FR2828490A1 - New anti-idiotypic antibodies of Class I human leucocyte antigen molecules, useful for preventing apoptosis and preservation of grafts - Google Patents

Abstract

Anti-idiotypic antibodies (AAb) of a Class I human leucocyte antigen (HLA) molecule (I), and their fragments or derivatives, is new. Independent claims are also included for the following: (1) nucleic acid (II) comprising, or containing, a sequence that encodes AAb; (2) conjugate (III) of AAb with a therapeutic or diagnostic agent; and (3) determining (I) or AAb.

Description

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 ANTI-IDIOTYPIC ANTIBODIES OF CLASS I HLA MOLECULES AND THEIR USE FOR THE PREPARATION OF COMPOSITIONS FOR INHIBITING VASCULAR ACTIVATION.

 The present invention relates to the field of inhibition of angiogenesis involved in numerous proliferative pathological syndromes.

The invention relates to anti-idiotypic antibodies to molecules of the Major Histocompatibility Complex (MHC) and more particularly to antiidiotypic 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 curbing 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 the endothelial cells involved in a process of pathological or non-pathological angiogenesis.

 Endothelial cells line the inside of all blood vessels in the body. These cells have a quiescent phenotype, they hardly proliferate, only 0.01% of endothelial cells are involved 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,

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 synthesis of cytokines, construction of new vessels irrigating neighboring territories during hypoxia. All these functions require or are accompanied by a change in the phenotype of the 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 transitory state common to endothelial cells, prior to any functional response.

 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 to say that they unwind antennas to immobilize killer lymphocytes. During transplants, for example, keeping the graft cold is sufficient 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.

 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

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 and become able to carry out the angiogenesis program, i.e. 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).

 Endothelial cells have been shown to express HLA class I molecules on their surface, the role of which 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. It has also been shown that peptides derived from HLA class I molecules can regulate the function of insulin receptors. However, to date no description of any non-immunological role of the soluble form of HLA G1 has been reported and there are also no experimental data demonstrating the presence of

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 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) stimulates expression VEGF receptors. Knowing that certain HLA-I molecules have a significant polymorphism and that they bind to distinct receptors which are still poorly understood, a versatile HLA-I mime has been prepared thanks to the strategy of manufacturing anti-idiotypic antibodies.

 Consequently, 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) 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.

 They are preferably antiidiotypic 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.

 Thus, the invention relates to anti-idiotypic antibodies of molecules derived from HLA class 1 molecules, soluble or not, having anti-angiogenic properties that a person skilled in the art is able to test. It can be fragments, truncated in shape,

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 of 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.

 Such a nucleic acid molecule is useful for the preparation of the antiidiotypic antibodies according to the invention by genetic engineering techniques.

 The invention contemplates, as a nucleic acid molecule comprising a polynucleotide sequence coding for 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.

 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.

 A subject of the invention is therefore also 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.

 Such a nucleic acid molecule is useful in gene therapy or cell therapy protocols consisting in administering the said

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 nucleic acid molecule or cells transformed by said nucleic acid molecule has an individual so as to express the anti-idiotypic antibody and thus inhibit angiogenesis.

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. Thus, these anti-idiotypic antibodies and the corresponding nucleic acid molecules are particularly suitable for the preparation of medicaments intended to prevent or treat angiogenesis and therefore the proliferation of cells.

Endothelial. Among the pathologies whose treatment requires the inhibition of endothelial activation, one 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.

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 The demonstration within the framework of the invention of the attachment of HLA class 1 molecules to a receptor on the surface of endothelal cells also makes it possible to use the antiidiotypic 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 ovarian yellow body, perfusion of the ischemic areas during vascular thrombosis such as in arteritis of the lower limbs and l 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 W6 / 32.

 A method of identifying receptors for class 1 HLA molecules on the surface of endothelal cells according to the invention comprises the following steps: - Purification of the monoclonal antibody according to techniques known to those skilled in the art.

 - Preparation of an affinity chromatography matrix in which the purified monoclonal antibody is conjugated to the solid phase.

 - Chromatography of a cell (eg cultured endothelial cells) or tissue (eg human placenta) lysate.

 - Elution of the receptor by a chaotropic agent (NaCl, urea, guanidine chloride, acid or basic shock) or specific ligand (example Fab fragment of the above antibody).

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 - Sequencing of the protein by conventional techniques.

 A method of identifying ligands for class 1 HLA molecules receptors 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.

 The invention also relates to the determination of HLA class I molecules or antiidiotypic 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-idyotipic 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 antiidiotypic labeled antibody is proportional to the quantity of anti-idiotypic antibody or of HLA class I molecules in the biological environment. Thus, the subject of the invention is a method of assaying HLA class I molecules or anti-idyotipic antibodies of 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-idyotipic antibodies of the invention,

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 advantageously labeled and attached to class I HLA antimolecule antibodies, - measuring the decrease in the binding of labeled anti-idyotipic antibodies with class I HLA anti-molecule antibodies

 Advantageously, 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. Thus, the said anti-idiotypic antibodies are useful for vectorizing substances d interest at the level of pathological or non-pathological endothelial activation foci, as well as 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 active substance such as 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.

