody is conjugated to the solid phase.
- Chromatographie d'un lysat cellulaire (exemple cellules endothéliales cultivées) ou tissulaire (par exemple placenta humain) . - Elution du récepteur par un agent chaotropique- Chromatography of a cell (eg cultured endothelial cells) or tissue (eg human placenta) lysate. - Elution of the receptor by a chaotropic agent
(NaCl, urée, chlorure de guanidine, choc acide ou basique) ou ligand spécifique (exemple fragment Fab du susdit anticorps) .(NaCl, urea, guanidine chloride, acid or basic shock) or specific ligand (example Fab fragment of the above antibody).
- Séquençage de la protéine par les techniques classiques.- Sequencing of the protein by conventional techniques.
Une méthode d'identification de ligands des récepteurs des molécules HLA de classe 1 à la surface des cellules endothéliales selon l'invention comprend des étapes similaires, excepté que la matrice de la chromatographie d'affinité présentera le récepteur purifié précédemment ou un fragment. Alternativement le récepteur pourra être remplacé par un récepteur recombinant obtenu par les techniques de génie génétique.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. Alternatively, the receptor can be replaced by a recombinant receptor obtained by genetic engineering techniques.
L'invention s'intéresse encore au dosage des molécules HLA de classe I ou d'anticorps anti-idiotypiques, comme marqueurs de pathologies angiogéniques . Par exemple l'anticorps W6/32 ou tout autre anticorps anti molécule HLA de classe I est immobilisé sur un support. Il est mis en contact une quantité fixe de l'anticorps anti-idiotypique marqué par de la biotine ou un élément radioactif tel que l'iode 125 et un volume connu du liquide biologique contenant la molécule à tester qui rentre donc en compétition avec la liaison de l'anticorps anti-idiotypique marqué sur l'anticorps anti HLA de classe I. Ainsi donc la diminution de la liaison de l'anticorps marqué anti- idiotypique est proportionnelle à la quantité d'anticorps anti-idiotypique ou de molécules HLA de classe I dans le milieu biologique. Ainsi, l'invention a pour objet une méthode de dosage de molécules HLA de classe I ou d'anticorps anti-idiotypiques de ces molécules éventuellement présentes dans un échantillon biologique comprenant : la mise en contact desdites molécules ou anticorps de l'échantillon avec une quantité déterminée d'anticorps anti-idiotypiques de l'invention, avantageusement marqués et fixés à des anticorps antimolécule HLA de classe I,The invention also relates to the assay of HLA class I molecules or of anti-idiotypic antibodies, as markers of angiogenic pathologies. For example, 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. Thus, 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. Thus, 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,
- la mesure de la diminution de la liaison des anticorps anti-idiotypiques marqués avec les anticorps anti-molécule HLA de classe I.- measuring the decrease in the binding of labeled anti-idiotypic antibodies with anti-HLA class I molecule antibodies.
Avantageusement, cette méthode est réalisée en immobilisant les anticorps anti-molécule HLA de classe I sur un support .Advantageously, this method is carried out by immobilizing the anti-HLA class I molecule antibodies on a support.
Les anticorps anti-idiotypiques selon l'invention sont remarquables en ce qu' ils peuvent se fixer à la surface des cellules endothéliales grâce aux récepteurs des molécules HLA de classe 1. Ainsi, les dits anticorps anti- idiotypiques sont utiles pour vectoriser des substances d'intérêt au niveau de foyers d' activation endothéliale pathologique ou non, comme de sites angiogéniques . L'invention a donc pour objet l'utilisation d'un anticorps anti-idiotypique d'une molécule HLA de classe 1 seul ou associé à une substance d'intérêt pour la préparation d'une composition destinée à inhiber 1 ' angiogénèse ou à détecter des sites angiogéniques.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. Thus, 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.
La substance d' intérêt peut être une substance chimique ou biologique active comme un médicament. On peut citer à titre d'exemple de tels médicaments : une toxine animale ou végétale, un composé radioactif, une drogue cytostatique ou cytolytique, un gène suicide ou une anti- sens .The substance of interest can be a chemical or biological substance active like a drug. Examples of such 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.
La substance d' intérêt peut être aussi une substance de diagnostic comme un traceur radioactif ou un produit de contraste pour l'imagerie par résonance magnétique permettant de marquer les sites angiogéniques.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.
