WO2004026311A2

WO2004026311A2 - Use of specific inhibitors of tyrosine kinases for immunomodulation

Info

Publication number: WO2004026311A2
Application number: PCT/FR2003/002744
Authority: WO
Inventors: Laurence Zitvogel; Christian Auclair; Thomas Tursz
Original assignee: Institut Gustave Roussy (Igr)
Priority date: 2002-09-18
Filing date: 2003-09-17
Publication date: 2004-04-01
Also published as: FR2844452A1; AU2003276351A1; WO2004026311A3

Abstract

The invention relates to the use of inhibitors of tyrosine kinases for immunomodulation, i.e. for regulation of the activity of immune cells in patients. The invention more particularly relates to the use of specific inhibitors of tyrosine kinases in the preparation of a composition for preventing or treating viral infections sensitive NK tumors, immune diseases and/or septic shock in mammals. Said inhibitors question are more particularly inhibitors of tyrosine kinases c-abl (bcr/abl), c-kit and /or tyrosine kinase associated with the PDGF receptor. The invention also relates to compositions comprising one such inhibitor, combined with other active agents, i.e. agents which can potentiate the inhibiting effect. The invention can be used in a preventative or curative manner, in vivo ou ex-vivo.

Description

USE OF SPECIFIC TYROSINE KINASE INHIBITORS

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 tyrosine kinase inhibitors 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 tyrosine kinases c-abl (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, in particular agents capable of potentiating the effect of the inhibitor. The invention can be used preventively or curatively, in vivo or ex vivo.

Tyrosine kinases are key enzymes for the positive control of cell multiplication. Frequently, mutations of proto-oncogenes to oncogenes are due precisely to mutations in the kinase domain making the tyrosine kinase activity constitutive and unregulated. This is notoriously the case in Chronic Myeloid Leukemia (CML), where translocation (9; 22) results in the expression of a bcr-abl fusion protein which 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 interstitial cell of Cajal (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 molecular pathogenesis (Hirota S, Science 279 (1998) 577, Miettinen M, Human Pathol 30 (1999) 1213). These mutations lead to 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 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 presenting a mutation in the corresponding genes (CML, GIST).

The best characterized inhibitor is STI571 (Novartis), also known as Gleevec, Glivec or Imatinib Mesylate. STI571 (STI for “Signal Transduction Inhibitor”) is a selective inhibitor compound for certain protein tyrosine kinases: 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; Heinrich MC, Blood 96 (2000) 925). This compound would act on the mechanisms of signal transduction.

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

Phases I and II of STI571 in GIST (van Oosterom, Lancet 358 (2001) 1421; Joensuu H, N Engl. J. Med. 344 (2001) 1052) also showed more than 50% of objective clinical responses, including some spectacular in their speed of installation. Unlike the CML model, in GIST, the molecular action site of STI571 is not known.

STI 571 also demonstrated in vitro inhibitory activity against tyrosine kinases other than those of the abl family proteins, in particular vis-à-vis those of c-Kit and PDGF. Kit is a growth factor receptor, a ligand of which is the “Stem Cell Factor” (SCF) or Steel factor, and has a crucial role in the multiplication of the 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 tyrosine kinase inhibitors, such as STI571, are capable of acting on the activity of dendritic cells, and of modifying their properties. The invention shows in particular that such compounds make it possible to modulate the activity of immature dendritic cells to induce the activation of NK cells, in vitro and in vivo. The invention also shows that such compounds are also capable of inhibiting the maturation of dendritic cells induced by LPS and, therefore, of limiting the inflammatory response. These results are completely unexpected since no immunomodulatory activity for such inhibitors had been previously considered. This activity is also very surprising since it is effective on cells which do not necessarily have a mutation in a receptor associated with a tyrosine kinase. This activity is also surprising due to its amplitude, since, as illustrated in the examples, the in vivo use of such compounds makes it possible to activate 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 previously envisaged for these compounds, and also makes it possible to potentiate or optimize their properties. In particular, the new biological properties of these inhibitors make it possible, for the first time, to envisage their use to treat multiple cancers sensitive to NK, cancers which have become secondarily resistant to STI571, to treat cancers which do not contain the c mutation -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 therefore offers new 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 resides 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 tyrosine kinase associated with the PDGF receptor, for the preparation of a composition intended to modulate the activity of dendritic cells in a mammal.

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

Another object of the invention lies in the use of a specific tyrosine kinase inhibitor for the prophylactic or curative treatment of tumors or viral infections, in particular of sensitive NK tumors.

Another object of the invention lies in the use of a specific tyrosine kinase inhibitor to increase NK activity in patients suffering from cancer or viral infection. Another object of the invention lies in the use of a specific tyrosine kinase inhibitor to reduce the inflammatory response, in particular in subjects suffering from chronic inflammatory pathologies or autoimmune diseases.

Another object of the invention lies in the use of a specific tyrosine kinase inhibitor to promote GVL during allogenic transplants.

