FR2844452A1 - Modulating dendritic cell activity, e.g. for treatment of viral infections, NK-sensitive tumors or autoimmune diseases, using inhibitors or specific tyrosine kinases, e.g. c-abl, bcr/abl and c-kit - Google Patents

Abstract

The use of inhibitors (I) of the tyrosine kinases c-abl, bcr/abl or c-kit and/or the tyrosine kinase associated with PDGF (platelet-derived growth factor) is claimed in the production of compositions for modulating the activity of dendritic cells in mammals (specifically humans). Independent claims are included for: (a) compositions comprising (I) and a potentiating agent (II); and (b) compositions comprising dendritic cells (specifically immature dendritic cells) treated with (I).

Description

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USE OF SPECIFIC INHIBITORS OF TYROSINE KINASES
FOR IMMUNOMODULATION.

The invention relates to the use of tyrosine kinase inhibitors for immunomodulation, in particular for regulating the activity of immune cells in patients. It relates more particularly to the use of specific inhibitors of tyrosine kinases for the preparation of a composition intended for the prevention or treatment of viral infections, sensitive NK tumors, immune diseases and / or septic shock in a mammal . The inhibitors concerned are more particularly inhibitors of c-abl tyrosine kinases (bcr / abl), c-kit and / or tyrosine kinase associated with the PDGF receptor. The invention also describes compositions comprising such an inhibitor, in combination with other active agents, including agents capable of potentiating the effect of the inhibitor. The invention can be used in a preventive or curative manner, in vivo or ex vivo.

Tyrosine kinases are key enzymes in the positive control of cell multiplication. Frequently, mutations of proto-oncogenes into oncogenes are precisely due to mutations in the kinase domain making the tyrosine kinase activity constitutive and uncontrollable. This is notoriously the case in chronic myeloid leukemia (CML), where the translocation (9; 22) results in the expression of a bcr-abl fusion protein that has a permanent tyrosine kinase activity. It has been clearly demonstrated that this constitutive tyrosine kinase activity is the primum movens of CML.

In gastrointestinal stromal tumors (GIST), tumors originating from the Cajal interstitial cell (CD34 +, c-kit +, desmin-, S-100-), mutations in the proto-oncogene c-kit (21-88 % point mutations or deletions in the juxtamembrane domain -exon 11> exon 9 or 13-) are also responsible for the molecular pathogenesis (Hirota S, Science 279 (1998) 577,

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Miettinen M, Human Pathol 30 (1999) 1213). These mutations result in the constitutive activation of c-kit, independent of the SCF ligand.

Given the role of tyrosine kinases in the control of cell multiplication, and the detection of cancers linked to the presence of a mutation in these proteins, it has been proposed to use specific inhibitors of these enzymes in the cell. treatment of these particular cancers.

In particular, it has been proposed to use specific inhibitors of c-kit or c-abl tyrosine kinases to treat tumors with a mutation in the corresponding genes (CML, GIST).

The best characterized inhibitor is ST1571 (Novartis), also referred to as Gleevec, Glivec or Imatinib Mesylate. STI571 (STI for Signal Transduction Inhibitor) is a selective inhibitory compound of certain tyrosine kinase proteins: PDGF receptor, ABL tyrosine kinases (p210, p185, p190) and c-kit (CD117, SCF receptor) (Buchdunger E, J Pharmacol Exp Ther 295 (2000) 139, Druker BJ, Nat Med 2 (1996) 561, MC, Blood 96 (2000) 925). This compound would act on the mechanisms of signal transduction.

STI571 is indicated for the treatment of Philadelphia chromosome positive Chronic Myeloid Leukemia (CML) in blast crisis, accelerated phase or chronic phase, particularly in patients with intolerance, refractoriness or failure of interferon alfa therapy. In CML, STI571 works by blocking the binding of ATP to the bcr / abl tyrosine kinase, thereby inhibiting its phosphorylation activity of the CRKL main substrate. This inhibitory action is based on competition with ATP at the level of the binding pocket of the protein with the latter.

Phases # and II of ST1571 in GISTs (van Oosterom, Lancet 358 (2001) 1421, Joensuu H, N Engl J. Med 344 (2001) 1052) also showed more than 50% of objective clinical responses, of which some spectacular in their

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 speed of installation. Unlike the CML model, in the GIST, the STI571 molecular action site is unknown.

STI 571 has also demonstrated in vitro inhibitory activity against tyrosine kinases other than those of proteins of the abl family, especially with respect to those of c-Kit and PDGF. Kit is a growth factor receptor of which a ligand is the Stem Cell Factor (SCF) or Steel factor, and has a key role in the proliferation of differentiation of early hematopoietic precursors.

The present application results from the demonstration of particularly remarkable and unexpected immunomodulatory properties of tyrosine kinase inhibitors. The present application shows in particular that, surprisingly, specific inhibitors of tyrosine kinase, such as ST1571, are capable of acting on the activity of dendritic cells, and of modifying their properties. The invention notably shows that such compounds make it possible to modulate the activity of immature dendritic cells in order to induce the activation of NK cells, in vitro and in vivo. The invention also shows that such compounds are also capable of inhibiting LPS-induced dendritic cell maturation and thereby limiting the inflammatory response. These results are totally unexpected in that no immunomodulatory activity for such inhibitors has been considered previously. This activity is also very surprising since it is effective on cells that do not necessarily have a mutation in a receptor associated with a tyrosine kinase. This activity is also surprising in its amplitude, since, as is illustrated in the examples, the in vivo use of such compounds makes it possible to achieve activation of NK cells in 30 to 56% of patients with GIST.

The demonstration of the immunomodulatory properties of these specific inhibitors opens up new therapeutic applications not envisaged until now for these compounds, and also makes it possible to potentiate or

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 optimize their properties. In particular, the new biological properties of these inhibitors make it possible, for the first time, to consider their use to treat multiple NK-sensitive cancers, cancers that are secondarily resistant to ST1571, to treat cancers that do not contain the mutation c -abl, c-kit or PDGF-R, to treat infectious diseases or reduce inflammation. By showing the action of inhibitors on dendritic cells, the invention also makes it possible to strongly potentiate the therapeutic effect of these compounds by combining them, for example, with growth factors of dendritic cells.

The present invention thus provides novel effective approaches in the treatment of cancers, infectious or immune diseases, based on an original and effective modulation of the immune response.

A first object of the invention thus lies in the use of a specific tyrosine kinase inhibitor for modulating the activity of dendritic cells in vivo, ex vivo or in vitro, in particular in the use of a tyrosine kinase inhibitor. c-abl, bcr / abl, c-kit and / or PDGF receptor-associated tyrosine kinase, for the preparation of a composition for modulating the activity of dendritic cells in a mammal.

