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

Abstract

Interleukin-34 is a cytokine involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. IL-34 has been shown to be involved in a wide range of areas, such as neuronal protection, autoimmune diseases, infection, cancer, and transplantation. Recent studies have also demonstrated that IL-34 acts as a regulator of Foxp3. + New possible therapeutic effects of cytokines secreted by Tregs that mediate transplant tolerance. The inventors have now generated new mutant IL-34 polypeptides that can be used as agonists or antagonists.

Description

Technical Field

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

[0002] Interleukin-34 is a cytokine whose function is only partially understood and was first described in 2008. Although IL-34 has very low homology to colony stimulating factor 1 (CSF-1 or M-CSF), they share a common receptor CSF-1R (CD115, c-Fms) and IL-34 also has two different 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 some tissues different from those expressing CSF-1, and that it is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. IL-34 has been shown to be involved in a wide range of areas, such as neuronal protection, autoimmune diseases, infection, cancer, and transplantation (Guillonneau C. Cell Mol Life Sci. 2017). Recent studies have also demonstrated that IL-34 acts as a Foxp3 + New possible therapeutic roles of cytokines secreted by regulatory T cells that mediate transplant tolerance (Bézie et al., JCI, 2015). Summary of the invention

[0003] As defined in the claims, the present invention relates to mutant interleukin-34 (IL-34) polypeptides and their use in therapy. DETAILED DESCRIPTION

[0004] As used herein, the term "IL-34" has the general meaning in the art and refers to interleukin-34 characterized by the amino acid sequence as shown in SEQ ID NO:1.

[0005] SEQ ID NO: 1: Interleukin-34 (Homo sapiens), comprising the leader sequence MPRGFTWLRYLGIFLGVALG from position 1 to position 20 NEPLEMWPLTQNEECTVTGF LRDKLQYRSR LQYMKHYFPI NYKISVPYEG VFRIANVTRL QRAQVSEREL RYLWVLVSLS ATESVQDVLL EGHPSWKYLQ EVETLLLNVQ QGLTDVEVSP KVESVLSLLN APGPNLKLVR PKALLDNCFR VMELLYCSCC KQSSVLNWQD CEVPSPQSCS PEPSLQYAAT QLYPPPPWSP SSPPHSTGSV RPVRAQGEGL LP

[0006] As used herein throughout, amino acid numbering is given with reference to the amino acid sequence as shown in SEQ ID NO:1.

[0007] As used herein, the term "CSF-1R" has the general meaning of the art and refers to the colony stimulating factor 1 receptor. CSF-1R is also referred to as FMS, FIM2, C-FMS, M-CSF receptor and CD115 in the art. This receptor is a single-pass transmembrane receptor with an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain, and has tyrosine kinase activity. CSF-1 or interleukin-34 ligands bind to the ligand of CSF-1R, resulting 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, CSF-1 and IL-34 both stimulate monocyte survival, proliferation, and differentiation into macrophages and other monocytic lineages such as osteoclasts, dendritic cells, and microglia.

[0008] As used herein, the term "PTP-ζ" has the general meaning in the art and refers to the receptor-type tyrosine protein phosphatase zeta. PTP-ζ is also known in the art as "phosphacan". This receptor is a single-pass type I membrane protein with two cytoplasmic tyrosine protein phosphatase domains, one α-carbonic anhydrase domain and one fibronectin type III domain.

[0009] As used herein, the term "CD138" has the general meaning in the art and refers to 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 syndecan-1 core protein consists of an extracellular domain that can be substituted by heparan sulfate and chondroitin sulfate glycosaminoglycan chains, a highly conserved transmembrane domain, and a highly conserved cytoplasmic domain containing two constant regions separated by a variable region.

[0010] As used herein, the term "ligand" refers to a molecule, such as a polypeptide, that has an affinity for binding to a receptor. One skilled in the art will appreciate that a molecule can be both a receptor and a ligand. Ligands for CSF-1R are well known in the art and include IL-34 and CSF-1. Ligands for PTP-ζ and ligands for CD138 are well known in the art and include IL-34.

[0011] As used herein, the term "affinity" means the strength with which a ligand binds to its receptor. The affinity of a ligand is expressed as the dissociation constant, Kd. Preferred methods for determining ligand affinity 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), which are incorporated herein by reference in their entirety. A preferred standard method for determining ligand affinity known in the art is to use a Biacore instrument.

[0012] As used herein, the term "binding" refers to the direct binding between two molecules, which is due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Specifically, as used herein, the term "binding" refers to the context of a ligand binding to its receptor, corresponding to about 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or smaller, about 10 -10 M or less, or about 10 -11 M or even smaller K D affinity.

[0013] As used herein, the term "agonist" refers to a mutant IL-34 polypeptide that mimics the biological activity of a ligand of CSF-1R, a ligand of PTP-ζ, and / or a ligand of CD138, or has the same or similar biological activity as a ligand of CSF-1R, a ligand of PTP-ζ, and / or a ligand of CD138.

[0014] As used herein, the term "antagonist" refers to a mutant IL-34 polypeptide that partially or completely blocks, inhibits or neutralizes the biological activity of a ligand for CSF-1R, a ligand for PTP-ζ and / or a ligand for CD138.

[0015] The ligands of CSF-1R include CSF-1 and IL-34. Specifically, the ligand of CSF-1R described in the present invention is a CSF-1 polypeptide, or preferably a natural or wild-type IL-34 polypeptide disclosed herein.

[0016] Ligands for PTP-ζ include heparin-binding growth factors, pleiotrophin (PTN) / HB-GAM and midkine (MK), fibroblast growth factor-2, extracellular matrix proteins such as tenascin-C and tenascin-R, and cell adhesion molecules such as Nr-CAM, L1 / Ng-CAM, F3 / contactin, NCAM, and TAG1 / axonin-1.

[0017] Ligands for CD138 include matrix proteins, proteases, adhesion receptors, cytokines and growth factors, complement and coagulation proteins.

[0018] Preferably, the CSF-1R ligand, PTP-ζ ligand or CD138 ligand of the present invention is a natural or wild-type IL-34 polypeptide disclosed herein.

[0019] Preferably, the agonist of the present invention is a mutant IL-34 polypeptide that mimics the biological activity of a ligand of CSF-1R or has the same or similar biological activity as a ligand of CSF-1R.

[0020] Preferably, the antagonist of the present invention is a mutant IL-34 polypeptide that partially or completely blocks, inhibits or neutralizes the biological activity of the ligand of CSF-1R.

[0021] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation to a labeling component. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics.

[0022] As used herein, the term "dimer" generally refers to a molecule comprising two or more subunits, wherein the subunits are formed by the mutant IL-34 polypeptides of the present invention. The dimers of the present invention generally function as agonists or antagonists.

[0023] The term "fusion polypeptide" or "fusion protein" means a protein produced by linking two or more polypeptide sequences together. Fusion polypeptides encompassed in the present invention include the translation product of a chimeric gene construct linking a nucleic acid sequence encoding a first polypeptide with a nucleic acid sequence encoding a second polypeptide. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by a peptide bond or by several peptides. The fusion protein may also include a peptide linker between the two domains.

[0024] As used herein, the term "linker" refers to a sequence of at least one amino acid that connects a first polypeptide and a second polypeptide in a fusion protein.

[0025] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or Pro230 to its carboxyl terminus.

[0026] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein ("adhesin") with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence having the desired binding specificity (i.e., being "heterologous") and an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence that comprises at least the binding site for a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can 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.

[0027] As used herein, "nucleic acid molecule" or "polynucleotide" refers to a DNA molecule (such as, but not limited to, cDNA or genomic DNA). Nucleic acid molecules can be single-stranded or double-stranded.

[0028] The term "isolated" when referring to a nucleic acid molecule or a 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 in nature.

[0029] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include mRNA splicing in eukaryotic cells.

[0030] As used herein, the terms "wild type" or "native" are terms of art understood by the skilled artisan and refer to the typical form of an organism, strain, gene or trait that occurs in nature as distinct from mutant forms.

[0031] As used herein, the term "mutation" has the general meaning in the art and refers to substitution, deletion or insertion. Specifically, the term "substitution" means removing a specific amino acid residue at a specific position and inserting another amino acid residue into the same position. The term "deletion" means removing a specific amino acid residue at a specific position.

[0032] As used herein, the term "variant" refers to a first composition (e.g., a first molecule) that is related to a second composition (e.g., a second molecule, also referred to as a "parent" molecule). The variant molecule can be derived from, isolated from, based on, or homologous to a parent molecule. The variant molecule can have complete sequence identity with the original parent molecule, or can have less than 100% sequence identity with the parent molecule. For example, a variant of a sequence can 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 as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of 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., 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., Proc. Natl. Acad. Sci. USA, 85:2444, 1988 et al., Meth. Mol. Biol., 24:307-31, 1994). Altschul et al., Nat. Genet., 6:119-129, 1994, detailed sequence alignment methods and homology calculations. For example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) can be used for sequence comparison (Internet 1996, WRPearson and the University of Virginia, fasta20u63 version2.0u63, published in December 1996). ALIGN compares the entire sequence to each other, while LFASTA compares local similar regions. For example, these alignment tools and their respective tutorials can be found on the NCSA website. Alternatively, for amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function can be used, using the default BLOSUM62 matrix settings to preset parameters (gap existence cost is 11, and the gap penalty for each residue is 1). When aligning short peptides (less than about 30 amino acids), the Blast 2 sequence function should be used for alignment, using the PAM30 matrix settings to preset parameters (open gap is 9, and the extension gap is 1 penalty). For example, the BLAST sequence comparison system is available 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.

[0033] The terms "vector", "cloning vector" and "expression vector" mean a vector that can introduce DNA or RNA sequences (eg, foreign genes) into host cells to transform the host and promote the expression (eg, transcription and translation) of the introduced sequences.

[0034] The term "transformation" means the introduction of a "foreign" (i.e., foreign 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."

