FR2783325A1 - MEANS FOR CONTROLLING THE NEGATIVE REGULATION OF AN ACTIVATION TRANSMITTED BY A RTK - Google Patents

Abstract

The present application relates to means for controlling the negative regulation of an activation transmitted by an RTK, and in particular for controlling the negative regulation of a proliferation and / or cell differentiation induced by an RTK. Such means include in particular the use of compounds capable of co-aggregating an RTK to a receptor comprising at least one ITIM motif, and of antagonist compounds of these co-aggregating compounds.

Description

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MEANS FOR CONTROL OF REGULATION
NEGATIVE OF AN ACTIVATION TRANSMITTED BY A RTK
The present invention relates, in general, to means for controlling the negative regulation of an activation transmitted by an RTK (receptor with tyrosine kinase activity), and for controlling the negative regulation of a proliferation and / or cell differentiation induced by a particular RTK.

 RTKs are a family of receptors with specific tyrosine kinase enzymatic properties. Among the RTKs, there may be mentioned in particular the receptors for growth factors, for differentiation factors, or for honestones such as erb B1 (or EGF-R, receptor for growth factor of epithelial cells), erb B2 ( or HER2, or neu, receptor for differentiation factor neu), PDGF-R (receptor for growth factor derived from platelets, in form a or P), NGF-R (receptor for growth factor for the nervous system) , FGF-R (receptor for fibroblast growth factor), Kit (receptor for stem cell factor or SCF, "Stem Cell Factor"), the receptor for insulin (or IR).

 RTKs are expressed by many cell types: hematopoietic cells (for Kit and CSF-R, for example), just like non-hematopoietic cells such as epithelial cells (EGF-R), melanocytes (Kit) cells of the nervous system central (NGF-R), liver and adipocyte cells (Insulin-R), intestinal cells and germ cells (Kit).

 RTKs control the stimulation of the proliferation and / or differentiation of the cells which express them: their activation induces non-immune cellular responses, such as triggering of

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 cell cycle of mitosis in particular, induction of cell differentiation, cell survival or transformation, initiation of meiosis, activation of metabolisms crucial for the life of the cell such as cellular metabolism of glucose, nucleosides, amino acids. The activation of RTK can lead to physiological or pathological responses: RTKs are indeed proto-oncogenes (form c-), and many oncogenic forms, constitutionally activated (form v-, mutant), have been identified.

 Controlling the negative regulation of RTKs would therefore make it possible to control crucial events in cell life in a physiological as well as a pathological state. If it is known that an RTK can be activated by stimulation of its ligand and aggregation, the means ensuring the negative regulation of this activation, which therefore ensure control functions on the inhibition of proliferation and cell differentiation, remained undetermined.

 The inventors have precisely developed means which allow a control of the negative regulation of an activation transmitted by an RTK, that is to say means making it possible to analyze this negative regulation, as well as, if necessary, modulate it in order to stimulate it (in order to decrease the activation of the RTK considered) or in order to inhibit it (in order to release the activation of the RTK considered).

 The control means according to the invention take advantage of the discovery that, quite surprisingly, an immunoreceptor with ITIM (immunoreceptor inhibition pattern based on residue (s) tyrosineI / V / L / xYxxL / V), such as, for example, RFc [gamma] IIB (immunoreceptor inhibitors for the Fc portion of IgG), KIR (immunoreceptor inhibitor for MHC or HLA molecules), Ly49, NKG2A, CD22, CD72, PIR-B , gp49Bl; MAFA,

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 ILTs, LIRs, SIRPa, LAIRs, is capable, by co-aggregation, of inhibiting the activation of a receptor which is not an immunoreceptor, which therefore does not interact with the antigen and does not include ITAM (immunoreceptor activation motif based on residue (s), namely that such an ITIM receptor is capable, by co-aggregation, of inhibiting the activation transmitted by an RTK, and in particular, of inhibiting cell proliferation and / or differentiation induced by RTK.

 The present invention thus relates to methods and kits for the identification of agents useful for the control of a negative regulation of the activation of an RTK (stimulating agents or inhibitors of such a negative regulation), artificial compounds as obtained by such methods, methods and kits for in vitro diagnosis of inappropriate negative regulation of RTK activation, as well as uses of such agents or artificial compounds for the manufacture of medicaments .

 One aspect of the present invention thus relates to a method for the identification of an agent useful for stimulating a negative regulation of the activation of an RTK, characterized in that it comprises the detection of at least one compound capable, under conditions suitable for cell physiology, of co-aggregating a receptor with tyrosine kinase activity (RTK) with a receptor comprising at least one ITIM motif, and / or in that it comprises the detection of constituent entities of such a compound. A co-aggregating compound thus detected, or the combination of the constituent entities thus detected essentially corresponds, according to the invention, to an agent useful for stimulating a negative regulation of the activation of an RTK. Advantageously, said detection is carried out by functional screening of chemical and / or biological banks according to conventional techniques. Examples of suitable biological banks include in particular

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 phage banks expressing, after genetic recombination, human antibodies or autoantibodies, and / or fragments of such antibodies or autoantibodies. Chemical libraries can also be screened to identify mimetopes of such compounds.

 Conditions suitable for cell physiology may correspond to in vivo conditions such as in particular after injection into a man or an animal such as a mouse, as well as to m vitro conditions mimicking the conditions in vivo, such as in particular in the presence of a culture medium of RPMI or DMEM type supplemented with 10% of fetal calf serum (FFS) in which the cells express the RTKs and the ITIM receptors considered are incubated, for example in a humidified incubator at 37 C.

 When a co-aggregating compound detected according to the invention effectively co-aggregates an ITIM receptor to an RTK, the negative regulation stimulation thus induced leads to phosphorylation of the ITIM motif (s) of said ITIM receptor, and the recruitment of phosphatases such as SHIP (case of RFcylIB), SHP-1, SHP-2 (case of other ITIM receptors).