Another object of the invention is the use of a specific tyrosine kinase inhibitor to stimulate NK cell activity in vivo, in vitro or ex vivo.

Another subject of the invention is the use of a specific inhibitor of tyrosine kinase for the preventive or curative treatment of tumors or viral infections, in particular of sensitive NK tumors.

Another object of the invention is the use of a specific tyrosine kinase inhibitor to increase NK activity in patients with cancer or viral infection.

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 Another subject of the invention is the use of a specific inhibitor of tyrosine kinase to reduce the inflammatory response, particularly in subjects suffering from chronic inflammatory diseases or autoimmune diseases.

Another subject of the invention is the use of a specific tyrosine kinase inhibitor to promote GVL during allogeneic transplants.

The invention also relates to methods for the preventive or curative treatment of cancerous, infectious, immune or inflammatory pathologies, comprising the administration to a patient of (i) a specific tyrosine kinase inhibitor, alone or in combination with one or more other active ingredients, or (ii) dendritic cells (preferably autologous) treated with such a compound.

The invention also relates to a method for modulating dendritic cell activity in a patient comprising administering to said patient an effective amount of a specific tyrosine kinase inhibitor.

The invention also relates to a method for stimulating NK cell activity in a patient comprising administering to said patient an effective amount of a specific tyrosine kinase inhibitor or dendritic cells treated with such an inhibitor.

The tyrosine kinase inhibitor may be used alone or, preferably, combined with a potentiating agent. In this regard, the invention also relates to a composition comprising a specific inhibitor of tyrosine kinases and a potentiating agent, preferably selected from a dendritic cell growth factor and a factor (co-) stimulation of NK cells.

It also relates to a composition comprising dendritic cells, in particular immature, treated with a specific inhibitor of tyrosine kinase.

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 It also relates to a therapeutic combination between a specific inhibitor of tyrosine kinases and a potentiating agent, preferably selected from a dendritic cell growth factor and a (co-) stimulation factor of NK cells, for separate use, simultaneous or spaced in time.

It also relates to a kit comprising a specific tyrosine kinase inhibitor and an agent stimulating the production of dendritic cells.

The invention can be used in the fields of immunology, immunotherapy or medical biotechnology. As indicated below, the use of these inhibitors as immunomodulatory agents is varied. They find applications both in vitro, ex vivo and in vivo, to control the activity of dendritic cells or NK cells, to regulate the immune or inflammatory response in different pathological contexts. The invention is usable in all mammals, in particular in humans, even if an animal use is also possible. The term treatment refers to preventive or curative treatment.

Specific tyrosine kinase inhibitors The present application therefore relates to the use of specific tyrosine kinase inhibitors as immunomodulators. For the purposes of the invention, the term "specific tyrosine kinase inhibitors" means compounds capable of preferentially inhibiting the activity of c-abl, bcr / abl, c-kit tyrosine kinases and / or tyrosine kinase associated with the PDGF.

The inhibition can be partial or total. They may be compounds capable of inhibiting the activity of the mutated form of the tyrosine kinases identified above, and in particular the constitutively active mutant form, or of inhibiting the non-mutated or overexpressed form in cells. normal no

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 transformed as dendritic cells. The compounds are preferably without substantial direct effect on other tyrosine kinases.

The preferred inhibiting compounds of the invention are chemical molecules of the pyrimidine family, such as the compounds described in Applications Nos. W001 / 64200, WO99 / 03854, EP 0564409 and US Pat. No. 6,258,812. It may also be pyrido [2-3a] -pyrimidine compounds described by Wisniewski D. et al. (Cancer Res 62 (2002) 4244).

A preferred inhibitor is a compound of the 2-phenylaminopyrimidine family, such as ST1571 or any derivative or analog thereof (an exemplary derivative is AB1003). STI571 is a specific inhibitor of the aforementioned protein tyrosine kinases, with no substantial effect on the EGF receptor, FLT1 and FLT3. The half-life of ST1571 is 16 hours in humans, and this compound is metabolized by cytochrome P450 (CYP3A4, CYP2D6). This compound is generally well tolerated by humans.

Other specific tyrosine kinase inhibitors may be used within the scope of the invention, although those having a selective activity for c-abl, bcr / abl, c-kit and / or receptor-associated tyrosine kinases are preferred. PDGF. Similarly, inhibitors can be identified or selected in vitro or in cell assays by measuring the binding or activity of test compounds on isolated tyrosine kinase or a cell containing it.

Activation of NK Cells - Production of DAK (DC-Activated Killer) Cells and Therapeutic Effects A particular object of the invention resides in the use of a specific tyrosine kinase inhibitor for the preparation of a composition intended to activate cells. NK of a subject. The invention shows that such

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 Compounds are able to increase NK function in patients, in vivo, leading to a powerful anti-tumor or anti-infectious action. The invention shows that this increase is related to a modulation of dendritic cells, giving them the ability to activate NK cells.

Natural killer cells (NK cells) are a population of lymphocytes that represent a very early line of defense against viruses and tumor cells. NK cells can be characterized by the presence of CD56 and CD16 markers, and by the absence of the CD3 marker.

NK cells have in particular been implicated in the non-specific anti-tumor immunity of antigens, for the prevention of establishment of primary or metastatic tumors in the immunocompetent or immunosuppressed host.

NK cells in particular appear to have a key role against tumor cells or class # negative MHC variants. The role of NK cells in anti-tumor immunosurveillance has been extensively documented in mice (Diefenbach, Nature 413 (2001) 165) and in humans (A. Velardi, Science 2002). The role of NK cells in the prevention of viral infections is also well documented, particularly for viruses of the Herpes Viridae group (C. Biron, N.Eng.J.Med.320 (1989) 1731; Welsch et al., J. Med Exp.

 2001).

Because of their non-specific antigen-specific cytotoxic properties and their efficacy, NK cells therefore constitute an effector cell population of particular interest for the development of adoptive immunotherapy approaches to cancers or infectious diseases. In this respect, in certain indications, such as patients with intensified malignant lymphoma, the administration of NK cells associated with low doses of IL-2 has given promising results in an adjuvant situation. NK cells have also been used for the experimental treatment of different types of tumors and some clinical studies have been initiated (Kuppen et al., Int.

Cancer. 56 (1994) 574; Lister et al., Clin. Cancer Res. 1 (1995) 607; Rosenberg et al., N. Engl. J. Med. 316 (1987) 889). It has also been shown

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 that NK cells promote GVL in allogeneic marrow transplants.

