Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/736—Glucomannans or galactomannans, e.g. locust bean gum, guar gum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/36—Blood coagulation or fibrinolysis factors
  • A61K38/37—Factors VIII
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

Definitions

• the invention relates to inhibiting the anti-FVIII immune response by blocking the endocytosis of the 'FVIII (Factor VIII) by the cells are capable of endocytosing the antigen the immune system.
• Hemophilia A is a hereditary condition related to an abnormality of the X chromosome, which results in an inability to coagulate in people with this disease, This disease is the result of mutations on the gene of a protein involved in coagulation, the factor HIV (FVIII), which determine either a complete absence of FVIIl in the blood, or a partial deficit.
• Hemophilia A is the most common defect affecting blood coagulation: it affects 1 in 5000 men in France, and represents 80% of patients with hemophilia.
• the other type of hemophilia, hemophilia B affects 20% of patients with hemophilia; it is caused by a deficiency in another factor of coagulation, factor IX.
• Treatment current hemophilia ⁇ type A or B) is to be administered intravenously deficient clotting factor or missing.
• FVIiI Fractionation and Biotechnologies (LFB) or international pharmaceutical laboratories, as well as in the form of recombinant drugs derived from genetic engineering.
• DNA coding for FVIiI has been isolated and expressed in mammalian cells ( Wood et al., Nature (1984) 312: 330-337), and its amino acid sequence deduced from the cDNA.
• the secreted FVIIII is a glycoprotein with a molecular weight of 300 Kda (2332 amino acids) playing a key role. in the activation of the intrinsic pathway of coagulation.
• Inactive PVIII consists of six domains: Al (residues 1-372), A2 (residues 373-740), B (residues 741-1648), A3 (residues 1649-2019), C1 (residues 2020-2172), and C2 (residues 2173-2332), from the N-terminus to the Cterminal end.
• FVIII interacts with von Willebrand factor (VWF), which protects it from plasma proteases. It is in this form that FVIII circulates in the blood.
• FVTiI dissociates from VWF after cleavage with thrombin.
• FViII circulates in the plasma
• This heterodimer is composed of a heavy chain (Al, A2) and a light chain (A3, Cl, C2)
• Activated factor X converts prothrombin to thrombin
• thrombin converts fibrinogen to fibrin
• a clot forms
• the major problem encountered with the administration of FVIII is the appearance in the patient of antibodies directed against FVIII, called "inhibitory antibodies.” These antibodies neutralize the procoagulant activity of FVIiI, which is rendered inactive as soon that it is perfused, so the coagulation factor administered is destroyed before it can stop the bleeding, which is a serious complication of hemophilia, the treatment becoming ineffective.
• some genetically non-haemophiliac patients may develop inhibitors against endogenous PVIII: this is an acquired hemophilia.
• FVII1 The mechanisms by which anti-FVIII antibodies interfere with the function of FVII1 are numerous, and include interference in proteolytic cleavage of FVII and in the interaction of FVIII with different partners such as Von Willebrand Factor (VWF), pho ⁇ pholipids. (PL), factor IX, activated factor X (FXa) or APC (Activated Frotein C).
• VWF Von Willebrand Factor
• PL pho ⁇ pholipids.
• FXa activated factor X
• APC Activated Frotein C
• the first step in initiating a specific immune response of FVIII is endocytosis.
• Dendritic cells (DCs) are the most potent antigen presenting cells (APCs), and one of the few types of APCs capable of activating primary naive T cells. DCs are therefore able to initiate a specific immune response of the antigen (1,2), CD endocytosent the antigen via a receptor or by macropinocytosis; receptor mediated endocytosis being advantageous for in vivo DCs.
• the surface of the CD presents many endocytic receptors most of which is dependent on divalent ions, mainly calcium ( Figure 1).