[0035] As used herein, "Treg" or "regulatory T cells" refers to cells that are functionally committed, i.e., capable of suppressive activity (i.e., inhibiting the proliferation of conventional T cells), either through cell-cell contact or through MLR inhibition (Mixed Lymphocytes Reaction). Tregs are characterized by the expression of FoxP3 and include CD8 + T cells and CD4 + T cells. As used herein, the term "Foxp3" has its ordinary meaning in the art and refers to a transcriptional regulator that is critical for the development and suppressive function of Tregs. Foxp3 plays an important role in maintaining immune system homeostasis by allowing the acquisition of full suppressive function and stability of the Treg lineage, as well as by directly regulating the expansion and function of conventional T cells. As used herein, the term "CD4 + "T cell" has its ordinary meaning in the art and refers to a subpopulation of T cells that express CD4 on their surface. + T cells are T helper cells that coordinate macrophages and CD8 + Activation of T cells (Th-1 cells), coordination of antibody production by B cells (Th-2 cells), or they are thought to play an important role in autoimmune diseases (Th-17 cells). As used herein, the term "CD8 + "T cells" have a general meaning in the art and refer to a subpopulation of T cells that express CD8 on their surface. They are MHC class I restricted and can function as cytotoxic T cells. "CD8 + "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 an associative recognition element in the interaction of the type I restricted major histocompatibility complex. Specifically, regulatory T cells are usually "forkhead box P3 (Foxp3 + ) regulatory T cells" and "CD45RC 低 Cells. As used herein, the term "Foxp3 + ) regulatory T cells" or "Foxp3 + Treg cells refer to 2-10% of CD4 + and CD8 + T cells, whose characteristic marker is the transcription factor Foxp3.

[0036] As used herein, the term "medium" refers to a medium (e.g., "culture medium") for maintaining a cell population or culturing a cell population, containing nutrients that maintain cell viability and support proliferation. The culture medium may contain any of the following in a suitable combination: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components, such as growth factors, cytokines, and the like. Culture media commonly used for specific cell types are known to those skilled in the art. The culture media of the present invention may be based on commercially available culture media, such as RPMI1640 from Invitrogen.

[0037] As used herein, the term "expand" refers to the process of converting and / or expanding a given cell population (e.g., immune cells, such as T cells). The expansion of T cells is preferably carried out by culturing a cell population comprising T cells in the presence of an antigen-specific stimulator, such as an antigen, a cell, an antibody, a lectin, etc. The expansion may also require culturing T cells in the presence of cytokines.

[0038] 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. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine responsive cells such as macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4 + 、CD8 + , Th1 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 occur in transplant rejection and the attendant physiological consequences of such immune responses, such as interstitial fibrosis, chronic graft arteriosclerosis or vasculitis. Immune responses also occur in autoimmune diseases and the attendant physiological consequences of such immune responses, 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 leading to the production of autoantibodies.

[0039] As used herein, the term "immune tolerance" refers to a state in which the immune system does not respond to substances or tissues that have the ability to elicit an immune response. As used herein, the term "specific" immune tolerance occurs when immune tolerance is preferentially elicited against certain antigens compared to other antigens.

[0040] As used herein, the term "unleashing" refers to achieving or increasing an immune response against 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, for example, inducing CD4 + CD25 - T cell expansion.

[0041] As used herein, the term "reducing immune tolerance" refers to a state of reduced unresponsiveness of the immune system to substances or tissues that have the ability to elicit an immune response.

[0042] The term "transplant" and its variations refer to the insertion of a transplant (also referred to as a graft) into a recipient, whether the transplant is isogenic (the donor and recipient are genetically identical), allogenic (the donor and recipient are different in genetic origin but belong to the same species), or xenogeneic (the donor and recipient are from different species). Thus, in a typical case, the host is a human, and the graft is an isograft derived from a human of the same or different genetic origin. In another case, the graft is derived from a species different from the species into which it is transplanted, including animals from phylogenetically distant species, e.g., a baboon heart is transplanted into a human host.

[0043] 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, lung, kidney, liver, pancreas, skin, uterus, bone, cartilage, small or large intestine, bladder, brain, breast, blood vessel, esophagus, fallopian tube, gallbladder, ovary, pancreas, prostate, placenta, spinal cord, upper and lower limbs, spleen, stomach, testicles, thymus, thyroid, trachea, ureter, urethra, uterus.

[0044] As used herein, the term "tissue" refers to any type of tissue in humans or animals, and includes, but is 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.

[0045] As used herein, the term "cells" refers to a composition enriched in target cells, preferably a composition comprising at least 30%, preferably at least 50%, even more preferably at least 65% of said cells.

[0046] As used herein, the term "transplant rejection" encompasses both acute and chronic transplant rejection. "Acute rejection" is the rejection of the immune system of the recipient of a tissue transplant when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the recipient's immune cells into the transplanted tissue, exerting their effector functions and destroying the transplanted tissue. The onset of acute rejection is rapid, and typically occurs in humans within a few weeks after the transplant. "Chronic transplant rejection" typically occurs in humans within months to years after the transplant, even if the acute rejection is successfully immunosuppressed. Fibrosis is a common factor in chronic rejection of all organ transplant types.

[0047] As used herein, the term "preventing or reducing transplant rejection" is intended to encompass preventing or inhibiting immune transplant rejection, and delaying the onset or progression of immune transplant rejection. The term is also intended to encompass prolonging the survival of a transplant in a patient, or reversing the failure of a transplant in a patient. In addition, the term is intended to encompass improving the symptoms of immune transplant rejection, including, for example, improving immune complications associated with immune rejection, such as interstitial fibrosis, chronic graft atherosclerosis, or vasculitis.

[0048] As used herein, the term "autoimmune inflammatory disease" refers to a disease in which the immune system generates an immune response (e.g., a B cell or T cell response) against an antigen that is a normal part of the host (self antigen), thereby causing damage to tissue. In an autoimmune disease, the host's immune system fails to recognize a particular antigen as "self" and mounts an immune response against host tissues expressing this antigen.

[0049] As used herein, "allergic disorder" refers to any disorder resulting from antigenic activation of mast cells, which results in an "anaphylactic reaction" or hypersensitivity state and an influx of inflammatory and immune cells.

[0050] As used herein, the term "asthma" refers to an inflammatory disease of the respiratory tract characterized by airway obstruction, wheezing, and shortness of breath.

[0051] As used herein, the term "unwanted immune response to a therapeutic protein" refers to any unwanted immune response to a protein expressed during gene therapy and / or a therapeutic protein such as Factor VIII (haemophilia A) and other coagulation factors, enzyme replacement therapy, monoclonal antibodies (e.g., natalizumab, rituximab, infliximab), polyclonal antibodies, enzymes or cytokines (e.g., IFNβ).

[0052] As used herein, the term "immunosuppressive drug" refers to any substance that is capable of producing an immunosuppressive effect, such as preventing or reducing an immune response.

[0053] As used herein, the term "cancer" has the general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. The term "cancer" further encompasses primary and metastatic cancers.

[0054] As used herein, the term "chemotherapy" refers to treatment with a chemotherapeutic agent.

[0055] As used herein, the term "immunotherapy" has the ordinary meaning in the art and refers to treatment by administering an immunogenic agent, ie, an agent capable of inducing, enhancing, suppressing or otherwise altering an immune response.

[0056] As used herein, the term "immune checkpoint inhibitor" has the general meaning of the art and refers to any compound that inhibits the function of immunosuppressive checkpoint proteins. As used herein, the term "immune checkpoint protein" has the general meaning of the art and refers to a molecule expressed by a T cell, which either raises the signal (stimulatory checkpoint molecule) or lowers the signal (inhibitory checkpoint molecule). Immune checkpoint molecules are considered to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways in the art (see, e.g., Pardoll, 2012.Nature Rev Cancer 12: 252-264; Mellman et al., 2011.Nature 480: 480-489). The example of inhibitory checkpoint molecules includes A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. Inhibition includes reduced function and complete blocking.

[0057] As used herein, the term "treatment" refers to preventive or preventive treatment and curative or disease-modifying treatment, including treatment of patients who are at risk of contacting a disease or suspect that they have been exposed to a disease and patients who are sick or have been diagnosed with a disease or medical condition, and includes suppressing clinical recurrence. Treatment can be applied to a subject suffering from a medical condition or who may eventually suffer from a condition, to prevent, cure, delay its onset, reduce its severity or improve one or more symptoms of a condition or recurrent condition, or to extend the survival time of the subject to exceed the expected survival time in the absence of such treatment." therapeutic regimen" means the mode of treating a disease, such as the mode of administration used during treatment. The therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a therapeutic regimen (or a part of a therapeutic regimen) for the initial treatment of a disease. The usual goal of an induction regimen is to provide a high level of drug to the patient in the initial stage of a therapeutic regimen. The induction regimen may employ (in part or in whole) a "loading regimen" which may include administering a greater dose of the drug than the physician would administer during a maintenance regimen, administering the drug more frequently than the physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance phase" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during treatment of a disease, e.g., to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may employ continuous treatment (e.g., administration of the drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment after reaching specific predetermined criteria [e.g., disease manifestation, etc.]).

[0058] By "therapeutically effective amount" is meant an amount of a mutant IL-34 polypeptide sufficient to treat and / or prevent a disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily dosage 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 dosage level for any particular patient will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the specific compound employed; the specific composition employed, the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination or concomitantly with the specific polypeptide employed; and similar factors well known in the medical arts. For example, it is well known to those skilled in the art to start the compound dosage at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range of 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 symptomatic adjustment of the dosage to be treated. The drug usually contains about 0.01 mg to about 500 mg of active ingredient, preferably 1 mg to about 100 mg of active ingredient. The effective amount of the drug is usually provided at a dosage level of 0.0002 mg / kg to about 20 mg / kg body weight per day, about 0.001 mg / kg to 10 mg / kg body weight per day, and especially about 0.001 mg / kg to 7 mg / kg body weight per day.

[0059] The term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce harmful, allergic or other untoward reactions when properly administered to mammals, especially humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0060] The inventors generated novel mutants of the human IL34 polypeptide.

[0061] Therefore, an object of the present disclosure relates to a mutant IL-34 polypeptide (P1) having an amino acid sequence ranging from an asparagine (N) residue at position 21 to a proline (P) residue at position 242 in SEQ ID NO: 1, wherein a histidine (H) residue at position 56 and a glycine (G) residue at position 112 are both substituted with a cysteine ​​(C) residue.

