Mutated interleukin-34 (IL-34) polypeptides and their use in therapy - Patents.com

Abstract

Interleukin-34 is a cytokine involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. IL-34 involvement has been demonstrated in diverse areas, including neuroprotection, autoimmune diseases, infectious diseases, cancer, and transplantation. Recent studies have also demonstrated a novel and promising therapeutic role for IL-34 as a Foxp3+ Treg-secreted cytokine mediator of transplantation tolerance. Therefore, the present inventors have generated novel mutated IL-34 polypeptides that can be used as agonists or antagonists.

Description

[Technical field]

[0001] The present invention relates to mutated interleukin-34 (IL-34) polypeptides and their use in therapy. [Background technology]

[0002] Interleukin-34 is a cytokine whose function is only partially understood, first described in 2008. IL-34 shares little homology with colony-stimulating factor 1 (CSF-1 or M-CSF), but shares a common receptor, CSF-1R (CD115, c-Fms), and IL-34 has two distinct receptors, PTP-ζ and CD138 (syndecan-1). IL-34 has also been shown to pair with CSF-1 to form heterodimers. To date, studies have shown that this cytokine is released by several tissues distinct from those in which CSF-1 is expressed and is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuroprotection, autoimmune diseases, infectious diseases, cancer, and transplantation (Guillonneau C. Cell Mol Life Sci. 2017). Recent studies have also demonstrated a novel and promising therapeutic role for IL-34 as a Foxp3+ regulatory T cell-secreted cytokine mediator of transplantation tolerance (Bezie et al., JCI, 2015). Summary of the Invention

[0003] The present invention, as defined by the claims, relates to mutated interleukin-34 (IL-34) polypeptides and their use in therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] As used herein, the term "IL-34" has its general meaning in the art and refers to interleukin-34 characterized by the amino acid sequence set forth in SEQ ID NO:1. SEQ ID NO: 1: Interleukin-34 (Homo sapiens) containing a leader sequence at positions 1 to 20 [ka]

[0005] Amino acid numbering used throughout this specification is provided with reference to the amino acid sequence set forth in SEQ ID NO:1.

[0006] As used herein, the term "CSF-1R" has its general meaning in the art and refers to colony-stimulating factor 1 receptor. CSF-1R is also referred to in the art as FMS, FIM2, C-FMS, M-CSF receptor, and CD115. This receptor is a single-pass transmembrane receptor that contains an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain with tyrosine kinase activity. Ligand binding of CSF-1 or interleukin 34 to CSF-1R results in receptor dimerization, upregulation of CSF-1R protein tyrosine kinase activity, phosphorylation of CSF1R tyrosine residues, and downstream signaling events (i.e., "biological activity"). For example, both CSF-1 and IL-34 stimulate monocyte survival, proliferation, and differentiation into macrophages and other monocytic cell lineages such as osteoclasts, dendritic cells, and microglia.

[0007] As used herein, the term "PTP-zeta" has its common meaning in the art and refers to receptor tyrosine protein phosphatase zeta. PTP-zeta is also known in the art as "phosphacan." The 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.

[0008] As used herein, the term "CD138" has its common meaning in the art and stands for cluster of differentiation 138. CD138 is also known in the art as "syndecan-1". This receptor is a transmembrane (type I) heparan sulfate proteoglycan. The core protein of syndecan-1 consists of an extracellular domain that can be substituted with heparan sulfate and chondroitin sulfate glycosaminoglycan chains, a highly conserved transmembrane domain, and a highly conserved cytoplasmic domain that contains two constant regions separated by a variable region.

[0009] As used herein, the term "ligand" refers to a molecule, such as a polypeptide, that has an affinity for binding to a receptor. As will be understood by those skilled in the art, the molecule may be a receptor and a ligand. The ligands of CSF-1R are well known in the art and include IL-34 and CSF-1. The ligands of PTP-ζ and CD138 are well known in the art and include IL-34.

[0010] The term "affinity" as used herein means the strength of binding of a ligand to its receptor. The affinity of a ligand is given by the dissociation constant Kd. Preferred methods for determining the affinity of a ligand can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), the entire references of which are incorporated herein by reference. One of the preferred standard methods for determining the affinity of a ligand that is well known in the art is the use of a Biacore instrument.

[0011] The term "binding" as used herein refers to a direct bond between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding" refers to a bond that is greater than about 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M and even lower K D This is in the context of binding of a ligand to its receptor with an affinity corresponding to

[0012] As used herein, the term "agonist" refers to a mutant IL-34 polypeptide that mimics the biological activity of a ligand for CSF-1R, a ligand for PTP-zeta, and / or a ligand for CD138 or has the same or similar biological activity as a ligand for CSF-1R, a ligand for PTP-zeta, and / or a ligand for CD138.

[0013] As used herein, the term "antagonist" refers to a mutant IL-34 polypeptide that partially or fully blocks, inhibits, or neutralizes the biological activity of a ligand for CSF-1R, a ligand for PTP-ζ, and / or a ligand for CD138.

[0014] Ligands for CSF-1R include CSF-1 and IL-34. In particular, the ligand for CSF-1R according to the present invention is a CSF-1 polypeptide or, preferably, a native or wild-type IL-34 polypeptide as disclosed herein.

[0015] Ligands for PTP-ζ include heparin-binding growth factors, pleiotrophin (PTN) / HB-GAM and midkine (MK), extracellular matrix proteins such as fibroblast growth factor-2, tenascin-C and tenascin-R, and cell adhesion molecules such as Nr-CAM, L1 / Ng-CAM, F3 / contactin, NCAM, and TAG1 / axonin-1.

[0016] Ligands for CD138 include matrix proteins, proteases, adhesion receptors, cytokines and growth factors, complement, and coagulation proteins.

[0017] Preferably, the ligand of CSF-1R, the ligand of PTP-zeta, or the ligand of CD138 according to the present invention is a native or wild-type IL-34 polypeptide as disclosed herein.

[0018] Preferably, the agonists of the present invention are mutant IL-34 polypeptides that mimic the biological activity of a ligand of CSF-1R or have the same or similar biological activity as a ligand of CSF-1R.

[0019] Preferably, the antagonists of the present invention are mutant IL-34 polypeptides that partially or fully block, inhibit, or neutralize the biological activity of the ligand of CSF-1R.

[0020] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling moiety. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0021] The term "dimer" as used herein broadly refers to a molecule comprising two or more subunits (wherein the subunits are formed by the mutated IL-34 polypeptides of the invention). The dimers of the invention typically function as agonists or antagonists.

[0022] The term "fusion polypeptide" or "fusion protein" refers to a protein made by linking together two or more polypeptide sequences. Fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct linking a nucleic acid sequence encoding a second polypeptide with a nucleic acid sequence encoding a first polypeptide. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by peptide bonds or through several peptides. Fusion proteins may also include a peptide linker between the two domains.

[0023] As used herein, the term "linker" refers to a sequence of at least one amino acid that joins a first polypeptide to a second polypeptide in a fusion protein.

[0024] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, and includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.

[0025] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the effector functions of immunoglobulin constant domains with the binding specificity of a heterologous protein (an "adhesin"). Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity (i.e., is "heterologous") and an immunoglobulin constant domain sequence. The adhesin portion of the immunoadhesin molecule is usually a contiguous amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence of the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

[0026] As used herein, a "nucleic acid molecule" or "polynucleotide" refers to a DNA molecule (such as, but not limited to, cDNA or genomic DNA). A nucleic acid molecule can be single-stranded or double-stranded.

[0027] The term "isolated" when referring to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is substantially free from at least one other component with which it is associated or found together in nature.

[0028] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0029] As used herein, the terms "wild-type" or "native" are terms of the art that are understood by those of skill in the art and refer to a naturally occurring typical form of an organism, strain, gene, or characteristic that differs from a mutant form.

[0030] As used herein, the term "mutation" has its usual meaning in the art and refers to a substitution, deletion, or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted at the same position. The term "deletion" means that a specific amino acid residue at a specific position is removed.

[0031] As used herein, the term "variant" refers to a first composition (e.g., a first molecule) relative to a second composition (e.g., a second molecule, also referred to as a "parent" molecule). A variant molecule may be derived from, isolated from, based on, or homologous to a parent molecule. A variant molecule may have complete sequence identity with the original parent molecule, or may have less than 100% sequence identity with the parent molecule. For example, a variant of a sequence may be a second sequence that is at least 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100% identical in sequence to the original sequence. Sequence identity is often measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math., 2:482, 1981;Needleman and Wunsch, J. Mol. Biol., 48:443, 1970;Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988;Higgins and Sharp, Gene, 73:237-244, 1988;Higgins and Sharp, CABIOS, 5:151-153, 1989;Corpet et al. Nuc. Acids Res., 16:10881-10890, 1988;Huang et al., Comp. Appls Biosci., 8:155-165, 1992;and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994).Altschul et al., Nat. Genet., 6:119-129, 1994, presents a detailed discussion of sequence alignment methods and homology calculations. As an example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) can be used to perform sequence comparisons (Internet Program® 1996, WR Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares an entire sequence to another sequence, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet, for example, at the NCSA website. Alternatively, for comparison of amino acid sequences of more than about 30 amino acids, the Blas2 sequences function can be used with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and per residue gap cost of 1). When aligning shorter peptides (fewer than about 30 amino acids), the alignment should be performed using the Blas2 sequences function with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).The BLAST sequence comparison system is available, for example, from the NCBI website; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

[0032] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) may be introduced into a host cell in order to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.

[0033] The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell, so that the host cell expresses the introduced gene or sequence to produce a desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0034] As used herein, "Treg" or "regulatory T cells" refers to functionally committed cells, i.e., cells capable of suppressive activity (i.e., inhibiting the proliferation of conventional T cells), either through cell-cell contact or MLR (Mixed Lymphocyte Reaction) inhibition. Tregs are characterized by the expression of FoxP3 and CD8 +T cells and CD4+ T cells. As used, the term "Foxp3" has its general meaning in the art and refers to a transcriptional regulator important for the development and inhibitory function of Tregs. Foxp3 plays an essential role in maintaining immune system homeostasis by allowing acquisition of full suppressive function and stability of the Treg lineage and directly regulating the proliferation and function of conventional T cells. As used herein, the term "CD4 + "T cells" has its common meaning in the art and refers to the subset of T cells that express CD4 on their surface. + T cells are macrophages and CD8 + T helper cells are thought to orchestrate the activation of T cells (Th-1 cells), the production of antibodies by B cells (Th-2 cells), or have an essential role in autoimmune diseases (Th-17 cells). As used herein, the term "CD8 + "T cells" has its common meaning in the art and refers to the subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. + "T cells" are also called cytotoxic T lymphocytes (CTLs), T killer cells, cytolytic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is a recognition element that binds in interactions restricted to major histocompatibility complex class I. In particular, regulatory T cells usually express "forkhead box P3 (Foxp3) + ) Regulatory T cells" and "CD45RC low As used herein, the term "forkhead box P3 (Foxp3 + ) Regulatory T cells" or "Foxp3 + Treg cells are CD4+ T cells in humans and rodents (rats or mice) whose characteristic marker is the transcription factor Foxp3. + and CD8 + They represent 2-10% of T cells.

[0035] As used herein, the term "culture medium" refers to a medium for maintaining or culturing a cell population (e.g., a culture medium) that contains nutrients that maintain the viability and support the proliferation of the cells. The medium may contain any of the following in appropriate combinations: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components such as growth factors, cytokines, etc. Media commonly used for specific cell types are known to those skilled in the art. The medium of the present invention may be based on commercially available media such as RPMI 1640 from Invitrogen.

[0036] As used herein, the term "expanding" refers to the process of converting and / or amplifying a population of given cells (e.g., immune cells such as T cells). Expansion of T cells is preferably performed by culturing a cell population comprising T cells in the presence of an antigen-specific stimulant, e.g., antigen, cells, antibodies, lecithin, etc. Expansion may also require culturing T cells in the presence of cytokines.

[0037] As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity, and further, the term immune response includes immune responses indirectly influenced by activation of T cells, such as antibody production (humoral response), and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in an immune response include lymphocytes, such as B cells and T cells (CD4 + cells, CD8 +These include immune cells, Th1 cells, and Th2 cells; antigen-presenting cells (e.g., professional antigen-presenting cells such as dendritic cells); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes. For example, immune responses are involved in transplant rejection and the associated physiological consequences of the immune response, such as interstitial fibrosis, chronic graft arteriosclerosis, or vasculitis. Immune responses are also involved in autoimmune diseases and the associated physiological consequences of the immune response, including T cell-dependent infiltration and direct tissue damage, T cell-dependent recruitment and activation of macrophages and other effector cells, and T cell-dependent B cell responses that result in the production of autoantibodies.

[0038] As used herein, the term "immune tolerance" refers to a state of unresponsiveness of the immune system to substances or tissues that have the ability to induce an immune response. As used herein, the term "specific" immune tolerance occurs when immune tolerance is preferentially induced to a particular antigen compared to others.

[0039] As used herein, the term "unleashing" refers to the realization or augmentation of an immune response to a harmful substance (e.g., a molecule on the surface of a cancer cell, a virus, a fungus, or a bacterium). In the context of the present invention, unleashing refers to the realization or augmentation of an immune response to a harmful substance (e.g., a molecule on the surface of a cancer cell, a virus, a fungus, or a bacterium). + CD25 - Represents induction of T cell proliferation.

[0040] As used herein, the term "reducing immune tolerance" refers to a reduction in the state of unresponsiveness of the immune system to substances or tissues that have the ability to elicit an immune response.

[0041] The term "transplantation" and variations thereof refer to the insertion of a transplant (also called a graft) into a recipient, whether the transplant is syngenic (donor and recipient genetically identical), allogenic (donor and recipient of the same species but of different genetic origin), or xenogeneic (donor and recipient from different species). Thus, in a typical scenario, the host is a human and the graft is a syngeneic graft derived from a human of the same or different genetic origin. In another scenario, the graft is derived from a different species than the host being transplanted, including animals from phylogenetically distant species, for example, a baboon heart is transplanted into a human host.

[0042] As used herein, the term "organ" refers to a solid, vascularized organ that performs a specific function or group of functions in an organism. The term organ includes, but is not limited to, the heart, lungs, kidneys, liver, pancreas, skin, uterus, bone, cartilage, small or large intestine, bladder, brain, breast, blood vessels, esophagus, fallopian tubes, gallbladder, ovaries, pancreas, prostate, placenta, spine, limbs including upper and lower limbs, spleen, stomach, testes, thymus, thyroid, trachea, ureters, urethra, and uterus.

[0043] As used herein, the term "tissue" refers to tissue of any species, human or animal, including, but not limited to, vascular tissue, skin tissue, liver tissue, pancreatic tissue, nervous tissue, urogenital tissue, gastrointestinal tissue, skeletal tissue including bone and cartilage, adipose tissue, connective tissue including tendons and ligaments, amniotic tissue, chorionic tissue, dura mater, pericardium, muscle tissue, glandular tissue, facial tissue, and eye tissue.

[0044] As used herein, the term "cells" refers to a composition enriched in cells of interest, preferably a composition comprising at least 30%, preferably at least 50%, even more preferably at least 65% of said cells.

[0045] 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 immunologically foreign. Acute rejection is characterized by infiltration of the graft tissue by the recipient's immune cells, which perform their effector functions and destroy the transplanted tissue. The onset of acute rejection is rapid, generally occurring within a few weeks of transplantation in humans. "Chronic transplant rejection" generally occurs within months to years of engraftment in humans, even when immunosuppression of acute rejection is successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants.