Les foyers d' activation endothéliale peuvent ainsi être visualisés dans tout système d'imagerie médicale.The endothelial activation foci can thus be viewed in any medical imaging system.
L'invention concerne donc un composé constitué d'un anticorps anti-idiotypique d'une molécule HLA de classe 1, ou d'un dérivé de celui-ci, couplé à une substance d'intérêt comme défini ci-dessus, ledit composé étant capable de se fixer aux cellules endothéliales par l'intermédiaire d'un récepteur. Les anticorps de l'invention constituent donc un système de vectorisation de substances d' intérêt au niveau des cellules endothéliales impliquées dans des phénomènes angiogéniques particulièrement utiles pour traiter ou détecter les pathologies indiquées précédemment . Le couplage de l'anticorps anti-idiotypique avec la substance d' intérêt peut être réalisé par tout moyen de liaison clivable ou non clivable dans un milieu biologique comme par exemple :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:
- Couplage par liaison covalente Réaction d'oxydation aboutissant à la substitution d'un atome d'hydrogène par un atome d'iode sur un noyau indole- Coupling by covalent bond Oxidation reaction leading to the substitution of a hydrogen atom by an iodine atom on an indole nucleus
Ces composés de l'invention peuvent être aussi des protéines ou de gènes de fusion.These compounds of the invention can also be proteins or fusion genes.
D'autres avantages et caractéristiques de l'invention apparaîtront dans les exemples qui suivent où il sera fait référence aux dessins en annexe dans lesquels :Other advantages and characteristics of the invention will appear in the examples which follow where reference will be made to the appended drawings in which:
- La figure 1 représente le titre des souris sur cellules Fv et montre que Igld/HLA-I reconnaît des récepteurs sur les cellules endothéliales.- Figure 1 shows the titer of mice on Fv cells and shows that Igld / HLA-I recognizes receptors on endothelial cells.
- La figure 2 montre que l'effet chimiotactique du 6/32 n'est pas inhibé par les VEGFRls ou VEGFR2s alors que l'effet chimiotactique du VEGF est aboli.- Figure 2 shows that the chemotactic effect of 6/32 is not inhibited by VEGFRls or VEGFR2s while the chemotactic effect of VEGF is abolished.
La figure 3 montre l'inhibition de l'action chimiotactique du VEGF par Igld/HLA-I.FIG. 3 shows the inhibition of the chemotactic action of VEGF by Igld / HLA-I.
- La figure 4 montre qu' Igld/HLA-I n'a pas d'effet sur la prolifération basale des cellules HUVEC.- Figure 4 shows that Igld / HLA-I has no effect on the basal proliferation of HUVEC cells.
La figure 5 montre l'action des anticorps monoclonaux anti-idiotypique de HLA de classe 1 sur la prolifération des cellules endothéliales.FIG. 5 shows the action of the anti-idiotypic HLA class 1 monoclonal antibodies on the proliferation of endothelial cells.
A - METHODES D'ETUDE.A - STUDY METHODS.
I - Réactifs .I - Reagents.
1.1 - Protéines utilisées.1.1 - Proteins used.
Le VEGF (isoforme de 165 acides aminés) est produit par infection de cellules d'insecte SF9 par un baculovirus recombinant contenant le cDNA correspondant et le FGF2 est produit chez Escherichia Coli (Plouët et al., 1997).VEGF (165 amino acid isoform) is produced by infection of SF9 insect cells with a recombinant baculovirus containing the corresponding cDNA and FGF2 is produced in Escherichia Coli (Plouët et al., 1997).
L'anticorps 6/32 est produit par injection de l'hybridome dans le péritoine de souris Balb/c. Les ascites sont recueillies 10-15 jours après l'inoculation et les IgG purifiées par précipitation au sulfate d'ammonium suivie de chromatographie sur protéine A sepharose. Les fragments Fab sont préparés par clivage à la papaïne.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.