The invention also relates to methods of prophylactic or curative treatment of cancerous, infectious, immune or inflammatory pathologies, comprising the administration to a patient (i) of 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 the activity of dendritic cells 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 the activity of NK cells in a patient, comprising administering to said patient an effective amount of a specific tyrosine kinase inhibitor or of dendritic cells treated with such an inhibitor.

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

It also relates to a composition comprising dendritic cells, in particular immature cells, treated with a specific tyrosine kinase inhibitor. It also relates to a therapeutic association between a specific tyrosine kinase inhibitor and a potentiating agent, preferably chosen from a growth factor for dendritic cells and a (co-) stimulation factor for NK cells, for separate use, simultaneous or spaced over 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 can be used in all mammals, in particular in humans, even if use in animals 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. Within the meaning of the invention, the term “specific tyrosine kinase inhibitors” is understood to mean compounds capable of preferentially inhibiting the activity of tyrosine kinases c-abl, bcr / abl, c-kit and / or tyrosine kinase associated with the receptor for 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 mutated form which is constitutively active, or else of inhibiting the non-mutated or overexpressed form in cells. "Normal no transformed "like dendritic cells. The compounds are preferably without direct substantial effect on other tyrosine kinases.

The preferred inhibitor compounds of the invention are chemical molecules of the family of pyrimidines, such as the compounds described in patent applications No. WO01 / 64200, WO99 / 03854, EP 0564 409 and US 6,258,812. It can also be the pyrido [2-3α] -pyrimidine compounds described by Wisniewski D. et al. (Cancer. Res. 62 (2002) 4244).

A preferred inhibitor is a compound of the family of 2-phenylaminopyrimidines, such as compound STI571 or any derivative or analog thereof.

STI571 is a specific inhibitor of the protein tyrosine kinases mentioned above, without substantial effect on the EGF receptor, FLT 1 and FLT3. The half-life of STI571 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 inhibitors of tyrosine kinases can be used within the scope of the invention, although preference is given to those having selective activity for tyrosine kinases c-abl, bcr / abl, c-kit and / or associated with the receptor of the PDGF. Likewise, inhibitors can be identified or selected in vitro or in cellular tests, by measuring the binding or the activity of test compounds on the isolated tyrosine kinase or a cell containing it.

Another example of an inhibitor is the compound AB1003 or any derivative (eg, by substitution) thereof, as described in application No. US60 / 400064. The formula of the compound AB1003 [2- (2-methyl-5-amino) phenyl-4- (3-pyridyl) -thiazole] is shown below:

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 the NK cells of a subject. The invention indeed shows that such compounds are capable of increasing the NK function in patients, in vivo, leading to a powerful anti-tumor or anti-infectious action. The invention shows that this increase is linked 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 the CD56 and CD16 markers, and by the absence of the CD3 marker. NK cells have in particular been implicated in non-antigenic immunity of antigens, for the prevention of the establishment of primary or metastatic tumors in the immunocompetent or immunosuppressed host. NK cells in particular seem to have a key role against tumor cells or MHC class I negative variants. The role of NK cells in anti-tumor immunosurveillance has been widely documented, in mice (Diefenbach, Nature 413 (2001) 165) as in humans (A. Velardi, Science 2002). The role of NK cells in the prevention of viral infections is also well documented, especially for viruses of the Herpes group Viridae (C. Biron, N. Eng. J. Med. 320 (1989) 1731; Welsch et al., J. Exp. Med. 2001).

Due to their non-specific cytotoxic antigen properties and their efficiency, NK cells therefore constitute a population of effector cells which is particularly interesting for the development of adoptive immunotherapy approaches for cancers or infectious diseases. In this regard, in certain indications such as patients with intensified malignant lymphomas, the administration of NK cells associated with low doses of IL-2 has given promising results in the adjuvant situation. NK cells have also been used for the experimental treatment of different types of tumors and certain clinical studies have been initiated (Kuppen et al. Int. J. Cancer. 56 (1994) 574; Lister et al., Clin. Cancer Res. 1 (1995) 607; Rosenberg et al., N. Engl. J. Med. 316 (1987) 889). NK cells have also been shown to promote GVL in allogeneic marrow transplants. In addition, these cells can also be used in vitro for the non-specific lysis of cells which do not express MHC class-1 molecules and, more generally, of any cell sensitive to NK cells.

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

The methodologies for activating NK cells 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 I or II, IL-15 in combination or not with 1L-1b, IL-18, IL- 21 or chemokines). However, the pathophysiological 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 costs of preparation of the latter, the toxic nature of many of them, which cannot be used in in vivo applications, or else the non-specific character. numerous cytokines, the use of which in vivo risks being accompanied by many side effects. In addition, the "natural killing" function is often impaired in patients with tumors, the possibility of harvesting these cells for activation ex vivo can be considerably reduced.

