WO2025132479A1 - Flt3 inhibitor for modulating macrophages polarization - Google Patents

Abstract

The role of pro- and anti-inflammatory macrophages in disease depends on the specific disease and the balance between the two types of macrophages. Imbalances can lead to the development or progression of diseases, and targeting macrophage function may be a potential therapeutic approach for certain diseases. For example, by altering the phenotype and function of TAM-M2, it is possible to shift the immune response from an anti-inflammatory to a pro-inflammatory state, which may help reduce tumor progression. Here, the inventors demonstrate the ability of Gilteritinib to reprogram primary human macrophages. Gilteritinib is a targeted therapy used in the treatment of acute myeloid leukemia (AML) with a specific genetic mutation called FLT3-internal tandem duplication (FLT3-ITD) or a tyrosine kinase domain (TKD) mutation. Accordingly, the present invention relates to an FLT3 inhibitor to modulate the immune environment and enhance existing therapies by blocking immunosuppressive immune cells.

Description

FLT3 INHIBITOR FOR MODULATING MACROPHAGES POLARIZATION

FIELD OF THE INVENTION:

The present invention relates to methods and compositions for modulation of macrophages. More particularly, the invention relates to treat cancers and fibrosis by modulating macrophages polarization.

BACKGROUND OF THE INVENTION:

Macrophages are immune cells that play a crucial role in the body's response to infection and inflammation. They can be classified into two broad categories based on their function: pro- inflammatory macrophages and anti-inflammatory macrophages. Pro-inflammatory macrophages, also known as Ml macrophages, are involved in the early stages of the immune response. They are activated by various signals, including cytokines and pathogen-associated molecular patterns (PAMPs) released by bacteria and viruses. Once activated, Ml macrophages produce pro-inflammatory cytokines, such as TNF-alpha and IL-1 beta, and chemokines, which recruit other immune cells to the site of infection or inflammation. They also produce reactive oxygen species (ROS) and nitric oxide (NO), which are toxic to pathogens. On the other hand, anti-inflammatory macrophages, also known as M2 macrophages, are involved in the later stages of the immune response, particularly in the resolution of inflammation and tissue repair. They are activated by different signals than Ml macrophages, such as interleukin-4 (IL-4) and interleukin- 13 (IL-13). Once activated, M2 macrophages produce anti-inflammatory cytokines, such as IL-10 and TGF-beta, and growth factors, which promote tissue repair and regeneration. They also phagocytose apoptotic cells and debris, helping to clear the site of inflammation. Both pro-inflammatory and anti-inflammatory macrophages are important for maintaining the balance between inflammation and tissue repair. Pro- and anti-inflammatory macrophages play important roles in various diseases. An imbalance in their function can lead to the development or progression of diseases. Inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and atherosclerosis are characterized by an excess of pro-inflammatory macrophages. These macrophages produce high levels of pro-inflammatory cytokines and chemokines that can cause tissue damage and promote inflammation. In cancer, macrophages can have both pro- and anti-tumor effects. TAM-M1 (Tumor-associated macrophages) can help to initiate an immune response against the tumor cells, but chronic inflammation can also promote tumor growth. TAM-M2 can promote tumor growth by producing factors that favor angiogenesis and suppress the immune response.

In summary, the role of pro- and anti-inflammatory macrophages in disease depends on the specific disease and the balance between the two types of macrophages. Imbalances can lead to the development or progression of diseases, and targeting macrophage function may be a potential therapeutic approach for certain diseases. For example, by altering the phenotype and function of TAM-M2, it is possible to shift the immune response from an anti-inflammatory to a pro-inflammatory state, which may help reduce tumor progression.

In this context, the inventors demonstrate the ability of Gilteritinib to reprogram primary human macrophages. Gilteritinib is a targeted therapy used in the treatment of acute myeloid leukemia (AML) with a specific genetic mutation called FLT3 -internal tandem duplication (FLT3-ITD) or a tyrosine kinase domain (TKD) mutation.

SUMMARY OF THE INVENTION:

The invention provides a FLT3 inhibitor for use in the polarization of macrophages. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors have showed that disease burden was reduced and animals’ survival was significantly improved in mice receiving MH (MataHari) cells with Gilteritinib compared to other groups (Figures 4A and 4B). Importantly, Gilteritinib did not impact MLL-AF9 cell growth in the OT- I group, suggesting that its mechanism of action is independent of the intrinsic FLT3 signaling pathway in leukemic cells and may instead rely on a Gilteritinib-induced immune response.

Accordingly, in a first aspect, the present invention relates a FLT3 inhibitor for use in the polarization of macrophages.

