Mutant CSF-1r extracellular domain fusion molecules and therapeutic uses thereof

Abstract

The present invention provides fusion molecules comprising the extracellular domain of CSF-1R which contains a specific single amino acid mutation. Such fusion molecules are capable of efficiently binding both CSF-1 and IL-34. The present invention also provides the use of such fusion molecules, or pharmaceutical compositions thereof, as therapeutic agents, in particular in the treatment of cancers, neurodegenerative diseases, autoimmune diseases, and allergic diseases, or in the prevention or inhibition of transplant rejection.

Description

[0001] Mutant CSF-1R Extracellular Domain Fusion Molecules and Therapeutic Uses Thereof

[0002] Related Application

[0003] The present application claims priority to European Patent Application No. EP 24 168 387 filed on April 4, 2024, which is incorporated herein by reference in its entirety.

[0004] Background of the Invention

[0005] The human colony-stimulating factor 1 receptor (CSF-1R), which is also known as macrophage colony-stimulating factor receptor (M-CSFR) and Cluster of Differentiation 115 (CD 115), is an important receptor tyrosine kinase (RTK) encoded by the c-fms protooncogene (Roth and Stanley, Curr. Top. Microbiol. Immunol., 1992, 181: 141-167). CSF-1R exists as an auto-inhibited form and activates through dimerization and autophosphorylation of several tyrosine residues initiating a signaling cascade and internalization of the receptor.

[0006] Activation of CSF-1R is mediated by its endogenous ligands, macrophage-colony stimulating factor 1 (CSF-1) and interleukin-34 (IL-34) (Stanley etal., Stem Cells, 1995, 12 Suppl. 1, 15-24; Hume et al., Blood, 2012, 119: 1810-1820). CSF-1 and IL-34 share low primary sequence homology but show similar folding / tertiary structure. They bind to overlapping regions in the extracellular domain (ECD) of CSF-1R in a homodimeric form and activate similar signaling pathways (Stanley et al., J. Biol. Chem., 1977, 252; 4305-4312; Lin et al., Science, 2008, 320: 807-811; Ma et al., Structure, 2012, 20: 676- 687; Munoz-Garcia et al., Theranostics, 2021, 11(4): 1568-1593; Liu et al, Biochim. Bioplys. Acta, 2012, 1824(7): 938-945; Boularkirba et al., Sci. Rep., 2018, 8(1): 256; Freuchet et al., J Leukoc. Biol., 2021, 110(4): 771-796). However, they have a different ability to polarize macrophages (Boulakirba et al., Scientific Reports, 2018, 8: 256).

[0007] The main biological effects of CSF-lR-signaling are the differentiation, proliferation, migration, and survival of cells of the mononuclear phagocytic lineage (Stanley and Chitu, Cold Spring Harbor Perspectives in Biology, 2014, 6(6): a021857; Cannarile et al., J. Immunother. Cancer, 2017, 5(1): 53; Dai et al., Blood, 2002, 99: 111- 120; Raivich and Kr eutzberg, Glia, 1994, 11 : 129-146). CSF-lR-mediated signaling also plays a crucial role in chemotaxis, migration, and activation of several cells, especially immune cells, thus contributing to the development of various inflammatory diseases and cancer (Cannarile et al., J. Immunother. Cancer, 2017, 5(1): 53; Ries et al., Cancer Cell, 2014, 25(6): 846-859; Stanley and Chitu, Cold Spring Harbor Perspectives in Biology, 2014, 6(6): a021857; Kumari et al., Biomed. Pharm., 2018, 103: 662-679; Lelio et al., J. Exp. Med., 2020, 217(3): e20190290). Over the past decades, the promising therapeutic potential of CSF-lR-signaling inhibition has been widely studied for decreasing immune suppression and escape in tumors, owing to depletion and reprogramming of tumor-associated macrophages (TAMs). Agents against CSF-lR-signaling have been increasingly investigated in preclinical or clinical studies in cancer, inflammatory disorders and neurodegenerative diseases. Three main different blocking strategies have been tested (Freuchet et al., J Leukoc. Biol., 2021, 110(4): 771- 796): blocking antibodies against CSF-1R, blocking antibodies against IL-34 or CSF-1, and tyrosine kinase inhibitors specific for CSF-1R. The use of CSF-1R extracellular domain (ECD) fusion molecules has also been suggested for the treatment of osteolytic disorders and cancer (U.S. Pat. No. 8,183,207 B2) and for the treatment of rheumatoid arthritis and multiple sclerosis (U.S. Pat. No. 8,080,246 B2).

[0008] Although promising therapeutic approaches for CSF-lR-signaling inhibition are being developed and tested in clinical trials, there is still a need in the art for improved strategies for targeting the CSF-1R / IL-34 / CSF-1 axis.

[0009] Summary of the Invention

[0010] The present Inventors have hypothesized that potential limitation of current therapeutic approaches for CSF-lR-signaling inhibition is linked to the fact that no strategy simultaneously targets both cytokines. They have shown that specific singleresidue mutations of the extracellular domain (ECD) of CSF-1R lead to a mutant CSF- 1R ECD, which, in a soluble and dimeric form, is capable of binding both IL-34 and CSF- 1 more efficiently than the wild-type CSF-1R ECD. Using human monocytes sorted from fresh PMBCs obtained from healthy donors, the present Inventors have shown that the mutant (M149K) CSF-1RECD covalently linked to a human Fc ((M149K) CSF-1RECD- Fc) induces a significantly better inhibition of cell viability than the wild-type CSF-1R ECD linked to the same human Fc (WT CSF-1R ECD-Fc). The same was found to be true when the monocytes were incubated in the presence of recombinant human IL-34 or CSF-1. In an ex vivo model of malignant pleural mesothelioma (MPM), they showed that (M149K) CSF-1R ECD-Fc inhibits monocytes differentiation more efficiently than WT CSF-IRECD-Fc. Furthermore, in this ex vivo model, the mutant CSF-1RECD was found to be more efficient than blocking antibodies (anti-IL-34 antibody and anti-CSF-1 antibody alone or in combination), while the wild-type CSF-1R ECD was observed to be about as efficient as the blocking antibodies (alone or in combination).

[0011] Consequently, the present invention provides a mutant extracellular domain of CSF- 1R (CSF-1R ECD), wherein said mutant CSF-1R ECD consists of a wild-type CSF-1R ECD comprising a single amino acid mutation selected from M149K mutation, S172W mutation, Q173K mutation, and S172K mutation.

[0012] In certain embodiments, the wild-type CSF-1R ECD consists of the amino acid sequence set forth in SEQ ID NO: 7 or in SEQ ID NO: 8, preferably in SEQ ID NO: 7.

[0013] In certain embodiments, the mutant CSF-1R ECD is:

[0014] - the (M149K) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 9; or

[0015] - the (S172W) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 10; or

[0016] - the (Q173K) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 11, or

[0017] - the (S172K) mutation CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 16.

[0018] In certain embodiments, the fusion molecule consisting of a mutant CSF-1R ECD as defined above is linked to at least one fusion partner. For example, the fusion partner may be an immunoglobulin Fc domain, in particular a silent human immunoglobulin Fc domain, such as a silent human immunoglobulin Fc domain which consists of the amino acid sequence set forth in SEQ ID NO: 12.

[0019] In certain embodiments, the mutant CSF-1R ECD fusion molecule as defined above is such that :

[0020] - the mutant CSF-1R ECD is the (M149K) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 13; or - the mutant CSF-1R ECD is the mutant CSF-1R ECD is the (S172W) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 14; or

[0021] - the mutant CSF-1R ECD is the (Q173K) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 15; or

[0022] - the mutant CSF-1R CED is the (S172K) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 17.

[0023] The present invention further provides an isolated nucleic acid encoding a mutant CSF-1R ECD as defined above or a fusion molecule as defined above.

[0024] The present invention also provides a vector comprising such a nucleic acid.

[0025] The present invention yet provides a host cell comprising such a nucleic acid and / or such a vector.

[0026] The present invention further provides a mutant CSF-1R ECD fusion molecule as defined above, or an isolated nucleic acid as defined above, or a vector as defined above, or a host cell as defined above, for use as a therapeutic agent.

[0027] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a mutant CSF-1R ECD as defined above, or a mutant CSF-1R ECD fusion molecule as defined above, and at least one pharmaceutically acceptable carrier or excipient.

[0028] In certain embodiments, the pharmaceutical composition further comprises at least one additional biologically active agent.

[0029] The present invention further provides a mutant CSF-1R ECD as defined above, or a mutant CSF-1R ECD fusion molecule as defined above, or an isolated nucleic acid as defined above, or a vector as defined above, or a host cell as defined above, or a pharmaceutical composition as defined above, for use in the treatment of a disease in a subject, wherein the disease is a cancer, a neurodegenerative disease, an autoimmune disease, an allergic disease, or a bone disease, or in the prevention or inhibition of transplant rejection in a subject. These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

[0030] Brief Description of the Drawing

[0031] Figure 1. Prediction of the free energy of mutation (AAGmut) of some residues of hCSF-lR located in the interface with IL-34 or CSF-1. The AAGmut at pH 7.4 was calculated for the substitutions of some residues of hCSF-lR, using the method of Spassov and Yan (see Examples section) implemented under Discovery Studio (Dassault Systemes BIO VIA Release 2017, San Diego) in the protocol “Calculate Mutation Energy (Binding)”. The input data were the atomic coordinates of IL-34 / CSF-1R and CSF- 1 / CSF-1R complexes (PDB codes 4DKD and 4WRL, respectively). The Figure presents the results obtained for mutations at positions: (A) V143, M149, and 1170; (B) S172, QI 73 and R192; and (C) V231, Q248 and S250.

[0032] Figure 2. Wild-type (WT) CSF-IR-Fc does inhibit the activation by IL-34 of CSF- 1R pathways in a human monocytic THP-1 cell line, while a monomeric form of CSF-1R ECD does not. The cells were stimulated with IL-34 (1 nM) or a vehicle alone for 5 minutes in the presence or absence of increasing concentrations of (A) WT CSF-IR-Fc or (B) a monomeric form of CSF-1R ECD.

[0033] Figure 3. Mutant (M149) CSF-IR-Fc (also called mutant (M149K) CSF-1R ECD- Fc) traps soluble IL-34 (A) and soluble CSF-1 (B) and thereby inhibits activation of CSF- 1R pathways (pAkt and pERK) in a reporter cell line (human monocytis THP-1). No cytokine and IL-34 / CSF-1 alone were respectively used as negative and positive controls in this experiment. (n=8 different experiments). Mean ± SEM; two-way ANOVA; **** p<0.0001.

[0034] Figure 4. Mutant (M149) CSF-IR-Fc (also called mutant (M149K) CSF-1R ECD- Fc) traps IL34 and CSF-1 and inhibits human monocytes viability more efficiently than WT CSF-IR-Fc (also called WT CSF-1R ECD). WT CSF-IR-Fc and mutant (M149K) CSF-IR-Fc were mixed with fresh human monocytes and 3 days later, cells were stained with a viability dye (FVD450). No cytokine and IL-34 alone (4 nM) were respectively used as negative and positive controls in the experiments. (n=8 / 9). Mean ± SEM; two- way ANOVA; * p<0.05 ** p<0.01. Figure 5. Mutant (M149K) CSF-IR-Fc traps endogenous IL34 and CSF-1 in MPM pleural effusion (PE), and inhibits human monocytes viability and differentiation in M2 macrophages (CD 163+ cells). (A) PE containing only CSF-1 (n=5 with 3 different patients) and (B) PE containing both CSF-1 and IL-34 (n=7 with 5 different patients). In this experiment, no cytokine and PE alone were used as negative and positive controls, respectively. Neutralizing antibodies against IL-34 and against CSF-1 were also used as controls (alone or in combination). Mean ± SEM; One-way ANOVA; ns: not significant, * p<0.05 ** p<0.01.