• RLC type C lectin receptors
• MR mannose receptor
• CD206 mannose receptor
• CD209 the drug, PEC-205
• Mannan is a ligand for these mannose-sensitive RLCs, particularly for MR and DC-SiGN (3-5).
• the DC-SIGN molecule of dendritic cells binds to T-cell ICAM-3 molecules. This specific interaction appears to play an important role in the initiation of the immunological synapse between dendritic cells and LTs. Activation of the lymphocytes is inhibited by an anti-DC-SIGN blocking antibody.
• the FVIII molecule contains 25 consensus sequences (Asn-Xxx-Thr / Ser) which are potential N-linked glycosylation sites, of which 20 have been shown to be glycosylated (6). Some N-linked glycosylations are maintained on FVIU-BDD (recombinant FVIII lacking B domain, Refacto, Wyeth) (7). Both plasma-derived FVIII and recombinant FVIII showed similar glycosylation profiles with residues ending in galactose or mannose (8, 9),
• FViII and FVI11-BDD are thus candidate ligands for RLCs on the surface of the dendritic cell.
• Another strategy for controlling the more recent FViXI inhibitory antibodies is the administration of anti-idiotypic antibodies (antibodies with the ability to interact with the variable region of other antibodies) to neutralize the inhibitory antibodies (St. RemyJM et al, (1999) Vox Sang; 77 (suppl 1); 21-24) * Because of the complexity of the in vivo analysis of this polyclonal immune response, teams have isolated monoclonal antibodies directed against certain domains of the immune system. FVIII. Thus, a monoclonal human IgG4kappa antibody, LE2E9, was isolated.
• This antibody is directed against the Cl domain of FVIII and inhibits the cofactor activity of FVIII and its binding to VWF (Jacquemin et al (2000) Blood 95: 156-163).
• a human monoclonal antibody against the C2 domain of FVII named BO2C11 (IgG4kappa), produced from a memory B cell repertoire of a patient with hemophilia A with inhibitors, was isolated ( Jacquemin et al., Blood 1998 JuI 15 ; 92 (2): 496-506).
• BO2C11 recognizes the C2 domain of FViII and inhibits its binding to VWF and phospholipids. It completely inhibits the procoagulant activity of native and activated FVIII.
• Another example of a monoclonal antibody is the antibody B0HB2, directed against the A2 domain of FVIII.
• the BOIIB2 antibody inhibits the activity of FVIII by 99%. By binding to the A2 domain, it can interfere with and inhibit the binding of FIXa, which has a low affinity binding site in this region of FVIIi and therefore inhibits activity
• the second possible mode of action is its interference in the equilibrium between the hetero-dimer (A2: A1 and A3: Cl: C2) form of FVIII and the hetero-trimer form (A2 and Al and A3).
• the Applicant has surprisingly observed that it is possible to blog the endocytosis of PVIII by the cells of the immune system capable of endocyting the antigen and, by way of Consequently, to inhibit the formation of inhibitory anti-FVIII antibodies and to increase the half-life of FVIII.
• a deglycosylated wine Factor or a fragment thereof for which the ability to interact and the ability to be endocyted by cells capable of endocyting an antigen are decreased or inhibited compared to native Factor VIII.
• this deglycosylated factor VIII or the fragment thereof has a decreased or inhibited capacity to interact with the receptors present on the surface of said cells capable of endocyting an antigen, in particular with the specific receptors of the mannose and more particularly with the CD206 mannose receptor or the DC-SlGN CD209 receptor (Dendritic cell-Specific Inter ⁇ ellular adhesion molecule 3 (ICAM-3) - Grabbing Non-integrin).
• IAM-3 Dendritic cell-Specific Inter ⁇ ellular adhesion molecule 3
• said cells capable of endocyting an antigen are antigen presenting cells (APCs) and, in particular, dendritic cells or B lymphocytes.
• APCs antigen presenting cells
• said cells capable of endocyting an antigen are selected from macrophages, endothelial cells, sinusoidal endothelial cells of the liver, Kupffer cells of the liver.