Measuring the level of phosphorylation of the ITIM motif (s) of said ITIM receptor is therefore an indicator of the level of negative regulation exerted by a co-aggregating compound detected according to the invention on the activation of said RTK. A means for verifying that a compound is capable of stimulating the negative regulation of such activation, or for measuring its level of effectiveness, thus notably comprises measuring the level of phosphorylation of the ITIM motif (s) of said ITIM receptor in the presence and absence of said test compound, and verifying that the level of phosphorylation observed in the presence of this compound is lower than that observed, under otherwise comparable conditions, in the absence of this same compound. The statistical difference between these two levels of

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 phosphorylation gives an image of the level of efficacy of the test compound.

 A co-aggregating compound detected according to the invention has the remarkable property of being capable, under said conditions, of stimulating the downregulation of a cellular response of a non-immune nature with which activation of RTK can be associated. Means for inducing a non-immune response to which activation of RTK can be associated comprises activating said RTKs with a ligand of these RTKs, so as to aggregate them together. Such activation can lead to cellular responses of non-immune type (proliferation and / or cell differentiation in particular), responses which are inhibited in the presence of a co-aggregating compound detected according to the invention is capable of inhibiting (by stimulation of negative regulation). A means for verifying that a compound is capable of stimulating the negative regulation of the activation of an RTK, or for measuring its level of efficacy, thus notably comprises verifying an inhibitory effect of the compound tested on the non-response. immune induced by said RTK.

 Said cellular response induced by said RTK, which a coaggregating compound detected according to the invention can inhibit, is advantageously included in the group consisting of the initiation of the cell cycle (passage from the quiescent state Go to phase G1; passage from G1 to phase S of DNA synthesis; from S to phase G2 and from phase G2 to phase M of mitosis; return of M to the quiescent state Go) triggering of mitosis, induction d cell differentiation, triggering meiosis, activating a metabolism essential to cell life such as glucose metabolism, nucleoside metabolism, amino acid metabolism. A means for verifying that a co-aggregating compound detected according to the invention is capable of inhibiting the proliferation of cells expressing the RTKs and the ITIM receptors considered may for example consist of:

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 verify that said co-aggregating compound is capable, under said conditions, of inhibiting the incorporation, by said cells, of a nucleoside, and of thymidine in particular. A co-aggregating compound detected according to the invention is thus capable of inhibiting such non-immune responses, and, in a particularly remarkable manner, of blocking at the G1 stage the cells expressing the RTKs and ITIM receptors considered, without however killing these same cells.

 Advantageously, said detection of the method for identifying a stimulating agent according to the invention is carried out so as to detect a compound comprising at least one entity having, under said conditions, an affinity for a molecule chosen from the group constituted by said RTK, an extracytoplasmic part of this RTK, an intracytoplasmic part of this RTK, a ligand of this RTK or a molecule capable of binding to this ligand.

 Said detection is also advantageously carried out so as to detect a compound comprising at least one entity having, under said conditions, an affinity for the molecule chosen from the group consisting of said ITIM receptor, an extracytoplasmic part of this receptor, an intra part -cytoplasmic of this receptor, an ITIM motif of this receptor, a ligand of this receptor or a molecule capable of binding to this ligand.

 A co-aggregating compound detected according to the invention is therefore capable of binding directly to the RTK and / or to the ITIM receptor considered, or else indirectly, via a ligand of this RTK and / or of this ITIM receptor, or of a molecule that binds to this (these) ligand (s).

 Said RTK can be advantageously chosen from the group consisting of a receptor for a growth factor, a receptor for a differentiation factor, a receptor for a hormone. Examples of such

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 RTK include PDGF-R, CSF-R, EGF-R, NGF-R, Insulin-R, Kit receptors.

 Said ITIM receptor can advantageously be chosen from the group consisting of RFcyIIB (B cells, T cells), KIR (NK cells and mast cells), Ly49 and NKG2A, CD22 and CD72 (B cells), PIR-B cells (B cells) and myeloid cells), gp49B and MAFA (mast cells), ILTs and LIRs (mast cells and dendritic cells), LAIRs (lymphoid cells), SIRPa (hematopoietic and non-hematopoietic cells).

 A particular co-aggregating compound detected according to the invention is capable, under said conditions, of co-aggregating the receptor to ITIM RFcyIIB, to an RTK, and to Kit in particular. Such a particular co-aggregating compound for example essentially consists of all or part of a portion Fc of antibodies, and of all or part of a ligand of said RTK (in the case of the RTK Kit, the ligand is the factor stem cell growth (SCF)). The ITIM receptor considered here being RFcylIB, examples of such particular co-aggregating compounds detected according to the invention include antibodies, and isolated autoantibodies. They also include artificial compounds resulting from the artificial combination of Fv, Fab, F (ab ') 2 fragments, CDR of anti-RFc [gamma] IIB antibodies and Fv, Fab, F (ab') 2 fragments, or CDR of anti-RTK antibodies, and anti-Kit in particular.

 Another aspect of the invention relates to a method for the identification of an agent useful for the inhibition of a negative regulation of the activation of an RTK, characterized in that it comprises the detection of at least a compound which is an antagonist of a compound capable, under conditions suitable for cell physiology, of co-aggregating a receptor with tyrosine kinase activity (RTK) to a receptor comprising at least one ITIM motif, and / or in that '' it includes the detection of the constituent entities of such a

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 antagonistic compound. The antagonist compound thus detected according to the invention, or the combination of the constituent entities of such an antagonist compound corresponds essentially, according to the invention, to an agent useful for the inhibition of a negative regulation of the activation of a RTK. Examples of conditions suitable for cell physiology have been given above.