[0035] Figure 6. Mutant (M149K) CSF-1R traps IL-34 and CSF-1 naturally produced by Meso34 MPM, inhibits human monocytes viability and restores cytotoxicity of CD8+ T cells. Meso34 cells and monocytes from healthy donors were seeded in low adherence 96-round well plates for 5 days then: (A) spheroids were dissociated and stained to analyze macrophages viability. Results are expressed as percentage (%) of CD14+ among live cells (n= 8 using 5 different donors); or (B) CD8 T cell clone specific of HLA- A*0201 / MUCl(950-958) were added to spheroids at effector / target ratio 5 / 1 and 24 hours later supernatants were recovered, luciferase activity measured and % lysed cells calculated (n= 6 using 3 donors).

[0036] Definitions

[0037] Throughout the specification, several terms are employed that are defined in the following paragraphs.

[0038] As used herein, the term “subject’ refers to a human or another mammal (e.g., primate, mouse, rat, rabbit, dog, cat, horse, cow, goat, pig, camel, and the like), including laboratory animals, that may or may not have a disease or disorder, in particular a disease or disorder associated with CSF-1 and / or IL-34 or one of their receptors, z.e., CSF-1R, CD138, PTPzeta and TREM2. Non-human subjects may be transgenic or otherwise modified animals. In most embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an “individual” or a “patient” . The terms “individual” and “patient” do not denote a particular age. The term “patient” more specifically refers to an individual suffering from a disease or disorder, in particular any disease where IL-34, CSF-1, CSF-1R, CD138, PTPzeta, and / or TREM2 are detrimental, including cancers, neurodegenerative diseases, autoimmune diseases, and bone diseases such as osteoporosis. As used herein, the term “cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth, lack of differentiation and ability to invade local tissues and metastasize. Cancer can develop in any tissue of any organ. Examples of cancers include, but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, glioblastoma, mesothelioma, tenosynovial giant cell tumor, and pituitary adenoma.

[0039] The terms “aggressive” and “invasive” are used herein interchangeably. When used herein to characterize a cancer, they refer to the proclivity of a tumor for expanding beyond its boundaries into adjacent tissue. Invasive cancer can be contrasted with organ- confined cancer wherein the tumor is confined to a particular organ. The invasive property of a tumor is often accompanied by the elaboration of proteolytic enzymes, such as collagenases, which degrade matrix material and basement membrane material to enable the tumor to expand beyond the confines of the capsule, and beyond confines of the particular tissue in which that tumor is located.

[0040] The term “metastasis” , as used herein, refers to the spread of tumor cells from one organ or tissue to another location. The term also refers to tumor tissue that forms in a new location as a result of metastasis. A “metastatic cancer” is a cancer that spreads from its original, or primary, location, and may also be referred to as a “secondary cancer” or “secondary tumor”. Generally, metastatic tumors are named for the tissue of the primary tumor from which they originate. The process of tumor metastasis is a multistage event involving local invasion and destruction of intercellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival in the circulation, extravasation out of the vessels in the secondary site and growth in the new location. The term neurodegenerative disease , as used herein, refers to a disease that occurs as a result of neurodegenerative processes, i.e., progressive loss of structure or function of neurons and / or death of neurons. Neurodegenerative diseases are incurable and debilitating, and affected patients typically have problems with movement (ataxias) and / or mental functioning (dementias). Examples of neurodegenerative diseases include, but are not limited to, Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Lewy body dementia, frontotemporal dementia, and amyotrophic lateral sclerosis (ALS). Other examples include motor neuron diseases, demyelinating diseases (such as Guillain-Barre syndrome and multiple sclerosis), prion disease, spinocerebellar ataxia, and spinal muscular atrophy.

[0041] The terms autoimmune disease and autoimmune inflammatory disease are used herein interchangeably. They refer to a disease in which the immune system produces an immune response (for example, a B-cell or a T-cell response) against an antigen that is part of the normal host (that is an auto-antigen), with consequent injury to tissues. In an autoimmune disease, the immune system of the host fails to recognize a particular antigen as “self’ and an immune reaction is mounted against the host’s tissues expressing the antigen. Autoimmunity can affect any organ in the body including, for example, the brain, skin, kidney, lungs, liver, heart, intestine and thyroid. Therefore, autoimmune diseases are anatomically diverse and the clinical expression of the disease depends upon the site affected. The resulting inflammation and tissue damage can cause kidney failure, breathing problems, abnormal heart function, pain, deformity, delirium, and death. Autoimmune diseases can affect connective tissues, i.e., tissues that connect or separate, and support all the other types of tissues in the body. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, and psoriasis.

[0042] The term “allergic disorder , as used herein, refers to a disease, symptom, or condition that may be defined as an abnormal immune response to innocuous environmental substances (allergens). Allergens can enter the body through many routes, including through breathing, ingestion, skin contact or injection (including insect bites). Allergies disorders include, but are not limited to, allergic rhinitis (e.g., hay fever), sinusitis, rhinosinusitis, chronic or recurrent otitis media, drug reactions, insect sting reactions, latex reactions, conjunctivitis, urticaria, anaphylaxis and anaphylactoid reactions, atopic dermatitis, asthma, and food allergies.

[0043] The term “osteoporosis”, as used herein, has its art understood meaning and refers to a systemic skeletal disease, characterized by low bone mass and deterioration of bone tissue, which result in an increase in bone fragility and susceptibility to fracture. The elderly are at greatest risk of osteoporosis. The problem is therefore predicted to increase significantly with the aging of the population. There are a number of causes of osteoporosis. Hormone-deficiencies (estrogen in women and androgen in men) are the leading cause.

[0044] As used herein, the term “transplant rejection” encompasses both acute and chronic transplant rejection. “Acute rejection” is the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunological foreign. Acute rejection is characterized by infiltration of the transplant tissue by immune cells of the recipient, which carry out their effector function and destroy the transplant tissue. The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. “Chronic transplant rejection” generally occurs in humans within several months to years after engraftment, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants.

[0045] By “preventing or reducing / inhibiting transplant rejection”, as used herein, is meant to encompass prevention or inhibition of immune transplant rejection, as well as delaying the onset of the progression of transplant rejection. The term is also meant to encompass prolonging survival of a transplant in a patient, or reversing failure of a transplant in a patient. The term is further meant to encompass ameliorating a symptom of an immune transplant rejection, including, for example, ameliorating an immunological complication associated with immune rejection, such as for example interstitial fibrosis, chronic graft atherosclerosis, or vasculitis.

[0046] The term “treatment” is used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease, disorder or condition (here a cancer or a neurodegenerative disease or an autoimmune disease); (2) slowing down or stopping the progression, aggravation or deterioration of the disease, disorder or condition; (3) bringing about amelioration of the symptoms of the disease, disorder or condition; or (4) curing the disease, disorder or condition. A treatment may be administered after initiation of the disease, disorder or condition, for a therapeutic action. Alternatively, a treatment may be administered prior to the onset of the disease, disorder or condition, for a prophylactic or preventive action. In this case, the term “prevention is used.

[0047] A pharmaceutical composition is defined herein as comprising an effective amount of at least one biological active ingredient (e.g., a mutant CSF-1R ECD fusion molecule described herein), and at least one pharmaceutically acceptable carrier or excipient.

[0048] As used herein, the term “effective amount' refers to any amount of a compound (e.g., a mutant CSF-1R ECD fusion molecule), agent, or composition that is sufficient to fulfil its intended purpose(s), e.g., a desired biological, diagnostic, or medicinal response in a cell, tissue, system or subject. For example, an effective amount of a mutant CSF- 1R ECD fusion molecule is an amount that can elicit a measurable amount of a desirable outcome, e.g., inhibition of CSF-1R activation and signalling; or in a method of treatment, an amount that can reduce or ameliorate by a measurable amount, a symptom of the disease or condition that is being treated.

[0049] As used herein, the term pharmaceutically acceptable carrier or excipient' refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s) and which is not excessively toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion media, coatings, encapsulating material, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example “Remington ’s Pharmaceutical Sciences", E.W. Martin, 18thEd., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety). Preferably, the pharmaceutical carrier or excipient is acceptable in human medicine. In certain embodiments, the pharmaceutically acceptable carrier or excipient is a veterinary acceptable carrier or excipient.

[0050] The term “vector", as used herein, refers to a polynucleotide that may be engineered to contain a cloned polynucleotide or polynucleotides that may be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and / or enhancers) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that may be used in colorimetric assays, e.g., 3-galactosidase). The term “ expression vector ' refers to a vector that is used to express a polypeptide of interest in a host cell.

[0051] As used herein, the term “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells, fungal cells; plant cells; and insect cells. Certain exemplary mammalian cells include, but are not limited to, HEK293 and CHO cells, and their derivatives, such as HEK293-6E and DG44 cells, respectively.

[0052] The term “isolated”, as used herein, refers to a molecule that has been separated from at least some of the components with which it is typically found in nature. For example, a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide. Similarly, a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated” so long as that polynucleotide is not found in that vector in nature.

[0053] The terms “approximately” and about , as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).

[0054] Detailed Description of Certain Preferred Embodiments

[0055] As mentioned above, the present invention provides mutant CSF-1R ECD fusion molecules that efficiently capture both CSF-1 and IL-34, thereby being capable of inhibiting the activation of the CSF-lR-mediated signaling pathways. The present invention also provides pharmaceutical compositions comprising at least one mutant CSF-1R ECD fusion molecule described herein and the use of the fusion molecules, and pharmaceutical compositions thereof, as therapeutic agents, in particular in the treatment of cancers, neurodegenerative diseases, autoimmune diseases, and allergic disorders, and in the prevention or inhibition of transplant rejection.

[0056] I - Mutant CSF-1R ECD Fusion Molecules

[0057] 1 - CSF-1R

[0058] As used herein, the term “CSF-1R” has its art-understood meaning and refers to the colony-stimulating factor 1 receptor, which is also referred to as FMS, FIM2, C-FMS, M- CSF receptor and CD115. CSF-1R, which is encoded by the c-fms protooncogene (Roth and Stanley, Curr. Top. Microbiol. Immunol., 1992, 181 : 141-167) is known since 1986 (Coussens etal., Nature, 1986, 320: 277-280). Ligand binding of the colony-stimulating factor 1 (CSF-1) or the interleukin 34 ligand (IL-34) to CSF-1R leads to receptor dimerization, upregulation of CSF-1R protein tyrosine kinase activity, phosphorylation of CSF-1R tyrosine residues, and downstream signaling events (z.e., “biological activity”).