In addition, these cells can also be used in vitro for nonspecific lysis of cells not expressing class-1 MHC molecules and, more generally, any NK-sensitive cell.

The possibility of activating in vivo the NK cells of a patient thus offers multiple benefits and therapeutic applications.

The NK cell activation methodologies described in the prior art are essentially dependent on the use of cytokines. NK cells can be activated by cytokines in vitro or in systemic administration in vivo (IL-2, IL-12, IFN type 1 or II, IL-15 in combination or not with IL-1b, IL-18, IL-21 or chemokines). However, the physiopathological role of these cytokines for NK activation in vivo has never been demonstrated. In addition, the clinical application of cytokines is limited, in particular because of the preparation costs of the latter, the toxic nature of many of them, which can not be used in in vivo applications, or the non-specific nature. many cytokines, whose use in vivo may be accompanied by many adverse effects. In addition, since the natural killing function is often impaired in tumor patients, the ability to harvest these cells for ex vivo activation can be significantly reduced.

The inventors have previously demonstrated that dendritic cells (DCs) are cells electively capable of activating NK cells in vitro and in vivo, leading to antitumor effects (WO99 / 45102, Fernandez et al., Eur.

Cyt. Net. 13 (2002) 17-27; Zitvogel L. JEM 195 (2002) F9-14).

The inventors have now discovered that, surprisingly, tyrosine kinase specific inhibitors, such as ST1571, induce in vivo or in vitro a phenotypic modification of dendritic cells which confers on them the power to activate NK cells. In this context, the inventors

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 in particular demonstrated that the administration to a mammal of a specific tyrosine kinase inhibitor induces a very pronounced increase in the function and activity of NK cells. They have also observed that STI571-type tyrosine kinase inhibitors are able to activate NK functions in patients with GIST. Thus, in GIST patients treated with ST1571, NK cells are activated in 30 to 56% of patients and these biological data for NK activation seem to correlate with the clinical responses obtained under STI571 (with, in particular, no primary STI571 resistance). in biological responders). These results thus demonstrate, in vivo, in humans, that tyrosine kinase inhibitors, such as STI571, exhibit immunomodulatory activities in addition to their inhibitory functions tyrosine kinase activities already described.

These results therefore make it possible to broaden the therapeutic applications of these molecules and to optimize their effects.

The present invention thus relates to the use of a specific inhibitor of tyrosine kinase for the preparation of a composition intended to increase the NK activity in patients suffering from cancer or infection (viral, bacterial or parasitic).

A particular object of the invention resides in the use of a specific c-abl, bcr / abl, c-kit and / or PDGF receptor-associated tyrosine kinase inhibitor for the preparation of a composition intended for prevention. or the treatment of infections, especially viral infections.

Another particular object of the invention resides in the use of a specific c-abl, bcr / abl, c-kit and / or PDGF receptor-associated tyrosine kinase inhibitor for the preparation of a composition intended for the prevention or treatment of NK sensitive tumors unrelated to a constitutive mutation in a tyrosine kinase, or resistant to STI571.

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 It also relates to the use of a specific tyrosine kinase inhibitor for the preparation of a composition intended to induce in a mammal a phenotypic modification of the dendritic cells and to allow or increase the activation of the NK cells.

It also relates to a method for inducing a phenotypic modification of dendritic cells and / or for activating NK cells, comprising bringing into contact in vitro, in vivo or ex vivo dendritic cells, alone or co-cultured with NK cells, with a c-abl tyrosine kinase inhibitor, bcr / abl, c-kit and / or associated with the PDGF receptor.

The invention also relates to a method for the preventive or curative treatment of a patient suffering from a cancer or a viral infection, comprising the administration of a quantity of tyrosine kinase specific inhibitor effective to activate the NK function in the patient.

The invention further relates to a method for activating NK cells in a subject, comprising administering to a c-abl tyrosine kinase inhibitor, bcr / abl, c-kit and / or tyrosine kinase associated with a PDGF receptor.

The subject is preferably a human being but it can also be an animal.

The invention can also be used to increase the survival of NK lymphocyte populations, in vitro, ex vivo or in vivo, as well as to increase, if necessary, the proliferation of lymphocyte subpopulation (s) NK.

For the implementation of the invention, the tyrosine kinase specific inhibitor may be used alone. Nevertheless, in a preferred and particularly effective variant, the inhibitor is used in combination with another active agent. This active agent may be of various kinds, but it is preferably an agent potentiating the action of the inhibitor, such as in particular a dendritic cell growth factor or a (co) stimulation factor.

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 NK cells. Indeed, the invention shows that specific tyrosine kinase inhibitors can make it possible to stimulate NK function in vivo in vivo, and that this stimulation involves a phenotypic modification of the dendritic cells. By combining several active agents on the production, on the modification of the dendritic cells and / or on the activation of the NK, a more powerful biological effect can thus be obtained. In particular, by combining a dendritic cell growth factor and an inhibitor according to the invention, it is possible to produce a sufficient stimulation of NK functions to obtain a therapeutic effect, even with respect to pathological cells resistant to action of the inhibitor alone.

In a preferred variant of the invention, an inhibitor as defined above is therefore used in combination with a potentiating agent. The term combination indicates that both agents can be used simultaneously or spaced apart over time, as their biological effects are combined in vivo. The agents can therefore be packaged simultaneously or separately and administered concomitantly or staggered over time.

In a first embodiment, the potentiating agent is capable of amplifying the dendritic cells, that is to say of increasing their differentiation, growth or activity. It is preferentially a dendritic cell growth factor, advantageously chosen from Flt3Ligand (Flt3L) described by Lyman S.D. et al. (Blood 83 (1994) 2795-2801) and Maraskovsky E. et al. (J. Exp Med 184 (1996) 1953-1962), GM-CSF, interleukin-4, interleukin-13 and their derivatives or analogues. This may include progenipoietin (ProGP4, fusion molecule between G-CSF and Flt3L), GM-CSF / IL-4 fusion, etc. This agent can be used simultaneously or staggered over time with respect to the tyrosine kinase inhibitor.

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 In another embodiment, the potentiating agent is a costimulatory factor for NK cells, and in particular any lymphokine or cytokine capable of activating or promoting the activation of NK cells. For example, IL-2, IL-12, IL-15, interferon type I, etc. may be mentioned.

In a particular embodiment, an inhibitor as defined above is used in combination with a dendritic cell growth factor and / or an NK cell costimulatory factor. A particularly preferred combination comprises the inhibitor and Flt3L (or a variant or analog thereof).