One way of carrying out said detection consists in functionally screening chemical and / or biological banks according to conventional techniques. Examples of suitable biological libraries include in particular phage libraries expressing, after genetic recombination, human antibodies or autoantibodies, and / or fragments of such antibodies or autoantibodies, chemical libraries can also be screened so as to identify mimetopes.

 By antagonist compound of a co-aggregating compound, we mean, in the present application, any compound which is capable, under said conditions, of significantly reducing, or preventing, the co-aggregation action of said co compound -aggregating. Such an antagonist compound detected according to the invention is capable of inhibiting the down regulation of the activation of an RTK, and is thus indirectly capable of stimulating this activation. Said detection of the method for identifying an inhibitor agent according to the invention is advantageously carried out so as to detect a compound capable, under said conditions, of preventing the binding (direct or indirect) of said co-aggregating molecule to RTK considered, or else to prevent the binding (direct or indirect) of said coaggregating molecule to the ITIM receptor considered, or else to prevent these two types of binding.

 Such an antagonist compound detected according to the invention is advantageously chosen or constructed so as to block the site of binding to

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 ligand of said RTK, and / or to block said receptor to ITIM at its ligand binding site or at the level of its ITIM motif (s).

 Such an antagonist compound detected according to the invention can thus be chosen so as to be capable, under said conditions, of blocking the ligand binding site of said RTK. In the particular case where the ITIM receptor considered is RFcyIIB, said antagonist compound according to the invention can be derived from an antibody, or from an autoantibody, by deletion of the part (s) of this antibody recognized by RFcyIIB, and by deletion of its Fc fragment in particular, said antibody being capable, under said conditions, and when it comprises all of its entities (complete antibody), of stimulating the negative regulation of 'an activation of RTK. Such a particular antagonist compound detected according to the invention then essentially consists of a fragment of antibody or autoantibody, and of human antibody or autoantibody in particular, preferably chosen from the group consisting of a Fv fragment, Fab, F (ab ') 2, said fragment being directed against the RTK considered. An antibody, or autoantibody, considered as an inhibitor of the activation of an RTK can thus, according to the invention, if the inhibition observed with this antibody is exerted via the receptor to ITIM RFcylIB, present properties of stimulator of this same activity by removing from it the part or parts recognized by RFcyIIB, and by removing its position Fc in particular.

 Examples of such antagonist compounds according to the invention also include, in the case where the ITIM receptor considered is RFcylIB, any antibody fragment or autoantibody, which is directed against the RFcylIB, without being directed against the RTK considered, this in order to block the binding site of the RFcyIIB considered; advantageously, this antibody fragment is specific for the ITIM receptor considered. Preferred antibody fragments are of human origin.

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 An antagonist compound detected according to the invention can also be chosen so as to be capable, under said conditions, of blocking the ITIM receptor considered at its ligand binding site, and / or at its level (s) ITIM motif (s).

 When a compound co-aggregating according to the invention effectively co-aggregates an ITIM receptor to an RTK, the ITIM motif (s) of said ITIM receptor become phosphorylated, then recruit phosphatases such as SHIP (case of RFcylIB), SHP-1 SHP-2 (for other ITIM receptors). An antagonist compound detected according to the invention can therefore essentially consist of any entity capable, under said conditions, of blocking the motif (s) of said ITIM receptor, and / or capable of preventing the binding of phosphatases such than SHIP, SHP-1, SHP-2.

 Another aspect of the invention thus relates to any artificial compound capable, under conditions suitable for cell physiology, of coaggregating a receptor with tyrosine kinase (RTK) activity and a receptor comprising at least one ITIM motif, as well as any antagonistic artificial compound of such an artificial co-aggregating compound. By artificial compound according to the invention, we mean, in the present application, any compound obtained by chemical synthesis and / or genetic engineering such as, in particular, chemical molecule or simple peptide obtained by chemical synthesis, combination of antibody fragments or d autoantibody, and human antibodies or autoantibodies in particular, such as humanized antibodies, bispecific antibody, scFv fragment (single-chain fragment of variable fragments). An artificial compound co-aggregating according to the invention can for example be obtained by assembling fragments of anti-RTK antibody and anti-receptor to ITIM (or anti-ligand for these receptors, or anti-molecules binding to these ligands), so as to form a bispecific antibody or a

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 scFv (antibody which can be produced by recombinant expression by bacteria in a form associated with the GST protein, and purification using anti-GST antibodies). Such artificial compounds are capable, under conditions appropriate to cell physiology, of controlling the downregulation of stimulation transmitted by an RTK, and in particular of controlling the downregulation of cell proliferation and / or cell differentiation induced by RTK: when they are co-aggregating, said artificial compounds are thus capable of stimulating such negative regulation; when they are antagonistic, they are able to inhibit such stimulation. This aspect of the invention therefore also includes any artificial agent as obtained using an identification method according to the invention presented above.

 According to another aspect, the present invention also relates to the use of a stimulating or inhibiting agent identified according to the invention, and / or of an artificial compound according to the invention in the diagnosis (as target to be detected), and / or prevention, care, treatment (as active agent or adjuvant) of an inappropriate negative regulation (excessive or deficient) of the activity of an RTK.

 An agent identified according to the invention, or an artificial compound according to the invention, can thus advantageously be used, as a target, for the in vitro diagnosis of an inappropriate negative regulation (considered excessive or deficient) of the activation of 'an RTK. Preferred agents for such use as a diagnostic target include human antibodies or autoantibodies, and their fragments.

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 The invention thus relates to a method for the in vitro diagnosis of an inappropriate negative regulation of the activity of an RTK, characterized in that it comprises: - the detection, in a biological sample, of at least one agent , stimulator or inhibitor of said negative regulation, as identified according to the invention, and / or at least one artificial compound co-aggregating or antagonist according to the invention with, if necessary, measuring its titer (or their respective titles), and - the comparison of the result obtained for said sample with the results observed on average under physiological conditions.