[0046] As used herein, the term "preventing or reducing transplant rejection" is meant to encompass preventing or inhibiting immune transplant rejection, and delaying the onset or progression of immune transplant rejection. The term also encompasses prolonging graft survival in a patient, or reversing graft failure in a patient. The term further encompasses ameliorating symptoms of immune transplant rejection, including, for example, ameliorating immunological complications associated with immune rejection, such as interstitial fibrosis, chronic graft atherosclerosis, or vasculitis.

[0047] As used herein, the term "autoimmune inflammatory disease" refers to a disease in which the immune system produces an immune response (e.g., a B cell response or a T cell response) against antigens that are part of the normal host (i.e., self-antigens) with resultant damage to tissues. In autoimmune diseases, the host's immune system fails to recognize certain antigens as "self" and the immune response is mounted against host tissues that express the antigen.

[0048] As used herein, "allergic disorder" refers to any disorder resulting from antigen activation of mast cells resulting in an "allergic response" or a state of hypersensitivity and influx of inflammatory and immune cells.

[0049] As used herein, the term "asthma" refers to an inflammatory disease of the airways characterized by airflow obstruction, wheezing, and shortness of breath.

[0050] As used herein, the term "undesirable immune response against a therapeutic protein" refers to any undesirable immune reaction directed against a protein expressed during gene therapy and / or a therapeutic protein such as Factor VIII (hemophilia A) and other clotting factors, enzyme replacement therapy, monoclonal antibodies (e.g., natalizumab, rituximab, infliximab), polyclonal antibodies, enzymes, or cytokines (e.g., IFNβ).

[0051] As used herein, the term "immunosuppressant" refers to any substance that can produce an immunosuppressive effect, e.g., preventing or reducing an immune response.

[0052] As used herein, the term "cancer" has its general meaning in the art, including, but not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. The term "cancer" further encompasses primary and metastatic cancers.

[0053] As used herein, the term "chemotherapy" refers to treatment with a chemotherapeutic agent.

[0054] As used herein, the term "immunotherapy" has its general meaning in the art and refers to treatment consisting of administering an immunogen, i.e., an agent capable of inducing, enhancing, suppressing, or otherwise modifying the immune response.

[0055] As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound that inhibits the function of immune inhibitory checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule expressed by T cells that enhances signals (stimulatory checkpoint molecules) or reduces signals (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art as constituting immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, for example, Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, and VISTA. Inhibition includes reduction as well as complete blocking of function.

[0056] As used herein, the term "treatment" or "treat" refers to both preventative or prophylactic treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, and patients suffering from or diagnosed with a disease or condition, including suppression of clinical recurrence. Treatment may be administered to a subject with a condition or a subject who may eventually acquire a disorder to prevent, cure, delay onset, reduce severity, or ameliorate one or more symptoms of a disorder or recurrent disorder, or to extend the subject's survival beyond that expected in the absence of said treatment. "Therapeutic regimen" refers to a pattern of treatment of a disease, such as a pattern of administration used during treatment. Therapeutic regimen may include induction regimen and maintenance regimen. The term "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used in the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. The induction regimen may use (partially or entirely) a "loading regimen", which may include administering a higher dose of the drug than the doctor uses during the maintenance regimen, administering the drug more frequently than the doctor uses during the maintenance regimen, or both. The term "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment after achievement of a certain predetermined criterion (e.g., symptoms of disease, etc.).

[0057] "Therapeutically effective amount" means an amount of mutated IL-34 polypeptide sufficient to treat and / or prevent a disease at a reasonable benefit / loss ratio applicable to any medical treatment. It is understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will vary depending on a variety of factors, including the disease being treated and the severity of the disease; the activity of the particular compound used; the particular composition used, the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; duration of treatment; drugs used in combination or simultaneously with the particular polypeptide used; and similar factors well known in the medical art. For example, it is well known to those skilled in the art to start the compound at a dose level lower than that required to achieve the desired therapeutic effect and gradually increase this dose until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range, from 0.01 to 1,000 mg per adult per day. Preferably, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of active ingredient, for symptom-related dosage adjustment to the patient being treated. The medicament usually contains about 0.01 to about 500 mg of active ingredient, preferably 1 mg to about 100 mg of active ingredient. An effective amount of the drug is usually supplied at a dosage level of 0.0002 mg / kg to about 20 mg / kg body weight / day, about 0.001 mg / kg to 10 mg / kg body weight / day, particularly about 0.001 mg / kg to 7 mg / kg body weight / day.

[0058] The terms "pharmacologically" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to a mammal, particularly a human, as appropriate. A pharma-ceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type.

[0059] The present inventors have generated novel variants of the human IL34 polypeptide.

[0060] Thus, one subject matter disclosed herein relates to a mutated IL-34 polypeptide (P1) having an amino acid sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, in which the histidine (H) residue at position 56 and the glycine (G) residue at position 112 are both substituted with cysteine ​​(C) residues.

[0061] Preferably, the mutated IL-34 polypeptide (P1) of the present invention comprises: A mutation in which the threonine (T) residue at position 36 is replaced by a tyrosine (Y) or tryptophan (W) or proline (P) or phenylalanine (F) or arginine (R) or histidine (H) or asparagine (N) or cysteine ​​(C) or isoleucine (I) or proline (P) or leucine (L) residue; A mutation in which the serine (S) residue at position 100 is replaced with an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue; a mutation in which the threonine (T) residue at position 124 is replaced with a phenylalanine (F) residue or a tryptophan (W) residue; A mutation in which the asparagine (N) residue at position 128 is replaced with a tyrosine (Y) or phenylalanine (F) residue; A mutation in which the glutamine (Q) residue at position 131 is replaced with an arginine (R) residue, a histidine (H) residue, a valine (V) residue, a phenylalanine (F) residue, an isoleucine (I) residue, or a proline (P) residue; A mutation in which the serine (S) residue at position 147 is replaced with a glutamic acid (E) or aspartic acid (D) residue; a mutation in which the asparagine (N) residue at position 150 is replaced with a glutamic acid (E) or aspartic acid (D) residue, and A mutation in which the leucine (L) residue at position 186 is replaced with an arginine (R) or phenylalanine (F) residue. The antibody is further characterized by at least one mutation selected from the group consisting of:

[0062] Another subject matter relates to a mutated IL34 polypeptide (P2) having an amino acid sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, wherein the proline (P) residue at position 59 is substituted with an amino acid residue selected from the group consisting of aspartic acid (D), glutamic acid (E), histidine (H), serine (S), threonine (T), glutamine (Q), tyrosine (Y), cysteine ​​(C), alanine (A), glycine (G), leucine (L), and methionine (M).

[0063] Alternatively, the proline (P) residue at position 59 is substituted with a lysine (K) or arginine (R) residue.

[0064] Preferably, the mutated IL-34 polypeptide (P2) of the present invention comprises: A mutation in which the threonine (T) residue at position 36 is replaced by a tyrosine (Y) or tryptophan (W) or proline (P) or phenylalanine (F) or arginine (R) or histidine (H) or asparagine (N) or cysteine ​​(C) or isoleucine (I) or proline (P) or leucine (L) residue; A mutation in which the serine (S) residue at position 100 is replaced with an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue; a mutation in which the threonine (T) residue at position 124 is replaced with a phenylalanine (F) residue or a tryptophan (W) residue; A mutation in which the asparagine (N) residue at position 128 is replaced with a tyrosine (Y) or phenylalanine (F) residue; A mutation in which the glutamine (Q) residue at position 131 is replaced with an arginine (R) residue, a histidine (H) residue, a valine (V) residue, a phenylalanine (F) residue, an isoleucine (I) residue, or a proline (P) residue; A mutation in which the serine (S) residue at position 147 is replaced with a glutamic acid (E) or aspartic acid (D) residue; a mutation in which the asparagine (N) residue at position 150 is replaced with a glutamic acid (E) or aspartic acid (D) residue, and A mutation in which the leucine (L) residue at position 186 is replaced with an arginine (R) or phenylalanine (F) residue. The antibody is further characterized by at least one mutation selected from the group consisting of:

[0065] Another subject is a sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the threonine (T) residue at position 36 is replaced by a tyrosine (Y) or tryptophan (W) or proline (P) or phenylalanine (F) or arginine (R) or histidine (H) or asparagine (N) or cysteine ​​(C) or isoleucine (I) or proline (P) or leucine (L) residue; A mutation in which the serine (S) residue at position 100 is replaced with an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue; a mutation in which the threonine (T) residue at position 124 is replaced with a phenylalanine (F) residue or a tryptophan (W) residue; A mutation in which the asparagine (N) residue at position 128 is replaced with a tyrosine (Y) or phenylalanine (F) residue; A mutation in which the glutamine (Q) residue at position 131 is replaced with an arginine (R) residue, a histidine (H) residue, a valine (V) residue, a phenylalanine (F) residue, an isoleucine (I) residue, or a proline (P) residue; A mutation in which the serine (S) residue at position 147 is replaced with a glutamic acid (E) or aspartic acid (D) residue; a mutation in which the asparagine (N) residue at position 150 is replaced with a glutamic acid (E) or aspartic acid (D) residue, and A mutation in which the leucine (L) residue at position 186 is replaced with an arginine (R) or phenylalanine (F) residue. The present invention relates to a mutated IL34 polypeptide (P3) having an amino acid sequence comprising at least one mutation selected from the group consisting of:

[0066] Another subject matter disclosed herein ranges from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), a mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C); A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D), A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R), A mutation in which the proline residue at position 59 is replaced by a lysine residue (P59K), a mutation in which the threonine residue at position 124 is replaced by a phenylalanine residue (T124F), and and a mutation in which the asparagine residue at position 150 is replaced with a glutamic acid residue (N150E).

[0067] Another subject is a sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). and wherein the amino acid sequence has at least 80% identity with an amino acid sequence containing at least one mutation selected from the group consisting of:

[0068] Preferably, the mutated IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0069] Another subject is a sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D), and A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R) and wherein the amino acid sequence has at least 80% identity with an amino acid sequence containing at least one mutation selected from the group consisting of:

[0070] Preferably, the mutated IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0071] Another target is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). an amino acid sequence comprising at least one mutation selected from the group consisting of: b) an amino acid sequence having at least 80% identity with the sequence defined in a), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138, and c) A fragment of a sequence as defined in a) or b), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta and CD138. A mutated IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0072] Preferably, the mutated IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0073] The mutated IL34 polypeptide is A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). The mutation may comprise a combination of two, three, four, five, or six mutations selected from the group consisting of:

[0074] Another target is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, in which the serine residue at position 100 is replaced with a phenylalanine residue (S100F); and A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). an amino acid sequence further comprising at least one mutation selected from the group consisting of: b) an amino acid sequence having at least 80% identity with the sequence defined in a), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138, and c) A fragment of a sequence as defined in a) or b), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta and CD138. A mutated IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0075] Preferably, the mutated IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0076] the mutated IL34 polypeptide comprises an S100F mutation, A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). The mutations may further comprise a combination of two, three, four, or five mutations selected from the group consisting of:

[0077] Another target is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), and A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F) an amino acid sequence comprising at least one mutation selected from the group consisting of: b) an amino acid sequence having at least 80% identity with the sequence defined in a), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138, and c) A fragment of a sequence as defined in a) or b), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta and CD138. A mutated IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0078] Preferably, the mutated IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0079] Another target is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, with the following three mutations: A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), and A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F) an amino acid sequence comprising b) an amino acid sequence having at least 80% identity with the sequence defined in a), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138, and c) A fragment of a sequence as defined in a) or b), with the proviso that said polypeptide is an agonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta and CD138. A mutated IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0080] Preferably, the mutated IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0081] Another target is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D) and A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R) an amino acid sequence comprising at least one mutation selected from the group consisting of: b) an amino acid sequence having at least 80% identity with the sequence defined in a), with the proviso that said polypeptide is an antagonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138; and c) A fragment of a sequence as defined in a) or b), with the proviso that said polypeptide is an antagonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. A mutated IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0082] Preferably, the mutated IL34 polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0083] Another target is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, comprising the following two mutations: A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D) and A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R) an amino acid sequence comprising b) an amino acid sequence having at least 80% identity with the sequence defined in a), with the proviso that said polypeptide is an antagonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138; and c) A fragment of a sequence as defined in a) or b), with the proviso that said polypeptide is an antagonist of the ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. A mutated IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0084] Preferably, the mutated IL34 polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138. In some embodiments, the mutated IL34 polypeptide is a polypeptide for use as an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-zeta, and CD138.

[0085] In some embodiments, the glutamine (Q) residue at position 81 is deleted in a polypeptide of the invention.

[0086] The polypeptides of the present invention have biological activity. Some polypeptides of the present invention have agonist activity. Other polypeptides of the present invention have antagonist activity.

[0087] By "agonist activity" herein is meant activity that mimics, is similar to, is identical to, or exceeds the activity of a ligand of CSF-1R, a ligand of PTP-zeta, or a ligand of CD138, such as IL-34, CSF-1, or M-CSF, preferably IL-34. Preferably, "agonist activity" is activity that mimics, is similar to, is identical to, or exceeds the activity of a ligand of CSF-1R.

[0088] By "antagonist activity" herein is meant activity which partially or completely blocks, inhibits, or neutralizes the biological activity of a ligand of CSF-1R, a ligand of PTP-ζ, or a ligand of CD138, such as IL-34, CSF-1, or M-CSF, preferably IL-34. Preferably, "antagonist activity" is activity which partially or completely blocks, inhibits, or neutralizes the biological activity of a ligand of CSF-1R.

[0089] Biological activities of a ligand for CSF-1R, a ligand for PTP-zeta, or a ligand for CD138 include, but are not limited to, the property of binding to CSF-1R, PTP-zeta, or CD138, the property of inducing or enhancing cell survival, e.g., inducing or enhancing the survival, proliferation, and differentiation of monocytes, particularly CD14+ monocytes, into macrophages and other monocytic cell lineages, e.g., osteoclasts, dendritic cells, or microglia, or the property of inducing or enhancing the phosphorylation of certain kinases, including protein kinase B (PKB, also known as Akt) or ERK1 / 2 (extracellular signal-regulated kinase 1 / 2).

[0090] A polypeptide of the invention has the biological activity of a ligand for CSF-1R, a ligand for PTP-zeta, or a ligand for CD138, provided it possesses at least one of the above-mentioned activities.

[0091] A polypeptide is an "agonist" of a ligand for CSF-1R, a ligand for PTP-zeta, or a ligand for CD138, or has agonist activity of a ligand for CSF-1R, a ligand for PTP-zeta, or a ligand for CD138, as soon as the polypeptide has the property of binding to CSF-1R, PTP-zeta, or CD138 and has another biological activity of a ligand for CSF-1R, a ligand for PTP-zeta, or a ligand for CD138.

[0092] In some embodiments, the agonist polypeptides of the present invention have the properties of binding to CSF-1R or PTP-ζ, of inducing or enhancing survival of cultured glial cells, neural progenitor cells, or glioblastoma cells, and / or of inducing or enhancing tyrosine phosphorylation.

[0093] In other embodiments, the agonist polypeptides of the invention have the properties of binding to CSF-1R and of inducing or enhancing Langerhans cell survival and / or of inducing or enhancing phosphorylation of Akt or ERK1 / 2.

[0094] Preferably, the agonist polypeptides of the invention have the property of binding to CSF-1R and the property of inducing or enhancing survival of cultured monocytes, particularly CD14+ monocytes, and / or the property of inducing or enhancing phosphorylation of Akt or ERK1 / 2.

[0095] A polypeptide is an "antagonist" of a ligand of CSF-1R, a ligand of PTP-zeta, or a ligand of CD138, or has antagonist activity of a ligand of CSF-1R, a ligand of PTP-zeta, or a ligand of CD138, as long as the polypeptide has the property of binding to CSF-1R, PTP-zeta, or CD138 and partially or completely blocks, inhibits, or neutralizes another biological activity of the ligand of CSF-1R, the ligand of PTP-zeta, or the ligand of CD138.

