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• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a new lymphocyte antigen as well as antibodies corresponding to this antigen and their applications.
• Lymphocyte activation induces or increases the expression of several surface structures, some of which, such as the IL2 and transferrin receptors, are directly involved in cell growth.
• the activation of human lymphocytes leads to phenotypic modifications appearing either immediately or at variable times after the disturbance of the antigen recognition structures. Phenotypic changes can be attributed to the expression on the cell surface of inducible molecules such as CD25 (1, 2) and CD71 (3), or to the increased expression of surface antigens such as CD26 (4, 5), CD 29 (6.7), CD45RO (8.9), or to an epitopic modulation as in the case of CD2 (10.11), or to changes in affinity as reported for the molecules CD 1 1a / CD18 (12) and LAMK13).
• these inducible molecules include receptor subunits of high affinity for various cytokines (2). Most often, they are detected using a monoclonal antibody (mAb) recognizing a putative receptor for an unknown ligand.
• mAb monoclonal antibody
• the hgands of CD5 (14,15), CD43 (16) and CD69 (17,18) were very recently identified, whereas the use of mAb as agonists against these structures had made it possible to identify their crucial role in the activation of the T cell (15, 17, 18, 19).
• mAbs have been shown to increase the cytoplasmic concentration of free calcium, if they are "cross linked" on the surface of T cells (19).
• monoclonal antibodies have been defined according to the invention against functionally defined clones of human T cells.
• BB 18 a monoclonal antibody called BB 18 was first isolated, recognizing, on the cell surface, a new lymphocyte antigen composed of subunits of 150 ⁇ 10 kDa, the expression of which on T lymphocytes increases rapidly. after their activation by various stimuli including iectins.
• PMA phorbol 12-myristate 13-acetate
• the expression on the cell surface of this structure of 150 ⁇ 10 kDa undergoes an even earlier negative modulation than the molecules of CD3.
• Biochemical studies as well as phenotypic analysis have revealed that this structure is different from all the molecules previously identified on the cell surface of lymphocytes.
• BD 16, BM4, BM71 and BM63 monoclonal antibodies
• BD 16, BM4, BM71 and BM63 monoclonal antibodies
• IgG1 which were obtained after immunization of Balb / c mice with a human thymic clone and fusion with the NSI cell line. They immunoprecipitate from the surface of human peripheral blood lymphocytes or radio-labeled iodine T clones, the same molecular structure. These antibodies react with different epitopes of this structure. Thus, there is no inhibition of the binding of the antibody BB18 in the presence of an excess of the antibody BD 1 6 and vice versa.
• the present invention therefore firstly relates to an antigenic protein consisting of a phosphorylated glycoprotein with a molecular weight of 150 ⁇ 10 kDa determined by electrophoresis of SDS PAGE type under reducing conditions, protein expressed on the surface of human T lymphocytes preferably activated.
• the present invention also relates to an antigenic protein consisting of the same phosphorylated glycoprotein but in the form of a disulfide-linked dimer with a molecular weight of 300 ⁇ 20kDa determined by SDS PAGE electrophoresis under non-reducing conditions, protein expressed at surface of preferably activated human T lymphocytes, composed of subunits of 150 ⁇ 10 kDa as defined above.
• a subject of the invention is also derivatives of the protein in monomer or dimer form, characterized in that the protein is in non-glycosylated and / or non-phosphorylated form or in that it comprises non-natural glycosilations or phosphorylations or consisting of a fragment of this protein comprising the essential antigenic sites of the protein or non-glycosylated and / or non-phosphorylated derivatives and / or comprising non-natural glycosylations and / or phosphorylation.
• the present invention also relates to the monoclonal antibodies recognizing an epitope of a protein according to the invention, in particular the monoclonal antibodies BD 16, BB18, BM4, BM71 and BM63, as well as hybridoma cell lines producing these antibodies, in particular the lines deposited at ECACC under the numbers 92010801 for the antibody BD16, 92010802 for the antibody BB18, 93 01 1201 for the antibody BM4, 93 01 1202 for BM71 and 93 011203 for BM63.