Les récepteurs solubles du VEGF sont produits dans le milieu conditionné de cellules CHO transfectées par la construction correspondante. Les domaines extracellulaires des récepteurs VEGFR1 et VEGFR2 (boucles de type immunoglobuline 1-7) sont synthétisés par PCR, puis séquences pour vérifier qu'il n'y a pas eu de mutation. Les inserts sont ensuite insérés dans le vecteur pcDNA- 3/myc/His (Invitrogen) . Les plasmides sont transfectés dans des cellules CHO pgsA-745 et les clones sécréteurs sélectionnés par leur résistance à la généticine. Le milieu conditionné (1 litre) est ensuite précipité par 50% (poids/poids) de sulfate d'ammonium, dialyse contre du tampon Tris 0,01 M, NaCl 0,05 M puis chromatographie sur une colonne de Sp Sepharose. Les fractions actives sont collectées par un gradient discontinu de NaCl (0,2-0,5 M) puis chromatographiées sur une colonne de nickel-sepharose . La pureté des protéines VEGFR1 et VEGFR2 purifiées est ensuite attestée par gel de polyacrylamide-SDS (>90%) et conservés à -80°C jusqu'à l'utilisation. 1.2 - Cellules utilisées.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 (immunoglobulin 1-7 type loops) 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.
Les milieux de culture et les sérums proviennent de la compagnie Life Technologies™. Les cellules endothéliales de veines de cordon ombilical humain (HUVEC) sont cultivées sur plastique recouvert de gélatine diluée à 0,2% dans du PBS . Les cellules sont cultivées en milieu endothélial SFM supplémenté par 50 unités/ml de pénicilline, 50 μg/ml de streptomycine, et 20% de sérum de veau fœtal et maintenues dans une étuve à 10% C02. Ces cultures sont entretenues avec du VEGF à 2 ng/ml ajouté au milieu tous les deux jours .The culture media and the sera come from the company Life Technologies ™. 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 μg / 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.
Les cellules endothéliales d'aorte bovine fœtale (FBAE) sont cultivées sur plastique recouvert de gélatine diluée à 0,2% dans du PBS . Les cellules sont cultivées en milieu DMEM-glutamax supplémenté par 50 unités/ml de pénicilline, 50 μg/ml de streptomycine, et 10% de sérum de veau nouveau-né et maintenues dans une étuve à 10 % C02. Ces cultures sont entretenues avec du VEGF à 2 ng/ml ajouté au milieu tous les deux jours et exhibent un phenotype angiogenique .Fetal bovine aorta endothelial cells (FBAE) 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 μg / ml of streptomycin, and 10% of newborn 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 and exhibit an angiogenic phenotype.
II - Préparation d'anticorps anti-idiotypiques.II - Preparation of anti-idiotypic antibodies.
11.1 - Fabrication d'anticorps préimmuns (Ig PI) . Avant chaque immunisation, du sang est prélevé, le sérum est fractionné immédiatement après le recueil et 1,5 ml de sérum est chromatographie sur une colonne de protéine A. La colonne est lavée par du PBS et les immunoglobulines sont éluées par de la glycine 0,2 M, pH 2,5, immédiatement neutralisées par adjonction de 1/5 du volume de K2HP04 1 M, puis dialysées contre du PBS. Les immunoglobulines (Ig PI) s o n t c o n s e r v é e s à11.1 - Manufacture of pre-immune antibodies (Ig PI). Before each immunization, blood is taken, the serum is fractionated immediately after collection and 1.5 ml of serum is chromatographed on a protein A column. The column is washed with PBS and the immunoglobulins are eluted with glycine 0 , 2 M, pH 2.5, immediately neutralized by adding 1/5 of the volume of 1 M K2HPO4, then dialyzed against PBS. Immunoglobulins (Ig PI) s o n t c o n s e r v é e à
-80 °C jusqu'à leur utilisation.-80 ° C until used.
11.2 - Anticorps anti-idiotypiques de HLA-I (Ig2Id HLA-1) .11.2 - HLA-I anti-idiotypic antibodies (Ig2Id HLA-1).
Les souris reçoivent par voie sous-cutanée 10 μg de 6/32 mélangé volume à volume avec de l'adjuvant complet de Freund sous un volume final de 100 μl . Quatre rappels sont effectués toutes les deux semaines par injection par voie intapéritonéale du mélange, excepté que l'adjuvant complet est remplacé par de l'adjuvant incomplet.The mice receive subcutaneously 10 μg of 6/32 mixed volume to volume with complete Freund's adjuvant in a final volume of 100 μl. Four booster shots are given every two weeks by intraperitoneal injection of the mixture, except that the complete adjuvant is replaced by incomplete adjuvant.