[0096] For example, an antagonist polypeptide of the invention may compete with a ligand of CSF-1R for binding to CSF-1R and then partially or completely inhibit another biological activity of said ligand of CSF-1R, such as kinase phosphorylation.

[0097] In some embodiments, the antagonist polypeptides of the present invention have the property of binding to CSF-1R or PTP-ζ, the property of partially or completely blocking, inhibiting or neutralizing the survival of cultured glial cells, neural progenitor cells or glioblastoma cells, and / or the property of partially or completely blocking, inhibiting or neutralizing tyrosine phosphorylation.

[0098] In other embodiments, the antagonist polypeptides of the present invention have the properties of binding to CSF-1R, partially or completely blocking, inhibiting, or neutralizing Langerhans cell survival, and / or partially or completely blocking, inhibiting, or neutralizing phosphorylation of Akt or ERK1 / 2.

[0099] Preferably, the antagonist polypeptides of the invention have the property of binding to CSF-1R and the property of partially or completely blocking, inhibiting or neutralizing the survival of cultured monocytes, particularly CD14+ monocytes, and / or the phosphorylation of Akt or ERK1 / 2.

[0100] Preferably, the polypeptides of the invention have the property of binding to CSF-1R and are agonists or antagonists of the ligands of CSF-1R.

[0101] The binding activity of a polypeptide can be easily evaluated by a person skilled in the art in particular by surface plasmon resonance (SPR) assays, especially SPR assays performed on Biacore, by Western blot assays, by ELISA, by co-immunoprecipitation (co-ip) assays, by pull-down assays, by cross-linking assays or by label transfer techniques (FRET or HTRF assays) in vitro or in vivo.

[0102] For example, SPR experiments can be typically performed at 25°C on a Biacore. First, recombinant human CSF-1 receptor can be immobilized on the chip surface, typically by amine coupling. Thus, recombinant CSF-1 receptor can be typically diluted to 20 μg / mL in 10 mM sodium acetate solution at pH 5.0. The diluted CSF-1R can typically be covalently immobilized to a flow cell of a CM5 sensor chip, for example, via a primary amine group. IL-34 muteins can typically be analyzed with the "SCK (Single Cycle kinetics)" model on a chip with immobilized CSF-1R. Kinetic parameters of IL-34 polypeptide through CSF-1R can typically be determined by using a series of protein dilutions in the "SCK (Single Cycle Kinetics)" model. For example, IL-34 muteins as analytes can typically be diluted in HBS-P buffer at concentrations ranging from 25 nM to 400 nM. The flow rate over the immobilized CSF-1R can typically be 30 μL / min, for example, 120 seconds for binding and 600 seconds for dissociation. The sensor chip surface can then typically be regenerated with, for example, 10 mM NaOH for 30 seconds.

[0103] In particular, the binding activity of a polypeptide can generally be evaluated by the materials and methods described in Example 4 herein.

[0104] The ability of a polypeptide to induce or enhance survival of cultured monocytes, particularly CD14+ monocytes, can be readily assessed by one skilled in the art in vitro or in vivo, particularly by culturing isolated monocytes in vitro in a culture medium containing the polypeptide and assessing monocyte survival, for example by viability staining and phenotypic analysis by flow cytometry.

[0105] For example, the properties of a polypeptide that induces or enhances survival of cultured monocytes are typically determined by inducing or enhancing the survival of CD14 in complete medium with WT IL-34 or a variant at a final concentration in the range of 1.5-200 ng / ml, typically 100 ng / ml. + The monocytes can be cultured and assessed. Cells can be harvested, usually on day 3, and used for phenotypic analysis, for example, by viability staining or flow cytometry.

[0106] In particular, the property of a polypeptide to induce or enhance survival of cultured monocytes, particularly CD14+ monocytes, may be assessed generally by the materials and methods described in Example 5 herein.

[0107] The properties of a polypeptide to induce or enhance phosphorylation of Akt or ERK1 / 2 can be readily assessed in vitro or in vivo by one skilled in the art, inter alia, by Western blot, by ELISA phosphoarray, or by flow cytometry.

[0108] For example, the property of a polypeptide to induce or enhance phosphorylation of Akt or ERK1 / 2 can be assessed by culturing monocytes in FBS-free medium with WT IL-34 or a variant, typically for 1, 3, and 5 minutes. Analysis can typically be performed by flow cytometry, using, for example, anti-phospho-Akt or anti-phospho-Erk1 / 2 primary and secondary antibodies.

[0109] The property of a polypeptide to induce or enhance the phosphorylation of Akt or ERK1 / 2 can generally be evaluated by the materials and methods described in Example 6 herein.

[0110] A polypeptide is considered herein to have a detectable or detectable biological activity that mimics or is similar, identical, or greater than the biological activity of a ligand of CSF-1R, a ligand of PTP-zeta, or a ligand of CD138, e.g., a human IL-34 polypeptide, if the level of detectable or detectable biological activity of said polypeptide is equal to, or preferably is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 300% greater than, the level of detectable or detectable biological activity of said polypeptide.

[0111] A polypeptide is considered herein to have a detectable or detectable biological activity that partially or completely blocks, inhibits, or neutralizes the biological activity of a ligand of CSF-1R, a ligand of PTP-zeta, or a ligand of CD138 if the level of detectable or detectable biological activity of said polypeptide is preferably 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 300% lower than the level of detectable or detectable biological activity of a ligand of CSF-1R, a ligand of PTP-zeta, or a ligand of CD138, e.g., a human IL-34 polypeptide.

[0112] The polypeptide of the present invention ranges from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). The amino acid sequence may comprise a fragment of the sequence comprising an amino acid sequence containing at least one mutation selected from the group consisting of:

[0113] The polypeptide of the present invention also includes a polypeptide ranging from an asparagine (N) residue at position 21 of SEQ ID NO:1 to a proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). The amino acid sequence may consist of a fragment of a sequence having at least 80% identity to the amino acid sequence containing at least one mutation selected from the group consisting of:

[0114] Another polypeptide of the invention ranges from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D) and A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R) The amino acid sequence may comprise a fragment of the sequence comprising an amino acid sequence containing at least one mutation selected from the group consisting of:

[0115] Another polypeptide of the invention ranges from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D) and A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R) The amino acid sequence may consist of a fragment of a sequence having at least 80% identity to the amino acid sequence containing at least one mutation selected from the group consisting of:

[0116] A "fragment" of a reference sequence herein means a sequence consisting of a chain of consecutive amino acids of the reference sequence, the size of which is smaller than the size of the reference sequence. In the context of the present invention, a fragment may have a size of, for example, 6-222, 6-210, 6-200, 6-175, 6-150, 6-125, 6-100, 6-75, 6-50, 6-25, 6-15, 6-10 amino acids, or a size of 6-222, 10-222, 25-222, 50-222, 75-222, 100-222, 125-222, 150-222, 175-222, 200-222, 210-222, 220-222 amino acids. Most preferably, the polypeptide of the present invention may have a size of 222 amino acids.

[0117] The polypeptides of the present invention also include any polypeptides which are "variants," "homologs," or "derivatives" of the polypeptides described herein above, which exhibit the same biological activity.

[0118] Preferably, the polypeptide of the present invention is a variant of a human IL-34 polypeptide.

[0119] Thus, the polypeptides of the present invention include polypeptides having a sequence derived from the amino acid sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, or derived from a fragment of the amino acid sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, defined by a certain percentage of sequence identity with the sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1.

[0120] A "variant," "homolog," or "derivative" polypeptide is defined as comprising a sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence.

[0121] These derived sequences may differ from the reference sequence by substitution, deletion and / or insertion of one or more amino acids at several positions, such that these modifications have no significant effect on the biological activity of the polypeptide. Substitutions may in particular correspond to conservative substitutions or to the replacement of natural amino acids by non-natural or pseudo amino acids.

[0122] "An amino acid sequence having at least 80% identity to a reference sequence (for example)" as used herein means a sequence that is identical to the reference sequence, but that may contain up to 20 mutations (substitutions, deletions, and / or insertions) per each portion of the 100 amino acids of the reference sequence. Thus, for a 100 amino acid reference sequence, an 80 amino acid fragment and a 100 amino acid sequence that contains 20 substitutions compared to the reference sequence are two examples of sequences that have 80% sequence identity to the reference sequence.

[0123] The percentage of identity is generally determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). The amino acid sequences to be compared are aligned to obtain the maximum percentage identity. For this purpose, it may be necessary to artificially add gaps to the sequence. Alignment can be performed manually or automatically. Automated nucleotide sequence alignment algorithms are well known to those skilled in the art, and are described, for example, in Altschul et al. (1997) Nucleic Acids Res. 25:3389 and implemented by software such as Blast software. One algorithm that can be isolated is, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J Mol Biol. 48:443-53). Once an optimal alignment has been achieved, a percentage identity is established by recording all positions where the amino acids of the two compared sequences are identical, and compared to the total number of positions.

[0124] Thus, the polypeptide of the present invention is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). A sequence having at least 80%, 85%, 90%, 95%, or 100% identity to an amino acid sequence containing at least one mutation selected from the group consisting of: b) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the serine residue at position 100 is replaced by a phenylalanine residue (S100F), A mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y), A mutation in which the glutamine residue at position 131 is replaced by a phenylalanine residue (Q131F), A mutation in which the threonine residue at position 36 is replaced by a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced by a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced by cysteine ​​residues (H56C and G112C). A fragment of a sequence having at least 80%, 85%, 90%, 95%, or 100% identity to an amino acid sequence containing at least one mutation selected from the group consisting of: It may comprise or consist of a sequence selected from:

[0125] Another polypeptide of the invention is a) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D), and A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R) A sequence having at least 80%, 85%, 90%, 95%, or 100% identity to an amino acid sequence containing at least one mutation selected from the group consisting of: b) ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the serine residue at position 100 is replaced by an aspartic acid residue (S100D), and A mutation in which the glutamine residue at position 131 is replaced by an arginine residue (Q131R) A fragment of a sequence having at least 80%, 85%, 90%, 95%, or 100% identity to an amino acid sequence containing at least one mutation selected from the group consisting of: It may comprise or consist of a sequence selected from:

[0126] In one particular embodiment, the sequence of the polypeptide differs from the reference sequence only through the presence of conservative substitution.Conservative substitution is the substitution of the same class of amino acids, such as the substitution of amino acids with uncharged side chains (such as asparagine, glutamine, serine, cysteine ​​and tyrosine), the substitution of amino acids with basic side chains (such as lysine, arginine and histidine), the substitution of amino acids with acidic side chains (such as aspartic acid and glutamic acid), the substitution of amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan).

[0127] In the present invention, polypeptides may be modified chemically or enzymatically to improve their stability or bioavailability. Such chemical or enzymatic modifications are well known to those skilled in the art. The following modifications may be mentioned, but are not limited to: Modification of the C-terminus or N-terminus of the polypeptide, such as deamination or acylation (preferably acetylation) of the N-terminus, or amidation or esterification of the C-terminus; Modification of the amide bond between two amino acids, such as acylation (preferably acteylation) or alkylation at the nitrogen or alpha carbon; A change in chirality, e.g., replacement of a natural amino acid (L-enantiomer) with the corresponding D-enantiomer; this modification can optionally be achieved by inversion of the side chain (from the C-terminus to the N-terminus); a change to an azapeptide in which one or more alpha carbons are replaced by nitrogen atoms; and / or A change to a β-peptide in which one or more carbons are added to the N-α or C-α side of the main chain.

[0128] In this regard, in particular: Amidation: This modification is easily accomplished in which the positive charge of lysine is replaced with a hydrophobic group (e.g., acetyl or phenylacetyl); Amination: Primary amine R = (CH2)4-NH3, for example by forming an N-methyl, N-allyl, or N-benzyl group + by formation of a secondary amide from; and By formation of N-oxide, N-nitroso, N-dialkylphosphoryl, N-sulfenyl, or N-glycosidic groups It is possible to modify one or more of the lysine amino acids (K) of the polypeptide.

[0129] Alternatively, one or more threonine (T) and / or serine (S) amino acids of a polypeptide can be modified, particularly by adding an ester or ether group to the OH group of the side chain of threonine and / or serine. Simple manipulations such as esterification can be performed using carboxylic acids, anhydrides to form acetates or benzoates, by cross-linking, etc. Etherification, which provides more stable compounds, can be performed using alcohols, halides, etc. to form, for example, methyl ethers or O-glycosides.

[0130] Alternatively, one or more glutamine (Q) amino acids can be modified, whether functionalized or not, by forming secondary or tertiary amines, for example by amidation, particularly with groups of the methyl, ethyl type.

[0131] Or, for example, By esterification to form methyl esters, ethyl esters, benzyl esters, thiols (activated esters), whether substituted or not; and By amidation, especially to form N,N dimethyl groups, nitroanilides, pyrrolidinyls, It is possible for one or more of the glutamic acid (E) and / or aspartic acid (D) amino acids to be modified.

[0132] On the other hand, it is preferred not to modify proline amino acids involved in the secondary structure of the polypeptide, bearing in mind also that the amino acids G, A and M generally do not offer obviously interesting modification possibilities.

[0133] In some embodiments, the polypeptide of the present invention may comprise a tag. A tag is an epitope-containing sequence that may be useful for purifying the polypeptide. It may be bound by various techniques, such as affinity chromatography, localizing said polypeptide in cell or tissue samples using immunolabeling techniques, detecting said polypeptide by immunoblotting, etc. Examples of tags commonly used in this field are GST (glutathione-S-transferase)-tag, FLAG™-tag, Strep-tag™, V5 tag, myc tag, His tag (usually consisting of six histidine residues), etc.

[0134] The present invention also relates to fusion proteins consisting of a mutated IL-34 polypeptide of the present invention fused to a heterologous polypeptide (ie, a polypeptide that is not IL-34 or a variant thereof).

[0135] In some embodiments, the fusion protein is an immunoadhesin in which the mutated IL-34 polypeptide of the present invention is fused to an Fc region. In some embodiments, the Fc region is a native sequence Fc region. In some embodiments, the Fc region is a variant Fc region. In some embodiments, the Fc region is a functional Fc region. In some embodiments, the mutated IL-34 polypeptide is fused to the Fc region via a linker. Such linkers may be useful to prevent steric hindrance. In some embodiments, the linker has 4;5;6;7;8;9;10;11;12;13;14;15;16;17;18;19;20;21;22;23;24;25;26;27;28;29;30 amino acid residues. However, the upper limit is not critical, but is selected for reasons of convenience, for example, for biopharmaceutical products of the polypeptide. The linker sequence may be a naturally occurring or non-naturally occurring sequence. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the immunoadhesin is administered.One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies described in International Patent Publications WO 96 / 34103 and WO 94 / 04678.Another example is a poly-alanine linker sequence.Another preferred example of a linker sequence is the Gly / Ser linker of different lengths, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0136] In some embodiments, it is contemplated that the mutated IL-34 polypeptides of the present invention are modified to improve their therapeutic effects. Such modifications of therapeutic compounds can be used to reduce toxicity, increase circulation time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be significantly reduced by combination with various drug carrier vehicles that modify biodistribution. A strategy to improve drug viability is the use of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change permeability through physiological barriers, and modify the rate of clearance from the body. Water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain to achieve a targeting or sustained release effect. For example, pegylation is a well-established and validated technique for the modification of a range of polypeptides. These advantages include, among others: (a) significantly improving circulatory half-life in vivo by avoiding renal clearance as a result of the polymer increasing the apparent size of the molecule beyond the limits of glomerular filtration and / or through avoidance of cellular clearance mechanisms; (b) reducing the antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improving pharmacokinetics; (d) increasing the proteolytic resistance of the conjugated protein; and (e) improving the thermal and mechanical stability of PEGylated polypeptides.