[0059] In preferred embodiments of the invention, the CSF-1R is a human CSF-1R (Swiss Prot P07333, NCBI Reference Sequence: NP_001275634.1) having the amino acid sequence set forth in SEQ ID NO: 1 :

[0060] 1 MGPGVLLLLL VATAWHGQGI PVIEPSVPEL WKPGATVTL RCVGNGSVEW DGPPSPHWTL

[0061] 61 YSDGSSSILS TNNATFQNTG TYRCTEPGDP LGGSAAIHLY VKDPARPWNV LAQEVWFED

[0062] 121 QDALLPCLLT DPVLEAGVSL VRVRGRPLMR HTNYSFSPWH GFTIHRAKFI QSQDYQCSAL

[0063] 181 MGGRKVMSI S IRLKVQKVI P GPPALTLVPA ELVRIRGEAA QIVCSASSVD VNFDVFLQHN

[0064] 241 NTKLAI PQQS DFHNNRYQKV LTLNLDQVDF QHAGNYSCVA SNVQGKHSTS MFFRWESAY

[0065] 301 LNLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPK LANATTKDTY

[0066] 361 RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRY PPEVSVIWTF INGSGTLLCA

[0067] 421 ASGYPQPNVT WLQCSGHTDR CDEAQVLQVW DDPYPEVLSQ EPFHKVTVQS LLTVETLEHN

[0068] 481 QTYECRAHNS VGSGSWAFI P I SAGAHTHPP DEFLFTPVW ACMSIMALLL LLLLLLLYKY

[0069] 541 KQKPKYQVRW KI IESYEGNS YTFIDPTQLP YNEKWEFPRN NLQFGKTLGA GAFGKWEAT

[0070] 601 AFGLGKEDAV LKVAVKMLKS TAHADEKEAL MSELKIMSHL GQHENIVNLL GACTHGGPVL

[0071] 661 VITEYCCYGD LLNFLRRKAE AMLGPSLSPG QDPEGGVDYK NIHLEKKYVR RDSGFSSQGV

[0072] 721 DTYVEMRPVS TSSNDSFSEQ DLDKEDGRPL ELRDLLHFSS QVAQGMAFLA SKNCIHRDVA

[0073] 781 ARNVLLTNGH VAKIGDFGLA RDIMNDSNYI VKGNARLPVK WMAPESI FDC VYTVQSDVWS

[0074] 841 YGILLWEI FS LGLNPYPGIL VNSKFYKLVK DGYQMAQPAF APKNIYSIMQ ACWALEPTHR

[0075] 901 PTFQQICSFL QEQAQEDRRE RDYTNLPSSS RSGGSGSSSS ELEEESSSEH LTCCEQGDIA

[0076] 961 QPLLQPNNYQ FC, wherein amino acid residues 1-19 of SEQ ID NO: 1 constitute the signal peptide.

[0077] Topologically, CSF-1R comprises a N-terminal extracellular domain (ECD), a transmembrane domain, and a C-terminal cytoplasmic domain. These domains can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, e.g., Stryer, Biochemistry, 3rdEd., 1988) and any of a number of internet-based sequence analysis programs. Similar to other members of the platelet-derived growth factor (PDGF) family to which it belongs, the intracellular domain of CSF-1R (amino acid residues 538-972 of SEQ ID NO: 1) is comprised of a juxtamembrane domain (JDM), and an intracellular tyrosine kinase domain that is interrupted by a kinase insert domain; and the extracellular domain of CSF-1R is a highly glycosylated extracellular region comprised of five immunoglobulin domains (subdomains D1-D5) (Coussens et cd., Nature, 1986, 32: 277- 280; Rotherwell and Rohr schneider, Oncogene Res., 1987, 1 : 311-324; Hampe et al., Oncogene Res., 1989, 4: 9-17).

[0078] CSF-1 and IL-34 bind to CSF-1R within the subdomains DI to D3 of the extracellular domain of the CSF-1R (CSF-1R ECD). The subdomains D4 and D5 are not involved in the CSF-1 and / or IL34 binding (Wang et al., Molecular and Cellular Biology, 1993, 13: 5348-5359). The subdomain D4 is involved in CSF-1R dimerization (Yeung et al., Molecular and Cellular Proteomics, 2003, 2: 1143-1155; Pixley et al., Trends Cell. Biol., 2004, 14: 628-638).

[0079] Subdomain DI (Ig-like C2-type 1) has the sequence SEQ ID NO: 2, which corresponds to amino acid residues 21 to 104 SEQ ID NO: 1.

[0080] SEQ ID NO: 2:

[0081] PVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNA TFQNTGTYRCTEPGDPLGGSAAIHLYVKDP.

[0082] Subdomain D2 (Ig-like C2-type 2) has the sequence SEQ ID NO: 3, which corresponds to amino acid residues 107 to 197 of SEQ ID NO: 1.

[0083] SEQ ID NO: 3:

[0084] PWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSP WHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQK.

[0085] Subdomain D3 (Ig-like C2-type 3) has the sequence SEQ ID NO: 4, which corresponds to amino acid residues 203 to 290 of SEQ ID NO: 1.

[0086] SEQ ID NO: 4:

[0087] PALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNR YQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTS. Subdomain D4 (Ig-like C2-type 4) has the sequence SEQ ID NO: 5, which corresponds to amino acid residues 299 to 399 of SEQ ID NO: 1.

[0088] SEQ ID NO: 5:

[0089] AYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKL

[0090] ANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLR.

[0091] Subdomain D5 (Ig-like C2-type 5) has the sequence SEQ ID NO: 6, which corresponds to amino acid residues 402 to 502 of SEQ ID NO: 1.

[0092] SEQ ID NO: 6:

[0093] PEVSVIWTFINGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPY

[0094] PEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSVGSGSWAFIPIS.

[0095] 2 - CSF-1R ECD

[0096] The terms CSI -1R extracellular domain and CSI -1R EC!) are used herein interchangeably. They refer to a portion of the CSF-1R protein which is capable of interacting with the extracellular environment. In other words, the term “CSF-1R ECD” refers to a CSF-1R polypeptide that lacks the intracellular and transmembrane domains.

[0097] In certain embodiments, the CSF-1R ECD is the entire amino acid sequence of the CSF-1R protein which is external of a cell or cell membrane. The term [full-length CSF- 1R ECD”, as used herein, refers to a CSF-1R ECD that extends to the last amino acid residue of the extracellular domain, and may or may not include an N-terminal signal peptide. For example, the last amino acid of the full-length CSF-1R ECD is at position 512 of SEQ ID NO: 1 for the human CSF-1R ECD. Thus, the human full-length CSF- 1R ECD may consist of the amino acid sequence set forth in SEQ ID NO: 7 (mature form, i.e., without the signal peptide) or in SEQ ID NO: 8 (with the signal peptide), wherein:

[0098] SEQ ID NO: 7 is:

[0099] IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSKDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFIPISAGAHTHPPDE, and

[0100] SEQ ID NO: 8 is: MGPGVLLLLLVATAWHGQGIPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWT LYSDGSSS ILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWF EDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSKDYQC SALMGGRKVMS IS IRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVF LQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRV VESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANA TTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFING SGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLL TVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTHPPDE .

[0101] In certain embodiments, the CSF-1R ECD is a portion of an amino acid sequence of the CSF-1R protein needed for ligand binding as may be assayed using a method known in the art (e.g., in vitro ligand binding assay). Such a portion is called a CSF-1R ECD fragment. As used herein, the term “CSF-1R ECD fragment” refers to a CSF-1R ECD having one or more (e.g., 2, 3, or 4) amino acid residues deleted from the N or C terminus of the full-length ECD and that retains the ability to bind to the CSF-1 and IL- 34 ligands. The CSF-1R ECD fragment may or may not include an N-terminal signal peptide.

[0102] In certain preferred embodiments, the CSF-1R ECD is the human full-length CSF- 1R ECD that consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0103] 3 - Mutant CSF-1R ECD

[0104] As used herein, the term “mutant CSF-1R ECI) refers to an extracellular domain of the colony-stimulating factor 1 receptor, mainly human CSF-1R ECD, which comprises a single amino acid residue mutation, wherein the single amino acid residue mutation is a substitution, z.e., the replacement of a wild-type amino acid residue with a different amino acid residue.

[0105] Preferably, the single amino acid residue mutation is located in subdomain D2.

[0106] In preferred embodiments, the single amino acid residue mutation (z.e., the substitution) takes place at position 149, or position 172 or position 173 of the human full-length CSF-1R ECD which consists of SEQ ID NO: 8 (with the signal peptide). Thus, for example, the term “position 149” refers to the 149thlocation of an amino acid residue in the CSF-1R extracellular domain of SEQ ID NO: 8, as it appears in the sequence from left to right.

[0107] In certain embodiments, the mutation is selected from the group consisting of the M149K mutation (wherein the wild-type methionine (M) is replaced with a lysine (K)), the S172W mutation (wherein the wild-type serine (S) is replaced with a tryptophan (W)), the Q173K mutation (wherein the glutamine (G) is replaced with a lysine (K)), and the S172K mutation (wherein the wild-type serine (S) is replaced with a lysine (K)). Thus, the mutant CSF-1R ECD may be the human full-length CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 7 or in SEQ ID NO: 8, and which comprises one single amino acid residue mutation selected from the group consisting of the M149K mutation, the S172W mutation, the Q173K mutation, and the S172K mutation.

[0108] In particular, in certain preferred embodiments, the mutant CSF-1R ECD is the (M149K) mutant CSF-1R ECD, which comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 9: IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLKRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFI P I SAGAHTHPPDE, wherein the mutation appears in bold and is underlined. In certain embodiments, the mutant CSF-1R ECD is the (M149K) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 9.

[0109] In other preferred embodiments, the mutant CSF-1R ECD is the (S172W) mutant CSF-1R ECD, which comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 10:

[0110] IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQWQDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFI P I SAGAHTHPPDE, wherein the mutation appears in bold and is underlined. In certain embodiments, the mutant CSF-1R ECD is the (S172W) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 10. In yet other embodiments, the mutant CSF-1R ECD is the (Q173K) mutant CSF- 1R ECD, which comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 11 :

[0111] IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSKDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFI P I SAGAHTHPPDE, wherein the mutation appears in bold and is underlined. In certain embodiments, the mutant CSF-1R ECD is the (Q173K) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 11.

[0112] In yet other embodiments, the mutant CSF-1R ECD is the (S172K) mutant CSF-1R ECD, which comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 16:

[0113] IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQKQDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFI P I SAGAHTHPPDE, wherein the mutation appears in bold and is underlined. In certain embodiments, the mutant CSF-1R ECD is the (S172K) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 16.

[0114] As will be understood by one skilled in the art, the present invention encompasses a mutant CSF-1R ECD as described herein. The present invention further encompasses the use of a mutant CSF-1R ECD in therapeutic applications (see below).

[0115] 4 - Mutant CSF-1R ECD Fusion Molecules

[0116] As used herein, the term mutant CSF-1R ECD fusion molecule refers to a molecule comprising a mutant CSF-1R ECD, as defined above, linked to at least one fusion partner. As used herein, the term “linked”, and associated terms such as “link” and “linkage”, refer to a connection between two chemical groups or two molecules (e.g.. between the mutant CSF-1R ECD and the fusion partner). The connection can be accomplished covalently (by covalent chemical bonds) or non-covalently, for example, by physical forces such as electrostatic bonds, hydrogen bonds, ionic bonds, van der Waals bonds, or hydrophobic / hydrophilic interactions. The connection may be direct or indirect. When indirect, the connection may be via a linker or spacer. The terms “linker and spacer " are used herein interchangeably, and refer to a molecule that joins two other molecules (e.g., the mutant CSF-1R ECD and the fusion partner) either covalently or non- covalently. Linkers and spacers, in particular peptidic linkers and spacers, used in peptide modifications are known in the art.

[0117] Thus, in certain embodiments, the mutant CSF-1R ECD and the fusion partner are covalently linked (z.e., “fused”). If the fusion partner is also a polypeptide (then referred to as “fusion partner polypeptide), the mutant CSF-1R ECD and the fusion polypeptide may be part of a continuous amino acid sequence, and the fusion partner polypeptide may be linked to either the N-terminus or the C-terminus of the mutant CSF-1R ECD. In such cases, the mutant CSF-1R ECD and the fusion partner polypeptide may be translated as a single polypeptide from a coding sequence that encodes both the mutant CSF-1R ECD and the fusion partner polypeptide.