In a particular embodiment, the FLT3 inhibitor for use according to the present invention inhibits the polarization of macrophages type I. In a particular embodiment, the FLT3 inhibitor for use according to claim I inhibits the polarization of macrophages type 2. Method for macrophages polarization

As used herein, the term “macrophages” refers to cells that have the highest plasticity of the hematopoietic system. They are either resident in tissues or derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. The various macrophage functions are linked to the type of receptor interaction on the macrophage and the presence of cytokines. Two distinct states of polarized activation for macrophages have been defined: the classically activated (Ml) macrophage phenotype and the alternatively activated (M2) macrophage phenotype. Similar to T cells, there are some activating macrophages and some suppressive macrophages, therefore, macrophages should be defined based on their specific functional activities. Granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) are involved in the differentiation of monocytes to macrophages. Human GM-CSF can polarize monocytes towards the Ml macrophage subtype with a "proinflammatory" cytokine profile (e.g. TNF-alpha, IL-lbeta, IL- 6, IL-12 and IL-23), and treatment with M-CSF produces an "anti-inflammatory" cytokine (e.g. IL-10, TGF-beta and IL-lra) profile similar to M2 macrophages. Classically activated (Ml) macrophages have the role of effector cells in TH1 cellular immune responses. The alternatively activated (M2) macrophages appear to be involved in immunosuppression and tissue repair.

As used herein, the term “polarization” refers to the phenotypic features and the functional features of the macrophages. The phenotype can be defined through the surface markers expressed by the macrophages. The functionality can be defined for example based on the nature and the quantity of chemokines and/or cytokines expressed, in particular secreted, by the macrophages. Indeed, the macrophages present different phenotypic and functional features depending of their state, either pro-inflammatory Ml-type macrophage or anti-inflammatory M2 -type macrophage. M2 -type macrophages can be characterized by the expression of surface markers such as CD206, CD 163, PD-L1 and CD200R and then secretion of cytokines such as CCL17, IL-10, TGFb. Ml-type macrophages can be defined by the expression of surface markers such as CD86 and CCR7 and the secretion of cytokines such as IL-6, TNF-a and IL12p40. In the context of the invention, FLT3 inhibitor allows to modulate the polarization of macrophages population by inhibiting the M2 -type macrophages and/or favoring the Ml -type macrophages.

As used herein, the term “macrophages type 1” known as classically activated macrophages (Ml macrophages or TAM-M1), refers to cells activated by lipopolysaccharides (LPS) or by double signals from interferon (IFN)-y and tumor necrosis factor-a (TNF-a). This first type of macrophage are able to kill microorganisms and tumor cells.

As used herein, the term “macrophages type 2” also known as “immunosuppressive tumor- associated macrophages M2” or “M2 macrophages or Tumor-associated macrophages type M2 (TAM-M2)” refers to a type of blood-borne phagocytes, derived from circulating monocytes or resident tissue macrophages. Exposure to IL-4, IL-13, vitamin D3, glucocorticoids or transforming growth factor-b (TGF-b) decreases macrophage antigen- presenting capability and up-regulates the expression of macrophage mannose receptors (MMR, also known as CD206), scavenger receptors (SR- A, also known as CD204), dectin-1 and DC-SIGN.9 M2-polarized macrophages exhibit an IL-12<low>, IL-23<low>, IL- 10<Mgh>phenotype. This second type of macrophage plays an important role in stroma formation, tissue repair, tumor growth, angiogenesis and immunosuppression. In blood cancers, TAMs are the most abundant inflammatory cells and are typically M2-polarized with suppressive capacity (1) that stems from their enzymatic activities and production of antiinflammatory cytokines, such as TORb (Fuxe et al, Semin Cancer Biol, 2012, 22:455-461). High TAM levels have been associated with poorer BC outcomes (Zhao et al, Oncotarget, 2017, 8:30576-86. Therefore, several strategies are currently under investigation, such as the suppression of TAM recruitment, their depletion, or the switch from the pro-tumor M2 to the anti -turn or Ml phenotype in patients with TNBC (Georgoudaki et al, Cell Reports, 2016, 15:2000-11).

As used herein, the term “Fms-like tyrosine kinase 3” (FLT-3) also known as Cluster of differentiation antigen 135 (CD 135) is a tyrosine-protein kinase receptor and is encoded by the FLT3 gene. FLT3 is composed of five extracellular immunoglobulin-like domains, an extracellular domain, a transmembrane domain, a juxtamembrane domain and a tyrosine-kinase domain consisting of 2 lobes that are connected by a tyrosine-kinase insert. Cytoplasmic FLT3 undergoes glycosylation, which promotes localization of the receptor to the membrane. It is expressed on the surface of many hematopoietic progenitor cells. Signalling of FLT3 is important for the normal development of haematopoietic stem cells and progenitor cells.

As used herein, the term “FLT3 inhibitor” has its general meaning in the art and refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of FLT-3. In the context of the invention, such compound is able to modify macrophage polarization in order to induce a pro-inflammatory environment. The method consists in the use of a FLT3 inhibitor able to inhibit the polarization of anti-inflammatory M2 -type macrophages and/or favors pro-inflammatory Ml -type macrophages, for inhibiting the anti-inflammatory signal provided by M2 -type macrophages and favouring the pro-inflammatory signal provided by Ml-type macrophages.

In a particular embodiment, the FLT3 inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

As used herein, the term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

In a particular embodiment, the FLT3 inhibitor is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In a particular embodiment, the FLT3 inhibitor is a small organic molecule.

As used herein, the term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In a particular embodiment, the FLT3 inhibitor is a small molecule which is a selective inhibitor of FLT3 selected among the following compounds: Gilteritinib, Quizartinib, KW-2449, Midostaurin, Ponatinib, Sorafenib, Sunitinib, Lestaurtinib, Tandutinib and Crenolanib.