[0062] Preferably, the mutant IL-34 polypeptide (P1) of the present invention is further characterized by at least one mutation selected from the group consisting of:

[0063] - The threonine (T) residue at position 36 is substituted by a tyrosine (Y) residue, a tryptophan (W) residue, a proline (P) residue, a phenylalanine (F) residue, an arginine (R) residue, a histidine (H) residue, an asparagine (N) residue, a cysteine ​​(C) residue, an isoleucine (I) residue, a proline (P) residue, or a leucine (L) residue

[0064] - The serine (S) residue at position 100 is replaced by an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue

[0065] - The threonine (T) residue at position 124 is replaced by a phenylalanine (F) residue or a tryptophan (W) residue

[0066] - The asparagine (N) residue at position 128 is replaced by a tyrosine (Y) or phenylalanine (F) residue

[0067] - The glutamine (Q) residue at position 131 is substituted by 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

[0068] - The serine (S) residue at position 147 is replaced by a glutamic acid (E) residue or an aspartic acid (D) residue

[0069] - the asparagine (N) residue at position 150 is substituted by a glutamic acid (E) residue or an aspartic acid (D) residue, and

[0070] - The leucine (L) residue at position 186 is substituted by an arginine (R) residue or a phenylalanine (F) residue.

[0071] Another object relates to a mutant IL34 polypeptide (P2) having an amino acid sequence ranging from an asparagine (N) residue at position 21 to a proline (P) residue at position 242 in SEQ ID NO: 1, wherein the proline (P) residue at position 59 is substituted by 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).

[0072] Alternatively, the proline (P) residue at position 59 is substituted with a lysine (K) residue or an arginine (R) residue.

[0073] Preferably, the mutant IL-34 polypeptide (P2) of the present invention is further characterized by at least one mutation selected from the group consisting of:

[0074] - The threonine (T) residue at position 36 is substituted by a tyrosine (Y) residue, a tryptophan (W) residue, a proline (P) residue, a phenylalanine (F) residue, an arginine (R) residue, a histidine (H) residue, an asparagine (N) residue, a cysteine ​​(C) residue, an isoleucine (I) residue, a proline (P) residue, or a leucine (L) residue

[0075] - The serine (S) residue at position 100 is replaced by an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue

[0076] - The threonine (T) residue at position 124 is replaced by a phenylalanine (F) residue or a tryptophan (W) residue

[0077] - The asparagine (N) residue at position 128 is replaced by a tyrosine (Y) or phenylalanine (F) residue

[0078] - The glutamine (Q) residue at position 131 is substituted by 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

[0079] - The serine (S) residue at position 147 is replaced by a glutamic acid (E) residue or an aspartic acid (D) residue

[0080] - the asparagine (N) residue at position 150 is substituted by a glutamic acid (E) residue or an aspartic acid (D) residue, and

[0081] - The leucine (L) residue at position 186 is substituted by an arginine (R) residue or a phenylalanine (F) residue.

[0082] Another object relates to a mutant IL34 polypeptide (P3) having an amino acid sequence ranging from an asparagine (N) residue at position 21 to a proline (P) residue at position 242 of SEQ ID NO: 1, comprising at least one mutation selected from the group consisting of:

[0083] - The threonine (T) residue at position 36 is substituted by a tyrosine (Y) residue, a tryptophan (W) residue, a proline (P) residue, a phenylalanine (F) residue, an arginine (R) residue, a histidine (H) residue, an asparagine (N) residue, a cysteine ​​(C) residue, an isoleucine (I) residue, a proline (P) residue, or a leucine (L) residue

[0084] - The serine (S) residue at position 100 is replaced by an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue

[0085] - The threonine (T) residue at position 124 is replaced by a phenylalanine (F) residue or a tryptophan (W) residue

[0086] - The asparagine (N) residue at position 128 is replaced by a tyrosine (Y) or phenylalanine (F) residue

[0087] - The glutamine (Q) residue at position 131 is substituted by 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

[0088] - The serine (S) residue at position 147 is replaced by a glutamic acid (E) residue or an aspartic acid (D) residue

[0089] - the asparagine (N) residue at position 150 is substituted by a glutamic acid (E) residue or an aspartic acid (D) residue, and

[0090] - The leucine (L) residue at position 186 is substituted by an arginine (R) residue or a phenylalanine (F) residue.

[0091] Another object of the present disclosure is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence having at least 80% identity to 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, and comprising at least one mutation selected from the group consisting of:

[0092] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0093] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0094] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0095] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0096] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F),

[0097] - the histidine residue at position 56 and the glycine residue at position 112 are both substituted by cysteine ​​residues (H56C and G112C),

[0098] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D),

[0099] - the glutamine residue at position 131 is substituted by an arginine residue (Q131R),

[0100] - the proline residue at position 59 is substituted by a lysine residue (P59K),

[0101] - the threonine residue at position 124 is substituted by a phenylalanine residue (T124F), and

[0102] - The asparagine residue at position 150 is substituted by a glutamic acid residue (N150E).

[0103] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence having at least 80% identity to the 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, and comprising at least one mutation selected from the group consisting of:

[0104] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0105] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0106] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0107] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0108] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0109] - The histidine residue at position 56 and the glycine residue at position 112 were both substituted by cysteine ​​residues (H56C and G112C).

[0110] Preferably, the mutant IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0111] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence having at least 80% identity to 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, and comprising at least one mutation selected from the group consisting of:

[0112] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), and

[0113] - The glutamine residue at position 131 is substituted by an arginine residue (Q131R).

[0114] Preferably, the mutant IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0115] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0116] a) is 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, and comprises at least one mutation selected from the group consisting of:

[0117] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0118] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0119] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0120] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0121] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0122] - the histidine residue at position 56 and the glycine residue at position 112 are both substituted by cysteine ​​residues (H56C and G112C),

[0123] b) an amino acid sequence having at least 80% identity to the sequence defined in a), provided that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and

[0124] c) A fragment of a sequence as defined in a) or b), provided that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0125] Preferably, the mutant IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0126] The mutant IL34 polypeptide may comprise a combination of 2, 3, 4, 5 or 6 mutations selected from the group consisting of:

[0127] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0128] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0129] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0130] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0131] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0132] - The histidine residue at position 56 and the glycine residue at position 112 were both substituted by cysteine ​​residues (H56C and G112C).

[0133] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0134] a) 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 serine residue at position 100 is substituted by a phenylalanine residue (S100F), and further comprising at least one mutation selected from the group consisting of:

[0135] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0136] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0137] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0138] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0139] - the histidine residue at position 56 and the glycine residue at position 112 are both substituted by cysteine ​​residues (H56C and G112C),

[0140] b) an amino acid sequence having at least 80% identity to the sequence defined in a), with the proviso that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and

[0141] c) A fragment of a sequence as defined in a) or b), provided that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0142] Preferably, the mutant IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0143] The mutant IL34 polypeptide comprises an S100F mutation and may further comprise a combination of 2, 3, 4 or 5 mutations selected from the group consisting of:

[0144] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0145] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0146] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0147] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0148] - The histidine residue at position 56 and the glycine residue at position 112 were both substituted by cysteine ​​residues (H56C and G112C).

[0149] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0150] a) is 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, and comprises at least one mutation selected from the group consisting of:

[0151] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0152] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y), and

[0153] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0154] b) an amino acid sequence having at least 80% identity to the sequence defined in a), with the proviso that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and

[0155] c) A fragment of a sequence as defined in a) or b), provided that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0156] Preferably, the mutant IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0157] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0158] a) is 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, and comprises the following three mutations:

[0159] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0160] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y), and

[0161] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0162] b) an amino acid sequence having at least 80% identity to the sequence defined in a), provided that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and

[0163] c) A fragment of a sequence as defined in a) or b), provided that the polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0164] Preferably, the mutant IL34 polypeptide is an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an agonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0165] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0166] a) is 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, and comprises at least one mutation selected from the group consisting of:

[0167] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), and

[0168] - the glutamine residue at position 131 is substituted by an arginine residue (Q131R),

[0169] b) an amino acid sequence having at least 80% identity to the sequence defined in a), with the proviso that the polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and

[0170] c) A fragment of a sequence as defined in a) or b), provided that the polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0171] Preferably, the mutant IL34 polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0172] Another object is a mutant IL34 polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0173] a) is 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, and comprises the following two mutations:

[0174] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), and

[0175] - the glutamine residue at position 131 is substituted by an arginine residue (Q131R),

[0176] b) an amino acid sequence having at least 80% identity to the sequence defined in a), with the proviso that the polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and

[0177] c) A fragment of a sequence as defined in a) or b), provided that the polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0178] Preferably, the mutant IL34 polypeptide is an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138. In some embodiments, the mutant IL34 polypeptide is used as an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

[0179] In some embodiments, the glutamine (Q) residue at position 81 is missing from the polypeptides of the invention.

[0180] 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.

[0181] "Agonist activity" herein means an activity that mimics, is similar to, is identical to, or is superior to the activity of a ligand of CSF-1R or a ligand of PTP-ζ or a ligand of CD138, such as IL-34, CSF-1 or M-CSF, and preferably IL-34. Preferably, "agonist activity" is an activity that mimics, is similar to, is identical to, or is superior to the activity of a ligand of CSF-1R.

[0182] "Antagonist activity" herein means an activity that partially or completely blocks, inhibits or neutralizes the biological activity of a ligand of CSF-1R or a ligand of PTP-ζ or a ligand of CD138, such as IL-34, CSF-1 or M-CSF, and preferably IL-34. Preferably, "antagonist activity" is an activity that partially or completely blocks, inhibits or neutralizes the biological activity of a ligand of CSF-1R.

[0183] The biological activities of the ligands of CSF-1R or PTP-ζ or CD138 include, but are not limited to, the ability to bind to CSF-1R, PTP-ζ or CD138, to induce or enhance cell survival, for example, to induce or enhance the survival, proliferation and differentiation of monocytes, especially CD14+ monocytes, into macrophages and other monocytic lineages such as osteoclasts, dendritic cells or microglia, or to induce or enhance the phosphorylation of certain kinases including protein kinase B (PKB, also known as Akt) or ERK1 / 2 (Extracellular signal-regulated Kinase 1 / 2).