In this regard, another subject of the invention also resides in a composition comprising a specific tyrosine kinase inhibitor as defined above and a potentiating agent, preferably chosen from a dendritic cell growth factor and a co-factor. -stimulation of NK cells. The potentiating agent is specifically selected from cytokines or lymphokines activating NK cells or from Fit 3L, GMCSF, IL-4, IL-13 and their derivatives or analogues (ProGP4, GM / IL-13). 4).

The compositions according to the invention may, of course, also comprise any pharmaceutically acceptable carrier or excipient, such as isotonic solutes, buffered saline solutions, stabilizing agents, and the like.

The applications of the compositions and methods of the invention are numerous, because of the broad spectrum of action of NK cells. They preferentially include the prevention or the treatment of various pathologies such as cancers or infectious diseases, in particular of viral origin or resulting from other pathogens, and NK or related congenital immunodeficiencies (immune deficiency leading to a mutation in the IFNy gene). -R, IL-12R ...).

For applications in the field of cancer treatment, the compositions or methods according to the invention can in particular be used for

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 delay or suppress the growth of tumors, induce tumor size regression, slow down or prevent the formation of metastases, etc., including tumors weakly expressing MHC class 1 molecules or possessing ligands for NK activating receptors or other pathological cells. They also prevent the establishment of primary or metastatic tumors in an immunocompetent or immunosuppressed mammal for T lymphocyte functions.

The invention can be used to prevent or treat any sensitive NK tumor, including those selected from carcinomas, melanomas, leukemias, lymphomas and sarcomas. The invention is particularly suitable for the preventive or curative treatment of melanoma, kidney cancer, gastrointestinal sarcoma, chronic myeloid leukemia (CML) and neuroblastoma.

It is more particularly the preventive treatment of cancers that do not have a mutation in a tyrosine kinase, or that have a non-constitutive mutation, or tumors that are resistant in vitro to STI571. In the case of sensitive NK tumors with a mutation in a tyrosine kinase, the invention makes it possible to improve the therapeutic efficacy by using a combination therapy with a potentiating agent, as mentioned above.

In this respect, the invention specifically relates to the use of a tyrosine kinase inhibitor as defined above, in combination with a potentiating agent, for the preparation of a composition intended for the preventive or curative treatment of CML or of GIST. It also relates to a corresponding method of treatment.

The invention also relates to the use of a tyrosine kinase inhibitor as defined above, in combination with a potentiating agent, for the preparation of a composition intended for the preventive or curative treatment of a

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 STI571 resistant tumor. It also relates to a corresponding method of treatment.

In the field of infectious diseases, the invention can be used to prevent or treat in particular a viral infection caused by a virus of the Herpes Viridae group () Herpes virus type 1 or 2, the Epstein-Barr virus, the cytomegalovirus, Kaposi sarcoma-virus, HSV-6, etc.), HIV, Vaccinia virus, MVM, ECMV or Papilloma Virus. It can also be used to treat microbial or parasitic infections.

The invention can also be implemented using a population of dendritic cells treated ex vivo with a tyrosine kinase inhibitor as defined above. The injection of dendritic cells thus treated, preferably autologous, also makes it possible to induce an activation of the NK function of a subject.

In this context, the invention also relates to a composition comprising dendritic cells treated with a tyrosine kinase inhibitor as mentioned above. It also relates to a composition comprising a tyrosine kinase inhibitor as defined above and dendritic cells and / or NK cells. According to a particular aspect of the invention, the composition comprises a tyrosine kinase inhibitor as defined above and dendritic cells.

In another aspect, the composition comprises a tyrosine kinase inhibitor as defined above, dendritic cells and NK cells.

It also relates to a process for the preparation or in vitro or ex vivo treatment of dendritic cells, comprising contacting dendritic cells (or their precursors) in the presence of a specific tyrosine kinase inhibitor. The cells obtained can be collected and packaged for immediate or future use.

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The dendritic cells are typically treated by incubation in the presence of the inhibitor, for example in the presence of STI571, at about 10-6 to 10-8 molar. It is preferred to use non-mature dendritic cells. Preferably, CD34 + human dendritic cells are placed in the presence of SCF, Flt3L, GM-CSF and TNFα for about 7 to 15 days (preferably 12 days), then in the presence of STI571 for about 12 to 48 hours (preferably Around 24h). It is understood that the precise conditions can be adapted by those skilled in the art. The cells thus obtained have the ability to activate NK cells, and can be packaged for their therapeutic use, or used to activate NK cells in vitro or ex vivo, in particular by co-culture. In this context, optimal activation of NK cells is observed, for example, after co-cultivation for a period of between approximately 20 to 48 hours. The co-culture ratio is ideally a dendritic cell for 25 to 125 NK cells approximately.

For use in vitro or ex vivo, the invention can be implemented in any device suitable for cell culture, preferably under sterile conditions. This may include plates, culture dishes, flasks, vials, bags, etc. The culture or co-culture is also carried out in any medium suitable for culturing dendritic cells and / or NK cells. It may be more generally commercially available culture media for culturing mammalian cells, such as, for example, RPMI medium, DMEM medium, IMDM medium or GBEA mediums (AIMV, X-VIVO), etc.

In a particular variant, the activation of the NK cells is carried out by in vitro or ex vivo co-culture of immature dendritic cells with NK cells, in the presence of a c-abl tyrosine kinase inhibitor, bcr / abl, c-kit and / or tyrosine kinase associated with the PDGF receptor.

In another particular embodiment of the invention, the activation of NK cells is carried out by in vitro or ex vivo treatment of dendritic cells.

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 immatures in the presence of an inhibitor of c-abl tyrosine kinases, bcr / abl, ckit and / or tyrosine kinase associated with the PDGF receptor, and then co-culture of the dendritic cells thus obtained with NK cells.

In another embodiment of the invention, the activation of the NK cells is carried out by in vitro or ex vivo treatment of immature dendritic cells in the presence of a c-abl, bcr / abl, c-kit and tyrosine kinase inhibitor. / or tyrosine kinase associated with the PDGF receptor, then administration of the dendritic cells thus obtained to a subject.

When NK cells or dendritic cells have thus been activated, it is possible either to separate the NK cells from the dendritic cells or to directly harvest the NK / CD cells co-culture.