[0118] In other embodiments, the mutant CSF-1R ECD and the fusion partner are covalently linked through other means, such as, for example, a chemical linkage other than a peptide bond. Many known methods of covalently linking polypeptides to other molecules (for example, fusion partners) may be used.

[0119] In still other embodiments, the mutant CSF-1R ECD and the fusion partner are non- covalently linked, for example using binding pairs. Exemplary binding pairs include, but are not limited to, biotin and avidin or streptavidin, an antibody and its antigen, etc.

[0120] As used herein, the term “fusion partner refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) that can be linked to the mutant CSF-1R ECD. Certain exemplary fusion partners include, but are not limited to, an immunoglobulin Fc domain, albumin, polyethylene glycol, glutathione-S-transf erase, a His-6-tag, and a FLAG-peptide. In certain embodiments, the fusion partner is chosen for its ability to confer one or more desirable properties to the mutant CSF-1R ECD. Thus, the fusion partner may be selected for its ability to facilitate purification of the mutant CSF-1R ECD, to increase stability and half-life of the mutant CSF-1R ECD in therapeutic application, or to facilitate dimerization of the extracellular of CSF-1R (as occurs, for example, when the fusion partner is an immunoglobulin Fc region).

[0121] In certain preferred embodiments, the fusion partner is an immunoglobulin Fc domain. The terms “immunoglobulin Fc domain", “immunoglobulin Fc region and “Fc domain" are used herein interchangeably. They refer to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The terms include native sequence Fc regions and variant Fc regions. The amino acid sequences of immunoglobulin Fc domains that can be used as fusion partners are known in the art. In certain embodiments, the immunoglobulin Fc domain used as fusion partner is a silent Fc domain. For Fc-fusion molecules meant to act solely as blocking agents, receptor agonists, or through other mechanisms, the Fc effector function is unnecessary, and can create safety concerns and unwanted side effects by activating host immune defenses. Several amino acid modifications in the Fc region have been reported to silence or reduce the effector function of the Fc domain. Thus, the term “silent Fc domain" refers to a Fc domain comprising at least one amino acid modification that silences or reduces the effector function of the Fc domain Such amino acid modifications are known in the art (Wang et al., Protein Cell, 2018, 9(1): 63-73); Lazar et al., Proc. Natl. Acad. Sci. USA, 2006, 103: 4005-4010), including, but not limited to, the mutations L235G (LE) (Alegre el al., J. Immunol., 1992, 148: 3461-3468); L234A / L235A (LALA) (Wines et al., J. Immunol., 2000, 164: 5313-5318; Lund et al., J. Immunol., 1991, 147: 2657- 2662; Hezareh el al., J. Virol., 2001, 75: 12161-12168); L234A / L235A / P329G (LALA- PG) (134); P331S / L234E / L235F (TM) (Oganesyan et al., Acta Crystallogr. D Biol. Crystallogr., 2008, 64; 700-704; Xu etal, J. Biol. Chem., 1994, 269: 3469-3474), D265A (DA) (Lund et al., J. Immunol., 1996, 157: 4963-4969), G236R / L328R(GRLR) (Horton et al., Blood, 2010, 116: 3004-3012; Horton et al, Cancer Res., 2008, 68: 8049-8057), and the like.

[0122] In certain preferred embodiments, the fusion partner is a silent human immunoglobulin Fc domain having the amino acid sequence set forth in SEQ ID NO: 12:

[0123] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein the mutations (L234A, L235A, A330S, P331S) are underlined and in bold. In certain embodiments, the fusion molecule comprises a mutant CSF-1R ECD, as defined above, linked to a silent human immunoglobulin Fc domain of SEQ ID NO: 12 via a GS motif consisting of an N-terminal glycine residue (Gly or G) followed by a serine residue (Ser or S).

[0124] When the mutant CSF-1R ECD is the (M149K) mutant CSF-1R ECD, the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 13:

[0125] IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLKRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFIPISAGAHTHPPDEGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK .

[0126] When the mutant CSF-1R ECD is the (S172W) mutant CSF-1R ECD, the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 14:

[0127] IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQWQDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFIPISAGAHTHPPDEGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK .

[0128] When the mutant CSF-1R ECD is the (Q173K) mutant CSF-1R ECD, the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 15:

[0129] IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSKDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFIPISAGAHTHPPDEGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK .

[0130] When the mutant CSF-1R ECD is the (S172K) mutant CSF-1R ECD, the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 17: IPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSS ILSTNNATFQN TGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVWFEDQDALLPCLLTDPVLEAG VSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQKQDYQCSALMGGRKVMS IS IRLKVQ KVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNN RYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRWESAYLNLSSEQNLIQEVT VGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKP SEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSV GSGSWAFIPISAGAHTHPPDEGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK .

[0131] 5 - Preparation of Mutant CSF-1R ECDs and Mutant CSF-1R ECD Fusion Molecules

[0132] The mutant CSF-1R ECDs and mutant CSF-1R ECD fusion molecules disclosed herein may be prepared using any of a variety of art-recognized methods well-known in the art for designing and making proteins, including chemical synthesis (e.g., conventional automated peptide synthesis methods) and recombinant expression in a suitable host cell.

[0133] For example, the mutant CSF-lRECDs of the present invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols, as described by Stewart and Young, in Solid Phase Peptide Synthesis, Freeman, San Francisco, 1969, pp. 24-66; Tam et al., J. Am. Chem. Soc., 1983, 105: 6442; Merrifield, Science, 1986, 232(3748): 341-347. In particular, solid-phase peptide synthesis, which was initially described by R.B. Merrifield (J. Am. Chem. Soc. 1963, 85: 2149-2154), is a quick and easy approach to synthesizing peptides and peptidic molecules of short known sequences. A compilation of such solid-state techniques may be found, for example, in “ Solid Phase Peptide Synthesis’" (Methods in Enzymology, G.B. Fields (Ed.), 1997, Academic Press: San Diego, CA, which is incorporated herein by reference in its entirety). Most of these synthetic procedures involve the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Once the desired peptide is assembled, it is cleaved off from the solid support, precipitated, and the resulting free peptide may be analyzed and / or purified as desired. Solution methods as described, for example, in “The Proteins’" (Vol. II, 3rdEd., H. Neurath et al. (Eds.), 1976, Academic Press: New York, NY, pp. 105-237), may also be used to synthesize the mutant CSF-1R ECDs described herein.

[0134] As an alternative to automated peptide synthesis, recombinant DNA technology (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel etal., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, N.Y., 1994) may be employed to produce a mutant CSF-1R ECD or a mutant CSF-1R ECD fusion molecule. In such methods, a nucleotide sequence (e.g., DNA) which encodes the polypeptide of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression. The nucleotide coding sequence may be readily prepared synthetically using methods known in the art (see, for example, Edge et al., Nature, 1981, 292: 756-762). Insertion of the nucleotide coding sequence into the expression vector results in the coding sequence being operatively linked to the necessary regulatory sequences. Expression systems containing the requisite control sequences, such as promoters and polyadenylation signals, and preferably enhancers, are readily available for a variety of hosts (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., 1989, Cold Spring Harbor Press: Cold Spring, NY; and R. Kaufman, Methods in Enzymology, 1990, 185: 537-566). A variety of expression vector / host systems may be utilized to contain and express the polypeptide (i.e., mutant CSF-1RECD or mutant CSF-1RECD fusion molecule)-encoding nucleotide sequence. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of polypeptides. Mammalian cells that are useful in recombinant protein productions include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138; BHK, HepGe, 3T3, RIN, MDCK, A549, PC12, K562 and HEK293 cells. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be important for correct insertion, folding and / or function. Different host cells such as CHO, HeLa, MDCK, HEK293, W138, and the like have specific cellular machinery and characteristic mechanisms for such post- translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

[0135] The transformed host cells are then cultured and maintained under conditions favoring expression of the desired polypeptide. The polypeptide thus produced is recovered and isolated, either directly from the culture medium or by lysis of the cells.

[0136] Mutant CSF-1R ECDs and mutant CSF-1R ECD fusion molecules according to the present invention can be purified using a variety of standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, differential solubilization, and the like.

[0137] It is recognized that “purity” is a relative term, and not to be necessarily construed as absolute purity or absolute enrichment or absolute selection. In some embodiments, the purity of the retrieved mutant CSF-1R ECD or mutant CSF-1R ECD fusion molecule is at least or about 60%, at least or about 70%, at least or about 80%, or at least or about 90% (e.g., at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%) or is approximately 100%. In some preferred embodiments, the mutant CSF-1R ECDs or mutant CSF-1R ECD fusion molecules described herein are preferably retrieved in “substantially pure form”. As used herein, the term “substantially pure” refers to a purity that allows for the effective use of the mutant CSF-1R ECD or mutant CSF-1R ECD fusion molecule in the applications to which it is intended. In treatment applications in humans, substantially pure preferably refers to at least or about 97%, at least or about 98%, at least or about 99%, or approximately 100% pure. The purity of any given protein or polypeptide, generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well-known to those of skill the art.

[0138] If desired, after preparation, a mutant CSF-1R ECD or mutant CSF-1R ECD fusion molecule may be sterilized. Sterilization may be carried out using any of a wide variety of sterilization techniques known in the art, for example, by filtration through a bacteria- retaining filter, by gamma irradiation, by electron-beam irradiation, or by incorporating sterilizing agents in the form of a sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Alternatively, or additionally, the mutant CSF-1R ECD or mutant CSF-1R ECD fusion molecule may be lyophilized prior to use.

[0139] The mutant CSF-1R ECDs and mutant CSF-1R ECD fusion molecules of the present invention can be commercially synthesized by specialized companies, such as for example Proteogenix (Schiltigheim, France), Genepep (Montpellier, France), Genscript (Piscataway, N.J.), New England Peptide (Gardner, Mass.), and CPC Scientific (Sunnyvale, Calif.), Peptide Technologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems (San Diego, Calif.).

[0140] Mutant CSF-1R ECDs and mutant CSF-1R ECD fusion molecules may be stored under appropriate conditions, either in a solution or in a solid form. For example, after preparation / purification or upon receipt from a provider, the protein products may be kept in a cool, dark place. For best preservation, they can be stored under refrigeration at 4°C or colder, away from bright light. For storage longer than days or weeks, they may preferably be stored at -20°C or at -80°C.

[0141] As will be understood by one skilled in the art, the present invention encompasses a nucleotide encoding a mutant CSF-1R ECD or encoding a mutant CSF-1R ECD fusion molecule, as described herein; a vector, in particular an expression vector, comprising such a nucleotide; and a host cell comprising such a vector. The present invention further encompasses the use of such nucleotide, vector or host cell in therapeutic applications (see below). 6 - Properties of Mutant CSF-1R ECD Fusion Molecules

[0142] A mutant CSF-1R ECD fusion molecule according to the present invention exhibits a significant binding affinity for both CSF-1 and IL-34. Furthermore, it is more efficient than the corresponding wild-type CSF-1R ECD fusion molecule at binding both CSF-1 and IL-34.