• the protein, its fragments or derivatives according to the invention can be obtained from the natural protein itself obtained by purification from producer cells by immunoprecipitation techniques, in particular with the antibodies BB18, BD 16, BM4, BM71 and BM63 or other techniques.
• the protein according to the invention, its fragments and derivatives according to the invention can however also be obtained by recombinant DNA techniques of genetic engineering from the DNA coding for the said protein incorporated in expression vectors of that this in eukaryotic or prokaryotic cells, in which case the glycosilation of the recombinant protein obtained can differ from the natural protein as is known to those skilled in the art.
• a protein or an antibody according to the invention can be labeled with a detectable label with a view to their assay in vitro or their localization in vivo or ex-vivo.
• the invention indeed also relates to the use of proteins and antibodies according to the invention for the diagnosis of pathological states or the monitoring of the treatment of ailment or as a warning lamp for the appearance of these ailments.
• the present invention relates to the use of these proteins and antibodies for the rapid diagnosis of a state of early activation of lymphocyte T cells.
• the present invention relates to the use of a protein, antibody or antibody fragment according to the invention as a medicament in particular for the treatment of diseases with signs of immune deficiency or of inflammatory or autoimmune pathology and in in vivo or ex vivo treatment of malignant lymphoid proliferation.
• FIG. 1 shows the kinetics of expression on the cell surface of the structure recognized by BB18 after activation of PBMC (mononuclear cells of peripheral blood) using PHA.
• PBMC peripheral blood mononuclear cells of peripheral blood
• the peripheral blood mononuclear cells were labeled before (DO), 3 days (D3), or 9 days (D9) after coculture with PHA 1 ⁇ g / ml, for indirect immunofluorescence with BB18, CD25 "BC96" or an antibody monoclonal negative IgGl.
• the fluorescence profiles are shown as histograms.
• FIG. 2 shows the PMA inducing an early decrease in reactivity with BB18.
• Peripheral blood T cells were incubated with PMA 1 ⁇ g / ml for 6, 10, 24 or 144 h. The cells were washed and treated for indirect immunofluorescence with various monoclonal antibodies. At each measurement time, a negative control made of cells incubated under the same conditions but without PMA was carried out. As no modification of the control cells was observed, the control tested after 6 h of culture is presented. The fluorescence profiles are shown as histograms.
• FIG. 3 shows the SDS-PAGE profile of an immunopreopity BB 18 obtained from a lysate of cloned T cells.
• the human thymus T cell immunizing clone B12 was surface labeled and lysed in NP40 1%.
• the immunoprecipitates obtained with BB 18, CD 18 "M232" or an irrelevant IgGI monoclonal antibody were analyzed on a 7.5% SDS-PAGE gel under non-reducing conditions. The positions of the molecular weight markers have been indicated on the left in kDa.
• - Figure 4 shows the two-dimensional gel analysis (non-reducing / reducing) of a BB18 immunoprecipitate obtained from lysates of cloned T cells.
• the immunoprecipitates obtained with the monoclonal antibody BB18 were first separated under non-reducing conditions (N. Red.) On a 7.5% gel using a PHAST system. Then the gel strip containing the immunoprecipitate was cut and subjected to electrophoresis in the second dimension, as recommended by the manufacturer, after reduction (Reduction) on a 7.5% SDS-PAGE gel. The positions of the molecular weight markers have been indicated on the right in kDa.
• FIG. 5 shows the kinetics of PBMC proliferation induced by the BB18 monoclonal antibody.
• Mononuclear peripheral blood cells were incubated with the purified CD3 "X3" 5 ⁇ g / ml (white triangles), the purified monoclonal antibody BB18 5 ⁇ g / ml (empty squares) or 10 ⁇ g / ml (white circles) in the presence 0.2 ng / ml PMA for 2 to 8 days.