La veille de la fusion, soit à J 67, cinq souris Balbc non- immunisées sont sacrifiées pour l'obtention des macrophages servant de cellules nourricières aux hybridomes. Les macrophages sont récupérés par injection de 8ml de milieu de culture ISCOVE dans le péritoine des souris. Après centrifugation, les macrophages sont repris dans du milieu ISCOVE contenant 20% de sérum de veau foetal (Biochrom, lot 5612). Ces cellules sont ensuite distribuées dans 20 plaques de 96 puits à raison de lOOμl par puitsThe 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 μl per well
(environ 3 x 10 cellules/puits) .(approximately 3 x 10 cells / well).
Le lendemain, soit à J 68, les souris présentant les titres les plus élevés d'anticorps anti-Id sont sacrifiées. Leurs rates sont prélevées et dilacérées dans du milieu ISCOVE pour libérer les splénocytes. Le tissu conjonctif est dégagé et les splénocytes sont centrifugés et comptés. En parallèle, la lignée de myélome de souris Ag8X63 a été cultivée depuis 10 jours dans du milieu ISCOVE à 20% de sérum Myoclone. Ces cellules sont lavées et numérées .The next day, ie on D 68, the 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. In parallel, the mouse myeloma line Ag8X63 was cultured for 10 days in ISCOVE medium containing 20% Myoclone serum. These cells are washed and scanned.
Les deux types cellulaires, splénocytes et myélomes, sont mélangés de façon à obtenir un rapport de 1 cellule de myélome Ag8X63 pour 6 splénocytes. La fusion s'effectue par ajout de 20 fois 50 μl de polyethylene glycol (PEG) à 30 secondes d'intervalle. 4 ml de milieu ISCOVE préchauffé à 37°C sont alors ajoutés goutte à goutte sur la suspension cellulaire, puis après une période d'incubation de 4 minutes à 37°C, 4 ml sont ajoutés. La suspension est centrifugée puis le culot cellulaire est alors repris dans 100 ml de milieu ISCOVE complémenté avec 20% de sérum de veau fœtal et du HAT IX (50X: Hypoxanthine 5mM, Aminoptérine 20μM et Thymidine 0.8mM) et distribué à raison de lOOμl par puits sur les macrophages. Après 5 jours, 100 μl de milieu HAT est ajouté, et entre 8 et 14 jours le milieu conditionné de chaque hybridome est prélevé pour mesurer la quantité d'anticorps dirigés contre les fragments Fab de l'anticorps 6/32The 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 μl 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. 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 20μM and Thymidine 0.8mM) and distributed at the rate of 100 μl per well on macrophages. After 5 days, 100 μl 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
(Elisa anti-idiotype) et les cellules endothéliales. Les hybridomes sélectionnés pour leur capacité à sécréter des anticorps anti-Id sont alors clones, c'est-à- dire que les cellules sont ensemencées en condition de dilution limite (5 cellules/ml) sous un volume de 0,1 ml par puits. Le milieu est changé après 10 jours. Après 15 jours, certains puits contiennent des foyers de cellules qui se sont multipliées à partir de la cellule ensemencée au départ, donc toutes ces cellules sont identiques et sont issues du même clone. Quand la surface occupée par les cellules représente au moins la moitié de la surface totale du puits, le milieu est prélevé et analysé comme précédemment. A ce stade, on peut sélectionner les clones producteurs d'anticorps anti-Id et connaître leur spécificité.(Elisa anti-idiotype) and endothelial cells. 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. When 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.
Une fois les clones identifiés leur nature monoclonale a été confirmée par l'opération classique consistant à ensemencer une plaque de 96 puits avec des cellules issues du même clone diluées en conditions limites comme précédemment. Les clones sécréteurs doivent donc tous sécréter un anticorps de même spécificité pour que l'on déclare cet anticorps monoclonal.Once the clones have been identified, their monoclonal nature has been confirmed by the conventional operation consisting in seeding a 96-well plate with cells from the same clone diluted under boundary conditions as before. The secreting clones must therefore all secrete an antibody of the same specificity for this monoclonal antibody to be declared.