The inventors have previously demonstrated that dendritic cells (CD) are cells that are capable of activating NK cells in vitro and in vivo, leading to anti-tumor 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, specific inhibitors of Tyrosine Kinases, such as STI571, induce in vivo or in vitro, a phenotypic modification of the dendritic cells which gives them the power to activate NK cells. In this context, the inventors have 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 also observed that tyrosine kinase inhibitors, of the STI571 type, are capable of activating the NK functions in patients carrying GIST. Thus, in GIST patients treated with STI571, NK cells are activated in 30 to 56% of patients and these biological data for activation of NK seem to correlate with the clinical responses obtained with STI571 (with, in particular, no primary resistance to STI571 in biological responders). These results therefore demonstrate, in vivo, in humans, that tyrosine kinase inhibitors, such as STI571, exhibit immunomodulatory activities in addition to their inhibiting functions of the 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 tyrosine kinase inhibitor for the preparation of a composition intended to increase NK activity in patients with cancer or infection (viral, bacterial or parasitic).

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

Another particular object of the invention lies in the use of a specific inhibitor of tyrosine kinase c-abl, bcr / abl, c-kit and / or associated with the PDGF receptor for the preparation of a composition intended for the prevention or treatment of sensitive NK tumors not linked to a constitutive mutation in a tyrosine kinase, or resistant to STI571.

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 dendritic cells and to enable or increase the activation of NK cells.

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

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

The invention further relates to a method for activating NK cells in a subject, comprising administering to the subject a tyrosine kinase inhibitor. c-abl, bcr / abl, c-kit and / or tyrosine kinase associated with the 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 NK lymphocyte subpopulation (s).

For the implementation of the invention, the specific tyrosine kinase inhibitor can be used alone. However, in a preferred and particularly effective variant, the inhibitor is used in combination with another active agent. This active agent may be of various natures, but it is preferably an agent potentiating the action of the inhibitor, such as in particular a growth factor of dendritic cells or a (co-) stimulation factor of NK cells. In fact, the invention shows that specific inhibitors of tyrosine kinase can make it possible to stimulate the NK function in vivo in subjects, and that this stimulation involves a phenotypic modification of the dendritic cells. By combining several agents active on production, on the modification of dendritic cells and / or on the activation of NK, a more powerful biological effect can therefore be obtained. In particular, by combining a growth factor for dendritic cells and an inhibitor according to the invention, it is possible to produce a stimulation of the NK functions sufficient 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 the two agents can be used simultaneously or spaced over time, as long as their biological effects are combined in vivo. The agents can therefore be packaged simultaneously or separately, and administered concomitantly or over time. In a first embodiment, the potentiating agent is capable of amplifying the dendritic cells, that is to say of increasing their differentiation, their growth or their activity. It is preferably a growth factor for dendritic cells, advantageously chosen from Flt3Ligand ("Flt3L") described by Lyman SD et al. (Blood 83 (1994) 2795-2801) and by Maraskovsky E. et al. (J. Exp. Med. 184 (1996) 1953-1962), GM-CSF, interleukin-4, interleukin-13 and their derivatives or analogs. It may especially be progenipoietin (ProGP4, fusion molecule between G-CSF and Flt3L), a GM-CSF / IL-4 fusion, etc. This agent can be used simultaneously or offset in time with respect to the tyrosine kinase inhibitor.

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

In a particular embodiment, an inhibitor as defined above is used in combination with a dendritic cell growth factor and / or an NK cell co-stimulation factor. A particularly preferred combination includes 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 chosen on a specific basis from cytokines or lymphokines activating NK cells or from Fit 3L, GM-CSF, IL-4, IL-13 and their derivatives or analogs (ProGP4, GM / IL-4). The compositions according to the invention can naturally also comprise any pharmaceutically acceptable vehicle or excipient, such as isotonic solutes, buffered saline solutions, stabilizing agents, etc.

The applications of the compositions and methods of the invention are numerous, due to the broad spectrum of action of NK cells. They preferably include the prophylactic or curative treatment of various pathologies such as cancers or infectious diseases, in particular of viral origin or resulting from other pathogens, and congenital NK or related immunodeficiencies (immune deficiencies leading to a mutation in the IFNγ 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 to delay or suppress the growth of tumors, induce a regression in the size of the tumors, slow down or prevent the formation of metastases, etc. ., in particular tumors weakly expressing MHC class I molecules or having ligands for receptors activating NK or other pathological cells. They also make it possible to 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, in particular those chosen from carcinomas, melanomas, leukemias, lymphomas and sarcomas. The invention is particularly suitable for the preventive or curative treatment of melanomas, kidney cancer, sarcomas of the digestive tract, chronic myeloid leukemia (CML) and neuroblastomas.

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

In this regard, 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 prophylactic or curative treatment of CML or from GIST. It also relates to a corresponding processing method.

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 prophylactic or curative treatment of a tumor resistant to STI571. It also relates to a corresponding processing method.

In the field of infectious pathologies, the invention can be used for the prophylactic or curative treatment in particular a viral infection caused by a virus of the group of Herpes Viridae (Herpes virus type 1 or 2, Epstein-Barr virus , 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.

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

It also relates to a process for preparing or treating in vitro or ex vivo 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.