• the incorporation of (H) TdR was measured by "drawing" the culture for the last 16 hours. The results are expressed as the average of triplicate samples and the SEM has always been less than 10% of the average. Under these conditions, PMA alone did not induce significant proliferation at any of the measurement times.
• FIG. 6 represents the autoradiography of SDS-PAGE at 8.5% immunoprecipitates of BB18 and BD 16 under reducing conditions, and treated or having undergone a simulated treatment with endo-F.
• the positions of the molecular weight markers are indicated on the right in kilodaltons.
• FIG. 7 shows the autoradiography of SDS-PAGE at 12% of BB18 and BD 16 immunoprecipitates under reducing conditions, and treated or having undergone a simulated treatment with V8 protease directly in the gel.
• the positions of the molecular weight markers are indicated on the right in kilodaltons.
• FIG. 8 shows the co-modulation of the PBL cell surface structure at 37 ° C for 8 hours, with an excess of Acm BB 1 8 or 24 hours with mAb BD 16. Next, the cells were carefully washed and labeled with a biotiny mAb or an FITC-labeled goat anti-mouse Ig, as a control.
• FIG. 9 represents a competitive binding experiment between mAbs BB18 and BD 16.
• the second antibody (2nd mAb) was biotinyié, as indicated in the section "Materials and methods", and its binding was revealed by streptavidune- phycoerythrin.
• FIG. 10 shows the SDS-PAGE analysis of BB18 and BD16 immunoprecipitates obtained from 32 P metabolic labeling of cloned T cells.
• the immunoprecipitates were analyzed under non-reducing conditions (left-hand part) and under reducing conditions
• FIG. 11 represents the SDS-PAGE analysis of BB18 and BD 16 immunoprecipitates obtained from metabolic labeling with 32 P (left part) or surface labeling with 125 1 (right part) cloned T cells. Immunoprecipitates were analyzed under non-reducing conditions on both sides. The positions of the molecular weight markers are indicated on the right in kilodaltons. In addition, a CD 18 mAb was used as a control.
• FIG. 12 represents the effect of mAbs BB18 (curve a) and BD16 (curve b) on the intracellular Ca 2+ concentration of Jurkat cells.
• the arrows indicate the nature and time in addition to the MCA.
• Each trace is representative of at least three experiences.
• the Y axis represents the intracellular Ca 2+ concentration in nM, while the X axis corresponds to time in seconds.
• FIG. 13 represents the kinetics of the proliferative responses of PBL to a CD2 pair of mAb (CD2X11 + D66) in the presence or absence of mAb BB18 or BD 16.
• mAb BB18 or BD16 Acm BB18 or BD16, soluble and purified, with a final concentration of 5 ⁇ g / ml.
• incubation of PBMC with PMA induces rapid negative modulation followed by complete re-expression within 24 hours.
• This molecule is not specific for the T cell because it is also expressed in EBV-transformed B cell lines.
• cells proliferate when this dimer has been stimulated in the presence of submitogenic concentrations of PMA.
• the monoclonal antibodies used such as CD 1 "L404",
• CD8 "OKT8”, CD 18 "M232” and CD25 “BC96” were either locally produced (23) or purchased commercially.
• BD16 were prepared by immunization of Balb / c mice with the thymic clone B12, CD4 + CD8 + (22) (three intravenous injections with 20x10 6 cells). Spleen cells from immunized mice were fused to the NS I cell line five days after the last injection. The initial screening by indirect immunofluorescence and flow cytometry using a Facstar (Becton Dickinson, Mountain View, CA) retained all the supernatants of hybridomas reacting with the immunizing cells but not or weakly with the mononuclear cells of the peripheral blood at rest. Cultures containing the above-mentioned monoclonal antibodies were cloned twice by limiting dilution.
• Human PBMCs were prepared by Ficoll-Isopaque density gradient centrifugation. The unfractionated population was separated into E rosette-less (E-) and E rosette-plus (E-) populations by interaction with 5% sheep red blood cell (SRBC). The mixture was deposited on Ficoll-Isopaque and the E- cells were recovered from the interface while the E + cells were obtained from the pellet after hypotonic lysis of the SRBC.