The term activation of NK cells within the meaning of the invention more particularly refers to the early upregulation of CD69, especially on the CD56bright population of NK (the one that expands in patients with GIST treated with STI571), the increase in production of IFNγ and / or cytotoxic activity of NK cells. These three parameters can easily be measured according to the techniques known to those skilled in the art. For the purposes of the invention, the term "activated NK cells" refers to NK cells having at least one of the properties mentioned above. It is also possible to detect NK activation by the ability of these cells to release prolonged high levels of IFNy in the presence of "immature CDs" and, optionally, in the presence of LPS.

In the compositions according to the invention, which comprise dendritic cells and / or NK cells, the cell populations are preferably autologous, that is to say derived from the same organism. They may also consist of allogeneic cells. In addition, it may also be dendritic cells sensitized with one or more antigens or on the contrary with naive cells. These compositions are

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 advantageously consist of isolated cell populations, that is to say that each of the cell populations is composed of at least 10%, preferably at least 30%, especially at least 50% of the corresponding cell type (NK or dendritic). These compositions can be packaged in any suitable device such as bags, vials, ampoules, syringes, vials, etc., and can be stored (cold) or used extemporaneously, as described below. Advantageously, these compositions comprise from 104 to 109 treated dendritic cells, preferably from 105 to approximately 108.

The administration of the inhibitor or the cells (dendritic or NK) can be carried out by any known technique and pathway, and especially by injection or oral administration. It may be for example a subcutaneous injection, systemic (intravenous, intra-arterial, intraperitoneal) or intramuscular. Where possible, the inhibitor is advantageously administered orally.

The injection is preferably a local or regional injection, especially an injection at or near the site to be treated, particularly close to a tumor.

Injections are generally carried out on the basis of cell doses ranging from 104 to 109 dendritic cells, preferably between 105 and 107 inclusive. It is possible to carry out the passive transfer of dendritic cells by repeated administrations, for example 1 to 2 administrations per week for several months. The inhibitory compound may be administered in various doses, for example between 200 and 800 mg per os / day, preferably between 400 mg and 600 mg per os / day, especially when combined with a potentiating agent (for example Flt3L sc 10-50 μg / kg for 10-20 days). The use of the inhibitor in vitro or ex vivo on dendritic cells before passive transfer is carried out preferentially at the dose of 10-6M or 10-8M. Repeated administrations are of course possible. In addition, the administration protocol can be adapted by the skilled person according to the situations (preventive, curative, isolated tumors, metastases, important or localized infection, etc.).

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 The Applicants have also discovered that specific inhibitors of tyrosine kinase, as defined above, make it possible to block dendritic cell maturation induced by a microbial agent such as LPS or by a Toll-like signal 4.

This immunomodulatory property of the inhibitors makes it possible to envisage a negative modulation of the inflammatory response or of the immune response in different pathological contexts. Indeed, the maturation of dendritic cells actively participates in the development of the inflammatory reaction, and the possibility of controlling this property thus offers new therapeutic approaches in the treatment or control of inflammation.

The invention therefore relates to a method for preventing or inhibiting the maturation of dendritic cells, in particular that induced by a toxin such as LPS, comprising bringing in vitro, ex vivo or in vivo contact with dendritic cells with a tyrosine kinase inhibitor. -abl, bcr / abl, c-kit and / or tyrosine kinase associated with the PDGF receptor.

The invention also relates to the use of a specific inhibitor of tyrosine kinase as defined above for the preventive or curative treatment of immune and / or inflammatory diseases in a mammal.

The invention also relates to the use of a specific inhibitor of tyrosine kinase as defined above for the preparation of a composition intended for the treatment of inflammatory pathologies, in particular chronic pathologies, such as atherosclerosis, Crohn's disease, asthma (chronic), rheumatoid arthritis, dermatitis (chronic), septic or endotoxinic shock, etc.

In this context, the term treatment also includes a reduction of the

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 inflammatory response, even partial and / or temporary, leading to an improvement in the quality of life of patients.

The invention also relates to the use of a specific inhibitor of tyrosine kinase as defined above for the preparation of a composition intended for the treatment of autoimmune pathologies.

The invention also relates to the use of a specific inhibitor of tyrosine kinase as defined above for the preparation of a composition intended for allogeneic transplantation. The invention is particularly suitable for the improvement of allogeneic marrow grafts. Indeed, by stimulating the NK activity, the invention makes it possible to improve the GVL (graft versus Leukemia) activity, and therefore the therapeutic benefit. In addition, by reducing the maturation of dendritic cells and thus, potentially, the activation of alloreactive cytotoxic T lymphocytes, the inhibitor also makes it possible to reduce GVHD (Graft Versus Host Disease) and thus to improve the safety of the treatment. . The invention can therefore be advantageously used to treat a host receiving an allogeneic, non-compatible HLA bone marrow graft.

The invention can also be used to prevent or treat congenital immune diseases, such as IL-12 receptor deficiency, IFNy receptor deficiency, common gamma-chain (IL-2Ry) deficiency cytokine receptors (SCID syndrome).

For these different applications, the doses administered and the modes of administration described above are directly applicable.

Other advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.

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 LEGEND OF FIGURES Table 1: Biological correlation between STI571 and activation of NK.

We studied a cohort of patients treated at the Gustave Roussy Institute for the treatment of a GIST. Patients' NKs were purified by negative selection from peripheral blood. The functions of NK cells (interferon gamma secretion and cytotoxicity) were analyzed after culturing with allogeneic dendritic cells. In 14 cases, samples are taken before the introduction of STI 571. In 31 cases, samples are taken during treatment with STI 571. A cohort of 17 healthy volunteers is studied in the same way.

The cytotoxic functions are reported as percentage specific lysis of a target sensitive to NK lysis (K562). The results are reported as an average for each group studied.

Data analysis is performed using Fisher's exact tests that highlight statistically significant differences between groups.

Table 2: Correlation between biological and clinical effects of STI571.

NK cells activated by mature dendritic cells (presence of LPS) have the ability to secrete interferon gamma. We defined this gamma interferon production as significant if more than 100 μg / mL could be measured in the supernatants of cocultures. Patients meeting this criterion are defined as biological responders. A correlation is established between these patients and the clinical efficacy of STI 571. The distribution of patients whose disease progresses under STI 571 is studied according to the biological response.

No patient identified as a biological responder presented with progressive disease under STI 571, while 5 of the 16 non-responders biologically progressed clinically (p = 0.042, Fisher exact test).

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Figure 1: Longitudinal follow-up of seven GIST patients under STI571. Acquisition of NK functions.

For 7 patients, we have samples and measurements of interferon gamma production. Figure 1 reports the study of NK functions before and during treatment with STI 571. For two patients, we showed a clear increase of CD56bright NK cells (panel A). For the seven patients followed, interferon gamma secretions are increased upon exposure to STI 571 (panel B).