[0184] The polypeptide of the present invention has the biological activity of a ligand of CSF-1R, a ligand of PTP-ζ, or a ligand of CD138, as long as it has at least one of the above activities.

[0185] A polypeptide is an "agonist" of a ligand of CSF-1R, a ligand of PTP-ζ, or a ligand of CD138, or has agonist activity, as long as the polypeptide has the ability to bind to CSF-1R, PTP-ζ, or CD138 and has another biological activity of a ligand of CSF-1R, a ligand of PTP-ζ, or a ligand of CD138.

[0186] In some embodiments, the agonist polypeptides of the invention have the ability to bind to CSF-1R or PTP-ζ, and induce or enhance the survival of cultured glial cells, neural progenitor cells, or glioblastoma cells, and / or induce or enhance tyrosine phosphorylation.

[0187] In other embodiments, the agonist polypeptides of the present invention have the ability to bind to CSF-1R, and induce or enhance the survival of Langerhans cells, and / or induce or enhance the phosphorylation of Akt or ERK1 / 2.

[0188] Preferably, the agonist polypeptide of the present invention has the ability to bind to CSF-1R, and induce or enhance the survival of cultured monocytes, especially CD14+ monocytes, and / or induce or enhance the phosphorylation of Akt or ERK1 / 2.

[0189] A polypeptide is an "antagonist" of a ligand of CSF-1R, a ligand of PTP-ζ, or a ligand of CD138, or has antagonist activity, as long as the polypeptide has the ability to bind to CSF-1R, PTP-ζ, or CD138, and partially or completely blocks, inhibits, or neutralizes another biological activity of a ligand of CSF-1R, a ligand of PTP-ζ, or a ligand of CD138.

[0190] For example, an antagonist polypeptide of the invention may compete with a ligand for CSF-1R for binding to CSF-1R, and may subsequently partially or fully inhibit another biological activity of the ligand for CSF-1R, such as kinase phosphorylation.

[0191] In some embodiments, the antagonist polypeptides of the invention have the ability to bind to CSF-1R or PTP-ζ, and partially or completely block, inhibit or neutralize the survival of cultured glial cells, neural progenitor cells or glioblastoma cells, and / or partially or completely block, inhibit or neutralize tyrosine phosphorylation.

[0192] In other embodiments, the antagonist polypeptides of the invention have the ability to bind to CSF-1R and partially or completely block, inhibit or neutralize the survival of Langerhans cells and / or partially or completely block, inhibit or neutralize the phosphorylation of Akt or ERK1 / 2.

[0193] Preferably, the antagonist polypeptide of the present invention has the ability to bind to CSF-1R and partially or completely block, inhibit or neutralize the survival of cultured monocytes, especially CD14+ monocytes, and / or the phosphorylation of Akt or ERK1 / 2.

[0194] Preferably, the polypeptide of the present invention has the ability to bind to CSF-1R and is an agonist or antagonist of a ligand of CSF-1R.

[0195] The binding activity of a polypeptide can be easily assessed in vitro or in vivo by a person skilled in the art, in particular by surface plasmon resonance (SPR) assay (in particular performed on Biacore), by Western blot assay, by ELISA, by co-immunoprecipitation (co-ip) assay, by pull-down assay, by cross-linking assay or by label transfer methods (FRET or HTRF assay).

[0196] For example, SPR experiments can typically be performed on Biacore at 25°C. First, recombinant human CSF-1 receptors can typically be immobilized on the chip surface by amine coupling. Therefore, recombinant CSF-1 receptors can typically be diluted to 20 μg / mL in a 10 mM sodium acetate solution at pH 5.0. The diluted CSF-1R can typically be covalently immobilized to the flow cell of a CM5 sensor chip, for example, through primary amine groups. IL-34 mutant proteins can typically be analyzed on a chip immobilized with CSF-1R in a "single cycle kinetics" (SCK) model. The kinetic parameters of IL-34 polypeptides on CSF-1R can typically be determined by using a series of protein dilutions in a "single cycle kinetics" (SCK) model. For example, IL-34 mutant proteins (muteins) as analytes can typically be diluted in HBS-P buffer, and the concentration is typically 25nM to 400nM. The flow rate over immobilized CSF-1R is typically 30 μL / min, for example 120 s binding and 600 s dissociation.The sensor chip surface can then typically be regenerated, for example using 10 mM NaOH for 30 s.

[0197] Specifically, the binding activity of a polypeptide can generally be assessed using the materials and methods described in Example 4 herein.

[0198] The ability of a polypeptide to induce or enhance the survival of cultured monocytes, especially CD14+ monocytes, can be easily assessed in vitro or in vivo by a person skilled in the art, in particular by culturing isolated monocytes in vitro in a medium containing the polypeptide and assessing monocyte survival, for example by viability staining and phenotypic analysis by flow cytometry.

[0199] For example, the ability of a polypeptide to induce or enhance the survival of cultured monocytes can usually be determined by culturing CD14 + Monocytes are assessed with WT IL-34 or mutants at a final concentration of 1.5 to 200 ng / ml, or typically 100 ng / ml. Cells can typically be harvested on day 3 and used, for example, for viability staining or phenotypic analysis by flow cytometry.

[0200] Specifically, the ability of a polypeptide to induce or enhance the survival of cultured monocytes, especially CD14+ monocytes, can generally be assessed using the materials and methods described in Example 5 herein.

[0201] The ability of a polypeptide to induce or enhance phosphorylation of Akt or ERK1 / 2 can be easily assessed by a person skilled in the art in vitro or in vivo, in particular by Western blot, by ELISA phospho-array or by flow cytometry.

[0202] For example, the ability of a polypeptide to induce or enhance phosphorylation of Akt or ERK1 / 2 can typically be assessed by incubating monocytes in FBS-free medium containing WT IL-34 or a mutant (typically for 1 minute, 3 minutes, and 5 minutes). Analysis can typically be performed by flow cytometry, for example using anti-phospho-Akt or anti-phospho-Erk1 / 2 primary and secondary antibodies.

[0203] The ability of a polypeptide to induce or enhance phosphorylation of Akt or ERK1 / 2 can generally be assessed using the materials and methods described in Example 6 herein.

[0204] If the level of detectable or detectable biological activity of a polypeptide is equivalent to the level of detectable or detectable biological activity of said ligand of CSF-1R or ligand of PTP-ζ or ligand of CD138, such as human IL-34 polypeptide, or preferably 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or 300% higher, then the polypeptide is considered herein to have a detectable or detectable biological activity that mimics, is similar to, is the same as or is superior to the biological activity of a ligand of CSF-1R or a ligand of PTP-ζ or a ligand of CD138.

[0205] 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 for CSF-1R or a ligand for PTP-ζ or a ligand for CD138 if the level of detectable or detectable biological activity of the 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 the ligand for CSF-1R or a ligand for PTP-ζ or a ligand for CD138, such as a human IL-34 polypeptide.

[0206] The polypeptide of the present invention may consist of a fragment of a sequence consisting of 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 and comprising at least one mutation selected from the group consisting of:

[0207] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0208] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0209] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0210] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0211] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0212] - The histidine residue at position 56 and the glycine residue at position 112 were both substituted by cysteine ​​residues (H56C and G112C).

[0213] The polypeptide of the present invention may also consist of a fragment of a sequence having at least 80% identity to 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 and comprising at least one mutation selected from the group consisting of:

[0214] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0215] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0216] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0217] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0218] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0219] - The histidine residue at position 56 and the glycine residue at position 112 were both substituted by cysteine ​​residues (H56C and G112C).

[0220] Another polypeptide of the present invention may consist of a fragment of a sequence consisting of 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, and comprising at least one mutation selected from the group consisting of:

[0221] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), and

[0222] - The glutamine residue at position 131 is substituted by an arginine residue (Q131R).

[0223] Another polypeptide of the present invention may also consist of a fragment of a sequence having at least 80% identity to 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, and comprising at least one mutation selected from the group consisting of:

[0224] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), and

[0225] - The glutamine residue at position 131 is substituted by an arginine residue (Q131R).

[0226] A "fragment" of a reference sequence is herein meant to be composed of a continuous amino acid chain of the reference sequence and a sequence whose size is less than the size of the reference sequence. In the context of the present invention, the size of a fragment can be, for example, between 6 to 222, 6 to 210, 6 to 200, 6 to 175, 6 to 150, 6 to 125, 6 to 100, 6 to 75, 6 to 50, 6 to 25, 6 to 15, 6 to 10 amino acids, or a size between 6 to 222, 10 to 222, 25 to 222, 50 to 222, 75 to 222, 100 to 222, 125 to 222, 150 to 222, 175 to 222, 200 to 222, 210 to 222, 220 to 222 amino acids. Most preferably, the size of a polypeptide of the present invention is 222 amino acids.

[0227] The polypeptides of the present invention also include any polypeptides that are "variants", "homologues" or "derivatives" of the above-mentioned polypeptides and exhibit the same biological activity.

[0228] Preferably, the polypeptide of the invention is a human variant of the IL-34 polypeptide.

[0229] Therefore, the polypeptides of the present invention include polypeptides having the following sequence: a sequence derived from 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, or a sequence derived from a fragment of 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, which is defined by the percentage of sequence identity with a 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.

[0230] 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%, even at least 95%, 96%, 97%, 98% or 99% identical to a reference sequence.

[0231] These derived sequences may differ from the reference sequence by substitution, deletion and / or insertion of one or more amino acids, at positions such that these modifications do not have any significant effect on the biological activity of the polypeptide. The substitution may correspond in particular to a conservative substitution or a substitution of a natural amino acid by a non-natural amino acid or a pseudo amino acid.