Un troisième clonage est alors effectué pour s'assurer que les clones sont bien monoclonaux.A third cloning is then carried out to ensure that the clones are indeed monoclonal.
Le criblage des anticorps anti-Id est effectué par les 2 Elisa, comme décrit ci-après. III - Étude de la spécificité des anticorps.The screening of anti-Id antibodies is carried out by the 2 Elisa, as described below. III - Study of the specificity of antibodies.
III.1 - ELISA. a) anti - idiotype .III.1 - ELISA. a) anti - idiotype.
Les boîtes de plastique sont tapissées de fragments Fab de l'anticorps W6/32. Les puits sont incubés avec les immunoglobulines à tester (2 h, 37°C) . Après rinçages, les immunoglobulines fixées sont révélées à l'aide d'anticorps de chèvre conjugués à la peroxydase dirigés contre les fragments Fc immunoglobulines de souris.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.
Ainsi, si un anticorps se fixe sur les fragments Fab de 6/32, il est soit dirigé contre l' isotype soit contre l'idiotope. L'utilisation ultérieure d'un second ELISA dans lequel les boîtes sont tapissées de fragments Fab d'un anticorps de même isotype permettra d'éliminer les faux positifs et de ne conserver que les anticorps anti- idiotypiques, subséquemment dénommé Igld/HLA-I. La nature anti-idiotypique des surnageants d' hybridomes a été évaluée par ELISA sur plaques tapissées de fragments Fab de 6/32.Thus, if 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.
635 hybridomes ont été testés. 27 anticorps monoclonaux ont été obtenus, b) Cellules endothéliales.635 hybridomas were tested. 27 monoclonal antibodies were obtained, b) Endothelial cells.
Les cellules Fv sont incubées à 5000 cellules par puits de multiplaque 96. Après la confluence, les cellules sont rincées et fixées par un traitement n' induisant pas la perméabilisation (0,25% de glutaraldéhyde) , saturées par une solution de glycine 1M~ pH8 puis par une solution d' IgG de lapin. Les cellules sont ensuite abondamment rincées puis les anticorps incubés 2 h à température ambiante. Après rinçages, les immunoglobulines fixées sont révélées à l'aide d'anticorps de chèvre conjugués à la peroxydase dirigés contre les immunoglobulines de souris.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.
Ainsi, si un anticorps se fixe sur les cellules endothéliales non perméabil isées alors que les immunoglobulines préimmunes ne le font pas, c'est qu'il reconnaît un récepteur spécifique sur ces cellules.Thus, if 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.
Les 27 anticorps monoclonaux ont ensuite été soumis à un test ELISA sur cellules F/V.The 27 monoclonal antibodies were then subjected to an ELISA test on F / V cells.
22 anticorps Igld/HLA-I reconnaissent des récepteurs sur les cellules endothéliales. III.2 - Migration cellulaire.22 Igld / HLA-I antibodies recognize receptors on endothelial cells. III.2 - Cell migration.
Les cellules FV sont ensemencées à forte densité dans des plaques de 12 puits en milieu DMEM supplémenté de 10% de sérum de veau nouveau-né et de 1 ng/ml de VEGF (50 000 cellules par puits) . Une fois la confluence atteinte les cellules sont transférées en milieu sans sérum et sans VEGF. Le lendemain les cellules sont décollées par grattage à l'aide d'une pointe mousse et rincées 3 fois. Les modulateurs sont ajoutés au milieu et 24 h après, les cellules sont fixées et colorées au May Grunwald Giemsa puis les cellules qui ont migré sont comptées dans au moins 8 champs .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.
III.3 - Prolifération cellulaire.III.3 - Cell proliferation.
Les cellules HUVEC sont ensemencées à faible densité (2000 cellules/cm2) dans des boîtes de 12 puits dont la surface a été préalablement tapissée de gélatine. Les modulateurs sont ajoutés après l'adhésion des cellules, au plastique de culture, puis après 2 jours. Chaque condition est étudiée dans trois puits différents. Les cellules sont trypsinisées et comptées au cinquième jour. Le pourcentage d' inhibition est calculé comme le rapport :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:
(nombre de cellules de l'échantillon - nombre de cellules en absence de VEGF) / (nombre de cellules en présence de VEGF - nombre de cellules en absence de VEGF)(number of cells in the sample - number of cells in the absence of VEGF) / (number of cells in the presence of VEGF - number of cells in the absence of VEGF)
B - RÉSULTATS .B - RESULTS.