Figure 2. Dendritic cells incubated in the presence of STI 571 are able to activate NK cells.

Human dendritic cells are generated from hematopoietic stem cells in the presence of GM-CSF and TNFα for 12 days. These dendritic cells are then incubated with STI 571 for 24 hours. We report two representative experiments conducted on two different samples (A and B). The cytotoxic functions NK are studied by lysis tests using 51 chromium labeled targets and sensitive to NK lysis (K562). In both cases, STI 571 gives dendritic cells the ability to activate NK cells.

Figure 3: Dendritic cells incubated in the presence of STI571 are able to activate NK cells.

STI 571 gives dendritic cells the ability to activate NK cells. Dendritic cells of murine origin, derived from bone marrow or spleen are pre-incubated with STI 571 in increasing doses and then cultured with NK cells. The use of STI 571 makes it possible to increase the secretion of interferon gamma by NKs cultured in the presence of dendritic cells. In addition, there is a dose effect between doses of STI 571 used

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 and the observed biological effect. The previous experiment is repeated in the presence of a non-specific phosphorylation inhibitor (AG 957) and STI 571. This experiment reveals that the previous results are specific to STI 571.

Figure 4. The STI analogue, AB1003, increases the ability of dendritic cells to activate NK cells.

Previous experiments are reproduced using a synthetic analog of STI 571, AB1003. This molecule also confers on the dendritic cells an activating function of the NK cells.

Figure 5: In vivo activation of NK by injection of dendritic cells incubated in the presence of STI571.

Murine dendritic cells are preincubated with STI571 and then injected subcutaneously into the lower limbs of the mice. The draining lymph nodes are collected and the functions of the NK cells analyzed after purification. The use of dendritic cells pre-incubated with STI 571 allows the in vivo activation of NK in the draining ganglion as evidenced by the presence of CD69 on the membrane of the NKs studied.

Figure 6: In vivo activation of NK by injection of STI571 and a dendritic cell growth factor.

Nude mice are treated for 10 days by intraperitoneal injections of Fit3L. This allows the recruitment and differentiation of dendritic precursors. STI 571 is administered by gavage (per os), from D8 to D12. The spleens and livers of the mice are then removed for purification of NK cells. The functional tests carried out show activation of the NK cells in the group treated with the Flt3L + STI combination, in contrast to the control groups (mice treated with IP PBS, IL2 IP, IP PBS + STI gavages).

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571, Flt3-L IP + PBS feeds). Panels A and B present flow cytometric analysis of the presence of CD69 (activation membrane marker) on the surface of NK cells (DX5 +) in the spleen and liver of mice. Panels C and D show the ability of these NK cells, purified from the spleens of mice in each group, to secrete interferon gamma after a culture phase with dendritic cells.

Panel E represents the study of the cytotoxic functions of the NK cells of these same groups, against a sensitive NK target (YAC1) and according to two effector / target ratios (200/1 and 50/1).

Figure 7: STI571 blocks DC maturation by LPS (Fig. 7A) and prevents endotoxin shock in vivo (Fig. 7B).

Dendritic cells are generated from bone marrow of C57BL6 mice. These dendritic cells of medullary origin are then tested for their ability to induce proliferation of allogeneic lymphocytes (panel A).

Dendritic cells cultured with GM-CSF and IL4 (BM-DC), activated by LPS (BM-DC + LPS) or by STI 571 (BM-DC STI) or by a non-specific phosphorylation inhibitor (BM-DC AG), are cultured in the presence of allogeneic lymphocytes for 5 days. The proliferation of lymphocytes is evaluated by their ability to integrate tritiated thymidine.

STI does not stimulate the proliferation of allogeneic lymphocytes. Its immunomodulatory effect is specific to the NK lymphocyte population.

This ability to inhibit inflammatory signals such as LPS is also tested in the endotoxin shock model. C57BL6 mice are treated by oral ingestion of STI 571 morning and evening (150 mg / kg). The control groups are treated in the same way, by ingestion of PBS. After 24 hours of treatment, the mice are administered 1 mg of LPS intraperitoneally. The gavages of the mice with STI 571 or PBS are continued for 48 hours.

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Materials and Methods: Purification of NK Cells Circulating peripheral lymphocytes (PBL) are contained in the non-adherent fraction of peripheral blood mononuclear cells after two hours of adherence. NK cells were isolated from PBL by magnetic negative selection (MACS, NK cell isolation kit, Miltenyi biotec) using anti-CD3, anti-CD19, anti-CD14, anti-CD36, anti-IgE antibodies coupled to a hapten, then secondary labeling with an anti-hapten antibody coupled to a microbead. The negative fraction was obtained after passing through a MidiMACS magnetic column of the LS + NS + type (Miltenyi biotec).

The purity of the fraction thus depleted is evaluated by flow cytometry. Phenotyping after NK purification includes anti CD45, anti CD3, anti CD56 antibodies. The purity of the NK CD45 + / CD3- / CD56 + thus selected varies from 85 to 95%.

DC / NK co-cultures The method used was initially described by Fernandez et al [18].

The NK purified by negative selection are placed in culture in 96-well plates (105 NK / 100 L / well), melting U for 48 hours, according to 4 conditions: - alone in RPMI A 'medium [Gibco, 10% AB serum , 1% 100mM sodium pyruvate (Gibco-BRL, France), 0.5% penicillin-streptomycin
10000Ul / mL-10000g / mL (Gibco-BRL, France) and 1% L-glutamine
200mM (100X, Gibco-BRL, France)]. only in RPMI A 'medium containing 1000 IU / ml of interleukin 2 in coculture with dendritic cells (DC / NK coculture ratio of 1/1, 1/10, 1/25 or 1/125 depending).

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Evaluation of NK activation and functions: The NK functions (cytotoxicity, IFNy secretion, upregulation of CD69) can be evaluated directly from fresh blood after purification on columns, or preferably after reactivity with standardized dendritic cells. allogeneic in the presence or absence of LPS 10 g / ml.

Cytotoxic Functions NK cells are used as effectors in a cytotoxicity assay against Daudi or K562. The target cells are labeled with 51 chromium (100 Ci / 10 cells) for 1 hour, then washed twice. They were then divided into 2000 cells per well in 96-well V-bottom plates. The maximum release of chromium 51 (Cr5i) is obtained by the addition of cetrimide (Sigma). The effector cells are distributed according to different effector / target ratios and maintained at 37 ° C. for 4 hours. The supernatants are removed. The cytotoxicity is evaluated by the specific prolonged release of chromium 51 in the supernatants (reading by a gamma radiation counter: Topcount, Packard): specific release of Cr51 = [Cr51 release of the spontaneous Cr51 sample-release by the targets ] / [maximum release of Cr51-spontaneous release of targets].