[0143] The term “CSF-1”, as used herein, has its art-understood meaning and refers to the colony stimulating factor 1, which is also known as macrophage colony-stimulating factor (M-CSF). In the context of the present invention, the term “CSF-1” refers to human CSF- 1. Three isoforms of the human protein are produced by alternative splicing. The active form of CSF-1 is found extracellularly as a disulfide-linked homodimer, and is thought to be produced by proteolytic cleavage of membrane-bound precursors. CSF-1 is a hematopoietic growth factor that is involved in the proliferation, differentiation, and survival of monocytes, macrophages, and bone marrow progenitor cells. It promotes the release of proinflammatory chemokines, and thereby plays an important role in innate immunity and in inflammatory processes. Aberrant expression of CSF-1 or of its receptor CSF-1R, can promote cancer cell proliferation, invasion and formation of metastases. Overexpression of CSF-1 or CSF-1R is observed in a significant percentage of breast, ovarian, prostate, and endometrial cancers. Aberrant expression of CSF-1 or CSF-1R may play a role in inflammatory diseases.

[0144] As used herein, the term IL-34 refers to a cytokine called interleukin-34, also known as C16orf77, which was first described in 2008. In the context of the present invention, the term “IL-34” refers to human interleukin-34, which is encoded by the IL34 gene located on chromosome 16. In addition to CSF-1R, IL-34 has three other receptors: the receptor-type tyrosine-protein phosphatase zeta (PTP-zeta), syndecan-1 (CD 138, in fact a co-receptor), and triggering receptor expressed on myeloid cells 2 (TREM2). It has been shown that IL-34 binds to CSF-1R with higher affinity than to PTPz and CD138 wherein the affinities of IL-34 for TREM2 and for CSF-1R are similar. (M149K) CSF- 1R ECD-Fc and WT CSF-1R ECD-Fc were found to inhibit with similar affinity the binding of IL-34 to PTPz, TREM2 and CD138. IL-34 has been shown as pairing with CSF-1 to form a heterodimer. Until now, studies have demonstrated that IL-34 is released by some tissues that differ from those where CSF-1 is expressed, and is involved in the differentiation and survival of macrophages, monocyte, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation (Guillonneau et al., Cell Mol. Life Sci., 2017, 74(14): 2569-2586).

[0145] The terms “PTP-zeta” ,“ PTP-C and “PTP-z” are used herein interchangeably and refer to the receptor-type tyrosine-protein phosphatase zeta. PTP-zeta is also known in the art as phosphacan. This receptor is a single-pass type I membrane protein with two cytoplasmic tyrosine-protein phosphatase domains, an alpha-carbonic anhydrase domain and a fibronectin type III domain.

[0146] As used herein, the term “CD138” has its general meaning in the art and refers to the cluster of differentiation 138, also known as syndecan-1. This receptor is a transmembrane (type I) heparan sulfate proteoglycan. The syndecan-1 core protein consists of an extracellular domain which can be substituted with heparan sulfate and chondroitin sulfate glycosaminoglycan chains, a highly conserved transmembrane domain, and a highly conserved cytoplasmic domain, which contains two constant regions that are separated by a variable region.

[0147] As used herein, the term “TREM2” refers to triggering receptor expressed on myeloid cells 2, a protein that is encoded by the TREM2 gene in humans. TREM2 is expressed on macrophages, immature monocyte-derived dendritic cells, osteoclasts, and microglia, which are immune cells in the central nervous system. In the liver, TREM2 is expressed by several cell types, including macrophages, that respond to injury. In the intestine, TREM2 is expressed by myeloid-derived dendritic cells and macrophage. TREM2 is overexpressed in many tumor types and has anti-inflammatory activities.

[0148] The term “binding affinity” is the strength of the binding interaction between a single molecule to its ligand or binding partner. It is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of biomolecule interactions. An equilibrium dissociation constant may be measured using any appropriate method known in the art, such as those described by Harlow et al., “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1988, Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol., 1983, 92: 589-601, which are incorporated herein by reference in their entirety. In certain embodiments, a dissociation constant is determined using a Surface Plasmon Resonance (SPR) assay. The term “binding”, as used herein, refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges. In particular, in the context of the present invention, the term “binding” refers to the binding of a ligand (e.g., CSF-1 or IL-34) with the extracellular domain region of a mutant CSF-1R ECD fusion molecule according to the present invention with an affinity corresponding to a dissociation constant, KD, of about 10'7M or less, such as about 10'8M or less, or about 10'9M or less, or about IO'10M or less, or about 10'11M or less.

[0149] In certain embodiments, a mutant CSF-1R ECD fusion molecule binds to CSF-1 or IL-34 with a dissociation constant KD of less than 8 nM, for example about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM or less, for example about 1 nM or about 0.5 nM or less.

[0150] A mutant CSF-1R ECD fusion molecule according to the present invention is said to be more efficient that the corresponding wild-type CSF-1R ECD fusion molecule if the mutant CSF-1R ECD fusion molecule binds to each of CSF-1 and IL-34 with a dissociation constant ko that is at least 1.2 times lower than the dissociation constant ko of the corresponding wild-type CSF-1R ECD fusion molecule, for example about 1.3 times lower, about 1.4 times lower, about 1.5 times lower, about 1.6 times lower, about 1.7 times lower, about 1.8 times lower, about 1.9 times lower or at least 2 times lower, such as for example, about 2.1 times lower, about 2.2 times lower, about 2.3 times lower, about 2.4 times lower, about 2.5 times lower, about 2.6 times lower, about 2.7 times lower, about 2.8 times lower, about 2.9 times lower, or at least 3 or more times lower than the dissociation constant ko of the corresponding wild-type CSF-1R ECD fusion molecule.

[0151] II - Therapeutic Applications of the Mutant CSF-1R ECD Fusion Molecules

[0152] As already mentioned above, the mutant CSF-1R ECD fusion molecules described herein exhibit a higher binding affinity for CSF-1 and IL-34 than the corresponding wildtype CSF-1R ECD fusion molecule. Thus, the mutant CSF-1R ECD fusion molecules may be used as ligand traps in vivo to treat diseases associated with CSF-1 and / or IL-34 and / or with their receptors, i.e., CSF-1R, CD138, PTPzeta, and TREM2. In particular, the mutant CSF-1R ECD fusion molecules may be used in the treatment of cancer, neurodegenerative diseases, autoimmune diseases, allergic disorders, and bone diseases, and in the prevention or inhibition of transplant rejection.

[0153] In the above paragraph as well as in the following paragraphs which deal with therapeutic applications, the term mutant CSF-1R ECD fusion molecule is used. However, as already mentioned above, the present invention also encompasses the use of a mutant CSF-1R ECD, a nucleotide encoding a mutant CSF-1R ECD or encoding a mutant CSF-1R ECD fusion molecule, a vector, in particular an expression vector, comprising such a nucleotide; and a host cell comprising such a vector in the treatment of cancer, neurodegenerative diseases, autoimmune diseases and allergic disorders, or in the prevention or inhibition of transplant rejection.

[0154] 1 - Indications

[0155] A. Cancer

[0156] The present invention relates to a mutant CSF-1R ECD fusion molecule described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) for use in the treatment of a cancer in a subject. The present invention further relates to a method for the treatment of a cancer in a subject, the method comprising a step of administering to the subject in need thereof a therapeutically effective amount of at least one mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof. The present invention also relates to the use of a mutant CSF-1R ECD fusion molecule described herein for the manufacture of a medicament for the treatment of cancer.

[0157] In the practice of the present invention, the cancer to be treated using a mutant CSF- 1R ECD fusion molecule described herein may be any cancer developed in any tissue or organ. Thus, the cancer may be a carcinoma, lymphoma, blastoma, sarcoma, or leukemia.

[0158] Examples of cancers that may be treated using a method according to the present invention include, but are not limited to, cancer of the bone, lung, liver, pancreas, skin, head or neck, brain, gum, tongue, skin, eye, uterus, ovary, anus / rectum, stomach, colon, nasopharynx, breast, sexual and reproductive organs, Hodgkin’s Disease, esophagus, gastrointestinal tract, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, bone marrow, bladder, kidney, pelvis, central nervous system (CNS), neuroectodermal cancer, spinal axis, In some embodiments, the subject to be treated with a mutant CSF-1R ECD fusion molecule according to the present invention suffers from a cancer selected from the group consisting of acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS- related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B- cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone Cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, carcinoma of the penis, carcinoma of unknown primary site, carcinosarcoma, Castleman's Disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear-cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family of tumor, Ewing family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, hemangiosarcoma, hematological malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, Lentigo maligna melanoma, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant Glioma, mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, metastatic squamous neck cancer with occult primary, metastatic urothelial carcinoma, mixed Mullerian tumor, monocytic leukemia, mouth cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic disease, myelodysplasia, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasm, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, Non-Hodgkin lymphoma, nonmelanoma skin cancer, non-small cell lung cancer, non-small cell lung cancer (NSCLC) which coexists with chronic obstructive pulmonary disease (COPD), ocular oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath, meningioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget’s disease of the breast, pancoast tumor, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, pituicytoma, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastema, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular cancer, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract carcinoma involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Ley dig cell tumor, sex cord-stromal tumor, Sezary syndrome, Signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, soot wart, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, stomach cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial-stromal tumor, synovial sarcoma, T-cell acute, lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, tenosynovial giant cell tumor, teratoma, terminal lymphatic cancer, testicular cancer, thecoma, throat cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell cancer of renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital neoplasm, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom’s macroglobulinemia, Warthin’s tumor, Wilms’ tumor, and any combination thereof.

[0159] In certain embodiments, the cancer is a solid malignant tumor. In particular, the solid cancer may be selected from the group consisting of malignant pleural mesothelioma, glioblastoma, and tenosynovial giant cell tumor.

[0160] In certain embodiments, the cancer is metastatic cancer.

[0161] B. Neurodegenerative Diseases

[0162] The present invention relates to a mutant CSF-1R ECD fusion molecule described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) for use in the treatment of a neurodegenerative disease in a subject. The present invention further relates to a method for the treatment of a neurodegenerative disease in a subject, the method comprising a step of administering to the subject in need thereof a therapeutically effective amount of at least one mutant CSF- 1R ECD fusion molecule , or a pharmaceutical composition thereof. The present invention also relates to the use of a mutant CSF-1R ECD fusion molecule described herein for the manufacture of a medicament for the treatment of a neurodegenerative disease.

[0163] Examples of neurodegenerative diseases that may be treated using a method according to the present invention include, but are not limited to, Parkinson’s disease and related disorders including Parkinson-dementia, autosomal recessive PARK2 and PARK6-linked Parkinsonism, atypical parkinsonian syndromes, including, progressive supranuclear palsy, corticobasal degeneration syndrome, Lewy bodies dementia, multiple system atrophy, Guadeloupean Parkinsonism and Lytigo-bodig disease; motor neuron diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy and post-polio syndrome; neuro-inflammatory diseases; Alzheimer’s disease and related disorders including early stage of an Alzheimer’s disorder, mild stage of an Alzheimer’s disorder, moderate stage of an Alzheimer’s disorder, mild to moderate stage of an Alzheimer’s disorder, advanced stage of an Alzheimer’s disorder, mild cognitive impairment, vascular dementia, mixed dementia, Pick’s disease, argyrophilic grain disease, posterior cortical atrophy, Wernicke- Korsakoff Syndrome; prion diseases; lysosomal storage diseases; leukodystrophies; Huntington’s Disease; multiple sclerosis; Down syndrome; spinal and bulbar muscular atrophy; HIV- Associated Neurocognitive Disorder; Tourette Syndrome; autosomal dominant spinocerebellar ataxia; Friedreich’s Ataxia; Dentatorubral pallidoluysian atrophy; myotonic dystrophy; schizophrenia; age-associated memory impairment; autism and autism spectrum disorders; attention-deficit hyperactivity disorder; chronic pain; alcohol-induced dementia; progressive non-fluent aphasia; semantic dementia; spastic paraplegia; fibromyalgia; post-Lyme disease; neuropathies; withdrawal symptoms; Alpers’ disease; cerebro-oculo-facio-skeletal syndrome; Wilson’s disease; Cockayne syndrome; Leigh’s disease; neurodegeneration with brain iron accumulation; opsoclonus myoclonus syndrome; alpha-methylacyl-CoA racemase deficiency; Andermann syndrome; Arts syndrome; Marinesco-Sjogren syndrome; mitochondrial membrane protein-associated neurodegeneration; pantothenate kinase-associated neurodegeneration; polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy; riboflavin transporter deficiency neuronopathy; and ataxia telangiectasia.