[0137] In the present invention, the mutated IL-34 polypeptides of the present invention (i.e., P1, P2, or P3) are produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and producing proteins are well known to those skilled in the art. The mutated IL-34 polypeptides of the present invention can be synthesized in solution or on solid supports according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols such as those described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The mutated IL-34 polypeptides of the present invention can also be synthesized by solid-phase techniques using an exemplary peptide synthesizer such as the Model 433A manufactured by Applied Biosystems Inc. The purity of any given protein produced via automated peptide synthesis or recombinant methods can be determined using reverse-phase HPLC analysis. The chemical authenticity of each peptide can be established by any method well known to those skilled in the art. As an alternative to automated peptide synthesis, recombinant DNA technology can be used in which a nucleotide sequence encoding a selected protein is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression as described herein below. Recombinant methods are particularly preferred for the production of longer polypeptides. A variety of expression vector / host systems can be utilized that contain and express sequences encoding peptides or proteins.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 (Giga-Hama et al., 1999); insect cell systems infected with viral expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids, see, e.g., Babe et al., 2000); or animal cell systems. Those skilled in the art are aware of various techniques for optimizing mammalian protein expression. See, e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells useful for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g., COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Exemplary protocols for recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are known to those skilled in the art and are briefly described herein below. Mammalian host systems for expression of recombinant proteins are also well known to those skilled in the art. Host cell lines can be selected for specific properties that process the expressed protein or result in specific post-translational modifications useful for providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipid modification, and acylation. Post-translational processing that cleaves the "prepro" form of the protein can also be important for correct insertion, folding, and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, have unique cellular and characteristic machinery for such post-translational activities and can be selected to ensure correct modification and processing of the introduced foreign protein.

[0138] Thus, a further object of the present invention relates to isolated, synthetic or recombinant nucleic acids encoding the mutated IL-34 polypeptides of the present invention.

[0139] In some embodiments, a nucleic acid of the invention is a DNA or RNA molecule and can be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0140] Thus, another subject of the present invention relates to a vector comprising a nucleic acid according to the invention.

[0141] Such vectors may include control elements, such as promoters, enhancers, terminators, etc., for effecting or directing expression of the polypeptide after administration to a subject. The vector may further include one or more origins of replication and / or selectable markers. The promoter region may be homologous or heterologous to the coding sequence and may provide ubiquitous, constitutive, regulated, and / or tissue-specific expression in any suitable host cell, including those for use in vivo. Examples of promoters include bacterial promoters (such as T7, pTAC, Trp promoters), viral promoters (such as LTR, TK, CMV-IE), mammalian gene promoters (such as albumin, PGK), and the like. Examples of plasmids include replicative plasmids that include an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, and the like. Examples of viral vectors include adenoviral vectors, retroviral vectors, herpes viral vectors, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in International Patent Publication Nos. 95 / 14785, 96 / 22378, U.S. Patent Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056, and International Patent Publication No. 94 / 19478.

[0142] Another subject of the present invention relates to a host cell which has been transfected, infected or transformed with a nucleic acid molecule and / or a vector according to the invention.

[0143] The nucleic acid molecules of the present invention can be used to produce the mutated IL-34 polypeptides of the present invention in a suitable expression system. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (such as produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). The construction of the expression vectors of the present invention and the transformation of the host cells can be carried out using conventional molecular biology techniques. The mutated IL-34 polypeptides of the invention can be obtained, for example, by culturing the genetically transformed cells of the invention and recovering the polypeptides expressed by said cells from the culture. These can then be purified, if necessary, by conventional techniques known per se to those skilled in the art, such as fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins can be used to produce the polypeptides of the invention.

[0144] Thus, the present invention also provides a method for producing a recombinant host cell expressing a mutated IL-34 polypeptide of the present invention, comprising the steps of: i) introducing the recombinant nucleic acid or vector into a competent host cell, either in vitro or ex vivo; ii) culturing the resulting recombinant host cells in vitro or ex vivo; iii) optionally selecting cells that express and / or secrete a polypeptide of the invention; The present invention relates to a method comprising the steps of:

[0145] Such recombinant host cells can be used to produce the polypeptides and fusion proteins of the invention.

[0146] The present invention further provides a method for producing a mutated IL-34 polypeptide of the present invention, comprising the steps of: i) culturing a transformed host cell according to the invention under suitable conditions to allow expression of said polypeptide or fusion protein; ii) recovering the expressed polypeptide or fusion protein; The present invention relates to a method comprising the steps of:

[0147] The mutated IL-34 polypeptides of the present invention are particularly suitable for therapeutic purposes.

[0148] In particular, the mutated IL-34 polypeptide (P1) can be used as an agonist since it is capable of forming stable dimers.

[0149] In particular, a mutated IL-34 polypeptide (P2) still binds to CSF-1R but is unable to form a dimer and can therefore be used as an antagonist.

[0150] In particular, the mutated IL-34 polypeptide (P3) has a high affinity for CSF-1R. Thus, when this mutation of the polypeptide (P3) is combined with the mutation of the polypeptide (P1), the resulting mutated IL-34 polypeptide represents a "super" agonist of CSF-1R, i.e., an agonist with high affinity for CSF-1R. Conversely, when the mutation of the polypeptide (P3) is combined with the mutation of the polypeptide (P2), the resulting mutated IL-34 polypeptide represents a "super" antagonist of CSF-1R, i.e., an antagonist with high affinity for CSF-1R. Some mutated IL-34 polypeptides (P3) also have agonist activity by themselves. Other mutated IL-34 polypeptides (P3) have antagonist activity by themselves.

[0151] Thus, a further object of the present invention relates to a mutated IL-34 polypeptide, nucleic acid or vector of the present invention for use as a medicament. More specifically, the present invention provides a method of treatment in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of at least one mutant IL-34 polypeptide, nucleic acid or vector of the present invention. Also disclosed is the use of a mutated IL-34 polypeptide, nucleic acid or vector of the present invention for the manufacture of a medicament.

[0152] In some embodiments, the agonist of the present invention is particularly suitable for inducing and / or maintaining immune tolerance in a patient in need thereof. Thus, the subject of the present invention relates to an agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for use in inducing and / or maintaining immune tolerance in a patient in need thereof. More specifically, the present invention provides a method for inducing and / or maintaining immune tolerance in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of at least one agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention. Also disclosed is the use of an agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for the manufacture of a medicament for inducing and / or maintaining immune tolerance in a patient in need thereof.

[0153] In particular, patients treated with the agonists of the invention compared to untreated patients exhibited the following physiological characteristics: a) antigen-specific effector CD4 + T lymphocytes and CD8 + a) a decrease in the level of an immune response (specific or non-specific) believed to be at least partially mediated by lymphocytes; b) a delay in the onset or progression of an immune response (specific or non-specific); or c) a decrease in the risk of onset or progression of an immune response (specific or non-specific). Thus, the agonists of the present invention are particularly intended for inducing and maintaining the proliferation of Treg cells.

[0154] In some embodiments, the agonist of the present invention is particularly suitable for preventing or reducing transplant rejection in a patient in need thereof. Thus, a further object of the present invention relates to an agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for use in preventing or reducing transplant rejection in a patient in need thereof. More specifically, the present invention provides a method for preventing or reducing transplant rejection in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of at least one agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention. Also disclosed is the use of an agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for the manufacture of a medicament for preventing or reducing transplant rejection in a patient in need thereof.

[0155] In some embodiments, the donor of the graft can be a living donor or a deceased donor, i.e., a cadaveric donor. In some embodiments, the graft is an organ, tissue, or cell. In some embodiments, the transplant rejection is a cardiac allograft rejection. In some embodiments, the cells are selected from the group consisting of multipotent hematopoietic stem cells derived from bone marrow, peripheral blood, or umbilical cord blood; or pluripotent (i.e., embryonic stem cells (ES) or induced pluripotent stem cells (iPS)), or differentiated cells derived from multipotent stem cells of different cell lineages, such as cardiomyocytes, beta pancreatic cells, hepatocytes, neurons, etc. In some embodiments, the cells are used for allogeneic hematopoietic stem cell transplantation (HSCT), thus including multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. HSCT can provide a cure for patients with leukemia and lymphoma. However, a significant limitation of allogeneic HCT is the development of graft-versus-host disease (GVHD), which occurs in a severe form in approximately 30-50% of people who undergo this treatment. Thus, in some embodiments, the agonists of the present invention are particularly suitable for preventing or reducing graft-versus-host disease (GvHD). Thus, in some embodiments, the patient undergoing hematopoietic stem cell transplantation suffers from a disease selected from the group consisting of acute myeloid leukemia (AML); acute lymphocytic leukemia (ALL); chronic myeloid leukemia (CML); myelodysplastic syndrome (MDS) / myeloproliferative syndrome; lymphoma, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma.

[0156] In some embodiments, the agonist of the present invention is particularly suitable for treating a neurodegenerative disease in a patient. Thus, a further object of the present invention relates to an agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for use in treating a neurodegenerative disease in a patient. More specifically, the present invention provides a method for treating a neurodegenerative disease in a patient, comprising administering to said patient a therapeutically effective amount of at least one agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention. Also disclosed is the use of an agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for the manufacture of a medicament for treating a neurodegenerative disease in a patient.

[0157] Examples of neurodegenerative diseases include, but are not limited to, Parkinson's disease, Parkinson-dementia, autosomal recessive PARK2 and PARK6-associated parkinsonism, progressive supranuclear palsy, corticobasal degeneration syndrome, dementia with Lewy bodies, multiple system atrophy, Guadeloupean Parkinsonism, and Lytigo-bodig disease. Parkinson's disease and related disorders, including atypical parkinsonism including amyotrophic lateral sclerosis, frontotemporal dementia, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, and motor neuron diseases, including post-polio syndrome; neuroinflammatory diseases; early Alzheimer's disease, mild Alzheimer's disease, moderate Alzheimer's disease, mild-moderate Alzheimer's disease, advanced Alzheimer's disease, mild cognitive impairment, vascular dementia, mixed dementia, Pick's disease, argyrophilic grain disease, posterior cortical dementia, PCA atrophy), Alzheimer's disease and related disorders including Wernicke-Korsakoff syndrome;Prion diseases;Lysosomal storage diseases;Leukodystrophies;Huntington's disease;Multiple sclerosis;Down syndrome;Spinal-bulbar muscular atrophy;HIV-associated neurocognitive disorders;Tourette's syndrome;Autosomal dominant spinocerebellar ataxia;Friedreich's ataxia;Dentatorubral-pallidoluysian atrophy;Myotonic dystrophy;Schizophrenia;Age-related memory disorders;Autism and autism spectrum disorders;Attention deficit hyperactivity disorder;Chronic pain;Al ... Cole-induced dementia;Progressive non-fluent aphasia;Semantic dementia;Spastic paraplegia;Fibromyalgia;Post-Lyme disease treatment syndrome;Neurological disorders;Withdrawal symptoms;Alpers disease;Cerebro-ocular-facial-skeletal syndrome;Wilson's disease;Cockayne syndrome;Leigh disease;Neurodegeneration with cerebral iron deposition;Osoclonus-myoclonus syndrome;α-Methylacyl-CoA racemase deficiency;Andermann syndrome;Arts syndrome;Marinesco-Sjögren syndrome;Mitochondrial membrane-associated neurodegeneration;Pantothenate kinase-associated neurodegeneration;PLOSL (Nasu-Hakola disease: polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy); riboflavin transporter deficiency neuronopathy; and ataxia telangiectasia.

[0158] Preferably, the neurodegenerative disease is selected from the group consisting of multiple sclerosis, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease.

[0159] In some embodiments, the agonist of the present invention is particularly suitable for treating autoimmune inflammatory diseases, alloimmune response allergic disorders, and asthma in the patient. Thus, a further object of the present invention relates to the agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for use in treating autoimmune inflammatory diseases, alloimmune response allergic disorders, and asthma in the patient. More specifically, the present invention provides a method for treating autoimmune inflammatory diseases, alloimmune response allergic disorders, and asthma in the patient, comprising administering to the patient a therapeutically effective amount of at least one agonist mutant IL-34 polypeptide, nucleic acid, or vector of the present invention. Also disclosed is the use of the agonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for the manufacture of a medicament for treating autoimmune inflammatory diseases, alloimmune response allergic disorders, and asthma in the patient.

[0160] In some embodiments, the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, osteoarthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis, and juvenile onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis (arthritis chronica progrediente), osteoarthritis, chronic primary polyarthritis (polyarthritis chronica primaria), reactive arthritis, and ankylosing spondylitis, inflammatory hyperproliferative skin diseases, psoriasis, such as psoriasis vulgaris, guttate psoriasis (gutate psoriasis), and psoriasis purpura. psoriasis), pustular psoriasis, and psoriasis of the nail, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, X-linked hyper IgM syndrome, urticaria, chronic idiopathic urticaria including chronic allergic urticaria, and chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic sclerosis, sclerosis, systemic sclerosis, multiple sclerosis (MS), optic-spinal MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, ulcerative colitis ulcerosa, microscopic colitis, collagen colitis, colitis polyposa (CP), necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult respiratory distress syndrome or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylactic and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and / or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitisuveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranoproliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematodes (SLE) or systemic lupus erythematodes, e.g. cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus disseminatus), lupus (including nephritis, encephalitis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile-onset (type I) diabetes mellitus including insulin-dependent diabetes mellitus (IDDM) of childhood, adult-onset (type II) diabetes mellitus, autoimmune diabetes mellitus, idiopathic diabetes insipidus, immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, large vasculitis, rheumatoid polyposis Myalgia, giant cell (Takayasu) arteritis, medium-sized vasculitis, Kawasaki disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, including 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 perniciosa, Addison's disease, pure red cell anemia or pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases associated with leukocyte leakage, CNS inflammatory disorders, multi-organ injury syndromes, e.g. secondary to sepsis, trauma, or hemorrhage, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid, e.g. bullous pemphigoidbullous) and cutaneous pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid), pemphigoid erythematosus, autoimmune polyendocrine syndrome, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathy, chronic neuropathy, IgM polyneuropathy, 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, Graves' disease, polyglandular syndromes, such as autoimmune polyglandular syndrome (or polyendocrine syndrome), neuro-paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome Paraneoplastic syndromes including stiff-man syndrome 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 syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease, disease), celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosisautoimmune ear diseases, e.g. autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus-myoclonus syndrome (OMS), polychondritis, e.g. refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis, optionally benign M-proteinemia or monoclonal garnmopathy of undetermined encephalomyelopathy (MGUS). (genetic significance), peripheral neuropathy, paraneoplastic syndromes, channelopathies, such as epilepsy, migraine, cardiac arrhythmias, muscle disorders, hearing loss, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathies, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, chorioretinitis, autoimmune hepatopathy, fibromyalgia, multiple endocrine disorders, Schmidt's syndrome, adrenal inflammation, gastric atrophy, presenile dementia, demyelinating diseases, such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler'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 abortions, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird breeder's disease, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis, e.g. allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Kaplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum), erythroblastosis fetalis, eosinophilic fasciitisfaciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis, e.g. chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuchs' cyclitis, Henoch-Schönlein 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, Evans syndrome, autoimmune gonadal dysfunction, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthalmopathy, ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephrosis, benign familial and ischemia-reperfusion injury, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway disease, silicosis, aphtha, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hamman-Rich disease, sensorineural hearing loss, haemoglobinuria paroxysmatica), hypogonadism, ileitis regionalis, leukopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, de Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antibodies antobodies), non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-related diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion deficiency, immune responses related to acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte leakage, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmopathy inflammation, 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, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrative eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disorders, sclerosing cholangitis, scleral, episclerotic, chronic mucocutaneous candidiasis, Bruton's syndrome, transient infantile hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia-telangiectasia, autoimmune disorders associated with collagen diseases, rheumatism, neurological disorders, ischemia-reperfusion injury, reduced blood pressure response, vascular insufficiency, antgiectasis, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia and diseases complicated by angiogenesis, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of the myocardium or other tissues, skin diseases with an acute inflammatory component, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion associated syndrome, cytokine induced toxicity, acute severe inflammation, chronic refractory inflammation, pyelitis, pulmonary cirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0161] Allergic disorders typically include systemic allergic reactions, systemic anaphylaxis or hypersensitivity reactions, anaphylactic shock, drug allergies, and insect bite allergies; respiratory allergic diseases such as asthma, hypersensitivity lung disease, hypersensitivity pneumonitis, and interstitial lung disease (ILD) (e.g., idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, or other autoimmune conditions); rhinitis, hay fever, conjunctivitis, allergic rhinitis conjunctivitis, and vaginitis; psoriasis. and inflammatory skin diseases, such as skin and dermatological disorders including dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, herpetiform dermatitis, linear IgA disease, acute and chronic urticaria and scleroderma; vasculitis (e.g. necrotizing, cutaneous, and hypersensitivity vasculitis); spondyloarthropathy; and intestinal reactions of the gastrointestinal system (e.g. inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, ileitis, enteritis, non-tropical sprue, and celiac disease).