I - ELISA.I - ELISA.
La nature anti-idiotypique des immunoglobulines purifiées a été évaluée par ELISA sur plaques tapissées de fragments Fab de 6/32. Les immunoglobulines pré-immunes ne se fixent pas sur les Fab et Igld/HLA-I le fait.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.
Les immunoglobulines ont ensuite été évaluées par leur capacité à reconnaître des cellules endothéliales activées. Comme l'indique la figure 1, les immunoglobulines pré-immunes ne se fixent pas sur les cellules endothéliales et Igld/HLA-I le fait. La figure 1 montre donc que Igld/HLA-I reconnaît des récepteurs sur les cellules endothéliales .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.
II - Migration. II a été démontré que la dimérisation de moléculesII - Migration. It has been shown that the dimerization of molecules
HLA de classe I transmembranaires induite par la ligation à l'aide de l'anticorps 6/32 stimulait la prolifération des cellules HUVEC. Cet effet serait dû à la surexpression de récepteurs du FGF2 (Harris, 1997) . Pour déterminer si cette ligation mettait en jeu aussi le système VEGF, les cellules ont été incubées avec 10 μg/ml de 6/32 et 1 μg/ml de VEGFRls ou VEGFR2s.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 μg / ml of 6/32 and 1 μg / ml of VEGFRls or VEGFR2s.
Les cellules FV sont ensemencées à forte densité (50 000 cellules/puits) dans des boîtes de 12 puits en présence de 2 ng/ml de VEGF. 2 jours après la confluence, la prolifération cellulaire est arrêtée par le transfert en milieu ne contenant ni sérum ni VEGF pendant 24 heures. Une blessure est alors pratiquée dans la monocoque par grattage à l'aide d'une pointe mousse, les cellules rincées abondamment puis 10 μg/ml de 6/32 ou 50 ng/ml de VEGF sont ajoutés en présence ou absence de 1 μg/ml de VEGFRls ouThe 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 μg / ml of 6/32 or 50 ng / ml of VEGF are added in the presence or absence of 1 μg / ml of VEGFRls or
VEGFR2s. Chaque condition est étudiée dans deux puits différents. 24 heures après les cellules sont rincées avec du milieu DMEM, colorées au May Grunwald-Giemsa et les cellules ayant migré sont comptées dans 8 champs par condition.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.
La figure 2 montre que l'effet chimiotactique du W6/32 n'est pas inhibé par les VEGFRls ou VEGFR2s alors que l'effet chimiotactique du VEGF est aboli. En conséquence, la signalisation du 6/32 n'est pas liée à celle du VEGF.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.
La figure 3 montre l'inhibition de l'action chimiotactique du VEGF par Igld/HLA-I.FIG. 3 shows the inhibition of the chemotactic action of VEGF by Igld / HLA-I.
Les cellules FV sont ensemencées à forte densité (50 000 cellules/puits) dans des boîtes de 12 puits en présence de 2 ng/ml de VEGF. 2 jours après la confluence la prolifération cellulaire est arrêtée par le transfert en milieu ne contenant ni sérum ni VEGF pendant 24 heures. Une blessure est alors pratiquée dans la monocouche par grattage à l'aide d'une pointe mousse, les cellules rincées abondamment puis 50 ng/ml de VEGF sont ajoutés en présence ou absence de 5-50 μg/ml de Igld/HLA-I. Chaque condition est étudiée dans deux puits différents. 24 heures après les cellules sont rincées avec du milieu DMEM, colorées au May Grunwald-Giemsa et les cellules ayant migré sont comptées dans 8 champs par condition.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 μg / 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 n'a pas d'effet sur la migration spontanée des cellules FV. En revanche un excès molaire de 100 fois suffit à inhiber pratiquement totalement l'activité chimiotactique du VEGF. En conséquence, la liaison de Igld/HLA-I à ses récepteurs endothéliaux induit un effet inhibant l'activité du VEGF.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.