• E- E rosette-less
• E- E rosette-plus
• the monocytes were obtained from E- cells by adhering to the glass overnight.
• the peripheral blood mononuclear cells CD2 + CD3 were obtained by complement-dependent lysis with a CD3 mAb in the E + cell fraction.
• Normal and leukemic samples were obtained from the Saint-Louis Hospital Blood Bank and cryopreserved as previously described (23).
• Human T cell clones were obtained as described elsewhere (22) and cultured in RPMI-1640 medium (GIBCO, Paisley, Scotland) containing 2mmol / liter of L-glutamine, penicillin (100 U / ml) , streptomycin (100 ⁇ g / ml), 10% heat-inactivated human serum and recombinant interleukin 2, kindly provided by Roussel-Uclaf (Romainvilie, France) (30 U / ml.) The cloned cells have been restimulated every 7 days with feeder cells in the presence of purified PHA (Wellcome, Beckenham, UK) at 1 ⁇ g / ml and of recombinant interleukin 2.
• the feeder cells were a mixture of irradiated aliogenic LSPs from three donors (the same donors being used for several months). Cultures of EBV-transformed leukemia or B cell lines and hybridoma cell lines were mycoplasma free and maintained logarithmic in RPMI containing 10% selected heat-inactivated fetal calf serum and antibiotics.
• the cells were first incubated with an excess of a first mAb for 30 min., Then they were centrifuged and incubated with the appropriate dilution of a biotinylated mAb. Controls included an incubation during the first stage with the same unconjugated mAb or with an inadequate mAb. After the final washes, the cells were resuspended in 0.3 ml of a 1% formalin solution in PBS. The purified antibodies were labeled with biotin using a standard protocol. Briefly, after dialysis in carbonate buffer (pH 8.8), the antibody was incubated (1 mg / ml) for 15 min.
• the bands separated by autoradiography of the dried gel were visualized, they were excised, digested with protease V8 in a buffer sample (at 1 mg / ml) , and the fragments were analyzed in a second SDS-PAGE experiment.
• the nitrogen-bridged sugars were eliminated by treatment of the specific immunoprecipitates with endo-beta-N-acetylglucosaminidase-F (endo-F) (Boehringer Mannheim, Meylan France).
• the digestion was carried out by bringing the BB18 and BD 16 immunoprecipitates to the boil for 4 minutes in 50 ⁇ l of 100 mM potassium phosphate buffer, pH 6.5 containing 50 mM EDTA, 0.5% SDS and 1% beta-mercaptoethanol.
• 100 ⁇ l of 100 mM potassium phosphate buffer (pH 6.5) containing 50 mM EDTA, 1% NP40 and 1% beta-mercaptoethanol was added before the addition of 12 ⁇ l (0.6 IU) of endo-F. After 24 h of incubation at 37 ° C, the reaction was stopped by adding a buffer sample.
• 50,000 PBMC were cultured in triplicate in 96-well rounded bottom plates (Costar, Cambridge, MA) in a total volume of 0.2 ml RPMI 1640, supplemented with 10% fetal calf serum in one volume 0.15 ml total of RPMI 1640, supplemented with 10% heat-inactivated human serum.
• Various concentrations of purified antibodies were added with 0.2 ng / ml PMA.
• the phosphorus labeling was carried out as follows. Cloned T cells were washed twice in phosphate-free medium containing 5% dialyzed human AB serum and then incubated in the same medium at 50 x 10 6 cells in 1 ml for 30 minutes at 37 ° C.
• the cells were washed twice in PBS before their lysis in 2-3 ml of buffer containing 10 mM Tris, pH 8.2, containing 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mg / ml of BSA,
• the intracellular Ca 2+ concentration was measured as previously described (26). Briefly, Jurkat cells were washed twice in medium containing 25 mM HEPES (pH 7.2), 125 mM NaCl, 5 mM KCl, 1 mM Na 2 HPO 4 , 1 mM Ca Cl 2 , 0.5 mM MgCl 2 , and 1% glucose (all subsequent incubations and washes were performed in the same medium) and they were resuspended at 30x106 cells / ml in the presence of 3 ⁇ M of Fura 2-AM (Calbiochem, letdon,
• mAbs obtained by repeated immunizations with highly functional human T cell clones 21).