[0162] In some embodiments, the agonist of the present invention is administered to a patient in combination with an immunosuppressant. Immunosuppressants include, but are not limited to, thiopurine drugs, such as azathioprine (AZA) and their metabolites; nucleoside triphosphate inhibitors, such as mycophenolic acid (Cellcept) and its derivatives (Myfortic); their derivatives; their prodrugs; and combinations thereof. Other examples include, but are not limited to, 6-mercaptopurine ("6-MP"), cyclophosphamide, mycophenolic acid, prednisolone, sirolimus, dexamethasone, rapamycin, FK506, mizoribine, azothioprine, and tacrolimus.

[0163] In some embodiments, the immunosuppressant is selected from the group consisting of cytostatic agents, such as mammalian target of rapamycin (mTOR) inhibitors and rapamycin (sirolimus); alkylating agents (cyclophosphamide) and antimetabolites (azathioprine, mercaptopurine, and methotrexate); therapeutic antibodies (such as anti-CD40L monoclonal antibodies, anti-IL-2R monoclonal antibodies, anti-CD3 monoclonal antibodies, anti-CD45RC monoclonal antibodies, anti-lymphocyte globulin (ALG), and anti-thymocyte globulin (ATG)); calcineurin inhibitors (cyclosporine); glucocorticoids, and mycophenolate mofetil.

[0164] In some embodiments, the immunosuppressant is a calcineurin inhibitor. As used herein, the term "calcineurin inhibitor" has its general meaning in the art and refers to a substance that inhibits calcineurin (i.e., a calcium / calmodulin-regulated protein phosphatase involved in intracellular signaling), which dephosphorylates appropriate substrates by targeting calcineurin phosphatase (PP2B, PP3), a cellular enzyme involved in gene regulation. Calcineurin inhibitors of the present invention are typically immunophilin-binding compounds with calcineurin inhibitory activity. Calcineurin inhibitors that bind to immunophilins are compounds that form calcineurin-inhibitory complexes with immunophilins, such as cyclophilins and macrophilins. Examples of calcineurin inhibitors that bind to cyclophilin are cyclosporine or cyclosporine derivatives (hereinafter cyclosporine), and examples of calcineurin inhibitors that bind to macrophilin are ascomycin (FR 520) and ascomycin derivatives (hereinafter ascomycin). A wide range of ascomycin derivatives are known, which occur naturally in fungal species or are obtained by engineering fermentation techniques or chemical derivatization. Ascomycin-type macrolides include ascomycin, tacrolimus (FK506), sirolimus, and pimecrolimus. Cyclosporines were originally extracted from the soil fungus Potypaciadium infilatum and have a cyclic 11 amino acid structure, including cyclosporine A-I, such as cyclosporine A, B, C, D, and G. Voclosporin is a next-generation calcineurin inhibitor that is a semi-synthetic derivative of cyclosporine A that is more potent and less toxic. In some embodiments, the calcineurin inhibitor of the present invention is trans-ISA247 (Cas number 368455-04-3), a trans version of voclosporin, e.g., as described in U.S. Patent Publication No. 2006 / 0217309, which is incorporated herein by reference.Further compositions of voclosporin are described, for example, in U.S. Patent No. 7,060,672, which is incorporated herein by reference. Tacrolimus (FK506) is another calcineurin inhibitor, also a fungal product, but with a macrolide lactone structure. Sirolimus (rapamycin) is a microbial product isolated from the actinomycete Streptomyces hygroscopicus. Sirolimus binds to immunophilin (FK binding protein 12, FKBP12) to form a complex and inhibits the mammalian target of rapamycin (mTOR) pathway via direct binding to mTOR complex 1 (mTORC1). Pimecrolimus is also a calcineurin inhibitor. Calcineurin inhibitors such as cyclosporine A, voclosporin, ascomycin, tacrolimus, pimecrolimus, analogs thereof, or pharma- ceutically acceptable salts thereof may be utilized in the mixed micelle compositions of the present disclosure.

[0165] In some embodiments, the immunosuppressant is a corticosteroid. As used herein, the term "corticosteroid" has its general meaning in the art and refers to a class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system that have anti-inflammatory activity. Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (11β,17-dihydroxy,21-(phosphonooxy)-pregn-4-ene,3,20-dione disodium), dihydroxycortisone, dexamethasone (21-(acetyloxy)-9-fluoro-1β,17-dihydroxy-16α-m-ethylpregna-1,4-diene-3,20-dione), and heavily derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-11-β,17,21,trihydroxy-16β-methylpregna-1,4-diene-3,20-dione 17,21-dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort, and betamethasone, corticosteroids such as cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

[0166] In some embodiments, the immunosuppressant is a CTLA4 molecule. In some embodiments, the CTLA4 molecule is abatacept. Abatacept is a soluble fusion protein consisting of the extracellular domain of human CTLA-4 bound to a modified Fc (hinge, CH2, and CH3 domain) portion of human immunoglobulin G1 (IgG1). Abatacept is produced by recombinant DNA technology in a mammalian cell expression system. The apparent molecular weight of abatacept is 92 kilodaltons. Abatacept was developed by Bristol-Myers Squibb and is disclosed, for example, in U.S. Pat. Nos. 5,851,795, 7,455,835, and EP 1962886. In some embodiments, the CTLA molecule is belatacept, which is the result of changing two amino acids in the CD80 / 86 binding portion of the abatacept compound (L104E and A29Y). This slight chemical change resulted in a 10-fold increase in the properties that inhibit T cell activation when compared in vitro. Belatacept (L104EA29YIg) is the first biological agent approved for initial maintenance immunosuppression that selectively inhibits the CD28 costimulatory pathway to block T cell activation (Larsen, CP. et al, Am. J. Transplant., 5:443-453 (2005)). In some embodiments, the CTLA4 molecule is MAXY-4, a protein also derived from abatacept, but with enhanced binding to the CTLA4 target, currently in preclinical development by Perseid Therapeutics, LLC and Astellas Pharma, Inc for the treatment of autoimmune diseases and transplant rejection.

[0167] In some embodiments, the agonists of the present invention are particularly suitable for preventing undesired immune responses against proteins or therapeutic proteins and allergies expressed in the patient during gene therapy. For example, this approach can in fact be applied to suppress immune responses, particularly to prevent immune reactions against certain proteins when their expression is restored by gene therapy in individuals with the corresponding gene defects. Thus, the agonists of the present invention may be used to prevent immune reactivity against proteins that are not normally present in the patient due to mutations, and their reconstitution is achieved by gene therapy. Furthermore, protein therapy is a field of medical innovation that is becoming more widespread and involves the direct application of proteins, such as enzymes, antibodies, or cytokines, as therapeutic products to patients. One of the main hurdles in the delivery of such drugs involves immune responses against the therapeutic protein itself. The administration of protein-based therapeutics is often achieved by the administration of immunosuppressants, which are used to facilitate a longer life span of the protein and thus an increased uptake of the protein into the cells and tissues of the organism. However, common immunosuppressants are disadvantageous due to the non-specific nature of the immunosuppression they provide and can result in undesirable side effects in patients. Thus, this approach can be applied to suppress immune responses to therapeutic proteins and peptides, such as therapeutic antibodies, cytokines, enzymes, or any other proteins administered to patients.

[0168] In some embodiments, the antagonist of the present invention is particularly suitable for treating bone disease with abnormal proliferation in the patient. Thus, a further object of the present invention relates to the antagonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for use in treating bone disease with abnormal proliferation in the patient. More specifically, the present invention provides a method of treating bone disease with abnormal proliferation in the patient, comprising administering to the patient a therapeutically effective amount of at least one antagonist mutant IL-34 polypeptide, nucleic acid, or vector of the present invention. Also disclosed is the use of the antagonist mutated IL-34 polypeptide, nucleic acid, or vector of the present invention for the manufacture of a medicament for treating bone disease with abnormal proliferation in the patient.

[0169] In some embodiments, the bone disease associated with abnormal proliferation is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, juvenile onset rheumatoid arthritis, spondyloarthritis, osteosarcoma, giant cell tumor, bone fractures, osteoporosis, bone infections, and Paget's disease.

[0170] The antagonists of the present invention are intended to inhibit Tregs (e.g., CD8 + and CD4 + The present invention is particularly suited to providing an effective and safe method for releasing a T cell immune response controlled (i.e. suppressed or inhibited) by CD4 Tregs. The present invention thus also provides a method for inducing a T cell response in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an antagonist of the present invention, wherein said T cell response is mediated by CD4 Tregs in said subject. + CD25 - In some embodiments, the present invention relates to a method of eliciting an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antagonist of the present invention, wherein the therapeutically effective amount is sufficient to reduce immune tolerance in the subject.

[0171] The present invention also provides a method for inducing an anti-tumor T cell response in a subject having a tumor, comprising administering to the subject a therapeutically effective amount of an antagonist of the present invention, wherein the T cell response is in the form of a CD4 + CD25 - In some embodiments, inducing an anti-tumor T cell response in a subject with a tumor or cancer allows for treating the tumor or cancer in the subject. In some embodiments, inducing an anti-tumor T cell response in a subject with a tumor or cancer prevents the establishment of metastasis in the subject. Thus, provided herein is a method of increasing / inducing / inducing an immune response in a subject in need thereof. For example, the subject may have developed or may develop immune tolerance to unwanted cells, such as cancer (e.g., tumor) cells and / or cells infected with a pathogen. The method comprises administering to the subject a therapeutically effective amount of an antagonist of the invention.

[0172] A further object of the present invention relates to the antagonist mutated IL-34 polypeptide, nucleic acid or vector of the present invention for use in the treatment of cancer. Or the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antagonist mutated IL-34 polypeptide, nucleic acid or vector of the present invention. Also disclosed is the use of the antagonist mutated IL-34 polypeptide, nucleic acid or vector of the present invention for the manufacture of a medicament for the treatment of cancer.

[0173] Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testicles, tongue, or uterus. In some embodiments, the subject is diagnosed with acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphocytic 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, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, 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, AT / RT (Atypical teratoid rhabdoid tumor), basal cell carcinoma, basal-like cell tumor, 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 lymphoma, cancer of unknown primary origin, carcinoid tumor, cell tumor, intraepithelial carcinoma, penile cell tumor, cell tumor of unknown primary origin, carcinosarcoma, Castleman disease, central nervous system embryonal tumor, cerebellar astrocytoma Follicular tumor, 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, DNT (dysembryoplastic neuroepithelial tumor), embryonal carcinoma, yolk sac tumor, endometrial cancer, uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, epidermoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing Family of TumorsTumor, Ewing Family Sarcoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's Disease, Fallopian Tube Cancer, Fetus in Inclusion Malformation fetu), fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, digestive tract 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, glucagon-producing tumor, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, hemangiosarcoma, blood tumor, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, HBOC (Hereditary Breast-Ovarian Cancer) cance syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, pancreatic islet cell carcinoma, pancreatic islet tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Kracken's tumor, Krukenberg's tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant, mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, MTT (Malignant Tritontumor), MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary site, metastatic urothelial carcinoma, mixed Mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, Mycosis fungoides, Myelodysplastic Disorders, Myelodysplastic Disorders, Syndromes, Myeloid Leukemia, Myelosarcoma, Myeloproliferative Disorders, Myxoma, Nasal Carcinoma, Nasopharyngeal Carcinoma, Neoplasms, Schwannoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular Melanoma, Non-Hodgkin's Lymphoma, Non-Melanoma Skin Cancer, Non-Small Cell Lung Cancer, Non-Small Cell Lung Cancer (NSCLC) Coexisting with Chronic Obstructive Pulmonary Disease (COPD), Ocular Tumors oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath, meningioma, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian tumor of low malignant potential, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papilloma, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma blastema, polygerminoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal carcinoma, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, tract carcinoma involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformationtransformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor tumor), small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, sooty wart, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial and stromal tumor, synovial sarcoma, T-cell acute lymphocytic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, capsular tumor, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Werner-Morrison syndrome The patient has a cancer selected from the group consisting of glaucoma, verrucous membrane necrosis syndrome, optic pathway glioma, vulvar cancer, Waldenstrom's hypergammaglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

[0174] In some embodiments, the antagonists of the invention are administered to the patient in combination with chemotherapy. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs; Cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189, and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuen bikinin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γll and calicheamicin ωll; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediynes, antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, de torubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin antimetabolites such as methotrexate, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgenic drugs such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;anti-adrenals such as aminoglutethimide, mitotane, trilostane;folic acid supplements such as frolinic acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabucil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A) A), Roridin A A) and anguidine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronic acid; irinotecan (e.g. CPT-1 1); the topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids, such as retinoic acid; capecitabine, as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the above.

[0175] In some embodiments, the antagonists of the invention are administered to a patient in combination with immunotherapy.

[0176] In some embodiments, the immunotherapy comprises administering at least one immune checkpoint inhibitor to the patient. A preferred immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. Many immune checkpoint inhibitors are known, and in analogy with these known immune checkpoint protein inhibitors, other immune checkpoint inhibitors may be developed in the (near) future. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules, and small molecules. Examples of immune checkpoint inhibitors include 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 antagonists, and BTLA antagonists.

[0177] In some embodiments, antagonists of the PD-1 (Programmed Death-1) axis include PD-1 antagonists (e.g., anti-PD-1 antibodies), PD-L1 (Programmed Death Ligand-1) antagonists (e.g., anti-PD-L1 antibodies), and PD-L2 (Programmed Death Ligand-2) antagonists (e.g., anti-PD-L2 antibodies). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg). In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105 is an anti-PD-L1 antibody described in International Patent Publication No. 2007 / 005874, also known as BMS-936559. Antibody YW243.55.S70 is an anti-PD-L1 antibody described in International Patent Publication No. 2010 / 077634. ​​MEDI4736 is an anti-PD-L1 antibody described in International Patent Publication No. 2011 / 066389 and US Patent Publication No. 2013 / 034559. MDX-1106 is an anti-PD-1 antibody described in US Patent No. 8,008,449 and International Patent Publication No. 2006 / 121168, also known as MDX-1106-04, ONO-4538, or BMS-936558. 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 International Patent Publication No. WO 2009 / 114335.CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in International Patent Publication No. 2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in International Patent Publication Nos. 2010 / 027827 and 2011 / 066342. Atezolizumab is an anti-PD-L1 antibody described in U.S. Patent Publication No. 8,217,149. Avelumab is an anti-PD-L1 antibody described in U.S. Patent Publication No. 20140341917. CA-170 is a PD-1 antagonist described in International Patent Publication Nos. 2015033301 and 2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Patent No. 8,609,089, U.S. Patent Publication Nos. 2010028330, and / or 20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the PD-L1 antagonist is selected from the group including avelumab, BMS-936559, CA-170, durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110, KY1003, and atezolizumab, and preferred PD-L1 antagonists are avelumab, durvalumab, or atezolizumab.