Secretion of cytokines Some activated NK cells secrete interferon γ (IFNy). Similarly, immature allogenic dendritic cells (standardized batches), placed in the presence of previously activated NK cells (by a classical cause (IL-2, IL-12, mature DC) or not (GIST under STI571)) secrete ng of TNFa. In order to assay these cytokines, the supernatants of the DC / NK cocultures are analyzed by assay ELISA (Enzyme Linked Immunoabsorbent Assay Kit OptEIA IFNy and Human TNFα-Pharmingen). Anti-IFNγ and anti-TNFα antibodies are then

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 incubated overnight at 4 C on a 96-well maxisorb plate. After washing and saturation of the antibodies with a solution of Assay dilute, the samples are incubated for 2 hours. The plate is then washed in PBS / Tween 20.0.05%. A detection antibody directed against a second epitope of IFNγ or TNFα, coupled to biotin, makes it possible to detect these cytokines. The revelation is carried out by a streptavidin biotin reaction. The reading of the optical density is carried out at 450 nm. A correction is made at 550 nm. A range of standardized IFNγ and TNFα allows the conversion of optical density to pg / mL.

Phenotypic study of activation markers When NK cells are activated, they can express on their surface the molecule CD69 (lectin type C). In order to evaluate the presence of this activation marker, 5x104 to 1x105 NK cells are labeled for flow cytometry analysis. The antibodies used are anti-CD56, anti-CD3, anti CD69. Isotype controls are performed for each manipulation.

Demonstration of elective amplification of in vivo NK subpopulations in treated GISTs After purification of peripheral NK of patients, immunostaining is performed with anti-CD3, CD56 and CD16 antibodies. The increase in the CD16dim, CD56bright subpopulation is highlighted, rising from 5% (pre-STI) to possibly 18% under STI571.

Example 1: Demonstration of activation of NK cells in STI571 treated GIST patients.

Blood and tumors were obtained from the CCPPRB in phase II GIST patients placed under STI 571.

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The function of freshly extracted NK cells by negative depletion (Miltenyi method) of the circulating blood of treated GIST patients (n = 31) or not (n = 14) by STI571 (400 or 600 mg / day since 2-4 or 6 days) months) or healthy VS volunteers (n = 17) was evaluated. The NK cells were incubated with standard allogeneic immature dendritic cells in the presence or absence of 10 g / ml LPS. After 48 hrs, a lysis test of K562, DAUDI or immature DCs at various effector / target ratios as well as an IFNγ and TNFα assay in coculture supernatants (according to methods similar to those suggested in Gerosa et al. (JEM 195 (2002) 327) or Piccioli et al (JEM 195 (2002) 335).

Interestingly, in 30-55% of cases, the NK cells of STI571 GIST patients have the ability to produce IFNy and to trigger the activation of immature DCs (secretion of TNFα ng / ml) in vitro. NK cells of healthy volunteers (VS) or untreated GIST patients (Table 1, Fig. 1). Such NK activations after coculture with DCs are also reflected in lytic capacity. The longitudinal study on 7 patients also clearly shows the acquisition of NK activation after administration of ST1571 (Fig. 1). The correlation between the immunomodulatory biologic effect and the clinical effect of ST1571 is shown in Table 2, which demonstrates that all patients responding to treatment show activated NK cells.

In a preliminary series involving some forty cases, the inventors have thus observed that the STI571 activates the NK functions in the presence of GIST and therefore has an immunomodulatory activity in addition to its inhibitory functions tyrosine kinase activities.

EXAMPLE 2 Demonstration of an Action of STI571 on Dendritic Cells, Which May Reflect Activation of NK Cells

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Since dendritic cells are, in contrast to macrophages, myeloid or lymphoid precursors that are able to activate NK at rest, we tested the capacities of STI571, administered at a rate of 10-6 M, 10-8 M and 10- 10 M, to modulate this property.

Human dendritic cells derived from CD34 + in SCF, GM-CSF, TNFα that do not activate human NK, strongly acquire this capacity when they are incubated for 24 hours from D12 to D13 with STI571 at 10-6M (Fig 2). .

In a murine system, IFNy secretion by NK is increased 2-3 fold at a co-culture CD: NK ratio of 1: 1. On the other hand, whereas at a CD: NK co-culture ratio of 1: 10, the untreated CDs no longer activate the NKs, CDSTI571 still strongly possesses this property. In addition, only ST1571 and not AG957 (Tyrphostin, a non-specific tyrosine kinase inhibitor) induces NK activation via in vitro DCs (Fig. 3). These data can be reproduced using a structural analogue of STI 171, AB1003 (Fig. 4).

Thus, dendritic cells or precursors, human or murine, are the potential target of STI571 in vivo in GIST patients under ST1571. Indeed, the applicants have demonstrated that STI571 does not activate NK directly in vitro.

Example 3 In Vivo Injection of Dendritic Cells Treated with STI571 Induces Activation of NK Cells In vivo CD administration can modulate the activation of NK cells of lymphoid organs (draining lymph nodes if injected subcutaneously, spleen if injected intravenously). Incubation for 24 hours of differentiated CD with STI571 before in vivo injection allows potentiation of NK recruitment and activation in vivo (percentage of Dx5 + / CD3 cells accumulating CD69) (Figure 5).

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 Experiments in mice demonstrate that STI571 increases by 10-30 the ability of DCs to induce IFNy secretion by resting NK in vitro and to activate NK in vivo.

Example 4: In Vivo Injection of STI571 and a CD Growth Factor Induces NK Cell Activation The inventors combined STI571 with Flt3L (hematopoietic growth factor of CD in vivo) in order to test whether this Association could lead to activation of NK cells, and eradication of sensitive NK tumors, regardless of a c-kit, abl or PDGF receptor mutation.

The NK functions of the livers and spleens of the treated animals were evaluated in various ways (% CD69 on CD3- / Dx5 +, direct lytic activities of the livers and spleens on YAC-1, secretion of IFNγ of the lymphoid organs treated in vitro 24hrs per day. IL-2). The spontaneous lytic activities of splenocytes of Nude mice treated with the Flt3L + STI571 combination were surprisingly superior to those of Nude mice treated with Flt3L alone, STI571 alone or PBS (Fig. 6). The increase of CD69 on the NK is clear with the combination Flt3L + STI, whereas it is not with each of the components taken in isolation. Production of IFNγ is most visible in ganglion cultures from animals that received Flt3L + STI571 (Fig6).