• Factor VIII or the fragment thereof is deglycosylated without resorting to one or more of the enzymes selected from the group consisting of a neuraiainida.se, a toeta- galactosidase and alpha-mannosidase.
• the wine factor or the fragment thereof is deglycosylated by the action of a single enzyme of the endoglucosidase type and, in particular, of the endo-beta-N-acetylglucosaininidase type,
• the enzyme used has the ability to cut oligomannose and hybrid-type hydrate structures, but does not have the ability to cut complex-type carbohydrate structures.
• said endo-beta-N-acetylglucosaminidase-type enzyme is selected from the group consisting of endo-beta-N-acetylglucosaminidase Fl and endo-beta-N-acetylglucosaminidase H, by For example, the endc-beta-N- acetylglu ⁇ osaminidase Fl of Cbrys & obacteri ⁇ m
• the deglycosylated factor VI11 of the invention or the fragment thereof is used as a medicament, in particular for the treatment of haemophiliac patients, and in particular for the treatment of haemophilia. type A or type B.
• the deglycosylated factor VIII of the invention or the fragment thereof is also used for the preparation of a medicament for the treatment of hemophilia patients or for the treatment of hemophilia type A or type B, and may advantageously be used in combination with exogenous wine factor.
• the Factor VIII deglycosylated of the invention or fragment thereof can be used for the preparation of a medicament for increasing 'the half-life of exogenous Factor Vlil or intended to decrease the immunogenicity of an exogenous factor VIII in haemophiliac patients.
• Another aspect of the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least minus the deglycosylated ViII Factor of the invention or a fragment thereof, as well as one or more pharmaceutically acceptable adjuvant (s) and / or excipient (s).
• a final subject of the invention concerns the use of a composition comprising mannan for the preparation of a medicament for the treatment of haemophiliac patients, or the treatment of hemophilia type A or B, according to one embodiment in particular, this composition comprising mannan is used to prepare a medicament intended to increase the half-life of an exogenous wine factor or to reduce the immunogenicity of an exogenous factor VIII in haemophiliac patients.
• the mannan composition further comprises an exogenous factor VIIi of native type and / or a deglycosylated factor VIII or a fragment thereof according to the invention,
• N residue which residue may be included in a polypeptide such as for example, native or deglycosylated FVIII
• N Asparagine
• N Asparagine
• the basic structure is formed by the linking of two N-acetylglucosamine residues (GIcNAc) bound in (31.4). glycoside with the protein (by asparagine), the other is linked to a mannose residue in ⁇ 1,4, itself linked to 2 mannose residues, in ⁇ 1,6 and ⁇ l, 3; for example :
• glycan structures are formed by the addition of monosaccharides to terminal mannoses. Depending on the nature of the sugar, there are three types of glycan structures:
• oligomannose type structures, which correspond to the addition of mannose residues to the basic structure; for example :
• FIG. 1 Schematic representation of FVII and CD membrane receptors
• Figure 2f Measurement of the intemalisation of ⁇ 2M in the presence or absence of RAP or mannan
• Figure 3a Measurement of relative intemalisation (%) of FVIII and FVIII-BDD in the presence of anti-MR and anti-DC antibodies -SlGN
• FIG. 3b Measurement of Internalization (%) of FVIII by HD420 Cells as a Function of FVIII Concentration ( ⁇ M)
• FIG. 3c Measurement of the Intensity of Bonding to CTI1D1-3 and CTLD4-7 Constructs as a function of FVIII concentration in FViIl,
• FIG. 5a Measurement of the inhibition of the pro-coagulant activity of FVIII (%)
• the main object of the present invention is a compound capable of inhibiting the interaction of Factor VIII with cells capable of endocyting the antigen and endocytosis of Factor VIII by said cells.