The dendritic cells are typically treated by incubation in the presence of the inhibitor, for example in the presence of STI571, at a rate of approximately 10 ^δ to 10 ^{8 8} molar. It is preferred to use immature dendritic cells. Preferably, human CD34 + dendritic cells are placed in the presence of SCF, Flt3L, GM-CSF and TNFα for approximately 7 to 15 days (preferably 12 days), then in the presence of STI571 for approximately 12 to 48 hours (preferably Around 24h). It is understood that the precise conditions can be adapted by a person skilled in the art. The cells thus obtained have the capacity to activate NK cells, and can be conditioned 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-culture for a period of between 20 to 48 hours approximately. The co-culture ratio is ideally one dendritic cell for approximately 25 to 125 NK cells.

For use in vitro or ex vivo, the invention can be implemented in any device suitable for cell culture, preferably under sterile conditions. These can in particular be plates, culture dishes, flasks, flasks, pockets, etc. The culture or co-culture is also carried out in any medium suitable for the culture of dendritic cells and / or NK cells. More generally, these may be commercially available culture media for the cultivation of mammalian cells, such as for example RPMI medium, DMEM medium, IMDM medium or GBEA media (AIMV, X-VIVO), etc.

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

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

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

The term “activation” of NK cells within the meaning of the invention more particularly designates the early upregulation of CD69, especially on the CD56 ^bnght population of ^NKs (that which spreads in GIST patients treated with STI571), l increase in IFNγ production and / or cytotoxic activity of NK cells. These three parameters can be easily measured according to techniques known to those skilled in the art. The term “activated” NK cells denotes, within the meaning of the invention, NK cells exhibiting at least one of the properties mentioned above. It is also possible to detect an NK activation by the capacity of these cells to release for a prolonged period of high levels of IFNγ in the presence of "immature CD" and, possibly, 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 from the same organism. They can also be made up of allogenic cells. In addition, it can also be dendritic cells sensitized to one or more antigens or, on the contrary, “naive” cells. These compositions 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%, in particular 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 (in the cold) or used extemporaneously, as described below. Advantageously, these compositions comprise from 10 ⁴ to 10 ⁹ treated dendritic cells, preferably from 10 ⁵ to 10 ⁸ approximately.

The administration of the inhibitor or of the cells (dendritics or NK) can be carried out by any technique and any known route, and in particular by injection or oral administration. It may for example be a subcutaneous, systemic (intravenous, intraarterial, intraperitoneal) or intramuscular injection. When possible, the inhibitor is advantageously administered orally. The injection is preferably a local or regional injection, in particular an injection at the site or close to the site to be treated, in particular close to a tumor. The injections are generally carried out on the basis of doses of cells which can range from 10 ⁴ to 10 ⁹ dendritic cells, preferably between 10 ⁵ and 10 ⁷ included. It is possible to passively transfer dendritic cells by repeated administrations, for example 1 to 2 administrations per week for several months. The inhibitor compound can be administered at various doses, for example between 200 and 800 mg per os / day, preferably between 400 mg and 600 mg per os / day, in particular in the event of association with a potentiating agent (for example Flt3L se 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 preferably carried out at a dose of 10 ^"6 M or 10 ^{" 8} M. Repeated administrations are of course possible. In addition, the administration protocol can be adapted by those skilled in the art according to the situations (preventive, curative, isolated tumors, metastases, significant or localized infection, etc.).

ANTI-INFLAMMATORY ACTION

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

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 therefore 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 the cells into contact in vitro, ex vivo or in vivo. dendritics with a tyrosine kinase inhibitor c-abl, bcr / abl, c-kit and / or tyrosine kinase associated with the PDGF receptor.

The invention also relates to the use of a specific tyrosine kinase inhibitor 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 tyrosine kinase inhibitor as defined above for the preparation of a composition intended for the treatment of inflammatory pathologies, in particular chronic, such as atherosclerosis, Crohn's disease, asthma (chronic), rheumatoid arthritis, dermatitis (chronic), septic or endotoxin shock, etc. In this context, the term treatment also includes a reduction in the inflammatory response, even partial and / or temporary, leading to an improvement in the quality of life of the patients.

The invention also relates to the use of a specific tyrosine kinase inhibitor 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 tyrosine kinase inhibitor as defined above for the preparation of a composition intended for allogenic grafting. The invention is particularly suitable for improving allogeneic marrow transplants. In fact, by stimulating NK activity, the invention makes it possible to improve GVL activity (“graft versus Leukemia”), and therefore the therapeutic benefit. But in addition, by reducing the maturation of dendritic cells and therefore, potentially, the activation of alloreactive cytotoxic T lymphocytes, the inhibitor also makes it possible to reduce GVHD ("Graft Versus Host Disease") and thus improve safety of treatment. The invention can therefore be used advantageously to treat a host receiving a non-compatible allogeneic marrow graft, HLA. The invention can also be used to prevent or treat congenital immune diseases, such as a deficiency in the receptor for IL-12, a deficiency in the receptor for IFNγ, a deficiency in the gamma chain (IL-2Rγ) common 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. The content of all references cited in the description and examples, including articles, patents and patent applications, is incorporated herein entirely by reference.