The STI571-Flt3L combination therefore makes it possible to induce in vivo a significant activation of the NK cell activity, allowing the efficient lysis of pathological cells in vivo.

Example 5: STI571 blocks LPS-induced CD maturation and prevents septic shock

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Bone marrow-derived dendritic cells (BM-DC) of H-2b mice (by culture in the presence of GM-CSF, IL-4 on culture day 7, according to the method described in Fernandez et al., Nat. 1999) 405) are incubated 24 hours in the presence of 10 g / ml LPS. They thus overexpress I-Ab, CD40, CD80, CD86 very significantly. They dramatically stimulate the proliferation of total alloreactive splenocytes (H-2d) at various E: T ratios (MLR-type reaction). Co-incubation of STI571 (and not AG957) with BM-DC in the concomitant presence of LPS prevents induced LPS maturation and alloproliferations (Figure 7A).

The property of these tyrosine kinase inhibitors to inhibit inflammatory signals such as LPS has also been investigated in vivo, in the mouse model of endotoxin shock (Fig. 7B). C57BL6 mice were treated by oral ingestion of STI 571 (150mg / Kg / 12h) or PBS. After the first 24 hours of treatment, endotoxin shock was induced by intraperitoneal injection of LPS (1 mg, serotype 026: B26, SIGMA). STI 571 or PBS treatments are continued for an additional 48 hours. Our preliminary results show a reduction in mortality in the STI 571 group (45% vs. 17.6%).

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Table 1

<Tb>
<tb> Secretion <SEP> Secretion <SEP> of <SEP> Lyse <SEP> Specific
<tb> Interferon <SEP> y <SEP> * <SEP> TNF <SEP> a <SEP> * <SEP> from <SEP> K562
<tb><SEP> Conditions of <SEP> - <SEP> LPS <SEP> + <SEP> LPS <SEP> - <SEP> LPS <SEP> - <SEP> LPS <SEP> + <SEP> LPS <SEP >
<tb> Culture <SEP> **
<tb> GIST <SEP> before <SEP> STI <SEP> 0% <SEP> 16.7% <SEP> 0% <SEP> 15.4% <SEP> 38.5%
<tb> 571 <SEP> (N = 14)
<tb> GIST <SEP> for <SEP> 30% * <SEP> 56.7% * 'it <SEP> 21.7% * <SEP> 45.8% <SEP> 62.5%
<tb> STI <SEP> 571 <SEP> (N = 31)
<tb> Volunteers <SEP> healthy <SEP> 11.1% <SEP> 11.1% <SEP> 10% <SEP> 30.8% <SEP> 42.9%
<tb> (N = 17)
<Tb>

Significant with a 95% safety interval according to Fisher's exact method, compared to GIST before STI 571
5 v Significant with a 95% safety interval according to Fisher's exact method, compared to healthy volunteers * Positivity threshold defined at 100 μg / ml ** NK cell functions are evaluated after incubation of NK CD3- / CD56 + cells peripheral blood with allogeneic immature dendritic cells in the presence or absence of LPS.

Table 2

<Tb>
<tb> Progressive <SEP> Diseases <SEP> Stabilized <SEP> Diseases <SEP> or <SEP> in
<tb> answer
<tb> Patients <SEP> biologically <SEP> 0 <SEP> 16
<tb> sensitive <SEP> at <SEP> STI <SEP> 571
<tb> Patients <SEP> biologically <SEP> no
<tb> sensitive <SEP> at <SEP> STI <SEP> 571 <SEP> 5 * <SEP> 11
<tb> 15 <SEP> * <SEP> exact <SEP> test of <SEP> Fisher <SEP>: <SEP> p = 0.042.
<Tb>

Claims (20)

1. Use of a c-abl tyrosine kinase inhibitor, bcr / abl, c-kit and / or PDGF receptor-associated tyrosine kinase, for the preparation of a composition for modulating dendritic cell activity in a mammal. 2. Use according to claim 1 for inducing a phenotypic modification of dendritic cells and enabling the activation of NK cells. 3. Use according to claim 2 for the preventive or curative treatment of viral infections. 4. Use according to claim 2, for the preventive or curative treatment of NK sensitive tumors not related to a constitutive mutation in a tyrosine kinase. 5. Use according to claim 1 for the preventive or curative treatment of immune and / or inflammatory diseases in a mammal. 6. Use according to claim 5 for the preventive or curative treatment of autoimmune diseases. 7. Use according to claim 5 for the preventive or curative treatment of chronic inflammatory diseases, including atherosclerosis, Crohn's disease, asthma, rheumatoid arthritis, dermatitis and septic shock or endotoxin. 8. Use according to claim 5 for the preventive or curative treatment of congenital immune diseases. <Desc / Clms Page number 34> 9. Use of a c-abl, bcr / abl, c-kit tyrosine kinase inhibitor and / or PDGF receptor-associated tyrosine kinase to increase NK activity in cancer patients. 10. Use according to one of claims 1 to 9, characterized in that the mammal is a human being. 11. Use according to any one of claims 1 to 10, characterized in that the inhibitor is a compound of the family of pyrimidines. 12. Use according to claim 11, characterized in that the inhibitor is 2-phenylaminopyrimidine (STI571) or a derivative or analogue thereof. 13. Use according to any one of the preceding claims, characterized in that the inhibitor is used in combination with a potentiating agent. 14. Use according to claim 13, characterized in that said potentiating agent is selected from a dendritic cell growth factor and a factor (co-) stimulation of NK cells. 15. Use according to claim 14, characterized in that said agent is chosen from Flt3L, GM-CSF, ProGP-4 and their derivatives or analogues. 16. Use according to one of claims 13 to 15, characterized in that said agent is used simultaneously or delayed in time relative to the inhibitor. <Desc / Clms Page number 35>  17. A composition characterized in that it comprises a c-abl tyrosine kinase inhibitor, bcr / abl, c-kit and / or PDGF receptor-associated tyrosine kinase and a potentiating agent. The composition of claim 17 comprising ST1571 and a dendritic cell growth factor or an NK cell (co) stimulation factor. 19. A composition comprising dendritic cells treated with a c-abl, bcr / abl, c-kit tyrosine kinase inhibitor, c-kit and / or PDGF receptor-associated tyrosine kinase. 20. Composition according to claim 19, characterized in that the dendritic cells are immature.