• the Applicant has shown that certain compounds are capable of inhibiting endocytosis of FVII1 by dendritic cells such as mannan and demannosylated FVIII *
• mannan thus inhibits the proliferation of FViII-specific T cells without blocking the pro-coagulant activity of FVIiI or the interaction of FVII1 with VWF.
• this compound may be capable of inhibiting the interaction of FVIXI with a receptor present on cells capable of endocyting the antigen and responsible for endocytosis of FVIII by said cells.
• the object of the present invention thus includes both compounds other than FVII1 that block receptors on cells responsible for endocytosis of FVIII, a complete modified FVTii molecule or a fragment thereof, of which the binding capacity with the cells responsible for its exidocytosis is lower than that of native FVIII.
• each of these two types of compound inhibits endocytosis of FVIII by immune system cells capable of endocyting the antigen.
• said FVIiI compound inhibits its own interaction and endocytosis by the cells capable of endocyting it: it is therefore a Modified FVIII or a fragment of it dorxt endocytosis is decreased compared to native FVIII.
• this FVI11 is modified relative to the native FVIII in its glycosylation.
• said receptor is specific for mannose and, in a particularly preferred manner, said mannose-specific receptor is the CD206 mannose receptor or the CD209 DC-SlGN receptor (dendritic ⁇ ell-specific intercellular adhesion molecule 3 (ICAM- 3) nonintegrin grabbing).
• CD206 mannose receptor or the CD209 DC-SlGN receptor (dendritic ⁇ ell-specific intercellular adhesion molecule 3 (ICAM- 3) nonintegrin grabbing).
• CD209 DC-SlGN receptor dendritic ⁇ ell-specific intercellular adhesion molecule 3 (ICAM- 3) nonintegrin grabbing
• said cells capable of endocyting the antigen are antigen presenting cells, for example dendritic cells or B cells.
• the compound according to the invention is then able to inhibit the endocytosis of FVIII by the antigen presenting cells, thus to inhibit the presentation of FVIII peptides which initiates the immune response. Therefore, the compound according to the invention then inhibits the production of anti-FVIII antibodies and decreases the immunogenicity of FVIII.
• said cells capable of endocyting the antigen are chosen from macrophages, endothelial cells, sinusoidal endothelial cells of the liver and liver Kesffer cells. These cells endocyte FVIII for the purpose of eliminating it.
• the compound according to the invention by inhibiting the endocytosis of FVIII by these cells decreases this route of elimination of FVIII, increases the amount of circulating FVII1 therefore increases the half-life of FVIII.
• Another object of the invention is the use of a compound according to the invention for the manufacture of a medicament for the treatment of hemophiliacs in association with exogenous FVII1.
• this drug is intended to decrease the immunogenicity of exogenous FVIII in hemophiliac patients and / or. to increase the half-life of exogenous FVIII in hemophiliac patients.
• the drug is administered with exogenous FVIII or a fragment thereof for its pro-coagulant activity.
• ⁇ iannan is used for the manufacture of a medicament for the treatment of hemophiliacs in association with exogenous FVIII, by decreasing the immunogenicity of exogenous FVIII in; hemophilia patients and / or increasing the half-life of FVIIi exxgene in hemophiliac patients.
• a further object of the invention is the use of a modified FVIII or a fragment thereof whose endocytosis by the cells capable of endocyting the antigen is decreased compared to the native FVIII for the manufacture of In particular, this FVIII or fragment of FVII1 is demannosylated.
• said drug consists of a FVIII less immunogenic than the native FVIII, whose half-life is increased compared to the native FVIII, but which has fully retained its pro-coagulant activity.
• a further object of the invention is a pharmaceutical composition
• a pharmaceutical composition comprising at least one compound according to the invention and one or more pharmaceutically acceptable adjuvant (s) and / or excipient (s).
• CDs were prepared from monocytes as previously described (29) with a change of culture media. Briefly, mononuclear cells were isolated from heparinized buffy coats of healthy adult donors by adherence to plastic cell culture dishes in RPMI 1S40 medium supplemented with 10% human AB serum, glutamine and antibiotics for 60 minutes. The non-adherent cells were removed by gentle washing with 3 times.