[0164] In certain embodiments, the neurodegenerative disease to be treated using a mutant CSF-1R ECD fusion molecule according to the present invention is selected from the group consisting of multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD).

[0165] C. Autoimmune Diseases and Allergic Disorders

[0166] The present invention relates to a mutant CSF-1R ECD fusion molecule described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) for use in the treatment of an autoimmune disease or an allergic disorder in a subject. The present invention further relates to a method for the treatment of an autoimmune disease or an allergic disorder in a subject, the method comprising a step of administering to the subject in need thereof a therapeutically effective amount of at least one mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof. The present invention also relates to the use of a mutant CSF-1R ECD fusion molecule described herein for the manufacture of a medicament for the treatment of an autoimmune disease or an allergic disorder.

[0167] Examples of autoimmune diseases that may be treated using a method according to the present invention include, but are not limited to, arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis, inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn’s disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE- mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen’s encephalitis, limbic and / or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener’s granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu’s) arteritis, medium vessel vasculitis, Kawasaki’s disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA- associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRC A), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex- mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet’s or Behcet’s disease, Castleman’s syndrome, Goodpasture’s syndrome, Reynaud’s syndrome, Sjogren’s syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter’s disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto’s disease, chronic thyroiditis (Hashimoto’s thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave’s disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan’s syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger’s disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig’s disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal garnmopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt’s syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressier’s syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas’ disease, rheumatic fever, recurrent abortion, farmer’s lung, erythema multiforme, post-cardiotomy syndrome, Cushing’s syndrome, birdfancier’s lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport’s syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampler’s syndrome, Caplan’s syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman’s syndrome, Felty’s syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch’s cyclitis, Henoch- Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer’s disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan’s syndrome, autoimmune gonadal failure, Sydenham’s chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck’s disease, cryoglobulinemia, Dupuytren’s contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich’s disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain’s thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Lesihmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex -mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loftier’ s syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0168] Allergic disorders that may be treated using a method according to the present invention include, but are not limited to, systemic allergic reactions, systemic anaphylaxis or hypersensitivity responses, anaphylactic shock, drug allergies, and insect sting allergies; respiratory allergic diseases, such as asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis and interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, or other autoimmune conditions); rhinitis, hay fever, conjunctivitis, allergic rhinoconjunctivitis and vaginitis; skin and dermatological disorders, including psoriasis and inflammatory dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiforms, linear IgA disease, acute and chronic urticaria and scleroderma; vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity vasculitis); spondyloarthropathies; and intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn’s disease, ulcerative colitis, ileitis, enteritis, nontropical sprue and celiac disease).

[0169] Other diseases that may be treated using a method according to the present invention include, but are not limited to, macrophage activation syndrome or hemophagocytic lymphohistiocytosis (HLH) (Paolino et cd., Front Oncol., 2022, 12 1016318; Summerlin et al., Ann. Pharmacother., 2023, 57(7): 867-879), which is associated to genetic diseases of immune cells or infections, cancer and autoimmune conditions, such as SARS-Cov-2 (Chen et al., Front Immunol., 2023, 14: 1200289) and Still’s disease (Ruscitti et al., Nature Rev. Rheumatol., 2024, 20(2): 116-132). HLH-like syndromes are associated with cytokine storm release in cancer therapy with monoclonal antibodies and CAR-T cells.

[0170] D. Bone Diseases

[0171] The present invention also relates to a mutant CSF-1R ECD fusion molecule described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) for use in the treatment of a bone disease, in particular osteoporosis, in a subject. The present invention further relates to a method for the treatment of a bone disease, in particular osteoporosis, in a subject, the method comprising a step of administering to the subject in need thereof a therapeutically effective amount of at least one mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof. The present invention also relates to the use of a mutant CSF-1R ECD fusion molecule described herein for the manufacture of a medicament for the treatment of a bone disease, in particular osteoporosis.

[0172] E. Transplant Rejection

[0173] The present invention also relates to a mutant CSF-1R ECD fusion molecule described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) for use in the prevention or reduction of transplant rejection in a subject. The present invention further relates to a method for the prevention or reduction of transplant rejection in a subject, the method comprising a step of administering to the subject in need thereof a therapeutically effective amount of at least one mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof. The present invention also relates to the use of a mutant CSF-1R ECD fusion molecule described herein for the manufacture of a medicament for the prevention or reduction of transplant rejection in a subject.

[0174] The donor of a transplant may be a living donor or a deceased donor, namely a cadaveric donor. The transplant may be an organ, a tissue, or cells. In some embodiments, the transplant rejection is cardiac allotransplant rejection. In some embodiments, the transplanted cells are selected from the group consisting of multipotent hematopoietic stem cells derived from bone marrow, peripheral blood, or umbilical cord blood; or pluripotent (z.e., embryonic stem cells (ES) or induced pluripotent stem cells (iPS)) or multipotent stem cell-derived differentiated cells of different cell lineages such as cardiomyocytes, beta-pancreatic cells, hepatocytes, neurons, etc. In some embodiments, the cells are used for allogeneic hematopoietic stems cell transplantation (HSCT) and thus comprise multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. HSCT can be curative for patients with leukemia and lymphomas. However, an important limitation of allogeneic HCT is the development of graft versus host disease (GVHD), which occurs in a severe form in about 30-50% of patients who receive this therapy. Accordingly, in certain embodiments, a mutant CSF-1R ECD fusion molecule described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) may be administered to a subject in need thereof for preventing or reducing Graft-versus-Host-Disease(GVHD). Preferably the GVHD is chronic GVHD. Thus, the patient who receives hematopoietic stem cell transplantation suffers from a disease selected from the group consisting of acute myeloid leukemia (AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia (CML); myelodysplasia syndrome (MDS) / myeloproliferative syndrome; lymphomas such as Hodgkin and non-Hodgkin lymphomas, chronic lymphatic leukemia (CLC) and multiple myeloma.

[0175] F. Other Diseases

[0176] It will be understood by one skilled in the art that a method of treatment according to the present invention may be used to treat any disease or disorder that has not been specifically listed above but that is known to be associated with CSF-1 and / or IL-34 and / or any of their receptors, i.e., CSF-1R, CD138, PTPzeta and TREM2.

[0177] 2 - Administration

[0178] A mutant CSF-1R ECD fusion molecule (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) can be administered to a subject in need thereof, in a desired dosage, by any suitable route. Various delivery systems are known and can be used to administer mutant CSF-1R ECD fusion molecules of the present invention, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and oral routes. A mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof, may be administered by any convenient or other appropriate route, for example, by infusion or bolus injection, by adsorption through epithelial or mucocutaneous linings (e.g., oral, mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Parenteral administration may be directed to a given tissue of the patient, such as by catheterization. As will be appreciated by those of ordinary skill in the art, in embodiments where a mutant CSF-1R ECD fusion molecule is administered along with an additional therapeutic agent, the mutant CSF-1R ECD fusion molecule and the therapeutic agent may be administered by the same route (e.g., orally) or by different routes (e.g., orally and intravenously).

[0179] A mutant CSF-1R ECD fusion molecule (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) may alternatively be administered incorporated in catheters, needles, or implants. 3 - Dosage

[0180] Administration of a mutant CSF-1R ECD fusion molecule (optionally after formulation with one or more appropriate pharmaceutically acceptable) will be in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend upon the therapeutic effect desired, the severity of the disease being treated, the age, sex, weight and general health condition of the patient as well as upon the potency, bioavailability and in vivo half-life of the mutant CSF-1RECD fusion molecule used, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable by the attending physician in the course of the therapy. Alternatively, or additionally, the dosage to be administered can be determined from studies using animal models. Adjusting the dose to achieve maximal efficacy based on these or other methods are well known in the art and are within the capabilities of trained physicians. As studies are conducted using mutant CSF-1R ECD fusion molecules of the invention, further information will emerge regarding the appropriate dosage levels and duration of treatment. For example, in certain embodiments, an effective amount is one that delays or prevents the onset of cancer, and / or one that slows down or stops the progression, aggravation, or deterioration of the symptoms of cancer, and / or one that brings about amelioration of the symptoms of cancer, and / or one that prevents, delays and / or reduces the likelihood of occurrence of metastases formation and / or one that reduces the number, growth rate, size, etc. of metastases if metastases are already present in the subject. The effects of a treatment according to the invention may be monitored using any of the diagnostic assays, tests and procedures known in the art.

[0181] A treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of a mutant CSF-1R ECD fusion molecule, or pharmaceutical composition thereof, may be constant for a certain period of time or periodic and at specific intervals, e.g., hourly, daily, weekly (or at some other multiple day interval), monthly, yearly (e.g., in a time release form). Alternatively, the delivery may occur at multiple times during a given time period, e.g., two or more times per week, two or more times per month, and the like. The delivery may be continuous delivery for a period of time, e.g., intravenous delivery.

[0182] In general, however, a suitable dose will be in the range of from about 0.001 to about 100 mg / kg body weight of the recipient per day, e.g., from about 0.01 to about 100 mg / kg of body weight per day, such as above about 0.1 mg / kg body weight per day, or in a range of from about 1 to about 10 mg / kg of body weight per day. For example, a suitable dose can be about 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, or 30 mg / kg of body weight per day.

[0183] In certain embodiments, the mutant CSF-1R ECD fusion molecule of the present invention may be conveniently administered in unit dosage form, for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form. In other embodiments, the mutant CSF-1R ECD fusion molecule of the present invention may be conveniently administered in unit dosage form, for example, containing about 0.0001 to 1 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1 mg or even about 10 mg per dose of the active ingredient per unit dosage form. In yet other embodiments, the dosage unit contains about 0.1 mg, about 0.5 mg, about 1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100 mg, of active ingredient.

[0184] 4 - Concomitant Therapies

[0185] A treatment according to the present invention may be administered alone or in combination with another therapy (z.e., a therapeutic agent and / or a therapeutic procedure), in particular a therapy known to be beneficial to a patient suffering from the disease to be treated. A mutant CSF-1R ECD fusion molecule described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) may be administered prior to administration of the additional therapeutic agent or procedure, concurrently with the therapeutic agent or procedure, and / or following administration of the additional therapeutic agent or procedure.

[0186] Therapeutic agents that may be administered in combination with the mutant CSF- 1R ECD fusion molecule, or a pharmaceutical composition thereof, may be selected among a large variety of biologically active compounds that are known to have a beneficial effect in the treatment of the disease to be treated (e.g., a cancer or a neurodegenerative disease or an autoimmune disease or an allergic disorder or a bone disease) or to have a beneficial effect to a patient in general (e.g., anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof).

[0187] Anti-cancer agents that may be administered in combination with a mutant CSF-1R ECD fusion molecule, or pharmaceutical composition thereof, include drugs conventionally classified into one of the following groups: alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, anti-metabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antihormones and anti-androgens. Examples of such anti-cancer agents include, but are not limited to, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane and amifostine.