 A kit for the implementation of this diagnostic method according to the invention comprises the reagents necessary for the detection of the targeted molecules. For example, a kit intended for the in vitro diagnosis of an inappropriate negative regulation of the activation of RTK by RFcyIIB receptors can comprise the reagents necessary for the detection of natural coaggregating molecules such as anti-RTK autoantibodies; such reagents can, for example, correspond to cells transfected with the cDNAs encoding the RTKs considered, and to indirect immunofluorescence reagents.

A kit intended for the detection of anti-growth factor autoantibodies may comprise the reagents necessary for carrying out an ELISA test on growth factors immobilized on 96-well plates.

 Another method according to the invention for the in vitro diagnosis of an inappropriate negative regulation of the activity of an RTK comprises: - the detection, in a biological sample, of the level of phosphorylation of the ITIM receptor, and - the comparison from the result obtained for said sample to the results observed on average under physiological conditions.

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 A kit for implementing this other diagnostic method according to the invention comprises in particular the reagents for detecting a level of tyrosine-phosphorylation, such as the material and the reagents necessary for an immunoblot using anti-phosphotyrosine antibodies. Such a kit may, in addition, also include the material and the reagents necessary for the immunoprecipitation of the targeted ITIM receptors, preferably using antibodies specific for the particular targeted ITIM receptors.

 The present application also relates to the use of an agent identified according to the invention and / or an artificial compound according to the invention, for the manufacture of a medicament intended to alleviate, prevent, and / or treat negative regulation. improper activation of an RTK.

 An agent stimulating said negative regulation, identified according to the invention, or an artificial co-aggregating compound according to the invention can indeed be used for the manufacture of a medicament intended to prevent, mitigate and / or treat a deficient negative regulation. activation of an RTK (proliferation and / or differentiation deemed excessive), and for the manufacture of an anti-cancer, contraceptive or abortifacient drug in particular.

 An inhibiting agent for said negative regulation, identified according to the invention, or an artificial antagonist compound according to the invention can, for its part, be used for the manufacture of a medicament intended to prevent, alleviate and / or treat excessive negative regulation of 'an RTK (deficiency in cell proliferation and / or differentiation), and, advantageously, for the manufacture of a medicament chosen from the group consisting of a medicament intended to re-stimulate an immune system, a medicament against aplasia, a medicine to stimulate cell proliferation, medicine to repair skin damage, medicine to

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 diabetes, a drug against infertility, and male infertility in particular. The term drug here includes the active agent, as well as the adjuvant which is used for its administration. The antagonistic molecules according to the invention can indeed be advantageously used for the manufacture of adjuvant for the above-mentioned drugs, and of a vaccine adjuvant in particular.

 The present invention is illustrated by the following examples, given by way of illustration: do not limit it in any way. Reference is made to them in Figures 1 to 4.

 FIGS. 1A, 1B, 1C represent the incorporation into mast cells deficient in [gamma] IIB - / - and wild-type thymidine RFc induced by anti-c-Kit antibodies.

 Figures 2A, 2B, 2C, 2D illustrate the incorporation into BALB / c mast cells of thymidine induced by anti-c-Kit antibodies.

 FIGS. 3A and 3B illustrate the phosphorylation of RFcyIIB and c-Kit, and the recruitment of phosphatases by RFcylIB after co-aggregation of c-Kit with RFcylIB.

 Figure 4 shows the induction and inhibition of mast cell proliferation (c-kit aggregation and co-aggregation of c-kit to RFcyIIB respectively).

EXAMPLES 1
We have studied the consequences of a co-aggregation of RFcyIIB with c-Kit which are co-expressed by mast cells. The protooncogene c-Kit is the transmembrane protein which is a tyrosine kinase receptor of the family of receptors CSF-1 / PDGF ("colony stimulating factor-1 / platelet-

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 derived growth factor ", colony stimulating factor 1 / growth factor derived from platelets). After dimerization by SCF (" Stem Cell factor "), c-Kit is autophosphorylated and induces cell proliferation. c-Kit thus controls the growth of hematopoietic cells, germ cells, melanocytes and intestinal and interstitial cells In transformed cells of rat, mouse and human mastocytomas, a point mutation in the kinase domain leads to constitutive activation of c- Kit and is at the origin of its oncogenic properties.

 Mast cells derived from growth factor-dependent bone marrow (BMMC) are obtained from mice deficient in RFcylIB and from normal mice (controls) C57BL / 6. BMMCs were obtained by cultivating mouse bone marrow cells in a culture medium constituted by RPMI (Seromed), 10% fetal calf serum (SVF), glutamine, penicillin and streptomycin ( Gibco), and supplemented with 20% of medium conditioned by culture of WEHI-3B cells. After 4 weeks of culture, it is found, by microscopic observation and by immunofluorescence using mouse IgE and F (ab ') 2 fragments of goat Ig anti-mouse conjugated to FITC (Jackson ImmuroResearch), cultures contain more than 90% mast cells.

 BMMCs are known to primarily express RFcylIB and traces of RFcylIIA. The absence of RFcylIB in BMMC RFc [gamma] IIB - / - was verified by analysis, using anti-peptide polyclonal antibodies specific for RFcyIIB, of Western blots of cell lysates. .