In a particular embodiment, the FLT3 inhibitor f is Gilteritinib, Crenolanib, or Midostaurin.

In a particular embodiment, the FLT3 inhibitor is the Gilteritinib and its derivatives. As used herein, the term “Gilteritinib” is also known as Xospata and is developed by Astellas Pharma. Gilteritinib has the following structure C29H44N8O3, the following CAS number 1254053-43-4, the following formula :

In a particular embodiment, the FLT3 inhibitor is the Crenolanib and its derivatives.

As used herein, the term “Crenolanib” is an orally bioavailable benzimidazoles and is developed by AROG Pharmaceuticals, LLC. Crenolanib has the following structure C26H29N5O2, the following CAS number 670220-88-9 and the following formula :

In a particular embodiment, the FLT3 inhibitor is the Midostorine and its derivatives. As used herein, the term “Midostaurin” is also known as Rydapt & Tauritmo both by Novartis, is a multi -targeted protein kinase inhibitor. Midostorine has the following structure C35H30N4O4, the following CAS number 120685-11-2 and the following formula:

In some embodiments, the FLT3 inhibitor is an antibody.

As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In particular, the FLT3 inhibitor is an intrabody having specificity for FLT3.

As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the FLT3 inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of FLT3.

In a particular embodiment, the inhibitor of JMY expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long doublestranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukaemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the FLT3 inhibitor is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics.l 13.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

Method for treating macrophage related disease

Accordingly, in a second aspect, the invention relates to a FLT3 inhibitor according to the invention for use as a drug.

In a particular embodiment, the FLT3 inhibitor for use according to the invention in the treatment of macrophage related disease.

As used herein, the term “macrophage related disease” refers to diseases related to an undesirable M2 activation.

In the context of the invention, the macrophage related disease refers to disease wherein the immune environment within the tumour has an immunosuppressor profile. Indeed, inventors have demonstrated that the presence of M2 -like tumor-associated macrophages (TAMs) is associated with tumor progression, immunosuppression, and resistance to therapy. Targeting these M2 -like TAMs is a valuable strategy to enhance the antitumor immune response. FLT3 inhibitors according to the invention are used to reprogram M2 -like macrophages in diseases where these anti-inflammatory macrophages have a detrimental effect.

In a particular embodiment, the present invention relates to an FLT3 inhibitor for use in the modulating the immune environment and enhance the efficacy of classical therapy.

More particularly, the FLT3 inhibitor for use in the modulating the immune environment blocks immunosuppressive immune cells. In a particular embodiment, the FLT3 inhibitor for use according to the invention wherein the macrophage related disease is selected from the group consisting of but not limited to: cancer, more particularly solid cancer, fibrotic diseases such as for example idiopathic pulmonary fibrosis (IPF), hepatic fibrosis or systemic sclerosis (Wynn and Barron, 2010, Semin. Liver Dis., 30, 245), Alzheimer’s disease, allergy, and inflammatory disease such as asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

In a particular embodiment, the FLT3 inhibitor for use according to the invention wherein the macrophage related disease is cancer.

As used herein, the term “cancer” refers to a malignant growth or tumor resulting from an uncontrolled division of cells. The term “cancer” includes primary tumors and metastatic tumors.

In a particular embodiment, the cancer is a solid cancer. In a particular embodiment, the solid cancer is selected from the group consisting of but not limited to: adrenal cortical cancer, anal cancer, bile duct cancer (e.g. peripheral cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astrocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenoacanthoma, papillary serous adenocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

In a particular embodiment, the solid cancer is melanoma.

In a further embodiment, the FLT3 inhibitor for use according to the invention wherein the macrophage related disease is fibrosis.

As used herein, the term “fibrosis” refers to the common scarring reaction associated with chronic injury that results from prolonged parenchymal cell injury and/or inflammation that may be induced by a wide variety of agents, e.g., drugs, toxins, radiation, any process disturbing tissue or cellular homeostasis, toxic injury, altered blood flow, infections (viral, bacterial, spirochetal, and parasitic), storage disorders, and disorders resulting in the accumulation of toxic metabolites. Fibrosis is most common in the heart, lung, peritoneum, and kidney.

In a particular embodiment, the FLT3 inhibitor for use according to the invention wherein the macrophage related disease is liver, lung or kidney fibrosis.