LEGEND OF FIGURES

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

A cohort of patients treated at the Gustave Roussy Institute has been studied for the treatment of GIST. The patient's NK were purified by negative selection from peripheral blood. The functions of the NK cells (gamma interferon secretion and cytotoxicity) were analyzed after culture with allogeneic dendritic cells. In 14 cases, the samples are taken before the introduction of TSI 571; in 31 cases, the samples are taken during STI 571 treatment. A cohort of 17 healthy volunteers is studied according to the same methods.

Cytotoxic functions are reported as a percentage of specific lysis of a target sensitive to NK lysis (K562). The results are reported as an average for each group studied. The analysis of the data is carried out using exact Fisher tests which make it possible to highlight the statistically significant differences between the 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 gamma interferon. This production of gamma interferon was defined as significant if it was possible to measure more than 100 μg / ml in the supernatants of cocultures. Patients who meet 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 as a function of the biological response.

No patient identified as a biological responder has progressive disease under STI 571, while 5 of 16 patients who are not biologically responder progress clinically (p = 0.042; Fisher exact test).

Figure 1: Longitudinal monitoring of seven GIST patients on STI571. Acquisition of NK functions.

Concerning 7 patients, samples and measurements of gamma interferon production are available. Figure 1 reports the study of functions

NK before and during treatment with STI 571. For two patients, a marked increase in CD56bright NK cells (panel A) could be demonstrated.

For the seven patients followed, the secretions of gamma interferon are increased during exposure to STI 571 (panel B).

Figure 2. Dendritic cells incubated in the presence of STI 571 are capable of activating 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.

Two representative experiments carried out on two different samples (A and B) are reported. The cytotoxic NK functions are studied by lysis tests using targets marked with chromium 51 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 capable of activating NK cells.

STI 571 gives dendritic cells the ability to activate NK cells. Murine dendritic cells 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 gamma interferon by NK cultured in the presence of dendritic cells. In addition, there is a dose effect between the doses of STI 571 used and the biological effect observed. The previous experiment is repeated in the presence of a non-specific phosphorylation inhibitor (AG 957) and of STI 571. This experiment reveals that the previous results are specific of STI 571.

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

The previous experiments are reproduced using a synthetic analogue of STI 571, AB1003. This molecule also gives dendritic cells an activating function for NK cells.

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

Murine dendritic cells are pre-incubated with ST1571 and then injected subcutaneously into the lower limbs of the mice. The draining nodes are collected and the functions of the NK cells analyzed. after purification. The use of dendritic cells preincubated 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 NK studied.

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

Nude mice are treated for 10 days with intraperitoneal injections of Flt3L. 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 the NK cells. The functional tests carried out show an activation of the NK cells in the group treated by the association Flt3L + STI, unlike the control groups (mice treated with PBS IP, IL2 IP, PBS IP + gavages of STI 571, Flt3-L IP + gavages of PBS). Panels A and B present the flow cytometry analysis of the presence of CD69 (membrane activation marker) on the surface of NK cells (DX5 +), in the spleen and liver of mice. Panels C and D present the capacities of these NK cells, purified from the spleens of the mice of each group, to secrete gamma interferon after a phase of culture with dendritic cells.

Panel E represents the study of the cytotoxic functions of 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 the maturation of DC 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 allogenic lymphocytes (panel A). Dendritic cells cultured in GM-CSF and IL4 (BM-DC), activated by LPS (BM-DC + LPS) or by STI 571 (BM-DC STI) or by a non-inhibitor specific phosphorylation (BM-DC AG), are cultured in the presence of allogenic lymphocytes for 5 days. Lymphocyte proliferation is assessed by their ability to integrate tritiated thymidine. STI does not stimulate the proliferation of allogenic lymphocytes. Its immunomodulatory effect is found to be 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. Gavages of mice with STI 571 or PBS are continued for 48 hours.

Figure 8: STI571 prevents the growth in vivo of tumors resistant to the antiproliferative effects of STI571 in vitro.

(AT). In vitro STI571 mouse tumor models.

AK7, B16F10, RMA-S, MCA 102 or BAF3p210 cells (which carry the bcr / abl translocation) were incubated for 24 hours with the indicated doses of STI571. The absolute number of surviving cells was determined using a trypan blue exclusion test. The absence of anti-proliferative effect of STI571 on all tumors except for BAF3p210 cells has been confirmed in two independent experiments. The signs of proliferation are apparent. The Wilcoxon test was used to compare the proliferation indexes (* p (0.05).

(B). STI571 inhibits, in vivo, the growth of AK7 and MCA102. ³ × ¹⁰ ⁶ AK7 tumor cells and ⁵ × 10 ⁵ MCA102 tumor cells were inoculated subcutaneously into the abdominal flank, on day 0. The oral supply of STI571 (150 mg / kg) or PBS (200 μl) was carried out from day 20 to day 27 and from day 3 to day 10 respectively. The results of an experiment involving 7-9 mice per group appear in this figure. The Wilcoxon test was used to compare the growth kinetics ( ^* p (0.05)). (VS). The anti-metastatic effects of STI571 against B16F10 are mediated by NK cells.