• an mAb was initially identified on the clone called B12 (22) of human thymus T cells. called BB18, recognizing a new disulfide-linked dimer composed of subunits of 150 ⁇ 10kDa. This structure, which is weakly expressed in normal peripheral blood lymphocytes, is quickly found to be strongly expressed during activation.
• BB18 mAbs react strongly with cloned T cells and weakly with resting human T cells by flow cytofluorometry.
• mice In order to obtain monoclonal antibodies defining the stages of T cell activation, six-week-old Balb / c mice were immunized with cloned human thymic cells (22). An mAb of the IgG1 isotype, named BB18, was thus isolated.
• Table 1 summarizes the reactivity of the aforementioned mAb with many normal human and hematopoietic malignant cells. The molecule recognized by this mAb is weakly expressed on resting T cells but not at all on B cells from peripheral blood or lympoid organs. Among the leukemic lymphoid cell lines tested, all T cells were strongly labeled, while the Burkitt lymphoma cell lines were not reactive.
• BB 18 strongly marked the YT cell line of activated NK, CD3 negative tumor cells and its CD 25 mutant, YT2C2. The structure recognized by BB18 appears to be strongly expressed immediately after cellular activation as shown by the representative kinetic experiment carried out with mononuclear cells from the peripheral blood of a normal individual stimulated with PHA (FIG. 1).
• the modulation of the CD3 molecule reaches its maximum after 24 hours while the modulation of CD4 is completed after 10 hours. However, as previously reported after 24 hours of culture in the presence of PMA, CD25 is induced and the expression of CD8 is not modified by this treatment. A.3. Biochemical analysis of the immunoprecipitated molecule by mAb BB18 after labeling the cell surface.
• mAb BB18 In order to define the molecule identified by mAb BB18, clones of human T cells were labeled with Iodine 125 on their external face, according to the lactoperoxidase method, and iysed. The BB 18 immunoprecipitates obtained from these lysates of labeled cells were then analyzed by SDS-PAGE. The autoradiography shown in Figure 3 indicates that mAb BB18 immunoprecipitates from a representative T cell clone. A predominant band migrating under non-reducing conditions with a molecular mass of approximately 300 kDa.
• CD18 "M232" mAbs used as immunoprecipitate control of three protein bands from the same lysate characteristic of CD 18 molecules and two different CD 11 molecules, while no protein band is obtained with an inappropriate IgG1 type mAb .
• the immunoprecipitate obtained with the BB18 Antibody consists mainly of homodimers linked by disulfide bridges of approximately 300 kDa in the first non-reducing dimension, which resolves in a second reducing dimension into a predominant protein band of approximately 150 kDa below the diagonal of the two-dimensional gel.
• Stimulation with soluble BB18 mAbs induces proliferation of LSPs in the presence of a submitogenic concentration of PMA.
• BB18 a new surface structure of a human lymphocyte cell with the mAb called BB 18.
• the immunoprecipitated molecule using BB18 from lysates of cloned T lymphocytes marked on the surface, appeared to be a homodimer linked by bridges. disulfides comprising subunits of approximately 150kDa. This structure is poorly detectable on resting peripheral blood T cells, while its expression increases rapidly after activation of the T cell and remains at this high level for some time after initial triggering.
• the BB18 Antibody reacts strongly with all the cloned T cells tested regardless of their CD4 / CD8 phenotype and their TcR (data not shown).
• mAb BB18 does not react with resting B cells, while long-standing EBV-transformed B cells are weakly labeled.
• Burkit's lymphoma cell lines (Daudi and Namalva) clearly lack responsiveness.
• leukemic but not myelomonocytic lymphoid cell lines are reactive to BB18.