[0232] "An amino acid sequence having, for example, at least 80% identity to a reference sequence" herein means a sequence that is identical to the reference sequence, but this sequence may contain up to 20 mutations (substitutions, deletions and / or insertions) in every portion of 100 amino acids of the reference sequence. Thus, for a reference sequence of 100 amino acids, a fragment of 80 amino acids and a sequence of 100 amino acids containing 20 substitutions compared to the reference sequence are two examples of sequences having 80% sequence identity to the reference sequence.

[0233] Sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705) is generally used to determine the percent identity. The amino acid sequences to be compared are aligned to obtain the maximum percent identity. To this end, it may be necessary to artificially add spaces in the sequence. The alignment can be performed manually or automatically. The automated alignment algorithm for nucleotide sequences is well known to those skilled in the art and is described, for example, in Altschul et al. (1997) Nucleic Acids Res. 25: 3389 and is implemented by software such as Blast software. An algorithm that can be separated is, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J Mol Biol. 48: 443-53). Once the optimal alignment is achieved, the percent identity is determined by recording all positions where the amino acids of the two compared sequences are identical and comparing them to the total number of positions.

[0234] Therefore, the polypeptide of the present invention may comprise or consist of a sequence selected from the group consisting of:

[0235] a) a sequence having at least 80%, 85%, 90%, 95% or 100% identity to 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, and comprising at least one mutation selected from the group consisting of:

[0236] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0237] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0238] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0239] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0240] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0241] - the histidine residue at position 56 and the glycine residue at position 112 are both substituted by cysteine ​​residues (H56C and G112C),

[0242] b) a fragment of a sequence having at least 80%, 85%, 90%, 95% or 100% identity to 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 and comprising at least one mutation selected from the group consisting of:

[0243] - the serine residue at position 100 is substituted by a phenylalanine residue (S100F),

[0244] - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y),

[0245] - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F),

[0246] - the threonine residue at position 36 is substituted by a tryptophan residue (T36W),

[0247] - the threonine residue at position 36 is substituted by a phenylalanine residue (T36F), and

[0248] - The histidine residue at position 56 and the glycine residue at position 112 were both substituted by cysteine ​​residues (H56C and G112C).

[0249] Another polypeptide of the present invention may comprise or consist of a sequence selected from the group consisting of:

[0250] a) a sequence having at least 80%, 85%, 90%, 95% or 100% identity to 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, and comprising at least one mutation selected from the group consisting of:

[0251] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), and

[0252] - the glutamine residue at position 131 is substituted by an arginine residue (Q131R),

[0253] b) a fragment of a sequence having at least 80%, 85%, 90%, 95% or 100% identity to 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 and comprising at least one mutation selected from the group consisting of:

[0254] - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), and

[0255] - The glutamine residue at position 131 is substituted by an arginine residue (Q131R).

[0256] In a specific embodiment, the sequence of the polypeptide differs from the reference sequence only by the presence of conservative substitutions. Conservative substitutions are substitutions of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (e.g., asparagine, glutamine, serine, cysteine, and tyrosine), substitutions of amino acids with basic side chains (e.g., lysine, arginine, and histidine), substitutions of amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), substitutions of amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan).

[0257] According to the present invention, the polypeptide may be chemically or enzymatically modified to improve its stability or bioavailability. Such chemical or enzymatic modifications are well known to those skilled in the art. The following modifications may be involved but are not limited thereto:

[0258] - 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;

[0259] - modification of the amide bond between two amino acids, such as acylation (preferably acetylation) or alkylation on the nitrogen or alpha carbon;

[0260] - a change in chirality, for example substitution of a natural amino acid (L-enantiomer) by the corresponding D-enantiomer; this modification may optionally be accompanied by an inversion of the side chain (from C-terminus to N-terminus);

[0261] - becomes an azapeptide in which one or more alpha carbons are replaced by a nitrogen atom; and / or

[0262] - becomes a beta peptide, where one or more carbons are added to the N-alpha or C-alpha side of the backbone.

[0263] In this regard, it is possible to modify one or more lysine amino acids (K) of the polypeptide, in particular by:

[0264] - Amidation: This modification is easy to achieve, where the positive charge of lysine is replaced by a hydrophobic group (e.g. acetyl or phenylacetyl);

[0265] - Amination: by reacting primary amine R = (CH2)4-NH3 + forming a secondary amide, for example by forming an N-methyl, N-allyl or N-benzyl group; and

[0266] - by forming an N-oxide, N-nitroso, N-dialkylphosphino, N-sulfinyl or N-glycosidic group.

[0267] Alternatively or additionally, one or more threonine (T) and / or serine (S) amino acids of the polypeptide may be modified, in particular by adding ester or ether groups at the OH groups of the threonine and / or serine side chains. Esterification is a simple operation that can be performed using carboxylic acids, anhydrides, by bridging, etc., to form acetates or benzoates. Etherification can produce more stable compounds and can be performed using alcohols, halides, etc., to form, for example, methyl ethers or O-glycosides.

[0268] Also or alternatively, one or more glutamine (Q) amino acids may be modified, for example by amidation, by formation of secondary or tertiary amines, in particular with groups of the methyl, ethyl type, whether functionalized or not.

[0269] One or more glutamic acid (E) and / or aspartic acid (D) amino acids may also or alternatively be modified, for example:

[0270] - by esterification to form methyl esters (whether substituted or not), ethyl esters, benzyl esters, thiols (activated esters); and

[0271] - by amidation, especially to form N,N-dimethyl, nitroanilide, pyrrolidinyl groups.

[0272] On the other hand, it is preferred that proline amino acids that participate in the secondary structure of the polypeptide are not modified, noting also that amino acids G, A, and M generally do not offer obvious interesting modification possibilities.

[0273] In some embodiments, the polypeptides of the present invention may include a tag. A tag is a sequence containing an epitope that can be used to purify a polypeptide. It is bound by various techniques such as affinity chromatography, used to locate the polypeptide in a cell or tissue sample using immunolabeling techniques, and to detect the polypeptide by immunoblotting, etc. Examples of tags commonly used in the art are GST (glutathione-S-transferase) tags, FLAG TM Tags, Strep-tag TM , V5 tag, myc tag, His tag (which usually consists of six histidine residues), etc.

[0274] The present invention also relates to a fusion protein consisting of the mutant IL-34 polypeptide of the present invention and a heterologous polypeptide (i.e., a polypeptide other than IL-34 or its mutant) fused thereto.

[0275] 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 can be used 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 important but is selected for reasons such as making biopharmaceutical production of such polypeptides more convenient. The linker sequence can be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is preferably non-immunogenic in a subject to which the immunoadhesin is administered. One group of useful linker sequences is a linker derived from a heavy chain antibody hinge region, as described in WO 96 / 34103 and WO 94 / 04678. Other examples are polyalanine linker sequences. Further preferred examples of linker sequences are Gly / Ser linkers of varying lengths, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0276] In some embodiments, modifications are included to the mutant IL-34 polypeptides of the present invention to improve their therapeutic efficacy. Such modifications to therapeutic compounds can be used to reduce toxicity, increase circulation time, or improve biodistribution. For example, the toxicity of potentially important therapeutic compounds can be significantly reduced by combining with a variety of drug carrier vehicles that improve biodistribution. One strategy to improve drug activity is the use of water-soluble polymers. A variety of water-soluble polymers have been shown to improve biodistribution, improve cellular uptake patterns, change permeability across physiological barriers; and improve clearance from the body. In order to achieve targeted or sustained-release effects, water-soluble polymers containing drug moieties as terminal groups on the polymer chain, as part of the backbone, or as side groups have been synthesized. For example, PEGylation is a mature and proven method for modifying a variety of polypeptides. Advantages include the following, among others: (a) significantly improved in vivo circulation half-life, due to escape from renal clearance due to the polymer increasing the apparent size of the molecule beyond the glomerular filtration limit, and / or by escape from cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the PEGylated molecules; (c) improved pharmacokinetics; (d) enhanced resistance to proteolysis of the binding protein; and (e) improved thermal and mechanical stability of the PEGylated polypeptides.

[0277] According to the present invention, the mutant 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. The general principles of designing and preparing proteins are well known to those skilled in the art. The mutant IL-34 polypeptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesis tools are commercially available and can be used according to known protocols described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The mutant IL-34 polypeptides of the present invention can also be synthesized by solid phase technology using an exemplary peptide synthesis tool such as model 433A from Applied Biosystems Inc. The purity of any given protein (produced by automated peptide synthesis or by recombinant methods) can be determined using reverse phase HPLC analysis. The chemical authenticity of each peptide can be established by any method known to those skilled in the art. As an alternative to automated peptide synthesis, recombinant DNA technology can be used, in which the nucleotide sequence encoding the 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 below. Recombinant methods are particularly preferred for producing longer polypeptides. A variety of expression vector / host systems can be used to contain and express peptide or protein coding sequences. 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 expression of proteins, see, e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that can be used 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 schemes for recombinantly expressing peptide substances or fusion polypeptides in bacteria, yeast and other invertebrates are known to those skilled in the art and are briefly described below. Mammalian host systems for expressing recombinant proteins are also well known to those skilled in the art. Host cell strains can be selected for processing the expressed protein or producing certain specific abilities of post-translational modifications that can be used to provide protein activity. Such modifications to polypeptides include but are not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing of the "prepro" form of cleaved protein is also important for correct insertion, folding and / or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular mechanisms and characteristic mechanisms for such post-translational activity, and can be selected to ensure the correct modification and processing of the exogenous protein introduced.

[0278] Therefore, another object of the present invention relates to an isolated, synthetic or recombinant nucleic acid encoding a mutant IL-34 polypeptide of the present invention.

[0279] In some embodiments, the nucleic acid of the invention is a DNA or RNA molecule, which can be contained in any suitable vector, such as a plasmid, a cosmid, an episome, an artificial chromosome, a phage, or a viral vector.

[0280] Therefore, another object of the present invention relates to a vector comprising the nucleic acid according to the invention.

[0281] Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to cause or guide the expression of the polypeptide after administration to a subject. The vector may further include one or more replication origins and / or selectable markers. Relative to the coding sequence, the promoter region may be homologous or heterologous, and provide ubiquitous, constitutive, regulated and / or tissue-specific expression in any suitable host cell, including for in vivo use. Examples of promoters include bacterial promoters (T7, pTAC, Trp promoters, etc.), viral promoters (LTR, TK, CMV-IE, etc.), mammalian gene promoters (albumin, PGK, etc.), etc. Examples of plasmids include replicating plasmids or integrating plasmids including replication origins, such as pUC, pcDNA, pBR, etc. Examples of viral vectors include adenovirus, retrovirus, herpes virus 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 virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found in, for example, WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056, and WO 94 / 19478.