[0178] In some embodiments, the CTLA-4 (cytotoxic T-lymphocyte antigen 4) antagonist is an anti-CTLA-4 antibody, a human anti-CTLA-4 antibody, a murine anti-CTLA-4 antibody, a mammalian anti-CTLA-4 antibody, a humanized anti-CTLA-4 antibody, a monoclonal anti-CTLA-4 antibody, a polyclonal anti-CTLA-4 antibody, a chimeric anti-CTLA-4 antibody, MDX-010 (ipilimumab), tremelimumab, an anti-CD28 antibody, an anti-CTLA-4 adnectin, an anti-CTLA- The antibody is selected from the group consisting of a four domain antibody, a single chain anti-CTLA-4 fragment, a heavy chain anti-CTLA-4 fragment, a light chain anti-CTLA-4 fragment, an inhibitor of CTLA-4 that recognizes the costimulatory pathway, an antibody disclosed in WO 2001 / 014424, an antibody disclosed in WO 2004 / 035607, an antibody disclosed in U.S. Patent Publication No. 2005 / 0201994, and an antibody disclosed in granted European Patent No. 1212422. Additional CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; International Patent Publication Nos. 01 / 14424 and 00 / 37504; and U.S. Patent Publication Nos. 2002 / 0039581 and 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in the methods of the invention include, for example, those disclosed in International Patent Publication No. 98 / 42752; U.S. Patent Nos. 6,682,736 and 6,207,156; Hurwitz et al., 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. Patent Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.A preferred clinical CTLA-4 antibody is the human monoclonal antibody disclosed in International Patent Publication No. WO 01 / 14424 (also called MDX-010 having CAS No. 477202-00-9 and ipilimumab, available from Medarex, Inc., Bloomsbury, NJ). As for CTLA-4 antagonists (antibodies), these are known and include tremelimumab (CP-675,206) and ipilimumab.

[0179] In some embodiments, the immunotherapy comprises administering to the patient a combination of a CTLA-4 antagonist and a PD-1 antagonist.

[0180] 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., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 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). Thus, 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 specific for TIM-3 are well known in the art and are generally described in International Patent Publication Nos. 2011155607, 2013006490, and 2010117057.

[0181] In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in International Patent Publication No. 2014150677. Examples of IDO inhibitors include, but are not limited to, 1-methyl-tryptophan (IMT), β-(3-benzofuranyl)-alanine, β-(3-benzo(b)thienyl)-alanine, 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyltryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-amino-naphthoic acid (naphthoic acid), pyrrolidine dithiocarbamate, 4-phenylimidazole, brassinin derivatives, thiohydantoin derivatives, β-carboline derivatives or brassilexin derivatives.Preferred IDO inhibitors are selected from 1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphtoic acid, and β-[3-benzo(b)thienyl]-alanine, or derivatives or prodrugs thereof.

[0182] It is also possible to choose to employ gene therapy by using or administering in place of the polypeptide of the invention a nucleic acid encoding the polypeptide, by administering to the patient a nucleic acid encoding the polypeptide of interest under conditions such that the polypeptide is expressed in vivo by the patient's cells to which the nucleic acid is delivered.

[0183] The present invention therefore also relates to a nucleic acid comprising or consisting of a sequence encoding a polypeptide of the invention, said nucleic acid being readily obtainable by cloning a fragment of a cDNA encoding a polypeptide of the invention.

[0184] Such nucleic acid encoding the polypeptide of the present invention may be in the form of a DNA vector, for example a plasmid vector.It is possible to administer one or more vectors, each of which possibly carries one or more sequences encoding at least one of the polypeptides of the present invention.In this vector, the sequence encoding at least one of the polypeptides of the present invention is functionally linked to one or more elements that allow its expression or the regulation of its expression, such as a transcription promoter, activator, and / or terminator.

[0185] In a preferred embodiment, a vector is used which carries a sequence encoding the polypeptide of the present invention.

[0186] One or more DNA vectors can be inserted in vivo using any technique known to those skilled in the art.In particular, it is possible to insert one or more DNA vectors in vivo in naked form, i.e. without the aid of any vehicle or system that can facilitate the transfection of the vector in cells (EP 465 529).

[0187] Gene guns can also be used, for example, by depositing DNA on the surface of "gold" particles and shooting these particles so that the DNA passes through the patient's skin (Tang et al., (1992) Nature 356:152-4). Injections using liquid gels can also transfect skin, muscle, adipose tissue, and mammary tissue simultaneously (Furth et al., (1992) Anal Biochem. 205:365-8).

[0188] Other available techniques include microinjection, electroporation, precipitation with calcium phosphate, formulation with nanocapsules or liposomes.

[0189] Particularly preferred are biodegradable nanoparticles of polyalkylcyanoacrylates.In liposomes, the use of cationic lipids promotes encapsulation of negatively charged nucleic acids and promotes fusion with negatively charged cell membranes.

[0190] Alternatively, the vector can be in the form of a recombinant virus containing a nucleic acid sequence encoding the polypeptide inserted into its genome.

[0191] The viral vector may preferably be selected from adenoviruses, retroviruses, particularly lentiviruses, and adeno-associated viruses (AAV), herpes viruses, cytomegaloviruses (CMV), vaccinia viruses, etc. Lentiviral vectors are described, for example, in Firat et al., (2002) J Gene Med 4:38-45.

[0192] Preferably, the recombinant virus is a defective virus. The term "defective virus" refers to a virus that cannot replicate in a target cell. In general, the genome of a defective virus lacks sequences necessary for the replication of said virus at least in the infected cell. These regions may be eliminated or rendered non-functional, or may be replaced by other sequences, in particular nucleic acids encoding a polypeptide of interest. Nevertheless, preferably, the defective virus maintains the sequences of its genome necessary for encapsulating viral particles.

[0193] The targeted administration of genes is described, for example, in WO 95 / 28 494.

[0194] A further object of the present invention relates to an in vitro / ex vivo method for obtaining a population of immunosuppressive macrophages, comprising culturing a population of monocytes in a medium containing an amount of an agonist of the present invention.

[0195] The population of monocytes that serves as the starting material can be isolated by any technique known in the art. For example, a population of human monocytes can be obtained from a variety of biological samples, including PBMCs. Typically, they are isolated from peripheral blood. They can be isolated by positive selection with beads labeled with different ligands (e.g., CD14). Such labeled cells can then be separated by a variety of techniques, such as cell sorting, as described below.

[0196] Thus, in some embodiments, the population of human monocytes is CD14 + A population of human monocytes. In some embodiments, the population of human monocytes is CD14 + CD16 + A population of human monocytes. In some embodiments, the population of human monocytes is CD14 + CD16 - A population of human monocytes.

[0197] A further subject of the present invention is an in vitro / ex vivo method for obtaining a population of human Treg cells, comprising: (a) culturing a population of monocytes in a medium containing an amount of an agonist of the invention to obtain a population of immunosuppressive macrophages (also called "IL34-differentiated macrophages"); (b) co-culturing a population of human peripheral blood mononuclear cells (PBMCs) and a population of immunosuppressive macrophages obtained in step (a); The present invention relates to a method comprising the steps of:

[0198] In some embodiments, the method of the invention comprises the step of isolating a population of human Treg cells from the population of PBMCs prior to step (b), and thus in this embodiment, step (b) comprises co-culturing the population of human Treg cells isolated from the population of PBMCs with the population of immunosuppressive macrophages obtained in step (a).

[0199] In some embodiments, the methods of the invention comprise a step of isolating a population of human Treg cells after the co-culture of step (b).

[0200] Generally, the agonist is added to the medium at a concentration in the range of 1 to 500 ng / ml, preferably 10 to 100 ng / ml, more preferably 50 ng / ml.

[0201] Generally, the agonist is added to the medium at a concentration in the range of 1 to 500 ng / ml, preferably 10 to 100 ng / ml, more preferably 20 ng / ml.

[0202] Step (a) of culturing a population of human monocytes in a medium containing an amount of the agonist of the present invention is carried out for a time required to obtain a population of immunosuppressive macrophages (IL34-differentiated macrophages). Usually, the culture of monocytes in the desired medium is carried out for at least 3 or 4 days and not more than 8 days, preferably 6 days. In some embodiments, the culture of monocytes in the desired medium is carried out for 3, 4, 5, 6, 7, or 8 days or more.

[0203] In step (b), a population of peripheral blood mononuclear cells (PBMCs) may be isolated by methods well known to those of skill in the art (eg, by density centrifugation, such as Ficoll-Paque™ density gradient centrifugation).

[0204] In general, isolation of a population of human Treg cells can be performed by a variety of methods for detecting specific immune cell populations available to one of skill in the art, including immunoselection techniques such as high throughput cell sorting using flow cytometry methods, affinity methods using antibodies labeled to magnetic beads, biodegradable beads, non-biodegradable beads, the use of bispecific antibodies specific for IL34 and CD proteins (e.g., CD4, CD8, CD25, CD127, or CD45RC, PD1, GITR, etc.), the use of bispecific antibodies specific for IL34, IFNγ, TGFβ and IL10, the use of trispecific antibodies, and combinations of such methods.

[0205] In some embodiments, the methods of the invention further comprise expanding the obtained population of Tregs.

[0206] In some embodiments, the medium suitable for the expansion of Tregs comprises an amount of at least one cytokine. Examples of cytokines that may be present in the medium suitable for the expansion of Tregs include, but are not limited to, IL-15, IL-12, IL-4, IL-7, IL-2, IFNγ, IL-34, and pro-inflammatory cytokines, such as IL-1 (particularly IL-1β), IL-6, and TNFα. In some embodiments, the medium suitable for the expansion of Tregs comprises an amount of interleukin-2 (IL-2) and / or an amount of interleukin-15 (IL-15). In some embodiments, the medium suitable for the expansion of Tregs comprises an amount of interleukin-2 (IL-2) and an amount of interleukin-15 (IL-15). Usually, IL-2 is added to the culture medium of the present invention at a concentration in the range of 1 to 250 ng / ml, preferably 10 to 100 ng / ml, more preferably 25 ng / ml. In some embodiments, IL-2 is added to the culture medium of the present invention at a concentration in the range of 1-1000 U / ml, preferably 10-500 U / ml, more preferably 25 U / ml.

[0207] The present invention further provides a method for expanding human Treg cells, comprising: (a) culturing a population of human monocytes in a medium containing an amount of an agonist of the invention to obtain a population of immunosuppressive macrophages; (b) co-culturing the population of Tregs and the population of immunosuppressive macrophages obtained in step (a) in a medium suitable for expanding the population of human Treg cells; (c) optionally isolating the population of human Treg cells obtained in step (b); The present invention relates to a method comprising the steps of:

[0208] In some embodiments, Treg cells or monocytes may be obtained from iPSCs (induced pluripotent stem cells).

[0209] In some embodiments, the population of Tregs is allogenic to immunosuppressive macrophages. Thus, Tregs may be isolated from the graft donor and immunosuppressive macrophages may be isolated from the recipient. Alternatively, Tregs may be isolated from patients suffering from autoimmune disease or allergy, or from patients in need of or waiting for organ transplantation, or from bone marrow donors (e.g., for treating GVHD) or healthy individuals. In some embodiments, Tregs are syngenic to immunosuppressive macrophages.

[0210] In some embodiments, the population of Tregs is CD4 + Foxp3 + Tregs and / or CD8 + Foxp3 + In some embodiments, the population of Tregs is CD4 + CD45RC low Tregs and / or CD8 + CD45RC low It is a group of Tregs.

[0211] Typically, Tregs are cultured for at least 12 days, e.g., 12 days to 6-8 weeks, preferably 15 days. In some embodiments, PBMCs are cultured in the medium of interest for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, PBMCs are cultured in the medium of interest for 1 week, 2 weeks, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or more.

[0212] In some embodiments, cytokines, preferably IL-2 and / or IL-15, are added to the culture medium on day 0 of the culture of Tregs. In some embodiments, cytokines, preferably IL-2 and / or IL-15, are further added to the culture medium once, twice, or three times, or more, for example, on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20. In some embodiments, cytokines, preferably IL-2 and / or IL-15, are added to the culture medium on day 0 and on days 5, 6, 7, or 8 of the culture of Tregs. In some embodiments, cytokines, preferably IL-2 and / or IL-15, are added to the culture medium on day 0 and every 2, 3, or 4 days until the culture is terminated.

[0213] In some embodiments of the method of the invention, the antibody anti-CD3 and / or the antibody anti-CD8 are added to the culture medium on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20, preferably on days 0 and / or 11, 12, 13, 14, and / or 15, of the culture of PBMCs or Tregs.

[0214] In some embodiments, 0.1 to 10 μg / ml, preferably 0.25 to 4 μg / ml, more preferably 1 μg / ml of anti-CD3 antibody and / or 0.1 to 10 μg / ml, preferably 0.25 to 4 μg / ml, more preferably 1 μg / ml of anti-CD28 antibody are added to the culture medium.

[0215] A further object of the present invention relates to a pharmaceutical composition comprising a mutated IL-34 polypeptide of the present invention together with a pharma- ceutically acceptable excipient and optionally a sustained release matrix, such as a biodegradable polymer.

[0216] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active principle can be administered to animals and humans in unit dosage forms, alone or in combination with another active principle, as a mixture with conventional pharmaceutical carriers (supports). Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms. Usually, pharmaceutical compositions contain a pharma- ceutically acceptable vehicle for injectable preparations. These can be, in particular, isotonic and sterile saline solutions (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dried, in particular lyophilized, compositions of sterilized water or saline, which, after addition, allow the constitution of an injectable solution, as the case may be. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage, and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. A solution containing the compound of the present invention as a free base or a pharmacologically acceptable salt can be prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils, can also be prepared. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0217] The mutated IL-34 polypeptides can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic acid, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0218] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0219] Sterile injectable liquid preparations are prepared by incorporating the effective polypeptide in the required amount in a suitable solvent together with some other components mentioned above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary components from above.In the case of sterile powder for preparing sterile injectable liquid preparations, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient plus any additional desired components from its solution that has been previously sterile-filtered.

[0220] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug release capsules and the like can be used as well.

[0221] For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used are known to those skilled in the art in view of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion therapy solution or injected at the proposed injection site. Some dosage variations will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in all cases determine the appropriate dose for the individual subject.

[0222] The mutated IL-34 polypeptide may be formulated in a therapeutic mixture to contain about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or even about 10 milligrams, etc. per dose. Multiple doses may also be administered.

[0223] The following items are also disclosed in this specification:

[0224] Item 1: A mutated IL-34 polypeptide (P1) having an amino acid sequence ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1, in which the histidine (H) residue at position 56 and the glycine (G) residue at position 112 are both substituted with cysteine ​​(C) residues.