In a particular embodiment, the fibrosis affects at least one organ selected from the group consisting of skin, heart, liver, lung, or kidney. Examples of fibrosis include, without limitation, dermal scar formation, keloids, liver fibrosis, lung fibrosis, kidney fibrosis, glomerulosclerosis, pulmonary fibrosis (e.g. idiopathic pulmonary fibrosis), liver fibrosis (e.g. following liver transplantation, liver fibrosis following chronic hepatitis C virus infection), renal fibrosis, intestinal fibrosis, interstitial fibrosis, cystic fibrosis of the pancreas and lungs, injection fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis. In some embodiments, the fibrosis is caused by surgical implantation of an artificial organ. In a particular embodiment, the fibrosis is lung fibrosis. In a further embodiment, the FLT3 inhibitor for use according to the invention wherein the macrophage related disease is an inflammatory disease. s used herein, the term “inflammatory disease” has its general meaning in the art and refers to the biological response of vascular tissues to harmful stimuli, including but not limited to such stimuli as pathogens, damaged cells, irritants, antigens and, in the case of autoimmune disease, substances and tissues normally present in the body. Examples of inflammatory disease include but are not limited to atherosclerosis, asthma, rheumatic disease such as rheumatoid arthritis (RA), systemic lupus erythematosus, Sjogren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome or Behcet's disease (2) type II diabetes (3) an autoimmune disease of the thyroid, such as Hashimoto's thyroiditis or Graves' Disease (4) an autoimmune disease of the central nervous system, such as multiple sclerosis, myasthenia gravis, or encephalomyelitis (5) a variety of phemphigus, such as phemphigus vulgaris, phemphigus vegetans, phemphigus foliaceus, Senear-Usher syndrome, or Brazilian phemphigus, (6) psoriasis, and (7) inflammatory bowel disease (e.g., ulcerative colitis or Crohn's Disease).

In a particular embodiment, the inflammatory disease is asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

In a particular embodiment, the FLT3 inhibitor for use according to the invention is Gilteritinib as described above. In a particular embodiment, the FLT3 inhibitor for use according to the invention is Crenolanib as described above. In a particular embodiment, the FLT3 inhibitor for use according to the invention is Midostaurin as described above

In a particular embodiment, the invention relates to a method for treating macrophage related disease in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of a FLT3 inhibitor.

As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with macrophages related disease. In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with a cancer. In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with a solid cancer. In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with melanoma. In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with a fibrosis. In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with inflammatory disease such as rheumatoid arthritis, inflammatory bowel disease or atherosclerosis.

In a particular embodiment, the subject is a human received or susceptible to receive a transplantation. In a particular embodiment, the subject transplanted is a human afflicted with or susceptible to be afflicted with acute myeloid leukemia (AML). In a particular embodiment, the subject is undergoing or susceptible to undergo hematopoietic stem cell transplantation.

The present invention also relates to a method for treating macrophages related disease in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of a FLT3 inhibitor. In a particular embodiment, the method according to the invention, wherein the FLT3 inhibitor and a classical treatment, as combined preparation for use simultaneously, separately, or sequentially in the treatment of macrophages related disease.

In another embodiment, the invention relates to a combined preparation comprising the FLT3 inhibitor for use according to the invention and a classical treatment. More particularly, the invention relates to a i) FLT3 inhibitor and a ii) classical treatment for simultaneous, separate or sequential use in the treatment of macrophages related disease, as a combined preparation.

In a particular embodiment, the invention relates to an i) FLT3 inhibitor and ii) a classical treatment for simultaneous, separate, or sequential use in the treatment of a solid cancer such as melanoma.

In a particular embodiment, the invention relates to an i) FLT3 inhibitor and ii) a classical treatment for simultaneous, separate, or sequential use in the treatment of inflammatory disease such as asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

In a particular embodiment, the invention relates to an i) FLT3 inhibitor and ii) a classical treatment for simultaneous, separate, or sequential use in the treatment of fibrosis.

As used herein, the term “classical treatment” refers to any compound, natural or synthetic, and immunotherapy, chemotherapy and radiotherapy used for the treatment of a cancer.

In a particular embodiment, the classical treatment refers to a treatment with a chemotherapeutic agent.

Typically, the invention relates to an i) FLT3 inhibitor and ii) a chemotherapeutic agent for simultaneous, separate, or sequential use in the treatment of a solid cancer such as melanoma. Typically, the invention relates to an i) FLT3 inhibitor and ii) a chemotherapeutic agent for simultaneous, separate, or sequential use in the treatment of an inflammatory disease such as asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

Typically, the invention relates to an i) FLT3 inhibitor and ii) a chemotherapeutic agent for simultaneous, separate, or sequential use in the treatment of a fibrosis.

As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In a particular embodiment, the classical treatment refers to a targeted therapy (TT).

Typically, the invention relates to an i) FLT3 inhibitor and ii) a targeted therapy for simultaneous, separate, or sequential use in the treatment of a solid cancer such as melanoma. Typically, the invention relates to an i) FLT3 inhibitor and ii) a targeted therapy for simultaneous, separate or sequential use in the treatment of an inflammatory disease such as asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

Typically, the invention relates to an i) FLT3 inhibitor and ii) a targeted therapy for simultaneous, separate, or sequential use in the treatment of a fibrosis.

As used herein, the term “targeted therapy” refers to targeting the cancer’s specific genes, proteins, or the tissue environment that contributes to cancer growth and survival. Example of targeted therapy: targeting human epidermal growth factor receptor 2 (HER2) for breast cancer; targeting epidermal growth factor receptor (EGFR), or vascular endothelial growth factor (VEGF) for colorectal cancer or lung cancer; targeting BRAF for melanoma.

In a particular embodiment, the classical treatment refers to a treatment with an immunotherapeutic agent.

Typically, the invention relates to an i) FLT3 inhibitor and ii) an immunotherapeutic agent for simultaneous, separate, or sequential use in the treatment of a solid cancer such as melanoma.