III - Mitogénicité . Igld/HLA-I n'a pas d'effet sur la prolifération basale des cellules HUVEC (Figure 4) . En revanche 2 classes d'anticorps Igld/HLA-I peuvent être distinguées :III - Mitogenicity. 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:
- Les anticorps inhibiteurs (sur un mode dépendant de la dose) de la prolifération des cellules HUVEC induite par le VEGF. En conséquence, la liaison de Igld/HLA-I à ses récepteurs endothéliaux induit un effet inhibant l'activité du VEGF.- Antibodies that inhibit (in a dose-dependent fashion) the proliferation of HUVEC cells induced by VEGF. Consequently, the binding of Igld / HLA-I to its endothelial receptors induces an effect inhibiting the activity of VEGF.
- les anticorps stimulateurs (sur un mode dépendant de la dose) de la prolifération des cellules HUVEC induite par le VEGF. En conséquence, la liaison de Igld/HLA-I à ses récepteurs endothéliaux induit un effet stimulant de l'activité du VEGF. - Stimulating antibodies (in a dose-dependent mode) of the proliferation of HUVEC cells induced by VEGF. Consequently, the binding of Igld / HLA-I to its endothelial receptors induces a stimulating effect on the activity of VEGF.
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- Ortéga N.,Hutchings H., Plouët J. , 1999. Signal relays in the VEGF System. Front. Biosc, 4, D141-D152.- Ortéga N., Hutchings H., Plouët J., 1999. Signal relays in the VEGF System. Forehead. Biosc, 4, D141-D152.
- Plouët J. , Moro F., Coldeboeuf N. , Bertagnolli S., Clamens S., Bayard F., 1997. Extracellular cleavage of the vascular endothelial growth factor 189 aa forrm by "urokinae is required for its mitogeriic activity. J. Biol . Chem., 272, 13390-13396.- Plouët J., Moro F., Coldeboeuf N., Bertagnolli S., Clamens S., Bayard F., 1997. Extracellular cleavage of the vascular endothelial growth factor 189 aa forrm by " urokinae is required for its mitogeriic activity. J. Biol. Chem., 272, 13390-13396.
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Claims

REVENDICATIONS
1) Un anticorps anti-idiotypique d'une molécule HLA de classe 1, un fragment ou un dérivé de celui-ci.1) An anti-idiotypic antibody of a class 1 HLA molecule, a fragment or a derivative thereof.
2) Un anticorps anti-idiotypique selon la revendication 1, choisi parmi un anticorps monoclonal, un anticorps polyclonal, un fragment comme Fab'2 ou Fab ou une combinaison de ceux-ci.2) An anti-idiotypic antibody according to claim 1, chosen from a monoclonal antibody, a polyclonal antibody, a fragment such as Fab'2 or Fab or a combination thereof.
3) Un anticorps anti-idiotypique selon l'une des revendications 1 ou 2, caractérisé en ce que la molécule HLA de classe 1 est choisie parmi : HLA-A, B, C, D, F, ou G.3) An anti-idiotypic antibody according to one of claims 1 or 2, characterized in that the HLA class 1 molecule is chosen from: HLA-A, B, C, D, F, or G.
4) Une molécule d'acide nucléique comprenant ou constituée par une séquence polynucléotidique codant pour un anticorps anti-idiotypique d'une molécule HLA de classe 1 selon l'une des revendications 1 à 3.4) A nucleic acid molecule comprising or consisting of a polynucleotide sequence coding for an anti-idiotypic antibody of a class 1 HLA molecule according to one of claims 1 to 3.
5) Une composition pharmaceutique utile pour prévenir ou inhiber 1 ' angiogénèse comprenant au moins un anticorps anti-idiotypique selon l'une des revendications 1 à 3 ou une molécule d'acide nucléique selon la revendication 4.5) A pharmaceutical composition useful for preventing or inhibiting angiogenesis comprising at least one anti-idiotypic antibody according to one of claims 1 to 3 or a nucleic acid molecule according to claim 4.
6) Utilisation d'anticorps anti-idiotypique selon l'une des revendications 1 à 3 ou d'une molécule d'acide nucléique selon la revendication 4 pour la préparation d'un médicament destiné à prévenir ou à traiter une pathologie impliquant une activation endothéliale.6) Use of anti-idiotypic antibodies according to one of claims 1 to 3 or of a nucleic acid molecule according to claim 4 for the preparation of a medicament intended for preventing or treating a pathology involving endothelial activation .