Aliquots of 5 x 105 BMMC were lysed for 5 minutes at 95 ° C in SDS (sodium dodecylsulfate) at 4% in RPMI. The proteins are precipitated with cold acetone for 30 minutes at 0 ° C., and lyophilized under vacuum (speedvac) for 20 minutes. The precipitates were dissolved in a reducing buffer by electrophoresis, subjected to a

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 electrophoresis in SDS and transferred to Immobilon-P membranes (Millipore). The membranes are saturated using 5% BSA (bovine serum albumin) diluted in a buffer containing 10 mM Tris, 150 mM NaCl pH 7.4 and 0.5% Tween 20 (Merk) (Buffer from Western), and incubated with polyclonal rabbit anti-RFc [gamma] IIB antibodies, then with goat anti-rabbit Ig conjugated with horseradish peroxidase (GAR) (Santa Cruz). Antibodies labeled with peroxidase were detected using the Amersham ECL kit. The results are illustrated in FIG. 1A, which shows the expression of RFcyIIB in BMMC RFc [gamma] IIB - / - and BMMC controls C57BL / 6.

 Comparable levels of thymidine incorporation are induced by SCF in control BMMCs and in BMMC RFc [gamma] IIB - / -. The results are illustrated in FIG. 1B which represents the incorporation of thymidine induced by the SCF ("Stem Cell factor"). The BMMCs were incubated for 24 hours with the indicated concentrations of SCF, and the incorporation of thymidine was measured as follows: aliquots of 3 × 10 4 BMMCs in 40 l RPMI containing 1% SVF and BSA at 0.5% (Sigma) were incubated in 96-well plates in the presence of recombinant SCF (R&D Systems), or in the presence of the indicated concentrations of the preformed immune complexes, for 24 hours at 37 C. 0.5uCi of [ 3H] thymidine were added to the wells, and the cells were incubated for an additional 4 hours at 37 C. The cells were harvested using a cell micro-harvester (Skatron) and the incorporated radioactivity was measured using an ss counter (Phannacia).

 When incubated with BMMC for 24 hours, the immune complexes, produced with IgG antibodies ACK2 rat antibody anti-mouse c-Kit and fragments F (ab ') 2 of MAR antibody (anti mouse antibody - rat Ig), do not induce thymidine incorporation in the

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 BMMC witnesses. The same complexes induce a dose-dependent incorporation of thymidine in BMMC RFc [gamma] IIB - / -. The results are illustrated in FIG. 1C which represents the incorporation of thymidine induced by complexes ACK2 F (ab ') 2 The BMMCs were incubated for 24 hours with preferred complexes constituted by the indicated concentrations of F (ab') 2 and IgG ACK2. The incorporation of thymidine was measured as described above in Figure 1B. The legend for Figure 1C is as follows: white circle 0 ug / ml MAR F (ab ') 2, black 1 g / ml MAR F (ab') 2, black square 3 ug / ml MAR F (ab ') 2, black 10 g / ml MAR F (ab') 2. This indicates that anti-c-Kit antibodies are capable of mimicking the effects of SCF in BMMCs provided that these BMMCs do not express RFcylIB which could be engaged with the Fc portion of the antibodies.

 To confine this hypothesis, BMMCs were produced from BALB / c mice and were used in all the experiments which follow.

BMMC BALB / c express c-Kit, as shown by immunofluorescence analyzes with ACK2, and express RFcy, as shown by analyzes with mouse anti-RFcyIIB / IIIA rat 2.4G2 antibody.

The analyzes relating to c-Kit with ACK2 were carried out by indirect immunofluorescence as follows. The BMMCs were incubated for 1 hour at 0 ° C. in the presence of 10 g / ml of rat antibody 2. 4G2 or ACK2 in HBSS (Harks isotonic saline solution) containing SVF at 5%, or in the presence of medium. alone. The cells were washed and labeled by incubating them for 30 minutes at 0 ° C. in the presence of 50 μg / ml of fragments F (ab ') 2 of anti-rat mice (MAR) with fluorescein isothiocyanate (Jackson ImmunoResearch). The fluorescence was analyzed by flow cytometry using a "FACScalibur" (Becton-Dickinson).

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 The analyzes relating to RFcyIIB were carried out by indirect immunofluorescence using the antibody 2. 4G2 described by J.C.

Unkeless, J. Exp. Med. 150, 580 (1979).

 The results are illustrated in FIG. 2A where the expression of c-Kit and RFcyIIB is represented by the BMMCs. The binding of the anti-c-Kit ACK2 antibody and that of the anti-RFcylIB 2. 4G2 antibody are analyzed by indirect immunofluorescence as described above. The histogram on the left shows the fluorescence of cells incubated only in the presence of MAR F (ab ') 2-FITC fragments. The histogram in thick line shows the fluorescence of cells incubated in the presence of MAR F (ab ') 2FITC fragments and of antibody 2. 4G2. The histogram in thin line shows the fluorescence of cells incubated in the presence of MAR F (ab ') 2-FITC fragments and of ACK2 antibodies.

 These BMMCs incorporate thymidine when they are incubated for 24 hours with SCF, but also when they are incubated with F (ab ') 2 fragments complexed with MAR F (ab') 2 fragments. These results are illustrated in Figure 2B. The incorporation of thymidine was measured as described above. The legend of FIG. 2B is as follows: small black diamond 0 g / ml of MAR F (ab ') 2 for the concentrations of ACK2 indicated on the abscissa, black circle 1 g / ml of MAR F (ab') 2 for the concentrations of ACK2 indicated on the abscissa, black square 10 g / ml of MAR F (ab ') 2 for the concentrations of ACK2 indicated on the abscissa. Although the ACK2 antibody blocks the binding of SCF to c-Kit, and has been described as a stimulation antagonist antibody, it can therefore function as a stimulation agonist antibody when it lacks its Fc portion.

All the data presented here suggest that the co-aggregation of cKit with the RFcyIIBs via the Fab and Fc portions of the antibodies

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 anti-c-Kit, respectively, can inhibit proliferative responses of BMMC induced by c-Kit.