Neutralizing anti-NK1.1 antibodies (300 μg of Acm PK136 per mouse) or normal mouse serum (SNS) was administered intraperitoneally on day -4, -2, 0 and on day +4 to C57BL / 6 mouse. 5χ10 ⁵ B16F10 tumor cells were injected into the tail vein on day 0. The oral supply of STI571 (150 mg / kg) or PBS (200 μl) was made from day +5 to day +11 and the mice were sacrificed to enumerate lung metastases on day +11. The results of an experiment involving 7-9 mice per group appear in this figure. The Kruskal Wallis multiple comparison test was used to compare the number of lung metastases (* p (0.05 between the SNS STI571 and PBS groups; ** p <0.05 between the SNS STI571 and anti-NK1.1 mAb groups; PBS versus STI571 is not significantly different in the anti-NK1.1 mAb groups).

Figure 9: STI571 stimulates the inhibition of N A cell-dependent R A-S tumor growth in vivo.

(A, B). FL and STI571 act in synergy to prevent the growth of the RMA-S tumor in vivo.

1 x 10 ^th deficient RMA-S cells, which are targets of NK (A), or TAP-sufficient RMA cells, which are not targets of NK (B), were inoculated into each of the opposite abdominal flanks of mice C57BL6 on day 0. From day -6 to day +3, FL (10 μg / day) or PBS alone (200 μl) were injected by ip. From day +1 to day +4, STI571 (150 mg / kg) or PBS (200μl) was administered orally. The number of mice without tumor (out of 5 mice) at the end of the experiment is indicated in brackets.

(C, D). The combination of FL + STI571 promotes the anti-tumor effects dependent on NK cells on established large tumors. The neutralizing anti-NK1.1 mAb antibody (300 μg of Acm PK136 per mouse) was administered on days + 8, +10, +13, +17, +22 and +29. 1 ^χ 10 ⁶ RMA-S cells were inoculated into the abdominal flank on day 0. Administration of FL has started on day +13 when the tumors reached a diameter of 50 + 20 mm ² . The administration of FL was maintained for 10 days and was combined with STI571 from days +20 to +23 (the same doses as in A, B). Each experiment included 5 mice per group, was repeated twice and gave similar results.

(E). The combination of FL + STI571 induces significant anti-tumor effects against AK7.

3 × 10 ⁶ AK7 tumor cells were inoculated on day 0 into the abdominal flank of C57BL6 mice. Administration of FL started on day +11 when AK7 tumors reached a diameter of 20 ± 20 mm ² . Administration was continued for 10 days and FL was combined with STI571 the day before discontinuation of administration. STI571 was then maintained for 9 consecutive days (the same doses as in A, B). Each experiment included 5 to 7 mice per group and was repeated twice with similar results. Kruskal Wallis' multiple comparison test was used for statistical analyzes and significant effects are indicated by asterisks.

EXPERIMENTAL PART

Materials and methods :

Purification of NK cells

Circulating peripheral lymphocytes (PBL) are contained in the non-adherent fraction of mononuclear cells of peripheral blood after two hours of adhesion. NK cells were isolated from PBL by negative magnetic 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 + / VS + type (Miltenyi biotec). The purity of the fraction thus depleted is evaluated by flow cytometry. Phenotyping after purification of NK 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 Femandez et al [18].

The NKs purified by negative selection are placed in culture in 96-well plates (10 ⁵ NK / 100 L / well), bottom U for 48 hours, under 4 conditions: only in RPMI A 'medium [Gibco, 10% serum AB , 1% sodium pyruvate 100mM (Gibco-BRL, France), 0.5% penicillin-streptomycin 10,000IU / mL-10,000g / 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 according to).

Evaluation of activation and NK functions:

The NK functions (cytotoxicity, secretion of IFNγ, positive regulation of CD69) can be evaluated directly from fresh blood after purification on columns, or preferably after reactivity with allogeneic standardized dendritic cells in the presence or not of LPS 10 μg / ml .

Cytotoxic functions

NK cells are used as effectors in a cytotoxicity test against Daudi or K562. The target cells are marked with chromium 51 (100 μCi / 10 ⁶ cells) for 1 hour, then washed 2 times. They were then distributed to 2000 cells per well in 96-well bottom V plates. The maximum release of chromium 51 (Cr ₅₁ ) 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. 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 Cr ₅ i = [release of Cr ₅ ι from the sample - spontaneous release of Cr ₅ ι by targets] / [maximum release of Cr ₅ ι - spontaneous release of targets].

Secretion of cytokines

Some activated NK cells secrete interferon (IFN). Similarly, allogeneic immature dendritic cells (standardized batches), put in the presence of NK cells previously activated [by a classic 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 in an ELISA assay (Enzyme Linked Immunoabsorbent Assay- kit OptEIA IFN and human TNFα-Pharmingen). The anti-IFN and anti-TNFα antibodies are then incubated overnight at 4 ° C. on a 96-well maxisorb plate. After washing and saturating the antibodies with a “dilute Assay” solution, 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 IFN or TNFα, coupled to biotin makes it possible to detect these cytokines. The revelation is carried out by a streptavidin biotin reaction. The optical density is read at 450 nm. A correction is made at 550 nm. A range of standardized IFN 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, 5 × 10 ⁴ to ¹ × ¹⁰ ⁵ NK cells are labeled for analysis in flow cytometry. The antibodies used are anti-CD56, anti-CD3, anti CD69. Isotypic controls are performed for each manipulation.