7) Utilisation selon la revendication 6, caractérisée en ce que la pathologie impliquant une activation endothéliale est choisie dans le groupe comprenant : le rejet d' allogreffes et de xénogreffes, les acrocyanoses , les sclérodermies , les cancers, les rétinopathies diabétiques, la polyarthrite rhumatoide, les angiomes, les angiocarcinomes, en particulier la maladie de Castelman et le sarcome de Kaposi.7) Use according to claim 6, characterized in that the pathology involving an endothelial activation is chosen from the group including: rejection of allografts and xenografts, acrocyanosis, scleroderma, cancer, diabetic retinopathy, rheumatoid arthritis, angiomas, angiocarcinomas, especially Castelman disease and Kaposi sarcoma.
8) Utilisation d'anticorps anti-idiotypique selon l'une des revendications 1 à 3 ou d'une molécule d'acide nucléique selon la revendication 4 pour la préparation d'une composition pharmaceutique utile pour la conservation de greffons entre le prélèvement et la transplantation.8) Use of anti-idiotypic antibody according to one of claims 1 to 3 or of a nucleic acid molecule according to claim 4 for the preparation of a pharmaceutical composition useful for the preservation of grafts between the collection and the transplantation.
9) Utilisation d'anticorps anti-idiotypique selon l'une des revendications 1 à 3 associé à une substance d'intérêt pour la préparation d'une composition destinée à inhiber 1 ' angiogénèse ou à détecter des sites angiogéniques .9) Use of anti-idiotypic antibody according to one of claims 1 to 3 associated with a substance of interest for the preparation of a composition intended to inhibit angiogenesis or to detect angiogenic sites.
10) Un composé constitué d'un anticorps anti- idiotypique d'une molécule HLA de classe 1 selon l'une des revendications 1 à 3 couplé à une substance d' intérêt en thérapie ou diagnostic.10) A compound consisting of an anti-idiotypic antibody of an HLA class 1 molecule according to one of claims 1 to 3 coupled to a substance of interest in therapy or diagnosis.
11) Méthode de dosage de molécules HLA de classe I ou d'anticorps anti-idiotypiques de ces molécules éventuellement présentes dans un échantillon biologique caractérisée en ce qu'elle comprend :11) Method for assaying HLA class I molecules or anti-idiotypic antibodies of these molecules possibly present in a biological sample characterized in that it comprises:
* la mise en contact desdites molécules ou anticorps de l'échantillon avec une quantité déterminée d'anticorps anti-idiotypiques selon l'une des revendications 1 à 3 marqués et fixés à des anticorps antimolécule HLA de classe I, * bringing said molecules or antibodies from the sample into contact with a determined quantity of anti-idiotypic antibodies according to one of claims 1 to 3 labeled and attached to HLA class I antimolecule antibodies,
- la mesure de la diminution de la liaison des anticorps anti-idiotypiques marqués avec les anticorps anti-molécule HLA de classe I. - measuring the decrease in the binding of labeled anti-idiotypic antibodies with anti-HLA class I molecule antibodies.
EP02772487A 2001-08-07 2002-08-06 Anti-idiotypic antibodies of hla class i molecules and the use thereof in the preparation of compositions that are designed to inhibit vascular activation Withdrawn EP1414862A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0110553A FR2828490A1 (en) 2001-08-07 2001-08-07 New anti-idiotypic antibodies of Class I human leucocyte antigen molecules, useful for preventing apoptosis and preservation of grafts
FR0110553 2001-08-07
PCT/FR2002/002813 WO2003014163A1 (en) 2001-08-07 2002-08-06 Anti-idiotypic antibodies of hla class i molecules and the use thereof in the preparation of compositions that are designed to inhibit vascular activation

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EP1414862A1 true EP1414862A1 (en) 2004-05-06

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EP (1) EP1414862A1 (en)
CA (1) CA2456287A1 (en)
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WO (1) WO2003014163A1 (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1025692A (en) * 1991-02-06 1992-08-13 Ciba-Geigy Ag Novel chimeric antiidiotypic monoclonal antibodies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03014163A1 *

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WO2003014163A1 (en) 2003-02-20
CA2456287A1 (en) 2003-02-20

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