• Adherent monocytes were cultured in X-VIVO medium (Cambrex Bio Sciences, Paris, France) supplemented with 1% human AB serum, antibiotics and in the presence of 500 IU / ml recombinant human interleukin-4 (rhIL- 4), R & D Systems (Lille, France) and 1000 IU / ml of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF), Immunotools (Friesoythe, Germany). Half of the medium, including all supplements, was replaced every other day. After 5 days of culture, the non-adherent and slightly adherent cells corresponding to the fraction enriched in CD were harvested, washed and used for subsequent experiments.
• FVIiI fluorescein-5-isothiocyanate
• EXAMPLE 3 Binding of the mannose receptor constructs to, FVIII in ELISA
• ELLA (Nunc; MAXISORP) plates were overlaid overnight with serial dilutions of FVIII forms (starting at 50 ⁇ g / ml) in 154 mM NaCl.
• TTBS buffer (10 mM Tris-HCl, pH 7.5, 10 mM Ca 2+, 154 mM NaCl and 0.05% Tween-20) was used.
• the non-reactive sites were blocked with TBS buffer (TTBS without Tween-20) containing 3% BSA.
• the plates were then incubated with the constructs at 2 or 10 ⁇ g / ml for 2 h at room temperature in TTBS buffer. containing 3% BSA.
• the binding of the MR constructs was detected using a murine antibody specific for the HRP-conjugated human IgG Fc portion (clone JDC-10, Southern Biotechnology Associates, Inc. AL, USA).
• the activity of HRP has been revealed with the OPD substrate (o- phenylenediamine, Sigma). Absorbance was measured at 492 run.
• the plates were coated with each form of FVIII (5.56 ⁇ g / ml). Different concentrations of mannan were incubated with 10 ⁇ g / ml of the CTLD (4-7) -Fc construct for 30 min at room temperature before incubation with FVII coated plates. The absorbance was measured as described herein. ⁇ above.
• CTLDs 4 to 7 have an affinity for mannosylated architectures not CTLDs 1 to 3.
• the mannan shows specific binding to CTLD (4-7) -Fc.
• Both FVIII and FVIII-BDD show a dose-dependent interaction specifically with CTLD (4-7) -Fc whereas they do not bind to the CTLD construct (1-3) indicating that glycosylation ( probably the exposed mannosylation) outside the B domain of FVIII allows the molecule to interact with the MR.
• CDs obtained in Example r 1 (0,4x10 5 cells / 96-well plate wells with 100 .mu.l / well) were incubated with different doses (0.029, 0.057, 0.143 and 0.358 uM) ligands fluorescent conjugates (FVIU- FITC, BDD-FVIU-FITC) obtained in Example 2 in the medium X-VlVO for 0, 15, 60 and 120 min at 4 ⁇ C or 37 0 C. After the incubation period, the cells were washed with cold PBS and analyzed by flow cytometry. To study the involvement of receptors in internalization, the cells were preincubated for 30 min at 37 ° C.
• FVIII The study of the internalization of FVIII was also carried out in the presence of 20 ug / IFIL monoclonal antibody anti ⁇ mannose receptor ⁇ P ⁇ M-1 isotype IgGl) or monoclonal anti-DC-SIGN (A2N-D1 isotype IgG1 ) or IgG j . , murine conjugated with PE-Cy (BD Pharmingen, France).
• ⁇ MFI calculated represent the differential labeling of the FVII-positive CDs after 2 hours of incubation at 37 ° C. and 40 ° C. The results demonstrate that the internalisation of FVII is an active process , an incubation period of 2 hours and a concentration, 0.143 uM ligand were selected for subsequent experiences.