Demonstration of the elective amplification of NK subpopulations in vivo in treated GISTs

After purification of the peripheral NK of the patients, an immunostaining is carried out with the anti-CD3, CD56 and CD16 antibodies. The increase in the CD16 ^dl , CD56 ^bright subpopulation is highlighted, going from 5% (pre-STI) to possibly 18% under STI571.

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

The blood and the tumors were obtained from the CCPPRB, in GIST phase II patients placed on STI 571.

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

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

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

EXAMPLE 2 Demonstration of an Action of STI571 on Dendritic Cells, Which Can Report on the Activation of NK Cells

Since dendritic cells are, unlike macrophages, myeloid or lymphoid precursors that are capable of activating NK at rest, we tested the capacities of STI571, administered at 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 which do not activate human NK, strongly acquire this capacity when they are incubated 24 hours from D12 to D13 with STI571 at 10 ^"6 M (Fig 2 ).

In a murine system, the secretion of IFNγ by the NKs is increased by 2 to 3 times at a CD: NK co-culture ratio of 1: 1. On the other hand, while at a CD: NK co-culture ratio of 1: 10, the untreated CDs no longer activate the NKs, CD-STI571 still has this property strongly. In addition, only STI571 and not IΑG957 (Tyrphostin, a non-specific tyrosine kinase inhibitor) induces the activation of NK via CD in vitro (Fig. 3). These data can be reproduced using a structural analogue of STI 171, AB1003 (Fig. 4). Thus, human or murine dendritic or precursor cells are the potential target of STI571 in vivo in GIST patients on STI571. Indeed, the applicants have demonstrated that STI571 does not directly activate NK in vitro.

EXAMPLE 3 The In Vivo Injection of Dendritic Cells Treated with STI571 Induces Activation of the NK Cells

The administration of CD in vivo makes it possible to modulate the activation of the NK cells of the lymphoid organs (draining nodes if injected subcutaneously, spleen if injected intravenously). The incubation for 24 hours of differentiated CD with STI571 before the injection in vivo makes it possible to potentiate NK recruitment and activation in vivo (percentage of Dx5 + / CD3- cells acquiring CD69) (Fig. 5).

Experiments in mice demonstrate that STI571 increases by a factor of 10-30 the capacity of DCs to induce secretion of IFNγ by NKs at rest in vitro and to activate NKs in vivo.

Example 4 Injection of STI571 and a CD Growth Factor In Vivo Induces Activation of NK Cells

The inventors combined STI571 with Flt3L (hematopoietic growth factor for DC in vivo) in order to test whether this association could lead to the activation of NK cells, and to the eradication of sensitive NK tumors, independently 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 livers and spleens on YAC-1, secretion of IFNγ from the lymphoid organs treated in vitro

24hrs by IL-2). Spontaneous lytic activities of mouse splenocytes Nude treated by the Flt3L + STI571 association were surprisingly found to be superior to those of Nude mice treated with Flt3L alone, STI571 alone or PBS (Fig. 6). The increase in CD69 on NK is clear with the Flt3L + STI combination, while it is not with each of the components taken in isolation. The production of IFNγ is especially visible in the lymph node cultures originating from animals having received Flt3L + STI571 (Fig6). The combination STI571 - Flt3L therefore makes it possible to induce in vivo a significant activation of the activity of NK cells, allowing efficient lysis of pathological cells in vivo.

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

Dendritic cells derived from bone marrow (“BM-DC”) from H-2b mice (by culture in the presence of GM-CSF, IL-4 to D7 culture, according to the method described in Fernandez et al. Nat. Med. 5 (1999) 405) are incubated for 24 hours in the presence of 10 μg / ml LPS. They thus overexpress L-Ab, CD40, CD80, CD86 in a very significant way. They dramatically stimulate the proliferation of alloreactive total splenocytes (H-2d) at various E: T ratios (MLR type reaction). Co-incubation of STI571 (and not AG957) with BM-DC in 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 (150 mg / Kg / 12 h) or by PBS. After the first 24 hours of treatment, endotoxin shock is induced by an intraperitoneal injection of LPS (1 mg, serotype 026: B26; SIGMA). Treatments with STI 571 or PBS are continued for an additional 48 hours. Our preliminary results show a reduction in mortality in the group treated with STI 571 (45% vs. 17.6%). Table 1

Secretion Specific Lyse secretion of interferon γ * TNF α * from K562

Conditions of - LPS + LPS LPS LPS + LPS

Culture **

GIST before STI 0% 16.7% 0% 15.4% 38.5%

571 (N = 14)

GIST for 30% ^* 56.7% _* ^: v 21.7% ^* 45.8% 62.5%

STI 571 (N = 31)

Healthy volunteers 11.1% 11.1% 10% 30.8% 42.9% (N = 17)

Significant with a 95% safety interval according to the exact Fisher method, compared to GIST before STI 571 ^v Significant with a 95% safety interval according to the exact Fisher method, compared to healthy volunteers * Positivity threshold defined at 100 pg / ml

** The functions of NK cells are evaluated after incubation of CD3- / CD56 + NK cells in peripheral blood with allogeneic immature dendritic cells in the presence or absence of LPS.