[0282] Another object 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 present invention.

[0283] The nucleic acid molecules of the present invention can be used to produce the mutant IL-34 polypeptides of the present invention in a suitable expression system. Common expression systems include Escherichia coli (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 (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or Saccharomyces, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced by lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). The construction of the expression vectors of the present invention and the transformation of the host cells can be performed using conventional molecular biology techniques. The mutant IL-34 polypeptides of the present invention can be obtained, for example, by culturing the genetically transformed cells of the present invention and recovering the polypeptides expressed by the cells from the culture. Then, if necessary, they can be purified by conventional methods known to those skilled in the art, for example by fractional precipitation, especially 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 present invention.

[0284] Therefore, the present invention also relates to a method for producing a recombinant host cell expressing a mutant IL-34 polypeptide of the present invention, the method comprising the steps consisting of:

[0285] i) introducing the recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo,

[0286] ii) a recombinant host cell obtained by in vitro or ex vivo culture, and

[0287] iii) optionally, selecting cells which express and / or secrete a polypeptide of the invention.

[0288] Such recombinant host cells can be used to produce the polypeptides and fusion proteins of the present invention.

[0289] The present invention further relates to a method of producing a mutant IL-34 polypeptide of the present invention, said method comprising the steps consisting of:

[0290] i) culturing the transformed host cell of the present invention under conditions suitable for the expression of the polypeptide or fusion protein; and

[0291] ii) recovering the expressed polypeptide or fusion protein.

[0292] The mutant IL-34 polypeptides of the present invention are particularly suitable for therapeutic purposes.

[0293] In particular, the mutant IL-34 polypeptide (P1) can be used as an agonist because the polypeptide is able to form a stable dimer.

[0294] Specifically, the mutated IL-34 polypeptide (P2) can be used as an antagonist because the polypeptide still binds to CSF-1R but is unable to form dimers.

[0295] Specifically, the mutated IL-34 polypeptide (P3) has an increased affinity for CFS-1R. Therefore, when the mutation of polypeptide (P3) is combined with the mutation of polypeptide (P1), the resulting mutated IL-34 polypeptide represents a "super" agonist of CFS-1R, i.e., an agonist with increased affinity for CSF-1R. Conversely, when the mutation of polypeptide (P3) is combined with the mutation of polypeptide (P2), the resulting mutated IL-34 polypeptide represents a "super" antagonist of CFS-1R, i.e., an antagonist with increased affinity for CSF-1R. Some mutated IL-34 polypeptides (P3) also have agonist activity themselves. Other mutated IL-34 polypeptides (P3) have antagonist activity themselves.

[0296] Therefore, another object of the present invention relates to a mutant 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 treating a patient in need thereof, comprising administering to the 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 mutant IL-34 polypeptide, nucleic acid or vector of the present invention for the preparation of a medicament.

[0297] In some embodiments, the agonists of the present invention are particularly suitable for inducing and / or maintaining immune tolerance in patients in need thereof. Therefore, one object of the present invention relates to an agonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for use in inducing and / or maintaining immune tolerance in patients in need thereof. More specifically, the present invention provides a method of inducing and / or maintaining immune tolerance in a patient in need thereof, 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 an agonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for the preparation of a medicament for inducing and / or maintaining immune tolerance in a patient in need thereof.

[0298] Specifically, patients treated with the agonists of the invention exhibit the following physiological characteristics compared to untreated patients: a) a reduction in the level of immune response (specific or nonspecific) (thought to be at least partially due to antigen-specific effector CD4 + T and CD8 +The invention provides a method for inducing and maintaining the proliferation of Treg cells, i.e., a) delaying the occurrence or development of an immune response (specific or non-specific); and c) reducing the risk of the occurrence or development of an immune response (specific or non-specific).

[0299] In some embodiments, the agonists of the present invention are particularly suitable for preventing or reducing transplant rejection in patients in need thereof. Therefore, another object of the present invention relates to an agonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for use in preventing or reducing transplant rejection in patients in need thereof. More specifically, the present invention provides a method for preventing or reducing transplant rejection in patients in need thereof, 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 an agonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention in the preparation of a medicament for preventing or reducing transplant rejection in patients in need thereof.

[0300] In some embodiments, the donor of the transplant can be a living donor or a dead donor, i.e., a cadaver donor. In some embodiments, the transplant is an organ, tissue, or cell. In some embodiments, transplant rejection is cardiac allogeneic transplant rejection. In some embodiments, the cell is selected from the group consisting of: pluripotent hematopoietic stem cells derived from bone marrow, peripheral blood, or umbilical cord blood; or pluripotent (pluripotent) (i.e., embryonic stem cells (ES) or induced pluripotent stem cells (iPS)) or differentiated cells of different cell lineages derived from pluripotent stem cells, such as cardiomyocytes, β-pancreatic cells, hepatocytes, neurons, etc. In some embodiments, the cell is used for allogeneic hematopoietic stem cell transplantation (HSCT), and therefore comprises pluripotent hematopoietic stem cells usually derived from bone marrow, peripheral blood, or umbilical cord blood. HSCT can cure leukemia and lymphoma patients. However, an important limitation of allogeneic HCT is the development of graft-versus-host disease (GVHD), which occurs in severe form in about 30-50% of the people receiving this treatment. Thus, in some embodiments, the agonists of the invention are particularly useful for preventing or reducing graft-versus-host disease (GvHD). Thus, in some embodiments, the patient receiving a hematopoietic stem cell transplant has 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; lymphomas, such as Hodgkin and non-Hodgkin lymphomas, chronic lymphocytic leukemia (CLL) and multiple myeloma.

[0301] In some embodiments, the agonists of the present invention are particularly useful for treating neurodegenerative diseases in patients thereof. Therefore, another object of the present invention relates to an agonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for use in treating a neurodegenerative disease in a patient thereof. More specifically, the present invention provides a method for treating a neurodegenerative disease in a patient with a neurodegenerative disease, 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 an agonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for the preparation of a medicament for treating a neurodegenerative disease in a patient with a neurodegenerative disease.

[0302] Examples of neurodegenerative diseases include, but are not limited to, Parkinson's disease and related disorders, including Parkinson's disease, Parkinson-dementia, autosomal recessive PARK2 and PARK6-related Parkinson's disease, atypical Parkinson's syndromes, including progressive supranuclear palsy, corticobasal degeneration syndrome, Lewy bodies dementia, multiple system atrophy, Guadeloupean Parkinsonism, and Lytigo-bodig disease; motor neuron diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, and post-polio syndrome. syndrome; neuroinflammatory diseases; Alzheimer's disease and related conditions, including early stage Alzheimer's disease, mild stage Alzheimer's disease, moderate stage Alzheimer's disease, mild to moderate stage Alzheimer's disease, advanced stage Alzheimer's disease, mild cognitive impairment, vascular dementia, mixed dementia, Pick's disease, argyrophilic grain disease, posterior cortical atrophy, Wernicke-Korsakoff syndrome; prion diseases; lysosomal storage diseases; leukodystrophy; Huntington's disease; multiple sclerosis; Down syndrome; spinal and bulbar muscular atrophy; HIV-associated neurocognitive disorder; Tourette syndrome Syndrome; autosomal dominant spinocerebellar ataxia; Friedreich's Ataxia; Dentatorubral pallidoluysianatrophy; myotonic dystrophy; schizophrenia; age-related memory impairment; autism and autism spectrum disorder; attention-deficit hyperactivity disorder; chronic pain; alcohol-induced dementia;progressive non-fluent aphasia; semantic dementia; spastic paraplegia; fibromyalgia; post-Lyme disease; neuropathy; withdrawal symptoms; Alpers' disease; cerebro-oculo-facio-skeletal syndrome; Wilson's disease; Cockayne syndrome; Leigh's disease; neurodegeneration with brain iron accumulation; opsoclonus myoclonus syndrome; alpha-methylacyl-CoA racemase deficiency; Andermann syndrome; Arts syndrome; Marinesco-; Syndrome (Marinesco- syndrome; mitochondrial membrane protein-associated neurodegeneration; pantothenate kinase-associated neurodegeneration; polycystic lipomembranousosteodysplasia with sclerosing leukoencephalopathy; riboflavin transporter deficiency neuronopathy; and ataxia telangiectasia.

[0303] Preferably, the neurodegenerative disease is selected from the group consisting of multiple sclerosis, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease.

[0304] In some embodiments, the agonists of the present invention are particularly useful for treating autoimmune inflammatory diseases, alloimmune response allergic disorders and asthma in patients. Therefore, another object of the present invention relates to the agonist mutant 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 patients. More specifically, the present invention provides a method for treating autoimmune inflammatory diseases, alloimmune response allergic disorders and asthma in patients, 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 mutant IL-34 polypeptide, nucleic acid or vector of the present invention for the preparation of a medicament for treating autoimmune inflammatory diseases, alloimmune response allergic disorders and asthma in patients.