• Figure 2d The specificity of mannan for mannose-sensitive RLCs was confirmed using dextran, a ligand typical of mannose-sensitive RLCs, and lucifer yellow (Iy), whose internalization proceeds exclusively by macropinocytosis independent of any receptor. The internalization of dextran was blocked at 80% in the presence of mannan while that of Iy was not affected.
• Figure 2e The internalization of dextran was blocked at 80% in the presence of mannan while that of Iy was not affected.
• FVII1 can interact with different endocytic receptors.
• Activated ⁇ 2M a mannosylated protein that specifically targets the CD91 / LRP endocytic receptor, has been used as a model antigen. It has been determined that the expression of mannosylated residues on ⁇ 2M does not influence the internalization of the antigen when other endocytic receptors are involved. Thus, the internalization of ⁇ 2M was completely inhibited in the presence of an excess of RAP while the mannan showed no effect. Indirectly, these results suggest that the mannosylated portions present on FViil are unique and make FVin more attractive to antigen presenting cells (APCs) than other mannosylated antigens.
• APIs antigen presenting cells
• the increased inhibition with mannan may result from the fact that the MR has several hydrocarbon recognition domains (CDRs): the inhibition is therefore more efficient by the polyhydrocarbon mannan than by the PAM-I antibody which is directed only against the 4th CDR of the MR.
• CDRs hydrocarbon recognition domains
• the AZN-D1 antibody did not inhibit the internalization of FVlil by immature CDs, whereas that of FVIII-BDD was inhibited by 17.6%.
• the expression of DC-SIGN by transfected B cells did not make it possible to increase endocytosis of FVIII.
• Example 5 In vitro test for the proliferation of autologous CD4 + T cells
• Example 1 The CDs of Example 1 were incubated with mannan (1 mg / ml) for 30 min at 37 ° C. FVIII dialyzed against RPMI-1640 medium (kogenate, Bayer) was added (40 ⁇ g / ml, 0.143 ⁇ M) for 2 hours. The cells were then washed and incubated with LPS (1 ⁇ g / 0.5 million cells) in complete medium for 48 hours. The cells were then washed and cultured (RPMI-1640, supplemented with 10% AB human male serum in 96-well round-bottomed cell dishes with 200 ⁇ l per well) with autologous CD4 + T cells.
• RPMI-1640 supplemented with 10% AB human male serum in 96-well round-bottomed cell dishes with 200 ⁇ l per well
• Figure 4a The CDs according to Example 1 are incubated alone or with mannan before the addition of FVIII. After ripening in the presence of LPS, the CDs are incubated with autologous CD4 + helper T cells. Mannan reduces the proliferation of T cells to levels comparable to negative controls.
• Example 1 After washing, the CDs of Example 1 were resuspended in DMEM: F12 (1: 1) medium containing 10% FCS and 10,000 CDs were added to each well of a 96-well cell culture plate thoroughly. round. After incubation with mannan (1 mg / ml) in each well for 30 min, the CDs were cultured with 5000 D9E9 (FViII-specific T cell clone) in DMEM: F12 (1: 1) medium containing 10 % FCS and 20 U / ml rhlL-2 with different doses of full FVTIi or FVin-BDD
• BO2C11 of EBV transformed B cells were cultured in the presence of 10 ⁇ g / ml of complete FVIII or FVIIi-BDD.
• the control conditions were preserved in the absence of FVIII, D9E9 or CD.
• hrFlX (Benefix, Baxter) was incubated with the cells in concentrations isomolar of FVII.
• the supernatants were harvested at the end of the incubation period and tested for their IFN-Y production using the human IEN-Y Duo Set (DY285, R & D Systems) according to the manufacturer's instructions. Results: Figure 4b
• FVII1 induces a dose-dependent activation of D9E9 cells which is inhibited up to 84%. presence of mannan (B). The proliferation of T cells is specific, as indicated by the absence of proliferation in the presence of FIX (A).