 The substance of interest can also be a diagnostic substance such as a radioactive tracer or a contrast product for magnetic resonance imaging enabling the marking of angiogenic sites.

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 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 at the level of 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 bonding means in a biological medium such as: - Coupling by covalent bond - Oxidation reaction leading to the recovery of d hydrogen atom by iodine atom on an indole nucleus
These compounds of the invention can also be proteins or fusion genes.

 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: FIG. 1 represents the title of the mice on Fv cells and shows that IgId / HLA-I recognizes receptors on endothelial cells.

 Figure 2 shows that the chemotactic effect of W6 / 32 is not inhibited by VEGFRls

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 or VEGFR2s while the chemotactic effect of VEGF is abolished.

 Figure 3 shows the inhibition of the chemotactic action of VEGF by 191d / HLA-1.

 - Figure 4 shows that 1g1d / HLA-1 has no effect on the basal proliferation of HUVEC cells.

 A-STUDY METHODS.

 1 - Reagents.

 1. 1-Proteins used.

 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).

 The W6 / 32 antibody is produced by injecting the hybridoma into the peritoneum of Balb / c mice. The ascites are collected 10-15 days after inoculation and the IgGs purified by precipitation with ammonium sulphate followed by chromatography on protein A sepharose. The Fab fragments are prepared by papain cleavage.

 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 type loops 1-7) 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, dialyzed against 0.01 Tris buffer

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 M, 0.05 M NaCl and then chromatographed 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.

ensuite attestée par gel de polyacrylamide-SDS ( > 90%) et conservés à -80oC jusqu'à l'utilisation. The purity of the purified VEGFR1 and VEGFR2 proteins is

then certified by polyacrylamide-SDS gel (> 90%) and stored at -80oC until use.

 I. 2-Cells used.

 The culture media and the sera come from the company Life TechnologiesTM.

 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 an 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% CO 2. These cultures are maintained with VEGF at 2 ng / ml added to the medium every two days.

 The 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-Preparation of antiidiotypic antibodies.

 II. 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

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 protein A column. The column is washed with PBS and the immunoglobulins are eluted with 0.2 M glycine, pH 2.5, immediately neutralized by adding 1/5 of the volume of 1 M K2HPO4, then dialyzed against PBS. Immunoglobulins (Ig PI) are stored at -80 C until they are used.

 II. 2-HLA-I anti-idiotypic antibodies (Ig2Id HLA-1).

 The mice receive subcutaneously 10 μg of W6 / 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. Blood is drawn between 5 and 8 weeks after the first injection and the IgGs are purified as described above.

 III-Study of the specificity of the antibodies.

 111. 1-ELISA. a) anti-idiotype.

 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 mouse immunoglobulins.

 Thus, if an antibody binds to the Fab fragments of W6 / 32, it is either directed against the isotype or against the idiotope. 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 IgId / HLA-I .

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 b) Endothelial cells.

 The Fv cells are incubated at 5000 cells per multiplaque 96 well. After the confluence, the cells are rinsed and fixed by a treatment which does not induce permeabilization (0.25% 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.

 So, if an antibody binds to non-permeabilized endothelial cells when the preimmune immunoglobulins do not, it recognizes a specific receptor on these cells.

 111. 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 which have migrated are counted in at least 8 fields.

 111. 3-Cell proliferation.

 HUVEC cells are seeded at low density (2000 cells / cm2) in boxes of 12 wells, the surface of which has been previously coated with gelatin. Modulators are added after cell adhesion. with culture plastic, then after 2

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days. Each condition is studied in three different wells. The cells are trypsinized and counted on the fifth day. The percentage inhibition is calculated as the ratio: (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-RESULTS.

 I-ELISA.

 The anti-idiotypic nature of the purified immunoglobulins was evaluated by ELISA on plates lined with Fab fragments of W6 / 32. Pre-immune immunoglobulins do not bind to Fab and IgId / 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 IgId / HLA-I does. FIG. 1 therefore shows that IgId / HLA-I recognizes receptors on endothelial cells.

 II-migration.

 The dimerization of HLA class I transmembrane molecules induced by ligation using the antibody W6 / 32 has been shown to stimulate 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 W6 / 32 and 1 g / ml of VEGFR1s or VEGFR2s.

 FV cells are seeded at high density (50,000 cells / well) in boxes of 12

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 well 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 made in the monolayer by scraping using a foam tip, the cells rinsed thoroughly and then 10 g / ml of W6 / 32 or 50 ng / ml of VEGF are added in the presence or absence of 1 g / ml of VEGFR1s 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 VEGFR1s or VEGFR2s while the chemotactic effect of VEGF is abolished. Consequently, the signaling of W6 / 32 is not linked to that of VEGF.