Table 2

Progressive diseases Stabilized or responding diseases

Biologically O 16 patients sensitive to STI 571

Patients biologically not sensitive to STI 571 1 1

* Fisher's exact test: p = 0.042. Example 6: STI571 exerts a therapeutic effect against NK-sensitive GIST tumors not linked to a constitutive mutation in a tyrosine kinase.

GIST tumors are the most common mesenchymal neoplasms of the gastrointestinal tract. Somatic mutations (providing a function) of KIT or PDGFA protooncogenes are present in 92% of GISTs [BP Rubin et al., Cancer Res. 61, 8118 (2001) and MC Heinrich, et al, Science, 299, 708 (2003)]. STI571 stabilizes the disease or induces partial responses in 80% of GIST patients [GD Demetri et al., N. Engl. J. Med. 347, 472 (2002)]. However, some GISTs do not respond in vitro to STI571 or become resistant. Longitudinal studies of GIST patients treated with STI571 have now revealed the therapy-induced increase in the production of IFNγ by NK cells, correlated with an increase in the anti-tumor response. Thus, in a phase II trial, several of the patients responding significantly to STI571 did not present any KIT or PDGFRA mutation (see Table 3). In addition to its anti-tumor activity, STI571 therefore acts indirectly on the host cells expressing KIT outside the tumor. Several tumor models resistant in vitro to the antiproliferative effects of STI571, but sensitive in vivo have thus been identified. Thus, melanoma B16F10, RMA-S lymphoma, fibrosarcoma MCA102 and mesothelioma AK7 resist STI571 in vitro (Fig. 8A) but their natural growth in C57BL6 mice is significantly prevented after 8 days of oral treatment using of STI571, both for the subcutaneous expansion of AK7 and MCA102 tumors (Fig. 8B), and for the establishment of metastases B16 of the lung (Fig. 8C). In this context, NK cells are critical mediators of the anti-tumor effects of STI571. Tumors refractory to the antiproliferative effects of STI571 in vitro respond to FL + STI571 in vivo in a manner dependent on NK cells. Thus, the combination of FL and STI571 completely suppresses the tumor in 100% mice (Fig. 9A). Depletion of NK1.1 ⁺ cells suppresses the therapeutic effect of FL + STI571 (Fig. 9C, D). FL + STI571 exerts a therapeutic effect against established tumors reaching a size of 150 + 20 mm ² (Fig. 9C). The synergistic therapeutic effects of FL + STI571 have also been demonstrated for AK7 mesothelioma (Fig. 9E).

* the target mutations are mutations affecting in KIT exons 9, 11, 13, 17 and in PDGFRA exons 12, 14 and 18 (6). ^§ Patients still alive at the time of the evaluation.

Claims

1. Use of an inhibitor of tyrosine kinases c-abl, bcr / abl, c-kit and / or tyrosine kinase associated with the PDGF receptor, for the preparation of a composition intended for the prophylactic or curative treatment of NK tumors sensitive not linked to a constitutive mutation in a tyrosine kinase.

2. Use of an inhibitor of tyrosine kinases c-abl, bcr / abl, c-kit and / or tyrosine kinase associated with the PDGF receptor, for the preparation of a composition intended for the prophylactic or curative treatment of inflammatory diseases .

3. Use according to claim 1 or 2, characterized in that the mammal is a human being.

4. Use according to one of claims 1 to 3, characterized in that the inhibitor is a compound of the family of pyrimidines.

5. Use according to claim 4, characterized in that the inhibitor is 2-phenylaminopyrimidine (STI571) or a derivative or analog thereof.

6. Use according to claim 5, characterized in that the analog of

STI571 is the AB1003.

7. Use according to any one of the preceding claims, characterized in that the inhibitor is used in combination with a potentiating agent.

8. Use according to claim 7, characterized in that said potentiating agent is chosen from a growth factor for dendritic cells and a (co-) stimulation factor for NK cells.

9. Use according to claim 8, characterized in that said agent is chosen from Flt3L, GM-CSF, ProGP-4 and their derivatives or analogs.

10. Use according to one of claims 7 to 9, characterized in that said agent is used simultaneously or deferred over time with respect to the inhibitor.

11. Composition characterized in that it comprises an inhibitor of tyrosine kinases c-abl, bcr / abl, c-kit and / or tyrosine kinase associated with the PDGF receptor and a potentiating agent.

12. Composition according to claim 11, comprising STI571 or AB1003 and a growth factor for dendritic cells or a (co-) stimulation factor for NK cells.

13. Composition comprising dendritic cells treated with an inhibitor of tyrosine kinases c-abl, bcr / abl, c-kit and / or tyrosine kinase associated with the PDGF receptor.

14. Composition according to claim 13, characterized in that the dendritic cells are immature.