• the CD28 molecule a 44 kDa disulfide-bridged homodimer, preferentially expressed on a T cell subpopulation, was the first structure described to induce costimulatory signals (25, 26). The effect has been shown to regulate the stability of the mRNA encoding lymphokines (27).
• costimulatory signal mediator molecules include the molecule CD26 (4, 5), identifying dipeptidylpeptidase IV, which is a 120 kDa structure; CD69, a very early activation heterodimer with disulfide bridges, of 28.32 kDa (17, 18); the specific T cell molecule identified by mAb 10D 1, which is a 90 kDa disulfide bridge homodimer (28); CD60 "UM4D4" recognizing a hydrocarbon structure expressed on various gangliosides (29).
• the CD43 mAbs are particularly interesting, identifying a highly sialylated glycoprotein of 95 kDa, which similarly to the CD3 and CD2 mAbs can directly trigger the activation of T cells in the presence of monocytes. It has been recently reported that a monoclonal antibody reactive with CD5, unlike the previously described CD5 mAb, is capable of inducing T cell proliferation in the presence of monocytes (15). This raised the possibility that only certain epitopes of the CD5 molecule, as in the case of CD2, are involved in the activation of T cells. Finally, it should be mentioned that, unlike the epitope recognized by the Antibody BB18, the surface expression of the majority of signal transduction structures is not increased during activation of the cell.
• T cells can be activated through several distinct surface molecules, these activation pathways appear to share common signal transduction mechanisms. It is well known that activation of the T cell via CD2 or CD5, but not via CD43 (16) is dependent on the expression of CD3-TcR. Although no physical link of the BB18 molecule with CD3-TcR has been demonstrated according to the invention, (the AMC has labeled YT2C2 CD3-TcR negative lymphocytes), links are possible with components such as CD3 chain.
• BD16 a second mAb, named BD16, presenting a similar reactivity motif in immunofluorescence studies and immunoprecipitating a dimeric structure with disulfide bridges of 300 kDa originating from the surface of the T cell.
• BB18 and BD 16 identified the same structure
• an additional biochemical analysis of the molecule immunoprecipitated by the two mAbs was performed after labeling the cell surface. Human T cell clones were labeled on their outer surface, according to the lactoperoxidase method, and lysed.
• FIG. 6 represents an autoradiography revealing an identical band of approximately 120 kDa in the two immunoprecipitates treated with endo-F, while the characteristic band of 150 kDa was obtained with the samples having received a simulated treatment.
• the 150 kDa cell surface molecule reactive to mAbs BB18 and BD16 is a phosphoprotein and the mAb BD16 co-precipitates a phosphorylated structure of 100 kDa.
• CD2-induced PBL proliferation is greatly increased by mAb BD 16 but not by mAb BB18.
• Freshly isolated PBLs were incubated for various durations with or without mAb, as described in the "Materials and Methods" section.
• the soluble and purified BB18, BD 16 and CD3 mAbs were used at final concentrations of 5 ⁇ g / ml.
• MAb BD 16 enhances the proliferative response of PBL to various pairs of
• the PBLs were cultivated for 5 days in the medium, CD2 mAbs alone with or without BB18 or BD16 as a control (results not exposed) or a combination of CD2 mAbs with or without BB18 or BD16 mAbs. Significant proliferation was observed only in wells containing pairs of CD2 mAbs. CD2 mAbs were used in the form of diluted ascites fluid, while BB18 and BD16 were purified mAbs used at final concentrations of 5 ⁇ g / ml.
• Sialophorin a sialoglycoprotein surface defective in the wiskott-aldrich synchrome, is involved in human T lymphocyte proliferation. 3. Exp. Med. 165: 1383.
• CD 28 activation pathway regulates the production of multiple T-cell derived lymphokmes / cytokmes. Proc. Natl. Acad. Sci. USA. 86: 1333.

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• 1992
  • 1992-01-13 FR FR9200255A patent/FR2686087A1/fr active Granted
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