[0225] Item 2: A mutation in which the threonine (T) residue at position 36 is replaced by a tyrosine (Y) or tryptophan (W) or proline (P) or phenylalanine (F) or arginine (R) or histidine (H) or asparagine (N) or cysteine ​​(C) or isoleucine (I) or proline (P) or leucine (L) residue; A mutation in which the serine (S) residue at position 100 is replaced with an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue; a mutation in which the threonine (T) residue at position 124 is replaced with a phenylalanine (F) residue or a tryptophan (W) residue; A mutation in which the asparagine (N) residue at position 128 is replaced with a tyrosine (Y) or phenylalanine (F) residue; A mutation in which the glutamine (Q) residue at position 131 is replaced with an arginine (R) residue, a histidine (H) residue, a valine (V) residue, a phenylalanine (F) residue, an isoleucine (I) residue, or a proline (P) residue; A mutation in which the serine (S) residue at position 147 is replaced with a glutamic acid (E) or aspartic acid (D) residue; a mutation in which the asparagine (N) residue at position 150 is replaced with a glutamic acid (E) or aspartic acid (D) residue, and A mutation in which the leucine (L) residue at position 186 is replaced with an arginine (R) or phenylalanine (F) residue. 2. The mutated IL-34 polypeptide (P1) according to item 1, further characterized by at least one mutation selected from the group consisting of:

[0226] Item 3: A mutated IL34 polypeptide (P2) having an amino acid sequence ranging from an asparagine (N) residue at position 21 of SEQ ID NO:1 to a proline (P) residue at position 242 of SEQ ID NO:1, wherein the proline (P) residue at position 59 is substituted with an amino acid residue selected from the group consisting of aspartic acid (D), glutamic acid (E), histidine (H), serine (S), threonine (T), glutamine (Q), tyrosine (Y), cysteine ​​(C), alanine (A), glycine (G), leucine (L), and methionine (M).

[0227] Item 4: A mutated IL34 polypeptide (P2) according to item 3, wherein the proline (P) residue at position 59 is substituted with a lysine (K) residue or an arginine (R) residue.

[0228] Item 5: A mutation in which the threonine (T) residue at position 36 is replaced by a tyrosine (Y) or tryptophan (W) or proline (P) or phenylalanine (F) or arginine (R) or histidine (H) or asparagine (N) or cysteine ​​(C) or isoleucine (I) or proline (P) or leucine (L) residue; A mutation in which the serine (S) residue at position 100 is replaced with an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue; a mutation in which the threonine (T) residue at position 124 is replaced with a phenylalanine (F) residue or a tryptophan (W) residue; A mutation in which the asparagine (N) residue at position 128 is replaced with a tyrosine (Y) or phenylalanine (F) residue; A mutation in which the glutamine (Q) residue at position 131 is replaced with an arginine (R) residue, a histidine (H) residue, a valine (V) residue, a phenylalanine (F) residue, an isoleucine (I) residue, or a proline (P) residue; A mutation in which the serine (S) residue at position 147 is replaced with a glutamic acid (E) or aspartic acid (D) residue; a mutation in which the asparagine (N) residue at position 150 is replaced with a glutamic acid (E) or aspartic acid (D) residue, and A mutation in which the leucine (L) residue at position 186 is replaced with an arginine (R) or phenylalanine (F) residue. The mutated IL-34 polypeptide (P2) according to item 2, further characterized by at least one mutation selected from the group consisting of:

[0229] Item 6: ranging from the asparagine (N) residue at position 21 of SEQ ID NO:1 to the proline (P) residue at position 242 of SEQ ID NO:1; A mutation in which the threonine (T) residue at position 36 is replaced by a tyrosine (Y) or tryptophan (W) or proline (P) or phenylalanine (F) or arginine (R) or histidine (H) or asparagine (N) or cysteine ​​(C) or isoleucine (I) or proline (P) or leucine (L) residue; A mutation in which the serine (S) residue at position 100 is replaced with an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue; a mutation in which the threonine (T) residue at position 124 is replaced with a phenylalanine (F) residue or a tryptophan (W) residue; A mutation in which the asparagine (N) residue at position 128 is replaced with a tyrosine (Y) or phenylalanine (F) residue; A mutation in which the glutamine (Q) residue at position 131 is replaced with an arginine (R) residue, a histidine (H) residue, a valine (V) residue, a phenylalanine (F) residue, an isoleucine (I) residue, or a proline (P) residue; A mutation in which the serine (S) residue at position 147 is replaced with a glutamic acid (E) or aspartic acid (D) residue; a mutation in which the asparagine (N) residue at position 150 is replaced with a glutamic acid (E) or aspartic acid (D) residue, and A mutation in which the leucine (L) residue at position 186 is replaced with an arginine (R) or phenylalanine (F) residue. A mutated IL34 polypeptide (P3) having an amino acid sequence comprising at least one mutation selected from the group consisting of:

[0230] Item 7: The mutated IL34 polypeptide according to items 1, 3, or 6, wherein the glutamine (Q) residue at position 81 is deleted.

[0231] Item 8: A fusion protein consisting of a mutated IL-34 polypeptide according to items 1, 3, or 6 fused to a heterologous polypeptide.

[0232] Item 9: The fusion protein of item 8, wherein the mutated IL-34 polypeptide is fused to an Fc region.

[0233] Item 10: An isolated, synthetic or recombinant nucleic acid encoding a mutated IL-34 polypeptide of items 1, 3, or 6.

[0234] Item 11: A vector comprising the nucleic acid according to Item 10.

[0235] Item 12: A host cell transfected, infected or transformed with the nucleic acid according to item 10 and / or the vector according to item 11.

[0236] Item 13: Use of a mutated IL-34 polypeptide according to items 1 to 6 also as an agonist.

[0237] Item 14: Use of a mutated IL-34 polypeptide according to item 3 as an antagonist.

[0238] Item 15: A mutated IL-34 polypeptide according to items 1, 3, or 6 for use as a medicament.

[0239] Item 16: A method of inducing immune tolerance in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agonist of item 13.

[0240] Item 17: A method of preventing or reducing transplant rejection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agonist of item 13.

[0241] Item 18: A method for preventing or treating autoimmune diseases, unwanted immune responses to therapeutic proteins, and allergies in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agonist of item 13.

[0242] Item 19: The method of items 16, 17, or 18, wherein the agonist is administered to the patient in combination with an immunosuppressant.

[0243] Item 20: A method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the antagonist of item 14.

[0244] Item 21: The method of item 20, wherein the antagonist is administered to the patient in combination with chemotherapy, immunotherapy, in particular an immune checkpoint inhibitor.

[0245] Item 22: An in vitro / ex vivo method for obtaining a population of immunosuppressive macrophages, comprising culturing a population of monocytes in a medium containing an amount of an agonist according to item 13.

[0246] Item 23: A pharmaceutical composition comprising a mutated IL-34 polypeptide according to items 1, 3 or 6 together with a mutated IL-34 polypeptide according to items 1, 3 or 6, together with a mutated IL-34 polypeptide, such as a mutated IL-34 polypeptide, and a mutated IL-34 polypeptide according to items 1, 3 or 6.

[0247] The invention is further illustrated in the following figures and examples, which should not, however, be construed in any way as limiting the scope of the invention. [Brief description of the drawings]

[0248] [Figure 1] Mutation free energies. Predicted effects of single mutations to cysteines at residues H56, G112, P59, L109, I60, and V108 at pH 7.5. [Diagram 2] Free energies of mutations. Predicted effect of mutation of residue P59 on all amino acids at pH 7.5. [Diagram 3] Free energies of mutations. Predicted effect of mutations of several residues in the IL-34 / CSF-1R interface at all amino acids at pH 7.5 (Figure 3A, T36; Figure 3B, Figure 3F40; Figure 3C, K44; Figure 3D, S100; Figure 3E, E103; Figure 3F, T124; Figure 3G, L125; Figure 3H, L127; Figure 3I, N128; Figure 3J, Q131; Figure 3K, S147; Figure 3L, N150; Figure 3M, L186, and Figure 3N, N187). [Figure 4] Interaction and kinetics of IL-34 muteins with CF1R. SPR experiments were performed on a Biacore T200 (GE Healthcare) with different IL-34 mutants (Figure 4A, wt IL-34; Figure 4B, S100F; Figure 4C, T36Y; Figure 4D, Q131F; Figure 4E, T36F; Figure 4F, T36W; Figure 4G, Q131R; Figure 4H, S100D; Figure 4I, P59K; Figure 4J, N150; Figure 4K, G112C / H56C, and Figure 4L, T124F). [Diagram 5] Binding parameters of different IL-34 mutant proteins. The affinity (KD), kinetic parameters (ka and kd) and resonance maximum (Rmax) of IL-34 through CSF-1R were determined by using a series of protein dilutions in the "SCK (Single Cycle Kinetics)" model. [Figure 6]Differential effects of IL-34 variants on the viability of cultured monocytes after sorting from human total PBMCs. Figure 6A. Percentage of live cells after 3 days of culture with wild-type IL-34 (IL-34 WT), each IL-34 variant, or medium control. Figure 6B, Absolute number of live cells expressed as percentage of live cells relative to the percentage of live cells obtained in the condition with WT IL-34 (set as 100% and indicated by dashed line) after 3 days of culture with WT IL-34, each IL-34 variant, or medium control. Figure 6C, Percentage of CD14+ cells in the monocyte population at a dose curve of 1.5 to 200 ng / ml of IL-34 WT, IL-34 variant, or medium control (no cytokine as negative control). [Figure 7] Differential phosphorylation of Akt and ERK1 / 2 in response to WT IL-34 or IL-34 mutants in CD14+ monocytes sorted from total human PBMCs. Figure 7A-B. Flow cytometry analysis of phosphorylation of Akt (Figure 7A) and ERK1 / 2 (Figure 7B) 1, 3, and 5 min after contact with IL-34 WT, S100F, T36W, T36Y, T36F, Q131F, Q131R, N150E, or IL-34-Fc IL-34 mutants (no cytokine as negative control). Results are shown as mean fluorescence intensity of pAkt-AF647 and pERK1 / 2-AF647 compared to T0. [Figure 8] Prediction of free energies (ΔΔGmut) of some residues of IL-34 located at the interface with CSF-1R. ΔΔGmut at pH 7.4 was calculated for substitution of IL-34 residue Thr36 (T36) with Tyr (Y) and for IL-34 residues Ser100 (S100) and Gln131 (Q131) with Phe (F). The input data were the atomic coordinates of the IL-34 / CSF-1R complex (pdb code 4DKD). The effects of double (T36Y / S100F, T36Y / Q131F, and S100F / Q131F) or triple (T36Y / S100F / Q131F) mutations are predicted as well. [Figure 9]Interaction, kinetics, and binding parameters of WT IL-34 and mutant S100F with CD138. Figure 9A-B. SPR experiments were performed on a Biacore T200 (GE Healthcare) for WT IL-34 (Figure 9A) and mutant S100F (Figure 9B). Figure 9C, affinity parameters (KD) of WT IL-34 and mutant S100F through CD138 were determined by using a series of protein dilutions in the "SCK (Single Cycle Kinetics)" model. [Figure 10] Interaction, kinetics, and binding parameters of PTP-ζ with WT IL-34 and mutant S100F. Figure 10A-B, SPR experiments were performed on a Biacore T200 (GE Healthcare) for WT IL-34 (Figure 10A) and mutant S100F (Figure 10B). Figure 10C, Affinity parameters (KD) of WT IL-34 and mutant S100F through CD138 were determined by using a series of protein dilutions in the "SCK (Single Cycle Kinetics)" model.

[0249] Working Example Example 1 method Prediction of the effect of mutations of several IL-34 residues to cysteine ​​on the stability of the dimeric form of IL-34 was performed by calculating the mutation energy (difference between the binding free energy of mutant and wild type) using the method developed by Spassov and Yan (Spassov and Yan, Proteins 2013; 81:704-714), accessible in the protocol "Calculate Mutation Energy (Binding)" performed under Discovery Studio (DS) (Dassault Systèmes BIOVIA software, San Diego, CA).

[0250] Briefly, the X-ray crystal structure of the dimeric form of IL-34 (Protein Data Bank code 4DKC) was used. First, the structure was prepared by adding hydrogen atoms, removing water molecules, and inserting missing atoms or loop regions using the Prepare Protein tool and CHARMm Polar H force field in DS2017. Mutation energies at residues in the IL-34 dimer interface and the IL-34 / CSF-1R interface were then calculated. The effect of a mutation was defined as stabilizing if the mutation energy was less than -0.5 kcal / mol, neutral if the mutation energy was between -0.5 and 0.5 kcal / mol, and destabilizing if the mutation energy was greater than 0.5 kcal / mol.

[0251] result Stabilization of IL-34 dimers Mutation energies were calculated for the following mutations: H56C, G112C, P59C, L109C, I60C, and V108C.

[0252] The mutations H56C, G112C, and V108C are predicted to be neutral, whereas the mutations P59C, L109C, and I60C are predicted to be destabilizing (Figure 1). The double mutation H56C / G112C is not predicted to have a detrimental effect on the stability of the IL-34 dimer, but may result in the formation of a disulfide bridge that stabilizes the dimer (Figure 1).

[0253] Destabilization of IL-34 dimers Mutation energies for P59 were calculated. Mutations P59K and P59R prevent the formation of IL-34 dimers (Figure 2).

[0254] Stabilization of the IL-34 / CSF-1R interface The following mutations potentially stabilize the complex IL-34 / CSF-1R (Figure 3): T36Y, T36F, or T36W S100D or S100F T124F or T124W N128Y or N128F Q131R, Q131F S147E N150E L186R

[0255] Example 2 PBMCs from blood of healthy volunteers (HV) were isolated by Ficoll gradient (CMSMSL01-01; Eurobio) and T, B and NK cells were depleted using magnetic beads with anti-CD3 (clone OKT3), anti-CD19 (clone HIB19; BD Biosciences) and anti-CD56 (clone MY31; BD Biosciences) antibodies. Monocytes were then sorted by morphological parameters of forward scatter (FSC), side scatter (SSC) and positive staining for CD14 (clone M5E2; BD Biosciences). Freshly sorted monocytes were washed and cultured at 1 × 10 in complete medium (RPMI 1640, 2 mM glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 10% FCS) supplemented with an amount of the polypeptides of the invention (P1), (P2) and (P3). 6Cells were seeded at 100 / ml. On day 2, cells were stimulated or not with 100 ng / mL LPS (L4391; Sigma-Aldrich) for 24 h. On day 3, cells were harvested and stained with anti-CD14 (clone M5E2; BD Biosciences), anti-CD16 (clone 3G8; BD Biosciences), anti-CD163 (clone GHI / 61; BD Biosciences), anti-CD206 (clone 19.2; BD Biosciences), anti-CD209a (clone DCN46; BD Biosciences), anti-CD169 (clone 7-239; BD Biosciences), anti-CD80 (clone L307.4; BD Biosciences), anti-CD86 (clone 2331; BD Biosciences), anti-CD40 (clone 5C3; BD Biosciences), and anti-HLA-DR (clone G46-6; BD Biosciences). Fluorescence was measured on a FACSCanto II flow cytometer (BD Biosciences) and data were analyzed using FlowJo software. Cells were first gated by their morphology and CD14 + CD16 - and CD14 + CD16 + To analyze the expression of different markers in monocytes, dead cells are excluded by DAPI live cell selection.

[0256] Example 3 For cell signaling experiments, cell lines are serum-starved (0.1% FBS) for 48 hours and treated with the polypeptides (P1), (P2), and (P3) of the present invention (10 ng / ml) for 20 minutes, at which time points samples are collected. Samples are then analyzed by Western blot using Tris / glycine buffer and transferred onto hybond-P membranes (Amersham, GE, Fairfield, CT, USA). All protein samples are quantified by using the BCA assay to ensure similar protein amounts in all lanes of the Western gel (Thermo Scientific Inc.). Antibodies used in the Western blot experiments are CSF-1R (sc692, 1:1000, Santa Cruz, Santa Cruz, CA, USA), phospho-tyrosine (sc-508, 1:1000, Santa Cruz), phospho-ERK (sc-7383, 1:1000, Santa Cruz), total ERK (sc-94, 1:1000, Santa Cruz), and β-actin (A2228, 1:10,000, Sigma-Aldrich). All antibodies are incubated with the blots overnight at 4°C in 5% BSA TTBS. Secondary antibodies mouse IgG-HPR (sc-2061, 1:10,000, Santa Cruz) or rabbit IgG-HPR (sc-2030, 1:10,000, Santa Cruz) were incubated in 5% milk TTBS for 1 h at room temperature. Signals were detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific Inc., Waltham, MA, USA).