Typically, the invention relates to an i) FLT3 inhibitor and ii) an immunotherapeutic agent for simultaneous, separate, or sequential use in the treatment of an inflammatory disease such as asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

Typically, the invention relates to an i) FLT3 inhibitor and ii) an immunotherapeutic agent for simultaneous, separate, or sequential use in the treatment of a fibrosis.

The term "immunotherapeutic agent" as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, immune checkpoint inhibitor, cytokines, cancer vaccines, monoclonal antibodies, and non-cytokine adjuvants. Alternatively, the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents. Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. Several cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins, and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), and IFN-beta (IFN-P). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Colony-stimulating factors (CSFs) contemplated by the present invention include sargramostim. Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. In addition to having specific or non-specific targets, immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e. comprise immune system components that were generated external to the body. Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor. Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject's immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins. Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campath®), and BL22. Other examples include anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PDl antibodies, anti-PDLl antibodies, anti-PLD2 antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies. In some embodiments, antibodies include B cell depleting antibodies. Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g. Rituximab (Roche), Ibritumomab tiuxetan (Bayer Schering), Tositumomab (GlaxoSmithKline), AME- 133v (Applied Molecular Evolution), Ocrelizumab (Roche), Ofatumumab (HuMax-CD20, Gemnab), TRU-015 (Trubion) and IMMU-106 (Immunomedics)], an anti-CD22 antibody [e.g. Epratuzumab, Leonard et al., Clinical Cancer Research (Z004) 10: 53Z7-5334], anti-CD79a antibodies, anti-CD27 antibodies, or anti-CD19 antibodies (e.g. U.S. Pat. No. 7,109,304), anti- BAFF-R antibodies (e.g. Belimumab, GlaxoSmithKline), anti -APRIL antibodies (e.g. antihuman APRIL antibody, ProSci inc.), and anti-IL-6 antibodies [e.g. previously described by De Benedetti et al., J Immunol (2001) 166: 4334-4340 and by Suzuki et al., Europ J of Immunol (1992) 22 (8) 1989-1993, fully incorporated herein by reference]. The immunotherapeutic treatment may consist of allografting, in particular, allograft with hematopoietic stem cell HSC. The immunotherapeutic treatment may also consist in an adoptive immunotherapy as described by Nicholas P. Restifo, Mark E. Dudley and Steven A. Rosenberg “Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews Immunology, Volume 12, April 2012). In adoptive immunotherapy, the subject’s circulating lymphocytes, NK cells, are isolated amplified in vitro and readministered to the subject. The activated lymphocytes or NK cells are most particularly be the subject’s own cells that were earlier isolated from a blood or tumor sample and activated (or “expanded”) in vitro.

In a particular embodiment, the classical treatment refers to a treatment with an immune checkpoint inhibitor.

Typically, the invention relates to an i) FLT3 inhibitor and ii) an immune checkpoint inhibitor for simultaneous, separate or sequential use in the treatment of a solid cancer such as melanoma.

Typically, the invention relates to an i) FLT3 inhibitor and ii) an immune checkpoint inhibitor for simultaneous, separate or sequential use in the treatment of an inflammatory disease such as asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

Typically, the invention relates to an i) FLT3 inhibitor and ii) an immune checkpoint inhibitor for simultaneous, separate or sequential use in the treatment of a fibrosis.

As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins.

As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte- Associated protein 4 also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD- 1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.

In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.

In a particular embodiment, the immune checkpoint inhibitor is an antibody. Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.

In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.

In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.

In a particular embodiment, the small organic molecules interfere with Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N'- hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide :

In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:

In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand- 1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

In some embodiments, the immune checkpoint inhibitor is an aptamer.

Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of FLT3 alone or in a combination with a classical treatment) into the subject, such as by, intravenous, intramuscular, enteral, subcutaneous, parenteral, systemic, local, spinal, nasal, topical or epidermal administration (e.g., by injection or infusion). When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. In a particular embodiment, Gilteritinib is administered orally between 5 and 50 mg twice per day. In a particular embodiment, Crenolanib is administered orally between 5 and 50 mg twice per day. In a particular embodiment, Midostaurin is administered orally between 5 and 50 mg twice per day.

In a third aspect, the invention relates to a pharmaceutical for use in the treatment of macrophages related disease.

In a particular embodiment, the pharmaceutical composition according to the invention comprises a FLT3 inhibitor.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising a FLT3 inhibitor and a classical treatment as described above.

In a particular embodiment, the pharmaceutical composition according to the invention wherein the FLT3 inhibitor and a classical treatment, as combined preparation for use simultaneously, separately or sequentially in the treatment of macrophages related disease (e.g. solid cancer, inflammatory disease or fibrosis). In another embodiment, the pharmaceutical composition according to the invention, wherein the FLT3 inhibitor is Gilteritinib or Crenolanib or Midostaurin.

The FLT3 inhibitor as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, of using a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In certain embodiments, the pharmaceutical formulation can be suitable for parenteral administration. The terms “parenteral administration” and “administered parenterally,” as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In certain embodiments, the present invention provides a parenteral formulation comprising a FLT3 inhibitor and a classical as a combined preparation.

In certain embodiments, the present invention provides a parenteral formulation comprising a FLT3 inhibitor and a classical treatment as a combined preparation. For example, and not by way of limitation, the present invention provides a parenteral formulation comprising Gilteritinib or Crenolanib or Midostaurine and a classical treatment as a combined preparation. In a particular embodiment, when the FLT3 inhibitor is combined with a classical treatment, the combination is formulated for oral, cutaneous or topical use.