 FIG. 3 shows the inhibition of the chemotactic action of VEGF by IgId / 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 eraser 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 IgId / 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 the cells which have migrated are counted in 8 fields by condition.

 IgId / HLA-I has no effect on the spontaneous migration of FV cells. On the other hand, a molar excess

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 100 times is sufficient to almost completely inhibit the chemotactic activity of VEGF. Consequently, the binding of IgId / HLA-I to its endothelial receptors induces an effect inhibiting the activity of VEGF.

 III-Mitogenicity.

 IgId / HLA-I has no effect on the basal proliferation of HUVEC cells (Figure 4). On the other hand, IgId / HLA-I inhibits, in a dose-dependent manner, the proliferation of HUVEC cells induced by VEGF (for example 15 μg / ml inhibits this proliferation by 40%. By comparison, the immunoglobulins purified from the preimmune serum do not affect the proliferation induced by VEGF.

 Consequently, the binding of IgId / HLA-I to its endothelial receptors induces an effect inhibiting the activity of VEGF.

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 BIBLIOGRAPHICAL REFERENCES - Bian H, Reed EF. Anti-HLA class antibodies transduce signals in endothelial cells resulting in FGF receptor translocation, down-regulation of ICAM-1 and cell proliferation. Transplant Proc 2001; 33 (1-2): 311 - Bian H, Harris PE, Reed EF. Ligation of HLA class I molecules on smooth muscle cells with antiHLA antibodies induces tyrosine phosphorylation, fibroblast growth factor receptor expression and cell proliferation. Int Immunol 1998; 10 (9): 1315-23 - Barnstable CJ., Bodmer WF., Brown G., Galfre G., Milstein C., Williams AF., Ziegler A. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigensnew tools for genetic analysis.

 - Blaschitz A., Lenfant F., Mallet V., Hartmann M., Bensussan A., Geraghty D. E., Le Bouteiller P. and Dohr G. (1997) Endothelial cells in chorionic fetal vessels of first trimester placenta express HLA-G.

Eur J Immunol 27,3380-8.

 - Brodsky FM, Parham P. Monomorphic anti-HLAA, B, C monoclonal antibodies detecting molecular subunits and combinatorial determinants. J Immunol 1982 128 (1): 129-35 - Kohler G, Howe SC, Milstein C. Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur J Immunol 1976 Apr; 6 (4): 292-5 - Lanier L. L. (1998) NK cell receptors.

Annu Rev Immunol 16,359-93.

 - Maejima M., Fujii T., Kozuma S., Okai T., Shibata Y. and Taketani Y. (1997) Presence of HLA-Gexpressing cells modulates the ability of peripheral

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 blood mononuclear cells to release cytokines. Am J Reprod Immunol 38,79-82.

 - Ortéga N., Jonca F., Vincent S., Favard C., Ruchoux M-M, Plouët J., 1997. Systemic activation of the vascular endothelial growth factor receptor flk-1 selectively triggers angiogenic endothelial cells. Am.

J. Pathol., 151, 1215-1224.

 - 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 mitogenic activity. J. Biol. Chem., 272,13390-13396.

 - Prats, H., Kaghah, M., Prats, A. C., Klagsbrun, M., Lelias, J. M., Liauzun, P., Chalon, P., Tauber, J. P., Amalric, F., Smith, J. A. (1989). High molecular mass forms of fibroblast growth factor are initiated by alternative CUG codons. Proc. Natl. Acad.

 Sci. USA. 86.1836-1840.

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N. (1997) Review: immunobiology of preeclampsia. Am J Reprod Immunol 37,79-86.

Claims (11)

1) An anti-idiotypic antibody of a class 1 HLA molecule, a fragment or a derivative thereof.  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) 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) 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) 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) 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 . <Desc / Clms Page number 21>  7) Use according to claim 6, characterized in that the pathology involving an endothelial activation is chosen from the group comprising: rejection of allografts and xenografts, acrocyanosis, scleroderma, cancer, diabetic retinopathy, rheumatoid arthritis , angiomas, angiocarcinomas, in particular Castelman's disease and Kaposi's sarcoma.  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) Use of anti-idiotypic antibodies according to one of claims 1 to 3 associated with a substance of interest for the preparation of a

 1 1 composition intended to inhibit angiogenesis or to detect angiogenic sites.  10) A compound consisting of an anti-idiotypic antibody of a HLA class 1 molecule according to one of claims 1 to 3 coupled to a substance of interest in therapy or diagnosis.  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: - bringing said molecules or antibodies from the sample into contact with a determined quantity of anti-idiotypic antibodies according to one of the <Desc / Clms Page number 22>  claims 1 to 3 marked and attached to anti-HLA class I molecule antibodies, - measuring the decrease in the binding of the labeled anti-idiotypic antibodies with the anti-HLA class I molecule antibodies