 To demonstrate this possibility, immune complexes were made with anti-biotin antibodies and biotinylated ACK2 IgGs, the complexes were used to stimulate BMMC BALB / c in which RFcyIIBs are free, or have been made inaccessible to using antibody 2. 4G2 which blocks the binding site of RFcylIB. (J.C. Unkeless, J. Exp. Med.

 150, 580 (1979)). When incubated with BMMC for 24 hours, the anti-biotin-ACK2-biotin complexes do not induce significant incorporation of thymidine. If, however, the BMMCs are preincubated with the 2.4G2 antibody, the same complexes induce a dose-dependent incorporation of thymidine. These results are illustrated in Figure 2C. BMMC preincubated with 2.4G2 (c-Kit aggregation, white symbols), or without 2.4G2 (RFcyIIB-c-Kit co-aggregation, black symbols) were incubated for 24 hours with the complexes constituted by the indicated concentrations of biotin-ACK2 and anti-biotin antibodies (a-biotin for short), and the incorporation of thymidine was measured as indicated above for Figure 2B. The legend of Figure 2C is as follows: - curves on the left: black circle: a-biotin 0 g / ml, 2. 4G2 0 g / ml, black triangle: a-biotin 3 g / ml, 2. 4G2 0 ug / ml, white circle: a-biotin 0 g / ml, 2. 4G2 10 g / ml, white triangle: a-biotin 3 g / ml, 2. 4G2 10 g / ml, - center curves: black circle: a -biotin 0 g / ml, 2. 4G2 0 g / ml, black triangle: a-biotin 10 g / ml, 2. 4G2 0 g / ml, white circle: a-biotin 0 ug / ml, 2. 4G2 10 g / ml, white triangle: a-biotin 10 g / ml, 2. 4G2 10 g / ml,

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- right curves: black circle: a-biotin 0 g / ml, 2. 4G2 0 g / ml, black triangle: a-biotin 30 g / ml, 2. 4G2 0 g / ml, white circle: a-biotine 0 ug / ml, 2. 4G2 10 ug / ml, white triangle: a-biotin 30 ug / ml, 2. 4G2 10 g / ml,
No binding of annexin V is detectable in cells treated with the biotin ACK2-anti-biotin complexes, whether or not they have been preincubated with the antibody 2. 4G2, which excludes the possibility that the defect d incorporation of thymidine by BMMCs not exposed to 2.4G2 can lead to apoptosis. To analyze apoptosis, the BMMCs were incubated with or without the antibody 2. 4G2, and stimulated with the immune complexes as described above. Cells treated for 1 hour at 37 ° C. with 0.5 g / ml of anti-Fas monoclonal antibody (Phanningen) served as positive controls. Aliquots of 5 x 105 cells were washed in PBS (Phosphate Buffer Sodium), incubated for 10 minutes at 0 ° C. in the presence of propidium iodide and annexin V conjugated to FITC as recommended by the manufacturer (Immunotech ). The fluorescence was analyzed using a "FACScalibur". The results are illustrated by FIG. 2D in which the absence of apoptosis can be observed after aggregation of cKit or after co-aggregation c-Kit-RFcyIIB. In this 2D figure, the pre-incubated BMMCs in the presence of 2. 4G2 (c-Kit aggregation, right curve) or in the absence of 2.4G2 (c-Kit-RFcylIB co-aggregation, left curve) were incubated for 24 hours in the presence of biotin ACK2anti-biotin complexes and the dead and apoptotic cells were visualized using, propidium iodide and annexin V as above indicated.

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 When they are co-engaged with c-Kit by the same antibody, the RFcylIBs can therefore inhibit the incorporation of thymidine induced by anti-c-Kit antibodies without killing the BMMCs.

 In order to analyze the contribution of RFcylIB in this inhibition, the BMMCs were preincubated with or without 2.4G2 before being stimulated for 5 minutes using biotin-ACK2-anti-biotin complexes. RFcyIIB and c-Kit were immunoprecipitated, and their phosphorylation was analyzed by immunoblotting using anti-phosphotyrosine antibodies.

 Briefly, the experiments were carried out as follows. The BMMCs, resuspended at 5 × 10 6 / ml, were incubated for 1 hour at 37 ° C. in the presence or absence of 10 g / ml of 2.4G2 in culture medium. They were washed, resuspended at the same concentration in the same medium, and stimulated for 5 minutes at 37 C for immune complexes or SCF. They were centrifuged, and the pellets containing 1 × 10 7 cells for immunoprecipitation by anti-c-Kit, or 3 × 10 7 cells for immunoprecipitation by anti-RFcylIB were lysed for 15 minutes at 0 ° C. at the concentration of 1 x 108 cells / ml in lysis buffer containing 10 mM Tris pH 7.4; mM NaCl; 1% NP40; 1 mM Na3VO4; 5mM NaF; 5 mM sodium pyrophosphate; 0.4 mM EDTA; 10 µg / ml aprotinin; 10 µg / ml leupeptin; 10 µg / ml pepstatin and 1 mM PMSF. The lysates were centrifuged at 10,000 RPM for 15 minutes at 4 ° C., and the supernatants were incubated for 1 hour at 4 ° C. with protein G-Sepharose conjugated with the anti-c-Kit 2B8 antibody (Pharmingen), or to antibody 2. 4G2. The immunoadsorbents were washed in the lysis buffer and brought to the boil for 3 minutes in the sample buffer. The eluted material was fractionated by SDS electrophoresis and transferred to Immobilon-P membranes. The membranes were saturated with 5% BSA or 5% skim milk (Régilait) diluted in

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 Western buffer. The membranes to which the anti-c-Kit immunoprecipitates were transferred were then incubated either with anti-PY antibodies conjugated to horseradish peroxidase (Chemicon) or with anti-c-Kit rabbit polyclonal antibodies (Upstate Biotechnology Inc .), then with goat anti-rabbit antibodies (GAR) conjugated to horseradish peroxidase. The membranes to which the anti-RFcyIIB immunoprecesses were transferred were incubated either with anti-PY antibodies conjugated to horseradish peroxidase or with anti-RFcylIB, anti-SHP-1 (Transduction Laboratories) or anti-SHIP ( Upstate Biotechnology Inc.), then with anti-rabbit goat antibodies (GAR) or goat anti-mouse Ig conjugated with horseradish peroxidase (Santa Cruz). Antibodies labeled with peroxidase were revealed using the Amersham ECL kit.