Method of screening of a FLT3 inhibitor

A further object of the present invention relates to a method of screening a drug suitable for the treatment of macrophage related disease comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity and/or expression of FLT3.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity of FLT3. In some embodiments, the assay first comprises determining the ability of the test compound to bind to FLT3. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of FLT3. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity of FLT3, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, peptidomimetics, small organic molecules, aptamers, or nucleic acids. For example, the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Evaluation of Gilteritinib toxicity on MO, Ml and M2-like macrophages. Human primary monocytes were differentiated during 5 days with 50 ng/mL CSF-1 and then polarized into MO-like macrophages (CSF-1), Ml -like macrophages (LPS+IFNy) or M2 -likes macrophages (IL-4/IL-13) for 4 days. Different concentrations of Gilteritinib were added two days after the CSF-1, LPS+IFNy, or IL-4-induced polarization. A) The evaluation of cell death was carried out by flow cytometry using DAPI labelling. Results are expressed in percentage of DAPI positives cells. B-C) Flow cytometry experiments were assessed using specific markers of Ml -macrophages (CD86) and M2-macrophages (CD209). Results are expressed in Mean of Fluorescence Index (MFI). Statistics were assessed using One-way ANOVA on 3 independent experiments. p<0,05 *, p<0,01 **, p<0,001 ***, p<0,0001 **** .

Figure 2: Gilteritinib inhibits the generation of M2-like macrophages. Human primary monocytes were differentiated during 5 days with 50 ng/mL CSF-1 and then polarized into MO- like macrophages (CSF-1), Ml -like macrophages (LPS+IFNy) or M2 -likes macrophages (IL- 4/IL-13) for 2 days. 0.3 pM Gilteritinib (Gilt) was added 16h before the polarization A-B) Macrophage polarization was assessed after 2 days of polarization by flow cytometry using specific markers of Ml -macrophages (HLA-DR, CD80 and CD86) and M2 -macrophages (CD163, CD206 and CD200R). Results are expressed in Mean of Fluorescence Index (MFI). C) The assessment of M2-like macrophage polarization was performed after 2 days of polarization by RT-qPCR measurement of anti-inflammatory chemokines Statistics were assessed by one-way ANOVA on 3 independent experiments. p<0,05 *, p<0,01 **, p<0,001 *** p<0 0001 ****

Figure 3: Gilteritinib reprograms M2-like macrophages. Human primary monocytes were differentiated during 5 days with 50 ng/mL CSF-1 and then polarized into MO-like macrophages (CSF-1), Ml-like macrophages (LPS+IFNy) or M2 -likes macrophages (IL-4/IL-13) for 4 days. 1 pM Gilteritinib (Gilt) was added 2 days after inducing polarization. A-B) Macrophage polarization was assessed after 4 days of polarization by flow cytometry using specific markers of Ml -macrophages (HLA-DR, CD80 and CD86) and M2-macrophages (CD163, CD206 and CD200R). Results are expressed in Mean of Fluorescence Index (MFI). C) The assessment of macrophage polarization was performed after 4 days of polarization by RT-qPCR measurement of pro- (IL-la, CCL14 and CXCL11) and anti-inflammatory chemokines (CLL13, CCL17, CCL26). Statistics were assessed by one-way ANOVA on at least 3 independent experiments. p<0,05 *, p<0,01 **, p<0,001 ***, p<0,0001 **** .

Figure 4: Gilteritinib enhances the anti-leukemic effect of CD8+ T cells. A) Flow cytometry analysis of the proportion of MLL-AF9-positive AML blasts in bone marrow of animals injected with either control OT-I or MataHari (MH) CD8 T cells, and treated with vehicle (Veh) or 30 mg/kg Gilteritinib. B) Kaplan-Meier analysis of mouse overall survival following the same experimental setting as in panel A. Statistics were assessed by one-way ANOVA. p<0,05 *, p<0,01 **, p<0,001 ***, p<0,0001 **** .

EXAMPLES:

EXAMPLE 1:

Material, Methods and Statistics:

To demonstrate the ability of the Gilteritinib compound to reprogram Ml or M2 macrophages, our experiments were performed on primary human macrophages. Macrophages are generated from primary human peripheral blood monocytes, purified by anti-CD14 magnetic sorting, and stimulated for 5 days with CSF-1 (100 ng/mL). The immature macrophages (M0) thus generated are then polarized for 2 or 4 days into pro-inflammatory macrophages (Ml) by adding LPS (100 ng/mL) + IFNg (20 ng/mL) or into anti-inflammatory macrophages (M2) by adding IL-4/IL-13 (20 ng/mL). The efficiency of Ml and M2 polarization is quantified by flow cytometry by regarding the expression level of pro- (CD80, CD86 and HLA-DR) and antiinflammatory (CD206, CD 163, CD200R, CD209) membrane markers. The results are expressed as mean of fluorescence index (MFI). The effect of Gilteritinib on macrophage polarization was assessed on the polarization induction and on the reprogramming of already polarized macrophages. To define the quantity of Gilteritinib to be added to M0, Ml and M2 macrophages, an evaluation of the compound toxicity was performed. For this, different concentrations of the molecule of Gilteritinib were added after two days of polarization and the evaluation of cell death was carried out by flow cytometry using DAPI labelling two days later. For reverse-transcription and real-time polymerase chain reaction, RNA was prepared from 6 x 10⁶ cells using the RNeasy Mini Kit according to manufacturer’s protocol (Qiagen). Each cDNA sample was prepared using AMV RT and random primers (Promega). Real-time polymerase chain reaction (PCR) was performed using the SyBR Green detection protocol (Life Technologies). Briefly, 5 ng of total cDNA, 500nM (each) primers, and 5pL SyBR Green mixture were used in a total volume of 10 pL. Detection of endogenous control L32 was used to normalize the results. Statistical analysis was performed using a one-way ANOVA test and significance was considered when P values were lower than 0.05. The results are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001, ns (not significant) according to a one-way ANOVA.