[0305] 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, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis and juvenile onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis and ankylosing spondylitis, inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis and atopic dermatitis, x-linked hyper IgM syndrome (x-linked hyper IgM syndrome), urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic sclerosis, sclerosis, systemic sclerosis, multiple sclerosis (MS), chordo-optic MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis (sclerosisdisseminata) and ataxia sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa), microscopic colitis, collagenous colitis, polypoidal colitis, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, acute respiratory distress syndrome (ARDS) in adults, meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disease, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and / or brainstem encephalitis, uveitis, anterior uveitisuveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), rapidly progressive glomerulonephritis, allergic conditions, autoimmune myocarditis, leukocyte adhesion defects, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous SLE, neonatal lupus syndrome (NLE), disseminated lupus erythematosus (lupus erythematosusdisseminatus), lupus (including nephritis, encephalitis, pediatric, non-renal, extrarenal, discoid, alopecia), juvenile-onset (type I) diabetes, including pediatric insulin-dependent diabetes mellitus (IDDM), adult-onset diabetes mellitus (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu) arteritis, medium vessel vasculitis, Kawasaki's disease), polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing cutaneous hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia, or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease (disease), pure red cell anemia or aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, leukocytopenia, diseases associated with leukocytic infiltration, CNS inflammatory disorders, multi-organ injury syndromes such as those secondary to sepsis, trauma, or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet or Behcet disease, Castleman syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathy, 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 (TTP), 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 polyglandular endocrine syndrome), paraneoplastic syndromes, including paraneoplastic syndromes of the nervous system such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndromesyndrome), encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, myasthenia gravis associated with thymoma, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphointerstitial pneumonitis, bronchiolitis obliterans (non-transplant) versus NSIP, Guillain-Barre syndrome, Berger's disease disease)(IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, celiac disease, coeliac disease, celiac sprue, refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease disease), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AGED), autoimmune hearing loss, optoclonus-myoclonus syndrome (OMS), polychondritis such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, noncancerous lymphocytosis, primary lymphocytosis, which includes monoclonal B-cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal gammopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndromes, channelopathies such as epilepsy, migraine, arrhythmias, muscle disorders, deafness, blindness, periodic paralysis and CNS channelopathies, autism, inflammatory myopathies, focal segmental glomerulosclerosis (FSGS), endocrine eye diseases (endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, Alzheimer's disease, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndromesyndrome), alopecia greatata, CREST syndrome (calcification, 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-fancier's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome, syndrome), alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue fever, endocarditis, endocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, syndrome), filariasis, cyclitis such as chronic cyclitis, metachronous cyclitis, iridocyclitis or Fuch's cyclitis, Henoch-Schonlein purpura, HIV infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, postvaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, poststreptococcal nephritis, thromboangitisubiterans, hyperthyroidism, tabes dorsalis dorsalis), choroiditis, giant cell polymyalgia, endocrine eye disease, chronic hypersensitivity pneumonitis, keratoconjunctivitis siccasicca), epidemic keratoconjunctivitis, idiopathic nephritis syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway disease, silicosis, aphthous stomatitis, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergic enteritis, erythema nodosum leprosum, idiopathic facial palsy, chronic fatigue syndrome, febris rheumatica, Hamman-Rich disease, sensorineural hearing loss loss), hemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephropathy, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, polyradiculitis acuta), pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, infertility due to antisperm antibodies, nonmalignant thymoma, vitiligo, SCID and Epstein-Barr virus-related diseases, acquired immunodeficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion defects, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte infiltration, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis totalis), dilated cardiomyopathy, acquired epidermolysis bullosaacquisita, 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 infiltrate eosinophilia, eosinophilic myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma or granuloma containing eosinophils, anaphylaxis, seronegative spondyloarthropathies, polyendocrine autoimmune diseases, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia, autoimmune disorders associated with collagen diseases, rheumatism, neuropathy, ischemic reperfusion disorders, decreased blood pressure response, vascular dysfunction, antgiectasis, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia and diseases associated with angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, reperfusion injury of the myocardium or other tissues, skin diseases with acute inflammatory elements, acute purulent meningitis or other central nervous system inflammatory disorders, inflammatory disorders of the eye and orbit, granulocyte transfusion-related syndrome, cytokine-induced toxicity, acute severe inflammation, chronic refractory inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic aortic disorder, intimal hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0306] Allergic conditions typically include anaphylaxis, allergic reactions 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 rhinoconjunctivitis, and vaginitis; skin diseases (skin and dermatologicaldisorder), including psoriasis and inflammatory skin diseases such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, linear IgA disease, acute and chronic urticaria, and scleroderma; vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity vasculitis); spondyloarthropathies; and intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, enteritis, non-tropical sprue, and celiac disease).

[0307] In some embodiments, the agonists of the present invention are administered to the patient in combination with immunosuppressive drugs. Immunosuppressive drugs include, but are not limited to, thiopurine drugs such as azathioprine (AZA) and its metabolites; nucleoside triphosphate inhibitors such as mycophenolic acid (Cellcept) and its derivatives (Myfortic); its derivatives; its prodrugs; and combinations thereof. Other examples include, but are not limited to, 6-mercaptopurine ("6-MP"), cyclophosphamide, mycophenolate mofetil, prednisolone, sirolimus, dexamethasone, rapamycin, FK506, mizoribine, azathioprine, and tacrolimus.

[0308] In some embodiments, the immunosuppressive drug is selected from the group consisting of: cytostatics, such as mammalian target of rapamycin (mTOR) inhibitors and rapamycin (sirolimus); alkylating agents (cyclophosphamide) and antimetabolites (azathioprine, mercaptopurine and methotrexate); therapeutic antibodies (e.g., anti-CD40L monoclonal antibody, anti-IL-2R monoclonal antibody, anti-CD3 monoclonal antibody, anti-CD45RC monoclonal antibody, anti-lymphocyte globulin (ALG) and anti-thymocyte globulin (ATG)); calcineurin inhibitors (cyclosporine); glucocorticoids and mycophenolate mofetil.

[0309] In some embodiments, the immunosuppressive drug is a calcineurin inhibitor. As used herein, the term "calcineurin inhibitor" has the general meaning of the art and refers to a substance that inhibits the dephosphorylation of calcineurin (i.e., protein phosphatase regulated by calcium / calmodulin involved in intracellular signal transduction) of a suitable substance by targeting calcineurin (PP2B, PP3), i.e., a cellular enzyme involved in gene regulation. The calcineurin inhibitor of the present invention is generally an immunophilin-binding compound with calcineurin inhibitory activity. An immunophilin-binding calcineurin inhibitor is a compound that forms a calcineurin inhibitory complex with an immunophilin such as cyclophilin and macrophage. An example of a cyclophilin-binding calcineurin inhibitor is cyclosporine or a cyclosporin derivative (hereinafter referred to as cyclosporin), and an example of a macrophage-binding calcineurin inhibitor is ascomycin (FR 520) and an ascomycin derivative (hereinafter referred to as ascomycin). A wide range of ascomycin derivatives are known, which occur naturally in fungal species or can be obtained by manipulating fermentation procedures or by chemical derivatization. Ascomycin macrolides include ascomycin, tacrolimus (FK506), sirolimus and pimecrolimus. Cyclosporin, originally extracted from the soil fungus Potypaciadium infilatum, has a cyclic structure of 11 amino acids and includes, for example, cyclosporin A to I, such as cyclosporin A, B, C, D and G. Voclosporin is a next generation calcineurin inhibitor that is a more potent and less toxic semisynthetic derivative of cyclosporin A. In some embodiments, the calcineurin inhibitor of the invention is the trans-version of voclosporin, trans-ISA247 (trans-ISA247) (Cas No. 368455-04-3), which is described, for example, in U.S. Patent Publication No. 2006 / 0217309, which is incorporated herein by reference. Other 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 that is also a fungal product, but has 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 that inhibits the mammalian target of rapamycin (mTOR) pathway by directly binding to mTOR complex 1 (mTORC1). Pimecrolimus is also a calcineurin inhibitor. Calcineurin inhibitors such as cyclosporin A, voclosporin, ascomycin, tacrolimus, pimecrolimus, analogs thereof, or pharmaceutically acceptable salts thereof can be used in the mixed micelle compositions of the present disclosure.

[0310] In some embodiments, the immunosuppressive drug is a corticosteroid. As used, the term "corticosteroid" has the general meaning in the art and refers to a class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system that is endowed with anti-inflammatory activity. Corticosteroid drugs generally include cortisone, cortisol, hydrocortisone (11β, 17-dihydroxy, 21-(phosphoryloxy)-pregnant-4-ene, 3,20-dione disodium), dihydroxycortisone, dexamethasone (21-(acetyloxy)-9-fluoro-1β, 17-dihydroxy-16α-m-ethylpregnant-1,4-diene-3,20-dione), and highly derived steroid drugs, such as beconase (beclomethasone dipropionate dipropionate, which is 9-chloro-11-β,17,21,trihydroxy-16β-methylpregna-1,4-diene-3,20-dione 17,21-dipropionic acid). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort, and betamethasone. Corticosteroids such as cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethasone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetate, acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone acetonide, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

[0311] In some embodiments, the immunosuppressive drug is a CTLA4 molecule. In some embodiments, the CTLA4 molecule is abatacept. Abatacept is a soluble fusion protein composed of the extracellular domain of human CTLA-4 and the modified Fc (hinge, CH2 and CH3 domains) of human immunoglobulin G1 (IgG1) connected in part. Abatacept is produced in a mammalian cell expression system by recombinant DNA technology. The apparent molecular weight of abatacept is 92 kilodaltons. Abatacept is developed by Bristol-Myers Squibb and is disclosed in, for example, U.S. Patents 5,851,795, U.S. Patents 7,455,835 and EP1962886. In some embodiments, the CTLA molecule is belatacept, which is obtained by changing two amino acids (L104E and A29Y) in the CD80 / 86 binding portion of the abatacept compound. This chemically subtle change results in a 10-fold increase in the ability to inhibit T cell activation compared to in vitro. Belatacept (L104EA29YIg) is the first biologic approved for primary maintenance immunosuppression that selectively blocks the CD28 co-stimulatory pathway to prevent T cell activation (Larsen, CP. et al, Am. J. Transplant., 5: 443-453 (2005)). In some embodiments, the CTLA4 molecule is MAXY-4, which is also a protein derived from abatacept but with increased binding to the CTLA4 target, and is currently in preclinical research by Perseid Therapeutics, LLC and Astellas Pharma, Inc. for the treatment of autoimmune diseases and transplant rejection.