 When co-aggregated with c-Kit, the RFcyIIB became phosphorylated, and SHIP, but not at SHP-1, co-precipitated with the phosphorylated RFcylIB. Both phosphorylation of RFcyIIB and co-precipitation of SHIP are prevented when BMMCs have been incubated with 2.4G2 before being stimulated for the biotin ACK2-anti-biotin complexes. The results are illustrated in FIG. 3A which represents the phosphorylation of RFcyIIB, and the recruitment of phosphatases. The RFcyIIBs were immunoprecipitated from BMMC stimulated with biotin ACK2-anti-biotin complexes after incubation for 5 minutes in the presence of 2.4G2 (c-Kit aggregation) or in the absence of 2.4G2 (c-Kit co-aggregation -RFcyIIB) as described above. The immunoprecipitates (Ip: a- RFcylIB) were subjected to electrophoresis and incubated sequentially with antiphosphotyrosine antibodies (a-PY), with anti-RFcylIB antibodies in order to verify that comparable quantities of materials were immunoprecipitated, and with antibodies. anti-SHIP and anti-SHP-1 so as to identify the co-precipitated phosphatases.

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The 4 tracks visible in FIG. 3A correspond to the immunoprecipitated material from cells treated as follows: track 1, reaction medium, track 2, biotin-ACK2-anti-biotin complexes alone, track 3, biotin-ACK2-anti complexes biotin and 2. 4G2, lane 4, whole cell lysates (WCL) served as positive controls for the detection of phosphatases. From top to bottom, this figure 3A shows the phosphorylated proteins (blot: a-PY) the RFcylIB (blot: a-RFcyIIB), SHIP (blot: aSHIP) and SHP-1 (blot: a-SHP-1) .

 The biotin-ACK2-anti-biotin complexes also induced the tyrosylphosphorylation of c-Kit. The phosphorylation is comparable intensity, whether the BMMCs were preincubated with or without 2. 4G2, indicating that the coaggregation of c-Kit with RFcylIB does not prevent its phosphorylation. These results are illustrated in FIG. 3B: was immunoprecipitated from BMMC incubated for 5 minutes with SCF, or with anti-biotin-ACK2-biotin complexes after incubation with 2. 4G2 (aggregation of c-Kit) or without 2. 4G2 (c-Kit-RFcylIB co-aggregation) as described above; the immunoprecipitates were fractionated by electrophoresis and analyzed by immunoblotting with anti-phosphotyrosine antibodies (a-PY), and with anti-c-Kit antibodies in order to verify that the comparable amounts of the material have been immunoprecipitated.

 Figure 3B shows the phosphorylated proteins at the top and c-Kit at the bottom. Lanes correspond to the immunoprecipitated material, namely cells treated as follows: lane 1, SCF, lane 2, medium alone, lane 3, anti-biotin-ACK2-biotin complexes, and 2.4G2; lane 4, biotinACK2-anti-biotin complexes.

 RFcyIIBs can therefore be phosphorylated when they are close to activated c-Kit, which allows them to recruit SHIP. As SHIP is not

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 me protein tyrosine phosphatase this does not affect the phosphorylation of c-Kit, but it can block signals downstream, which lead to a proliferative response.

 Finally, we examined the consequences of cKit aggregation and co-aggregation of c-Kit at RFcyIIB on BMMC proliferation.

BMMCs, preincubated with or without 2.4G2, were cultivated for 4 days in the presence of a limiting concentration of WEHI-3 conditioned medium, allowing the survival of BMMCs, but not their proliferation.

 The BMMCs were incubated in the presence or absence of 10 g / ml of 2.4G2 for 1 hour at 37 ° C. in culture medium supplemented to 2% with WEHI-3B conditioned medium. The cells were seeded at 3 x 105 cells / ml with the preformed immune complexes indicated in 96-well plates in the same medium. After 4 days of culture, the cells excluding Trypan blue were counted under the microscope.

 The biotin ACK2-anti-biotin complexes induce the proliferation of cells whose RFcyIIBs are blocked by 2. 4G2, but do not induce the proliferation of cells whose RFcyIIBs are accessible.

 These results are illustrated in Figure 4.

 BMMCs, preincubated with 10 g / ml of 2.4G2 (c-Kit aggregation, white bars) or without 2.4G2 (RFcylIB-c-Kit co-aggregation, black bars) were seeded at 3 x 105 cells per ml and cultured for 5 days with complexes consisting of 10 g / ml of biotin-ACK2 and the indicated concentrations (from left to right 1 g / ml; 3 g / ml; 10 g / ml) of antibiotic antibody as described above above.

 This figure 4 shows the increase in the concentration of viable cells after 5 days of cultures.