Results :

While gliteritinib's ability to kill FLT3-positive leukaemia cells is well established, its immunomodulatory role remains unclear. To explore this, we generated human monocyte- derived macrophages that we then ex vivo polarized into immature macrophages by adding CSF-1 (M-CSF), pro-inflammatory macrophages (Ml) by adding LPS+IFNg or into antiinflammatory macrophages (M2) by adding IL-4/IL-13. Thanks to this model, we demonstrated using dose response that Gilteritinib is non-toxic on different subpopulations of human macrophages tested (M0, Ml and M2), and this up to 1 pM (Figure 1A). Furthermore, Gilteritinib can significantly reduce the CD209 expression on M2 -like macrophages (Figure IB) without affecting CD86 on Ml -like macrophages (Figure 1C). This suggests that Gilteritinib has only the potential to reprogram M2-like macrophages at low micromolar concentrations. We next evaluated the ability of Gilteritinib to inhibit or reinforce the induction of Ml or M2 macrophage polarization. For this purpose, 0.3 pM Gilteritinib was added 16 hours prior to polarization induction and macrophage polarization was analyzed by flow cytometry focusing on the membrane expression level of pro-inflammatory (Figure 2A) and anti-inflammatory markers (Figure 2B). Gilteritinib significantly increased HLA-DR and downregulated CD80 on Ml macrophages (Figure 2A). More significantly, we established that Gilteritinib abrogates the expression of all analyzed M2 -markers (Figure 2B), demonstrating the ability of Gilteritinib to impair the induction of macrophage M2 polarization. The expression of anti-inflammatory chemokines was next analyzed by RT-qPCR (Figure 2C) to confirm our results. Gilteritinib addition before M2 -polarization induction blocked the mRNA expression of CCL13, CCL14, CCL17, CCL22, CCL23 and CCL26, confirming the ability of Gilteritinib to inhibit M2 macrophage polarization induction. In addition, we assessed the ability of Gilteritinib to reprogram macrophages that were already polarized. To do this, we added 1 pM of Gilteritinib after two days of polarization and the macrophages obtained were analyzed by cytometry (Figure 3A-B) and RT-qPCR (Figure 3C) two days later. Although Gilteritinib did not affect the expression of pro-inflammatory markers (Figure 3A), the FLT3 inhibitor distinctly reprogrammed M2 -like macrophages, resulting in a significant reduction in the expression of membrane M2-markers (Figure 3B) and anti-inflammatory chemokines (Figure 3C). Altogether, we have demonstrated the capacity of Gilteritinib to suppress M2- polarization and to reprogram M2 -like macrophages. In cancer, tumor-associated macrophages (TAMs) can exhibit both Ml and M2 characteristics. The presence of M2 -like tumor-associated macrophages (TAMs) is associated with tumor progression, immunosuppression, and resistance to therapy. Targeting these M2 -like TAMs could be a valuable strategy to enhance the antitumor immune response. We propose Gilteritinib as a new strategy to reprogram M2-like macrophages in diseases where these anti-inflammatory macrophages have a detrimental effect.

EXAMPLE 2:

Material and Methods:

For ELISA, we analyzed the secretome using simple plex automated immunoassays (Bio- techne). To establish the graft-versus-leukemia (GVL) mouse model, 8- to 16-month-old MataHari (MH) mice will be euthanized to collect their spleens. The cell suspension will be incubated with the UTY antigenic peptide. The cells will undergo Ficoll separation to isolate mononucleated live cells, which will then be washed and resuspended in fresh mIL-2. MataHari lymphocytes will be collected and antibody-stained for subsequent experiments. OT-I lymphocytes will be activated using a similar method, with lymph nodes also collected and incubated with the SL8 antigenic peptide. For all in vivo experiments, MLL-AF9-driven AML blasts will be injected into 5- to 10-week-old sublethally irradiated female C57BL/6 mice. Each animal will be divided into two subgroups (n=20 per group), which will subsequently be injected with either control OT-I or MH T lymphocytes. Each of these subgroups will then be treated with either a vehicle or a given small-molecule FLT3 inhibitor (n=10 mice per subgroup). Flow cytometry will be used to assess the proportion of DsRed-positive MLL-AF9 leukemic cells in bone marrow. The same experimental design will be used for the Kaplan- Meier survival analyses. Statistical analysis was performed using a one-way ANOVA test and significance was considered when P values were lower than 0.05. The results are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001, ns (not significant) according to a one-way ANOVA.