[0188] Other examples of such anti-cancer agents include therapeutic antibodies used in the treatment of cancer, including, but not limited to, anti-CD52 antibodies such as alemtuzumab (CAMPATH™), which is used in the treatment of chronic lymphocytic leukemia; anti-VEGF antibodies including bevacizumab (AVASTIN™) used in the treatment of colorectal cancer and breast cancer; anti-CD33 antibodies, including gemtuzumab ozogamicin (MYLOTARG™) used in the treatment of acute myeloid leukemia; anti-CD20 antibodies including ibritumomab (ZEVALIN™) used in the treatment of lymphoma, rituximab (RITUXAN™) used in the treatment of Hodgkin lymphoma, tositumomab (BEXXAR™) used in the treatment of Hodgkin lymphoma and of atumumab (ARZERRA™) used in the treatment of chronic lymphocytic leukemia; anti-EGFR antibodies such as cetuximab (ERBITUX™) used in the treatment of colorectal cancer, head and neck cancer, and squamous cell carcinoma, and panitumumab (VECTIBEX™) used in the treatment of colorectal cancer; anti-Her2 antibodies, including trastuzumab (HERCEPTIN™) used in the treatment of breast cancer and stomach cancer; anti-CTLA4 antibodies including Ipilimumab (YERVOY™) used in the treatment of melanoma; adnectins; and domain antibodies. Active fragments and fusions of these antibodies will also find use herein.

[0189] In certain embodiments, a mutant-CSF-lR ECD fusion molecule, or a pharmaceutical composition thereof, is administered in combination with immunotherapy. As used herein, the term immunotherapy has its art understood meaning and refers to the treatment that consists in administering an immunogenic agent, i.e., an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response. The immunotherapy may consist in the administration of a immune checkpoint inhibitor. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the near future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitors includes PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonist, and BTLA antagonists.

[0190] PD-1 (Programmed Death-1) axis antagonists that can be used in the context of the present invention include PD-1 antagonists (for example anti-PD-1 antibodies), PD-L1 (Programmed Death Ligand-1) antagonists (for example anti-PD-Ll antibodies) and PD- L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibodies). An anti-PD-1 antibody may be selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and OPDIIVO®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, KEYTRUDA®, and SCH- 900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). A PD-1 binding antagonist may be AMP -224 (also known as B7-DCIg). An anti-PD-Ll antibody may be selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in W02007 / 005874. Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010 / 077634 Al. MEDI4736 is an anti-PD-Ll antibody described in WO201 1 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and W02006 / 121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009 / 114335. CT- 011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP -224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Atezolimumab is an anti- PD-Ll antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-Ll antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in W02015033301 and WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and / or US 20120114649. A PD-1 inhibitor may be an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. A PD- L1 antagonist may be selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI- A1014, A110, KY1003 and Atezolimumab, and preferably Avelumab, Durvalumab or Atezolimumab.

[0191] CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonists that can be used in the context of the present invention may be selected from the group consisting of anti-CTLA- 4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti-CTLA- 4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the co-stimulatory pathway, the antibodies disclosed in WO 2001 / 014424, the antibodies disclosed in WO 2004 / 035607, the antibodies disclosed in U.S. Publication No. 2005 / 0201994, and the antibodies disclosed in EP Patent No. 1212422 B. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01 / 14424 and WO 00 / 37504; and in U.S. Publication Nos. 2002 / 0039581 and 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98 / 42752; U.S. Pat. Nos. 6,682,736 and 6,207, 156; Hurwitz etal., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301- 5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7, 132,281. A preferred clinical CTLA-4 antibody is a human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00-9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01 / 14424. CTLA-4 antagonists (antibodies) are known and include Tremelimumab (CP-675,206) and Ipilimumab. In certain embodiments, a mutant-CSF-lR ECD fusion molecule, or a pharmaceutical composition thereof, is administered in combination with a CTLA-4 antagonist and a PD-1 antagonist.

[0192] Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG- 3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al.. 2007, J. Immunol. 179: 4202-4211). Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., Clin. Cancer Res., 2012, 18: 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et aL, J. Exp. Med., 2010, 207: 2175-2186 and Sakuishi etal., 2010, J. Exp. Med., 2010, 207: 2187-94). As used herein, the term “TIM-3” has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM- 3 is galectin 9 (Gal9). Accordingly, the term“TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM- 3 signaling pathway and / or block the binding of TIM-3 to galectin-9. Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO201 1155607, WQ2013006490 and WO2010117057.

[0193] In some embodiments, the immune checkpoint inhibitor may be an IDO inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), beta-(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-m ethoxy-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 beta-carboline derivative or a brassilexin derivative.

[0194] In certain embodiments, the immunotherapy administered in combination with a mutant-CSF-lR ECD fusion molecule of the present invention, or a pharmaceutical composition thereof, is CAR-T cell therapy. CAR-T cell therapy refers to the use of genetic engineering techniques to activate T-cells collected from peripheral blood and produce Chimeric Antigen Receptor (CAR) on the T cells, followed by mass culture and amplification in vitro and then reinfusion into patients.

[0195] Anti-cancer therapeutic procedures that may be performed in combination with administration of a mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof, include, but are not limited to, surgery, radiation therapy, and the like.

[0196] As known in the art, treatment of neurodegenerative syndromes is limited and largely symptomatic. Anti -neurodegenerative agents that may be administered in combination with a mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof, generally include, but are not limited to agents from the two classes of medication approved for cognition: cholinesterase inhibitors (e.g., donepezil, rivastigmine, galatamine) and N-methyl-D-aspartate (NMD A) receptor antagonists (e.g., memantine, amantadine, ketamine, nitro-memantine, dextromethorphan, methadone, dextropropoxyphene, ketobemidone); levodopa, carbidopa, dopamine agonists (e.g., pramipexole, ropinirole, rotigotine, apomorphine); catechol O-methyltransferase (COMT) inhibitors (e.g., entacapone, tolcapone, opicapone, nebicapone); monoamine oxidase-B inhibitors (e.g., selegiline, rasagiline, safinamide); and other medications that may address behavorial issues such as irritability, anxiety or depression including: antidepressants, such as citalopram, fluoxetine, paroxetine, sertraline and trazodone; anxiolytics such as lorazepam and oxazepam; antipsychotics such as aripiprazole, clozapine, haloperidol, olanzapine, quetiapine, risperidone and ziprasidone; drugs for mood stabilization such as carbamazepine; medications for sleep changes such as tricyclic antidepressants (e.g., nortriptyline, trazodone), benzodiazepines (e.g., lorazepam, oxazepam and temazepam), zolpidem, zaleplon, chloral hydrate, risperidone, onlanzapine, quetiapine, and haloperidol, and any beneficial combination thereof.

[0197] In the case of neurodegenerative disorders, therapeutic procedures that may be used in combination with administration of a mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof, include, but are not limited to, speech therapy, which has shown some efficacy with aphasia sy dromes (acquired language dysfunctions due to neurological injury of disease); physical therapy, which can prove helpful in prolonging motor function; neuropsychological rehabilitation (amelioration of cognitive, emotional, psychosocial, and behavioral deficits caused by an insult to the brain); physiotherapy (for restoring movement and function); and occupational therapy (to develop, recover, or maintain the meaningful activities of everyday life).

[0198] While there is no known definitive cure to autoimmune diseases, symptoms can be alleviated and controlled with treatment. Anti-autoimmune agents that can be administered in combination with a mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof, include, but are not limited to, immunosuppressant drugs to reduce the immune response against the body’s own tissues, such as: nonsteroidal anti-inflammatory drugs (NSAIDs), which can help with inflammation, swelling, stiffness, fever, and pain (e.g., aspirin, ibuprofen, acetaminophen, and naproxen); glucocorticoids (e.g., hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, deflazacort, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, beclometasone); disease-modifying anti -rheumatic drugs (DMARDs) (e.g., methotrexate, sulfasalazine, hydroxychloroquine, leflunomide, azathioprine); calcineurin inhibitors such as cyclosporine; and immunosuppressive biologies (e.g., IL-1 blockers such as anakinra, canakinumab, and rilonacept, IL-6 blockers such as tocilizumab, and sarilumab, TNF- inhibitors such as adalimumab infliximab, golimumab, certolizumab, and etanercept, T- cell activity blockers such as abatacept, B-cells targeting agents such as rituximab, B-cell growth factor targeting agents such as belimumab, IL- 17 blockers such as secukinumab, ixekizumab and brodalumab, IL-12 / 23 blockers such as ustekinumab).

[0199] In the case of autoimmune diseases, therapeutic procedures that may be used in combination with administration of a mutant CSF-1R ECD fusion molecule, or a pharmaceutical composition thereof, include, but are not limited to, surgery (for example, to treat bowel blockage in the case of Crohn’s disease), physical therapy to encourage mobility in particular when the autoimmune disease impacts bones, joints, or muscles), blood transfusions if the disease is blood-related, and any beneficial combination thereof.

[0200] Ill - Pharmaceutical Compositions and Packs and Diagnostic Kits

[0201] 1 - Pharmaceutical Compositions

[0202] As mentioned above, in therapeutic applications, a mutant CSF-1R ECD fusion molecule described herein, may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides a pharmaceutical composition comprising an effective amount of at least one mutant CSF-1R ECD fusion molecule described herein and at least one pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition further comprises one or more additional biologically active agents.

[0203] The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “unit dosage form”, as used herein, refers to a physically discrete unit of at least one mutant CSF-1R ECD fusion molecule for the patient to be treated. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.

[0204] A. Formulation

[0205] A pharmaceutical composition described herein may be administered in any amount and using any route of administration effective for achieving the desired prophylactic and / or therapeutic effect. The optimal pharmaceutical formulation can be varied depending upon the route of administration (orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes, or by injection into tissue), and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient.

[0206] Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di -glycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

[0207] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be administered by, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by gradual infusion. Where necessary or desired, the composition may include a local anesthetic to ease pain at the site of injection.

[0208] In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming micro-encapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues.

[0209] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and pressurized compositions. In addition to at least one mutant CSF-1R ECD fusion molecule described herein, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or another solvent, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizes or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0210] Solid dosage forms for oral administration include, for example, lozenges, troches, tablets, capsules, effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches and sublingual tablets. In such solid dosage forms, a mutant CSF-1R ECD fusion molecule may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and one or more of: (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannital, and silicic acid; (b) binders such as, for example, carboxymethylcellulose, alginates, gelatine, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulphate, and mixtures thereof. Other excipients suitable for solid formulations include surface modifying agents such as non-ionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, pol oxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, magnesium aluminum silicate, and triethanolamine. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0211] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. Examples of embedding compositions which can be used include polymeric substances and waxes.

[0212] In certain embodiments, it may be desirable to administer an inventive composition locally. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, by injection, by means of a catheter, by means of suppository, or by means of a skin patch or stent or other implant.

[0213] For topical administration, the composition is preferably formulated as a gel, an ointment, a lotion, or a cream which can include carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulphate (5%) in water. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

[0214] In addition, in certain instances, it is expected that the inventive compositions may be disposed within transdermal devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the active ingredient by either passive or active release mechanisms. Transdermal administrations include all administration across the surface of the body and the inner linings of bodily passage including epithelial and mucosal tissues. Such administrations may be carried out using the present compositions in lotions, creams, foams, patches, suspensions, solutions, sprays, and suppositories (rectal and vaginal).