 These results demonstrate that the proliferation induced by c-Kit, growth factor-dependent mast cells can be regulated

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 negatively by IgG antibodies when these antibodies co-aggregate c-Kit with RFcyIIB. This inhibition does not affect the phosphorylation of c-Kit and is correlated with the tyrosylphosphorylation of RFcyIIB (phosphorylation probably carried out by c-Kit) and the subsequent recruitment of SHIP. The RFcylIB downregulation of cKit-induced cell proliferation demonstrated here in an experimental model can be induced in vivo by non-neutralizing anti-SCF or anti-c-Kit autoantibodies. These results call for research into the existence of such antibodies and the role they can play under normal or pathological conditions. The downregulation described here is not significantly restricted to mast cells or c-Kit, and ITIM receptors such as RFcyIIB are capable of controlling any cell proliferation that depends on a growth factor that binds to an RTK. ITIM receptors such as RFcyIIB can therefore control cell proliferation induced by receptors such as PDGF-R (receptor for platelet-derived growth factor), CSF-R (receptor for colony stimulating factor) , EGF-R (epithelial cell growth factor receptor), FGF-R (fibroblast growth factor receptor), NGF-R (nervous system growth factor receptor), insulin receptor.

 It follows that the main effector molecules of the immune system, namely IgG antibodies, can control the proliferation of most cells of the hematopoietic line. Since the SHIP-dependent inhibition exerted by RFcyIIB affects free signals downstream of the receptor phosphorylation, it can also affect the proliferation of transformed growth factor independent cells which carry an oncogenic, constitutively activated form of an RTK . Thus, the anti-RTK or anti-ligand antibodies (IgG in the case of RFcylIB) of such a receptor

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 (anti-growth factor for example) provide potential therapeutic tools to specifically control the proliferation of malignant cells expressing ITIM receptors such as RFcylIB.

EXAMPLE 2:
The functional screening of chemical and / or biological libraries makes it possible to identify other co-aggregating or antagonistic molecules according to the invention. Such screening is carried out according to standard screening techniques. An appropriate screening strategy is as follows: 1. Search for monovalent ligands capable of binding to ITIM immunoreceptors and to RTKs: - Construction of a library of monovalent molecules, - Construction and expression of ITIM immunoreceptors and RTK in cells, - Test for binding of monovalent molecules to transfected cells.

2. Search for homo-divalent ligands capable of aggregating RTKs: - Construction of a bank of homo-divalent molecules, - Construction of activating chimeras and expression in appropriate cells, - Construction and expression of reporter systems in the same cells , - Test and transfer in an automated system for large-scale screening,

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 - Screening of ligands for the extracellular domains of the receptors to be studied.

3. Search for hetero-divalent ligands capable of co-aggregating ITIM receptors and RTKs - Building a library of hetero-divalent molecules from the ligands identified during the second phase, - Building activating and inhibiting chimeras and cotransfection with a reporter system.

 - Screening of heterodimers.

Claims (10)

 CLAIMS 1. Method for the identification of an agent useful for the stimulation of a negative regulation of the activation of an RTK, characterized in that it comprises the detection of at least one capable compound, under conditions suitable for cell physiology, to co-aggregate a receptor with tyrosine kinase (RTK) activity to a receptor comprising at least one ITIM motif, and / or in that it comprises the detection of the constituent entities of such a compound. 2. Method according to claim 1, characterized in that said detection is carried out so as to detect a compound comprising at least one entity having, under said conditions, an affinity for a molecule chosen from the group consisting of said RTK, an extracytoplasmic part of this RTK, an intra-cytoplasmic part of this RTK, a ligand of this RTK or a molecule capable of binding to this ligand. 3. Method according to claim 1 or 2, characterized in that said detection is carried out so as to detect a compound comprising at least the entity having, under said conditions, an affinity for a molecule chosen from the group consisting of said ITIM receptor , an extracytoplasmic part of this receptor, the intracytoplasmic part of this receptor, an ITIM motif of this receptor, a ligand of this receptor or a molecule capable of binding to this ligand. 4. Method according to any one of the preceding claims, characterized in that said detected co-aggregating component is capable, under said conditions, of leading to the inhibition of a cellular response of <Desc / Clms Page number 29>  non-immune nature with which activation of RTK can be associated, such as inhibition of cell proliferation and / or differentiation. 5. Method for the identification of an agent useful for the inhibition of a negative regulation of the activation of an RTK, characterized in that it comprises the detection of at least one compound which is an antagonist of a compound capable, under conditions suitable for cell physiology, of co-aggregating a receptor with tyrosine kinase activity (RTK) to a receptor comprising at least one ITIM motif, and / or in that it comprises the detection of entities constitutive of such an antagonistic compound. 6. Method according to claim 5, characterized in that said detection is carried out so as to detect a compound capable, under said conditions, of blocking the ligand binding site of said RTK, and / or of blocking said ITIM receptor at the level of its ligand binding site or at the level of (or its) motifs) ITIM. 7. Artificial compound, characterized in that it is capable, under conditions suitable for cell physiology, of co-aggregating a receptor with tyrosine kinase (RTK) activity and a receptor comprising at least one ITIM motif, or of s' oppose such co-aggregation. 8. Method for the in vitro diagnosis of an inappropriate negative regulation of the activity of an RTK, characterized in that it comprises: - the detection, in a biological sample, of at least one agent, stimulator or inhibitor of said downregulation, as identified using a method according to any one of claims 1 to 6, and / or at least one co-aggregating or antagonistic artificial compound according to the <Desc / Clms Page number 30>  claim 7, with, if necessary, the measurement of its titer (or their respective titer), and - the comparison of the result obtained for said sample with the results observed on average under physiological conditions. 9. Method for the in vitro diagnosis of an inappropriate negative regulation of the activity of an RTK, characterized in that it comprises: - the detection, in a biological sample, of the level of phosphorylation of receptor to ITIM, and - comparison of the result obtained for said sample with the results observed on average under physiological conditions. 10. Use of an agent identified using a method according to any one of claims 1 to 6, and / or a synthetic compound according to claim 7, for the manufacture of a medicament intended to alleviate , prevent, or treat inappropriate negative regulation (deficient or excessive) of RTK activation.