Results:

To strengthen our hypothesis that FLT3 inhibitors represent a promising strategy for reprogramming anti-inflammatory macrophages, we validated the effects of Gilteritinib by using another FLT3 inhibitor, Crenolanib (Figure 4). Interestingly, Crenolanib demonstrated no toxicity across various human macrophage subpopulations (M0, Ml, and M2) at concentrations up to 10 nM (data not shown). Notably, this concentration was sufficient to significantly reduce CD209 expression on M2-like macrophages (data not shown) while leaving CD86 expression on Ml -like macrophages unaffected (data not shown). Similar to Gilteritinib, we assessed Crenolanib's ability to modulate macrophage polarization and reprogram them (data not shown). Similar to Gilteritinib, Crenolanib effectively inhibited M2- like macrophage polarization, as demonstrated by an increase in HLA-DR expression, a reduction in M2 macrophage markers (data not shown), and the downregulation of mRNA expression for the anti-inflammatory cytokines CCL14, CCL22, and CCL23 (data not shown). Furthermore, Crenolanib successfully reprogrammed M2 -like macrophages without affecting Ml -like macrophages (data not shown). This reprogramming was marked by a significant reduction in the expression of M2 markers (data not shown) and anti-inflammatory cytokines (data not shown). Altogether, our ex vivo findings demonstrate the ability of FLT3 inhibitors, Gilteritinib and Crenolanib, to suppress M2 polarization and reprogram M2 -like macrophages into an unpolarized state. To confirm in vivo our results, we used a graft-versus-leukemia (GVL) mouse model (Figure 4). In this model, CD8+ T cells targeting the male-specific H-Y antigen (encoded by the Uty gene) are transferred into sublethally irradiated mice bearing male AML blasts transformed by MLL-AF9. These CD8+ T cells, expressing the "MataHari" (MH) T-cell receptor, exhibit cytotoxicity specifically against male leukemic blasts, allowing targeted analysis of anti-leukemia responses. To evaluate the effect of the FLT3 inhibitor, Gilteritinib, MLL-AF9+ male AML cells were injected into female mice, which were treated with 30 mg/kg Gilteritinib for three days, followed by injection with either MH or non-targeting OT-I CD8+ T cells. Results showed that disease burden was reduced and animals’ survival was significantly improved in mice receiving MH cells with Gilteritinib compared to other groups (Figures 4A and 4B). Importantly, Gilteritinib did not impact MLL-AF9 cell growth in the OT-I group, suggesting that its mechanism of action is independent of the intrinsic FLT3 signaling pathway in leukemic cells and may instead rely on a Gilteritinib-induced immune response.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A FLT3 inhibitor for use in the polarization of macrophages.

2. The FLT3 inhibitor for use according to claim 1 in the modulating the immune environment and enhancing the efficacy of classical therapy.

3. The FLT3 inhibitor for use according to claim 1 wherein said inhibitor is Gilteritinib, Crenolanib, or Midostaurin.

4. The FLT3 inhibitor for use according to claim 1 wherein the subject is a human received or susceptible to receive a transplantation.

5. The inhibitor for use according to claims 1 to 5 in the treatment of macrophage related disease.

6. The inhibitor for use according to claims 1 to 5 wherein the macrophage related disease is a disease when the immune environment within the tumour has an immunosuppressor profile.

7. The inhibitor for use according to claims 1 to 6 wherein the macrophage related disease is selected from the group consisting of but not limited to: solid cancer, fibrotic diseases, hepatic fibrosis or systemic sclerosis, Alzheimer's disease, allergy, and inflammatory disease such as asthma, rheumatoid arthritis inflammatory bowel disease or atherosclerosis.

8. The inhibitor for use according to claims 1 to 7 wherein the macrophage related disease is liver, lung or kidney fibrosis.

9. The inhibitor for use according to claims 1 to wherein the solid cancer is selected from the group consisting of but not limited to: adrenal cortical cancer, anal cancer, bile duct cancer (e.g. peripheral cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astrocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer ( e.g. ductal carcinoma in situ, infiltrating ductal carcmoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), cervical cancer, colorectal cancer, endometrial cancer ( e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors ( e.g. choriocarcinoma, chorioadenoma destruens), Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer ( e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, nonsmall cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma ( e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer ( e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer ( e.g. follicular carcinoma, anaplastie carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

10. A combined preparation comprising the inhibitor FLT3 for use according to claims 1 to 8 and a classical treatment.

11. The combined preparation for use according to claim 10 in the treatment of macrophage related disease.

12. The combined preparation for use according to claimlO, wherein the classical treatment is a compound natural or synthetic, and immunotherapy, chemotherapy, and radiotherapy.

13. A pharmaceutical composition comprising a FLT3 inhibitor for use in the treatment of macrophage related disease in a subject in need thereof.

14. The pharmaceutical composition according to claim 13, wherein the FLT3 inhibitor is

Gilteritinib or Midostaurine or Crenolanib.

15. A method for treating macrophage related disease in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of a FLT3 inhibitor.