• Mannan does not prevent the activation of D9E9 cells by autologous B cells pulsed with FVII, LE2E9 (C). This suggests that the potential effect of mannan to decrease the immunogenicity of FVIII may only be therapeutically exploited in a situation where FViII-specific B cells have not yet been stimulated (i.e., in patients previously untreated), or n r have not yet developed inhibitor after treatment.
• Example 8 ELISA for the binding of FVIII to. VWF
• ELLA plates (Nunc, Roskilde, Denmark) were coated with VWF (VWF, Willefactin, LFB, Les Ulis, France) at 2 ⁇ g / ml per well in PBS (pH 7.4) at 37 ° C. for 1 hour. . Plates were saturated with PBS containing 1% skim milk and 0.1% tween 20 for 1 hour at 37 ° C. FVI11 (0.3 ⁇ g / ml) was preincubated alone or with mannan (0.01 to 4 mg / ml) or with BO2C11 (human monoclonal anti-FVlil IgG) (0.05 to 40 ⁇ g / ml) in blocking buffer for 1 hour at 37 ° C.
• VWF VWF, Willefactin, LFB, Les Ulis, France
• Kiannan does not interfere with the interaction of FVIII with VWF.
• EndoF1 is the only endoglycosidase capable of effectively cutting sugar residues on FVII1-BDD (see Figure 6).
• FVIII-BDD is deglycosylated with EndoF1 according to the manufacturer's instructions, separated by SDS-PAGE and revealed by Western blotting.
• 3.7 ⁇ g of FVII1-BDD are loaded into each well of 7.5% SDS-PAGE gels and transferred to nitrocellulose membranes. these are then revealed using 10 ⁇ g / ml of the CTLD (4-7) -Fc construct and anti-human IgG conjugated to alkaline phosphatase (left) or protogold (right) (see Figure 7).
• the modification of the molecular weight of FVIII-BDD after incubation with EndoF1 confirms an effective deglycosylation of EndoF1.
• FVI11-BDD Conjugate FITC treated or not treated with EndoF1 (0.143 ⁇ M) is incubated with CDs according to Example 1 (4 ⁇ 10 5 cells / well of 100 ⁇ l) in X-VIVO medium without serum at 37 ° C. C or 4 ⁇ C.
• the cells optionally been pre-incubated with mannan (1 mg / ml) for 30 min at 37 0 C.
• EndoF1 leaves N-acetylglucosamine residues which are also ligands of the mannose receptor.
• ELISA plates are coated with FVIIi-BDD (5.56 ⁇ g / ml, 0.033 ⁇ M).
• the VWF (0 to 1 ⁇ M) is incubated with 10 ⁇ g / ml of the CTLD (4-7) -Fc construct for 30 min at room temperature and added to the immobilized FVIII-BDD.
• the binding of the construct to FVIII-BDD is revealed using HRP conjugated human anti-Fc antibody and OPD substrate (cf.
• Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products, J Exp Me ⁇ 182: 389.

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• 2006
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  • 2006-12-22 JP JP2008546528A patent/JP4906873B2/ja not_active Expired - Fee Related
  • 2006-12-22 CA CA002634608A patent/CA2634608A1/fr not_active Abandoned
  • 2006-12-22 AU AU2006328809A patent/AU2006328809B2/en not_active Ceased
  • 2006-12-22 EP EP06847152A patent/EP1976875A2/de not_active Withdrawn
  • 2006-12-22 US US12/086,851 patent/US20090317373A1/en not_active Abandoned
  • 2006-12-22 BR BRPI0621020-1A patent/BRPI0621020A2/pt not_active IP Right Cessation
  • 2006-12-22 KR KR1020087017294A patent/KR20090003152A/ko not_active Abandoned
  • 2006-12-22 CN CNA2006800486723A patent/CN101356193A/zh active Pending
• 2008
  • 2008-06-15 IL IL192171A patent/IL192171A0/en not_active IP Right Cessation

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