[0215] Transdermal administration may be accomplished through the use of a transdermal patch containing an active ingredient (i.e., a mutant CSF-1R ECD fusion molecule) and a carrier that is non-toxic to the skin, and allows the delivery of the ingredient for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be suitable. A variety of occlusive devices may be used to release the active ingredient into the bloodstream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

[0216] Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository’s melting point, and glycerine. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used. For administration by intranasal delivery (e.g., nasal mucosal delivery or intranasal mucosal delivery) by inhalation or insufflation, at least one mutant CSF-1R ECD fusion molecule described herein may be combined with a fine inert powdered carrier or with a liquid carrier. One or more mucosal delivery-enhancing agents may also be present. Examples of absorption enhancers for the intranasal adsorption of peptidic drugs include, but are not limited to, bile salts, surfactants, fluidic acid derivatives, phosphatidylcholines, cyclodextrins, and cell-penetrating peptides (CPPs).

[0217] Materials and methods for producing various formulations are known in the art and may be adapted for practicing the subject invention. Suitable formulations can be found, for example, in “Remington ’s Pharmaceutical Sciences”, E.W. Martin, 18thEd., 1990, Mack Publishing Co.: Easton, PA.

[0218] B. A dditional Biologically A ctive Agents

[0219] In certain embodiments, a mutant CSF-1R ECD fusion molecule described herein is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biologically active agents. Examples of suitable biologically active agents include, but are not limited to, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof. Other examples of suitable biologically active agents include, but are not limited to, the therapeutic agents described above.

[0220] In such pharmaceutical compositions, the mutant CSF-1RECD fusion molecule and the at least one additional biologically active agent may be combined in one or more preparations for simultaneous, separate or sequential administration of the mutant CSF- 1RECD fusion molecule and biologically active agent(s). More specifically, an inventive composition may be formulated in such a way that the mutant CSF-1R ECD fusion molecule and biologically active agent(s) can be administered together or independently from each other. For example, the mutant CSF-1RECD fusion molecule and biologically active agent can be formulated together in a single pharmaceutical composition. Alternatively, they may be maintained (e.g., in different compositions and / or containers) and administered separately, thereby constituting a pharmaceutical kit or pack. 2 - Pharmaceutical Packs or Kits

[0221] In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of at least one mutant CSF-1R ECD fusion molecule described herein, to a subject in need thereof, for therapeutic purpose.

[0222] Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Packs or kits according to the invention may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale.

[0223] In certain embodiments, a pharmaceutical pack or kit can include a device for administering a mutant CSF-1R ECD fusion molecule, or composition thereof, e.g., syringe needle, pen device, jet injector or another needle-free injector.

[0224] In certain embodiments, a pharmaceutical pack or kit includes one or more additional therapeutic agent(s), as described above. Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein.

[0225] Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0226] An identifier, e.g., a bar code, radio frequency, ID tags, and the like may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc. 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.

[0227] Examples

[0228] The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually carried out or data were actually obtained.

[0229] Identification of Suitable Mutations in the Extracellular Domain of CSF-1R

[0230] The first aim of the study was to design a CSF-1R ECD-based molecule capable of efficiently capturing both IL-34 and CSF-1. Binding affinity was modified by single residue mutation of the extracellular domain of CSF-1R.

[0231] First, the effect of single mutations of different residues of CSF-1R ECD on the stability of the complexes IL-34 / CSF-1R ECD and CSF-1 / CSF-1R ECD was estimated by in-silico prediction, using the method of Spassov and Van (Proteins, 2013, 81 : 704- 714) and the crystal structure of CSF-1R ECD complexed with CSF-1 (PDB AWRL) or IL-34 (PDB 4DKD).

[0232] Figure 1 presents the results obtained for mutations by D (aspartate), E (glutamate), K (lysine), R (arginine), H (histidine), S (serine), T (threonine), N (asparagine), Q (glutamine), Y (tyrosine), C (cysteine), A (alanine), G (glycine), V (valine), L (leucine), I (isoleucine), P (proline), M (methionine), F (phenylalanine), and W (tryptophane) at residue positions V143, M149, 1170, S172, Q173, R192; V231, Q248 and S250, of CSF- 1R ECD.

[0233] The mutant CSF-lRs predicted to display a better affinity for both IL-34 and CSF- 1 were found to be those having the M149K mutation, the S172W mutation, the Q173K mutation, or the S172K mutation. The mutant CSF-1R ECD having the M149K mutation was selected for the rest of the study. Preparation of Mutant (M149K) CSF-1R ECD-Fc and of WT CSF-1R ECD-Fc

[0234] A pilot production of mutant (M149K) CSF-1R and WT CSF-1R was carried out. For each molecule, only the extracellular domain (ECD) of the human CSF-1R was used and linked to a human Fc to allow dimerization. As shown in Figure 2, IL-34 trapping is only possible for WT CSF-IR-Fc (also called WT CSF-1R ECD-Fc), which can undergo dimerization, while WT CSF-1R (also called WT CSF-1R ECD) cannot inhibit the activation by IL-34 of CSF-1R pathways in a human monocytic THP-1 cell line.

[0235] Affinity Properties of Mutant (M149K) CSF-IR-Fc and WT CSF-IR-Fc

[0236] The affinity of mutant (M149K) CSF-IR-Fc (also called (M149K) CSF-1R ECD- Fc) and of WT CSF-IR-Fc was determined using a Surface Plasmon Resonance (SPR) assay. WT or mutant (M149K) CSF-IR-Fc was immobilized on a SPR chip. Then, increasing concentrations of soluble human IL-34 or CSF-1 were added. The results obtained are presented in Table 1.

[0237] Table 1. KD values obtained by SPR affinity assay.

[0238] These results confirm the in-silico prediction of an increased affinity of mutant (M149K) CSF-IR-Fc vs. WT CSF-IR-Fc for both IL-34 and CSF-1.

[0239] In vitro Properties of Mutant (M149K) CSF-IR-Fc and WT CSF-IR-Fc

[0240] In vitro experiments were performed to check the inhibition of IL-34 and CSF-1 signalization through CSF-1R by mutant (M149K) CSF-IR-Fc. Towards this aim, the THP-1 human monocytic cell line was used and two intracellular pathways downstream of CSF-1R were analyzed (Akt and ERK). To measure the activation of each signalization pathway, an intracellular staining of phosphorylated Akt and ERK was performed. Mutant (M149K) CSF-IR-Fc was mixed at a molecular ratio of 1 :1 (4 nM) with CSF-1 and IL-34, and the mixture was then added to THP-1 cells and 3 minutes later cells were fixed, permeabilized and stained. The results obtained, which are presented on Figure 3, confirm that mutant (M149K) CSF-IR-Fc traps IL-34 or CSF-1 and avoid their fixation to CSF-1R naturally expressed by the THP-1 cells, and thereby inhibits activation of the signalization pathways. This inhibition was confirmed in a monocyte viability assay. Human monocytes sorted from fresh PMBCs from healthy donors were plated with WT CSF-IR-Fc or mutant (M149K) CSF-IR-Fc, and 3 days later cell viability was measured by flow cytometry. Human monocytes naturally produce CSF-1, but do not produce IL-34. Therefore, the experiments were carried out in the presence of recombinant IL-34 added to the cell culture. As shown in Figure 4 (black), mutant (M149K) CSF-IR-Fc induces a significantly better inhibition of cell viability than WT CSF-IR-Fc. Mutant (M149K) CSF-IR-Fc also results in higher inhibition of monocytes viability induced by incubation with recombinant human IL-34 (Figure 4, white).

[0241] Ex vivo Properties of Mutant (M149K) CSF-IR-Fc and WT CSF-IR-Fc on Naturally Produced IL-34 and / or CSF-1 in MPM

[0242] The efficacy of both CSF-IR-Fc molecules was tested in an ex vivo model of malignant pleural mesothelioma (MPM). Similar to the monocyte viability assay, human monocytes sorted from PBMCs were cultured 3 days with pleural effusions from MPM patients containing CSF-1 but no IL-34 (Figure 5(A)) or both CSF-1 and IL-34 (Figure 5(B)). Monocytes polarization (immunosuppressive macrophages, CD14+CD163+) was analyzed and as observed above, mutant (M149K) CSF-IR-Fc was found to induce a significantly better inhibition of monocytes differentiation than WT CSF-IR-Fc. Interestingly, mutant (M149K) CSF-IR-Fc was found to be more efficient than blocking antibodies (alone or in combination). In contrast, WT CSF-IR-Fc was observed to be about as efficient as blocking antibodies (alone or in combination).

Claims

ClaimsWhat is claimed is:

1. A mutant extracellular domain of CSF-1R (CSF-1R ECD), wherein said mutant CSF-1R ECD consists of a wild-type CSF-1R ECD comprising a single amino acid mutation selected from M149K mutation, S172W mutation, Q173K mutation, and S172K mutation.

2. The mutant CSF-1R ECD according to claim 1, wherein the wild-type CSF-1R ECD consists of the amino acid sequence set forth in SEQ ID NO: 7 or in SEQ ID NO: 8, preferably in SEQ ID NO: 7.

3. The mutant CSF-1R ECD according to claim 2, wherein the mutant CSF-1R ECD is:- the (M149K) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 9; or- the (S172W) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 10; or- the (Q173K) mutant CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 11; or- the (S172K) mutation CSF-1R ECD, which consists of the amino acid sequence set forth in SEQ ID NO: 16.

4. A mutant CSF-1R ECD fusion molecule, said fusion molecule consisting of a mutant CSF-1R ECD according to any one of claims 1 to 3 is linked to at least one fusion partner.

5. The mutant CSF-1R ECD fusion molecule according to claim 4, wherein the fusion partner is an immunoglobulin Fc domain, in particular a silent human immunoglobulin Fc domain.

6. The mutant CSF-1R ECD fusion molecule according to claim 5, wherein the human immunoglobulin Fc domain is a silent human immunoglobulin Fc domain which consists of the amino acid sequence set forth in SEQ ID NO: 12.

7. The mutant CSF-1R ECD fusion molecule according to any one of claims 4 to 6, wherein:the mutant CSF-1R ECD is the (M149K) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 13; or- the mutant C SF - 1 R ECD i s the mutant C SF - 1 R ECD i s the ( S 172 W) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 14; or the mutant CSF-1R ECD is the (Q173K) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 15; or- the mutant CSF-1R CED is the (S172K) mutant CSF-1R ECD, and the mutant CSF-1R ECD fusion molecule consists of the amino acid sequence set forth in SEQ ID NO: 17.

8. An isolated nucleic acid encoding a mutant CSF-1R ECD according to any one of claims 1 to 3 or a fusion molecule according to any one of claims 4 to 7.

9. A vector comprising the nucleic acid according to claim 8.

10. A host cell comprising the nucleic acid according to claim 8 and / or the vector according to claim 9.

11. A mutant CSF-1R ECD according to any one of claims 1 to 3, or a mutant CSF- 1R ECD fusion molecule according to any one of claims 4 to 7, or an isolated nucleic acid according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, for use as a therapeutic agent.

12. A pharmaceutical composition comprising a therapeutically effective amount of a mutant CSF-1R ECD according to any one of claims 1 to 3, or a mutant CSF- 1R ECD fusion molecule according to any one of claims 4 to 7, and at least one pharmaceutically acceptable carrier or excipient.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition further comprises at least one additional biologically active agent.

14. A mutant CSF-1R ECD according to any one of claims 1 to 3, or a mutant CSF- 1R ECD fusion molecule according to any one of claims 4 to 7, or an isolatednucleic acid according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, or a pharmaceutical composition according to claim 12 or claim 13, for use in the treatment of a disease in a subject, wherein the disease is a cancer, a neurodegenerative disease, an autoimmune disease, an allergic disease, or a bone disease, or for use in the prevention or inhibition of transplant rejection in a subject.

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