[0257] Example 4 material and method Reagents and materials Series S CM5 sensor chips and HBS-P buffer (10 mM HEPES buffer, pH 7.4, with 2.7 mM KCl, 150 mM NaCl, and 0.05% surfactant P20), Amine Coupling Kit [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); 1.0 M ethanolamine, pH 8.5)], immobilization buffer (sodium acetate pH 5.0), and regeneration solution (NaOH 10 mM, analytical grade) were purchased from GE Healthcare Life Science (Uppsala, Sweden).

[0258] Surface plasmon resonance (SPR) analysis SPR experiments were performed at 25°C on a Biacore T200 (GE Healthcare). HBS-P buffer was filtered through a 0.45 μm membrane filter and degassed before use. First, recombinant human CSF-1 receptor (CSF-1R, ref. K10161-H08H) from Sino Biological was immobilized on the chip surface by amine coupling. Briefly, recombinant CSF-1 receptor was diluted to 20 μg / mL in 10 mM sodium acetate solution at pH 5.0. Using a standard Amine Coupling Kit, the diluted CSF-1R was quickly covalently immobilized on the flow cell of a CM5 sensor chip via the primary amine groups. Then, IL-34 muteins were analyzed in the "SCK (Single Cycle kinetics)" model on the chip with immobilized CSF-1R.

[0259] Interaction and kinetics of IL-34 muteins with CSF-1R The affinity (KD), kinetic parameters (ka and kd) and resonance maximum (Rmax) of IL-34 through CSF-1R were determined by using a series of protein dilutions in the "SCK (Single Cycle Kinetics)" model. IL-34 muteins as analytes were diluted in HBS-P buffer at concentrations ranging from 25 nM to 400 nM and flowed over the immobilized CSF-1R and the resulting response units (RU) were recorded. The flow rate was 30 μL / min with 120 s for association and 600 s for dissociation. The sensor chip surface was then regenerated with 10 mM NaOH for 30 s. The dissociation equilibrium constant KD, kinetic parameters kd and ka, and Rmax were determined by direct curve fitting of the sensorgrams to the Langmuir 1:1 model of interaction.

[0260] result Biacore analysis showed that several IL-34 mutants, especially mutants S100F, T36Y, Q131F, T36F, T36W, Q131R, and S100D, have higher affinity for CSF-1R than WT IL-34, suggesting that these mutants are more efficient than WT IL-34 (Figures 4A-H and 5). In contrast, IL-34 P59K, N150E, G112 / H56C, and T124F mutants have reduced or similar affinity for CSF-1R compared to WT IL-34 (Figures 4I-L and 5).

[0261] Example 5 material and method Buffy coats were obtained from anonymous healthy individuals by the Etablissement Francais du Sang (EFS, Nantes, France). PBMCs were obtained by Ficoll gradient and monocytes were isolated by magnetic bead separation (Classical Monocyte Isolation Kit, Miltenyi Biotec). CD14 +Monocytes were cultured in flat-bottom 96-well plates at 10 °C in complete medium (RPMI 1640, 10% FBS, 1% penicillin-streptomycin, 1% glutamine, 1% AANE, 1% Hepes, 1% sodium pyruvate) with WT IL-34 or mutants: S100F, S100D, T36W, T36Y, T36F, Q131F, Q131R, N150E, C112 / C56, P59K, T124F, and IL-34 Fc at a final concentration of 100 ng / ml (Figure 6A and Figure 6B) and at final concentrations ranging from 1.5 to 200 ng / ml (Figure 6C). 6 The cells were cultured at 123 cells / ml. Medium without cytokines (no cytokines) was used as a negative control. On day 3, the cells were harvested by flushing in PBS 2% FBS 2mM EDTA and further used for viability staining and phenotypic analysis by flow cytometry. The absolute cell count was analyzed using counting beads (123count eBeads™ Counting Beads, ThermoFisher Scientific).

[0262] result Mutants T36F, S100F, Q131F, T36W, T36Y, N150E, and IL-34-Fc are as efficient as WT IL34 in inducing monocyte / macrophage survival (Figure 6A). S100F, T36Y, Q131F, and IL-34-Fc were of particular interest because they displayed more potent properties in maintaining cell survival (Figure 6B).

[0263] Interestingly, Q131R and S100D, which have better binding affinity for CSF-1R than WT IL34, inhibited macrophage survival (Figure 6A and B), thus acting as antagonists. Furthermore, analysis of CD14 expression by monocytes in the presence of decreasing concentrations of IL-34 mutants showed that the S100F, Q131F, and T36Y mutants were less efficient at differentiating CD14+ monocytes than the same concentrations of WT IL-34 (Figure 6C).

[0264] Example 6 material and method Freshly sorted CD14 cells were used for analysis of Akt and ERK1 / 2 phosphorylation (pAkt and pERK1 / 2, respectively). + Monocytes were cultured with WT IL-34 or different S100F, T36W, T36Y, T36F, Q131F, Q131R, N150E, and IL-34-Fc mutants at a concentration of 100 ng / ml in FBS-free medium (RPMI 1640, 1% penicillin-streptomycin, 1% glutamine, 1% AANE, 1% Hepes, 1% sodium pyruvate) in low-attachment 96-well plates for 1, 3, and 5 min. Analysis was performed by flow cytometry using phospho-Akt (Ser473) and phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) primary antibodies (refs. #4060 and #4370, Cell Signalling) and goat anti-rabbit IgG(H+L)-AF647 (ref A21245, Life Technologies) secondary antibodies according to the BD Biosciences Phosflow protocol using BD Cytofix Fixation buffer and BD Phosflow Perm Buffer III (BD Biosciences).

[0265] result Next, flow cytometric analysis of pAkt and pERK1 / 2 was performed to functionally assess signaling induced by binding of each mutant of IL-34 to CSF-1R. Flow cytometric analysis of pAkt shows increased phosphorylation of the T36F, IL-34-Fc, S100F, Q131F, T36W, and T36Y mutants compared to WT IL-34 after 3 min, and similarly for most of these (T36F, IL-34-Fc, Q131F, T36W, and T36Y) after 5 min (Figure 7A). In contrast, the N150E mutant is less efficient than WT IL-34 in inducing phosphorylation of Akt after 3 min, and as efficient as WT IL-34 after 5 min. Finally, the Q131R mutant of IL-34 does not induce any phosphorylation of Akt after 3 or 5 min. Flow cytometric analysis of pERK1 / 2 shows increased phosphorylation for IL-34-Fc, S100F, Q131F, and T36Y mutants compared to wt IL-34 after 3 min, and similarly for Q131F and T36Y after 5 min (Figure 7B). Furthermore, the T36W and T36F mutants show the same kinetics as WT IL-34. In contrast, the N150E mutant is again less efficient than WT IL-34 after 3 and 5 min. Moreover, as already observed with pAkt, the Q131R mutant does not induce any phosphorylation of ERK1 / 2 (Figure 7B).

[0266] Example 7 material and method ΔΔGmut at pH 7.4 was calculated for the substitution of IL-34 residue Thr36 (T36) with Tyr (Y) and for the substitution of IL-34 residues Ser100 (S100) and Gln131 with Phe (F) using the method of Spassov and Yan (Spassov and Yann, 2013) performed under Discovery Studio (Dassault Systèmes BIOVIA Release 2017, San Diego) with the protocol “Calculate Mutation Energy (Binding)”. The input data were the atomic coordinates of the IL-34 / CSF-1R complex (pdb code 4DKD).

[0267] result Consistent with the results obtained in the previous examples, the mutations T36Y, S100F, and Q131F are stable in silico (ΔΔGmut<-0.5) (Figure 8). It was therefore hypothesized that combining two or three of these mutations may be even more stabilizing than one mutation alone. Interestingly, the effect of double mutations was predicted to be the same or even slightly more stabilizing than a simple single mutation. The triple mutation combination was predicted to be more stable.

[0268] Example 8 material and method Reagents and materials Series S CM5 sensor chips and HBS-P buffer (10 mM HEPES buffer (pH 7.4) containing 2.7 mM KCl, 150 mM NaCl, and 0.05% surfactant P20), Amine Coupling Kit [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); 1.0 M ethanolamine (pH 8.5)], immobilization buffer (sodium acetate pH 5.0), and regeneration solution (NaOH 10 mM, analytical grade) were purchased from GE Healthcare Life Science (Uppsala, Sweden).

[0269] Surface plasmon resonance (SPR) analysis SPR experiments were performed at 25°C on a Biacore T200 (GE Healthcare). HBS-P buffer was filtered through a 0.45 μm membrane filter and degassed before use. First, recombinant human CD138 (SDC1, Syndecan-1, ref 11429-H08H) from Sino Biological and PTPz (ref OPCA02772) from Aviva System Biologic were immobilized on the chip surface by amine coupling. Briefly, recombinant CD138 or PTP-ζ was diluted to 20 μg / mL in 10 mM sodium acetate solution at pH 5.0. Using a standard Amine Coupling Kit, the diluted CD138 or PTP-ζ was quickly covalently immobilized on the flow cell of a CM5 sensor chip via the primary amine groups. Next, the IL-34 muteins were analyzed in the "SCK (Single Cycle kinetics)" model on chips immobilized with CD138 or PTP-ζ.

[0270] Interaction and kinetics of IL-34 muteins with CD138 or PTP-ζ The maximum resonance (Rmax) of IL-34 through CD138 or PTP-ζ was determined by using a series of protein dilutions in the "SCK (Single Cycle Kinetics)" model. IL-34 muteins as analytes were diluted in HBS-P buffer at concentrations ranging from 25 nM to 400 nM and flowed over immobilized CD138 or PTP-ζ, and the resulting response units (RU) were recorded. The flow rate was 30 μL / min with 120 s for association and 600 s for dissociation. The sensor chip surface was then regenerated with 10 mM NaOH for 30 s. Rmax was determined by direct curve fitting of the sensorgrams to the Langmuir 1:1 model of interaction.

[0271] result Biacore analysis showed that the S100F mutant of IL-34 has higher affinity for CD138 (Figure 9A-C) and PTP-ζ (Figure 10A-C) than WT IL-34, suggesting that this mutant is more efficient than WT IL-34.

[0272] References Throughout this application, various references are described that describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure.

Claims

1. 1. A mutated IL34 polypeptide, comprising: An amino acid sequence consisting of amino acids at positions 21 to 242 of SEQ ID NO: 1, wherein at least one mutation is introduced, and the at least one mutation is A mutation in which the serine residue at position 100 is replaced with a phenylalanine residue (S100F); a mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y); A mutation in which the glutamine residue at position 131 is replaced with a phenylalanine residue (Q131F); a mutation in which the threonine residue at position 36 is replaced with a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced with a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced with cysteine ​​residues (H56C and G112C). A mutated IL34 polypeptide comprising or consisting of an amino acid sequence, which is at least one mutation selected from the group consisting of:

2. The polypeptide, An amino acid sequence consisting of amino acids from position 21 to position 242 of SEQ ID NO: 1, in which the serine residue at position 100 is replaced with a phenylalanine residue (S100F); and a mutation in which the threonine residue at position 36 is replaced by a tyrosine residue (T36Y); A mutation in which the glutamine residue at position 131 is replaced with a phenylalanine residue (Q131F); a mutation in which the threonine residue at position 36 is replaced with a tryptophan residue (T36W); A mutation in which the threonine residue at position 36 is replaced with a phenylalanine residue (T36F), and A mutation in which the histidine residue at position 56 and the glycine residue at position 112 are both replaced with cysteine ​​residues (H56C and G112C).

2. The mutated IL34 polypeptide of claim 1, comprising or consisting of an amino acid sequence further comprising at least one mutation selected from the group consisting of:

3. 1. A mutated IL34 polypeptide, comprising: An amino acid sequence consisting of amino acids at positions 21 to 242 of SEQ ID NO: 1, wherein at least one mutation is introduced, and the at least one mutation is A mutation in which the serine residue at position 100 is replaced with an aspartic acid residue (S100D), and A mutation in which the glutamine residue at position 131 is replaced with an arginine residue (Q131R) A mutated IL34 polypeptide comprising or consisting of an amino acid sequence, which is at least one mutation selected from the group consisting of:

4. A fusion protein consisting of a mutated IL-34 polypeptide according to any one of claims 1 to 3 fused to a heterologous polypeptide.

5. The fusion protein of claim 4, wherein the mutated IL-34 polypeptide is fused to an Fc region.

6. 10. An isolated, synthetic or recombinant nucleic acid encoding a mutated IL-34 polypeptide according to any one of claims 1 to 3, or encoding a fusion protein according to claim 4 or 5.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell comprising a nucleic acid according to claim 6 and / or a vector according to claim 7.

9. A mutated IL-34 polypeptide according to any one of claims 1 to 3, a fusion protein according to claim 4 or 5, a nucleic acid according to claim 6 or a vector according to claim 7 for use as a medicament.

10. A mutated IL-34 polypeptide according to claim 1 or 2, or a fusion protein consisting of a mutated IL-34 polypeptide according to claim 1 or 2 fused to a heterologous polypeptide, for use as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ, and CD138.

11. 1. A pharmaceutical composition for use in inducing immune tolerance in a patient in need thereof, comprising a mutated IL-34 polypeptide according to claim 1 or 2, a fusion protein consisting of a mutated IL-34 polypeptide according to claim 1 or 2 fused to a heterologous polypeptide, a recombinant nucleic acid encoding a mutated IL-34 polypeptide according to claim 1 or 2, or a vector comprising said nucleic acid.

12. 1. A pharmaceutical composition for use in preventing or reducing transplant rejection in a patient in need thereof, comprising a mutated IL-34 polypeptide according to claim 1 or 2, a fusion protein consisting of a mutated IL-34 polypeptide according to claim 1 or 2 fused to a heterologous polypeptide, a recombinant nucleic acid encoding a mutated IL-34 polypeptide according to claim 1 or 2, or a vector comprising said nucleic acid.

13. 1. A pharmaceutical composition for use in the prevention or treatment of a neurodegenerative disease, an autoimmune disease, an unwanted immune response to a therapeutic protein, or an allergy in a patient in need thereof, comprising a mutated IL-34 polypeptide according to claim 1 or 2, a fusion protein consisting of a mutated IL-34 polypeptide according to claim 1 or 2 fused to a heterologous polypeptide, a recombinant nucleic acid encoding a mutated IL-34 polypeptide according to claim 1 or 2, or a vector comprising said nucleic acid.

14. A mutated IL-34 polypeptide according to claim 3, or a fusion protein comprising the mutated IL-34 polypeptide according to claim 3 fused to a heterologous polypeptide, for use as an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ, and CD138.

15. 10. A pharmaceutical composition for use in the treatment of cancer or a bone disease associated with abnormal proliferation in a patient in need thereof, comprising a mutated IL-34 polypeptide of claim 3, a fusion protein consisting of a mutated IL-34 polypeptide of claim 3 fused to a heterologous polypeptide, a recombinant nucleic acid encoding a mutated IL-34 polypeptide of claim 3, or a vector comprising said nucleic acid.

16. 10. An in vitro / ex vivo method for obtaining a population of immunosuppressive macrophages, comprising culturing a population of monocytes in a medium containing a mutated IL-34 polypeptide according to claim 1 or 2.

17. A pharmaceutical composition comprising at least one pharma- ceutical acceptable excipient and a mutated IL-34 polypeptide according to any one of claims 1 to 3, a fusion protein according to claim 4 or 5, a nucleic acid according to claim 6, or a vector according to claim 7.

18. 20. The pharmaceutical composition of claim 17, further comprising at least one sustained release matrix.

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