[0312] In some embodiments, the agonist of the present invention is particularly suitable for preventing unwanted immune responses and allergies for proteins or therapeutic proteins expressed during gene therapy in patients. For example, this method can indeed be used to suppress immune responses, especially for preventing immune responses for specific proteins (the expression of which is restored by gene therapy in individuals with corresponding genetic defects). Therefore, the agonist of the present invention can be used to prevent the immunoreactivity of proteins that are usually not present in patients due to mutations, and its reconstruction (reconstitution) is achieved by gene therapy. In addition, protein therapy is an increasingly extensive field of medical innovation, and involves applying proteins such as enzymes, antibodies or cytokines directly to patients as therapeutic products. One of the main obstacles to delivering such drugs involves immune responses for therapeutic proteins themselves. The administration of protein-based therapeutic agents is usually accompanied by the administration of immunosuppressants, the latter's purpose of use is to promote the life of the protein to be longer and therefore increase protein uptake into organism cells and tissues. However, general immunosuppressants may be disadvantageous due to the non-specific nature of the immunosuppression performed, resulting in unwanted side effects in patients. Thus, this method can be used to inhibit the immune response to therapeutic proteins and peptides, such as therapeutic antibodies, cytokines, enzymes, or any other protein, administered to a patient.

[0313] In some embodiments, the antagonists of the present invention are particularly useful for treating bone diseases involving abnormal proliferation in patients. Therefore, another object of the present invention relates to the antagonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for treating bone diseases involving abnormal proliferation in patients. More specifically, the present invention provides a method for treating bone diseases involving abnormal proliferation in patients, 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 mutant IL-34 polypeptide, nucleic acid or vector of the present invention for the preparation of a medicament for treating bone diseases involving abnormal proliferation in patients.

[0314] In some embodiments, the bone disease involving 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 infection, and Paget's disease.

[0315] The antagonists of the present invention are particularly useful in providing an effective and safe way to release Tregs (e.g., CD8 + and CD4 +Thus, the present invention also provides a method of eliciting a T cell response in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an antagonist of the present invention, wherein the T cell response comprises increasing CD4 + CD25 - Expansion of T cells. In some embodiments, the invention relates to a method of releasing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antagonist of the invention, wherein the therapeutically effective amount is sufficient to reduce immune tolerance in the subject.

[0316] The present invention also provides a method for eliciting a T cell response against a tumor in a subject having a tumor, comprising the step of administering to the subject a therapeutically effective amount of an antagonist of the present invention, wherein the T cell response comprises increasing CD4 + CD25 - Amplification of T cells. In some embodiments, inducing an anti-tumor T cell response in a subject with a tumor or cancer allows treatment of 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 metastasis in the subject. Therefore, provided herein is a method for increasing / inducing / eliciting an immune response in a subject in need thereof. For example, a subject may have developed or may develop immune tolerance to unwanted cells such as cancer (e.g., tumor) cells and / or cells infected by pathogens, etc. The method includes administering a therapeutically effective amount of an antagonist of the present invention to a subject.

[0317] Another object of the present invention relates to the antagonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for use in treating cancer. The present invention also provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antagonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention. Also disclosed is the use of the antagonist mutant IL-34 polypeptide, nucleic acid or vector of the present invention for preparing a medicament for treating cancer.

[0318] Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. In some embodiments, the subject has a cancer selected from the group consisting of acanthoma, acinar cell carcinoma, acoustic neuroma, acral melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantoma, adenocarcinoma, adenoid cystic leukemia, Cancer, adenoma, odontogenic adenoma, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor teratoid rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, bile duct cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioalveolar carcinoma, brown tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ situ), penile cancer, cancer of unknown primary site, carcinosarcoma, Castleman disease, embryonal tumor of the central nervous system, cerebellar astrocytoma, brain astrocytoma, cervical cancer, bile duct cancer, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelocytic 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 tumortumor), diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial carcinoma, endometrial uterine carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family of Sarcoma Sarcoma), Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, parasitic fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor tumor), germ cell tumor (germinoma), gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, glioma cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematological malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor tumor), laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid 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, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumortumor), mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous neck cancer with occult primary, metastatic transitional cell carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic disease, syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cancer, nasopharyngeal cancer, nasopharyngeal carcinoma), neoplasm, neurotheca, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, non-small cell lung cancer (NSCLC) with chronic obstructive pulmonary disease (COPD), eye tumors, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath, meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cell cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor), pancreatic cancer, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pinealoblastoma, pituicytoma, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastema, polyembryony, precursor T-lymphocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformationtransformation), sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland cancer, secondary neoplasms, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestinal cancer, soft tissue sarcoma, somatostatinoma, soot wart, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor Tumor), surface epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphoma, testicular cancer, thecoma, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, genitourinary tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Vemer Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

[0319] In some embodiments, the antagonists of the invention are administered to a patient in combination with chemotherapy. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimine and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins (particularly bullatacin) and acetogenins. ) and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 1I; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicins;and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, actinomycin D actinomycin, daunorubicin, detorubicin, 6-diaza-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, nestatin inostatin), zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadreners, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; epothilone; etoglucid; 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; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel and docetaxel; chloranbucil; 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; ibandronate; irinotecan (e.g., CPT-1 1) Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids and derivatives thereof. ;

[0320] In some embodiments, antagonists of the invention are administered to a patient in combination with immunotherapy.

[0321] In some embodiments, immunotherapy includes administering at least one immune checkpoint inhibitor to the patient.Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins.Many immune checkpoint inhibitors are known, and alternative immune checkpoint inhibitors similar to these known immune checkpoint protein inhibitors may be developed in the (near) future.Immunocheckpoint 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.

[0322] In some embodiments, PD-1 (programmed death-1) axis antagonists 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 ), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, 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, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. Antibody YW243.55.S70 is an anti-PD-L1 described in WO 2010 / 077634 A1. MEDI4736 is an anti-PD-L1 antibody described in WO2011 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and WO2006 / 121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and WO2009 / 114335. CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Atezolizumab is an anti-PD-L1 antibody described in U.S. Patent No. 8,217,149. Avelumab is an anti-PD-L1 antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in WO2015033301 and WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Patent No. 8,609,089, US 2010028330 and / or US20120114649. 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 consisting of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110, KY1003 and Atezolizumab, and preferably Avelumab, Durvalumab or Atezolizumab.

[0323] In some embodiments, the CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonist is selected from the group consisting of an anti-CTLA-4 antibody, a human anti-CTLA-4 antibody, a mouse 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-4 domain antibody, a single chain anti-CTLA-4 fragment, a heavy chain anti-CTLA-4 fragment, a light chain anti-CTLA-4 fragment, a CTLA-4 inhibitor that agonizes a co-stimulatory pathway, an antibody disclosed in PCT Publication No. WO 2001 / 014424, an antibody disclosed in PCT Publication No. WO 2004 / 035607, the antibodies disclosed in U.S. Publication No. 2005 / 0201994, and the antibodies disclosed in Granted European Patent No. EP 1212422 B. Other CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; described in PCT Publication Nos. WO 01 / 14424 and WO 00 / 37504; and described in U.S. 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 WO 98 / 42752; U.S. Pat. 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. Pat. 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 (also known as MDX-010 and ipilimumab with CAS number 477202-00-9 and available from Medarex, Inc., Bloomsbury, NJ) disclosed in WO 01 / 14424.With regard to CTLA-4 antagonists (antibodies), these are known and include tremelimumab (CP-675,206) and ipilimumab.

[0324] In some embodiments, the immunotherapy comprises administering to the patient a combination of a CTLA-4 antagonist and a PD-1 antagonist.

[0325] 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. Specifically, 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 the general meaning of the art and refers to a molecule 3 containing a T cell immunoglobulin and a mucin domain. The natural ligand of TIM-3 is galectin 9 (Gal9). Therefore, the term "TIM-3 inhibitor" used herein refers to a compound, substance or composition capable of inhibiting TIM-3 function. For example, the inhibitor can inhibit the expression or activity of TIM-3, regulate or block the TIM-3 signaling pathway and / or block TIM-3 from binding to galectin 9. Antibodies specific for TIM-3 are well known in the art and are generally those described in WO2011155607, WO2013006490 and WO2010117057.

[0326] In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO2014150677. 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-methyl-tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, and 5-Br-4-Cl-indoxyl Preferably, the IDO inhibitor is selected from 1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphthoic acid and β-[3-benzo(b)thienyl]-alanine or a derivative or prodrug thereof.

[0327] Alternatively, gene therapy may be used by using or administering a nucleic acid encoding a polypeptide of the present invention instead of the polypeptide. In this case, a nucleic acid encoding a polypeptide of interest is administered to the patient so that the polypeptide is expressed in vivo by the patient's cells into which the nucleic acid has been transferred.

[0328] Therefore, the present invention also relates to a nucleic acid comprising or consisting of a sequence encoding a polypeptide of the present invention. The nucleic acid can be easily obtained by cloning a cDNA fragment encoding a polypeptide of the present invention.

[0329] Such nucleic acids encoding polypeptides of the present invention may in particular be in the form of DNA vectors, such as plasmid vectors. One or more vectors may be used, each of which may carry one or more sequences encoding at least one polypeptide of the present invention. In this vector, the sequence encoding at least one polypeptide of the present invention is functionally linked to one or more elements, such as transcriptional promoters, activators and / or terminators, that allow its expression or regulate its expression.

[0330] According to a preferred embodiment, a vector carrying a sequence encoding a polypeptide of the invention is used.

[0331] The one or more DNA vectors may be inserted in vivo using any technique known to those skilled in the art. In particular, the one or more DNA vectors may be inserted in vivo in naked form, i.e., without the aid of any vector or system that facilitates transfection of the vector in the cell (EP 465 529).

[0332] Gene guns can also be used, for example, by depositing DNA onto the surface of "gold" particles and firing these particles to pass DNA through the patient's skin (Tang et al., (1992) Nature 356:152-4). Injections using liquid gels can also simultaneously transfect skin, muscle, adipose tissue, and breast tissue (Furth et al., (1992) Anal Biochem. 205:365-8).

[0333] Other available techniques include microinjection, electroporation, calcium phosphate precipitation, formulation using nanocapsules or liposomes.

[0334] Biodegradable nanoparticles in polyalkylcyanoacrylates are particularly advantageous. For liposomes, the use of cationic lipids facilitates the encapsulation of negatively charged nucleic acids and promotes fusion with negatively charged cell membranes.

[0335] Alternatively, the vector may be in the form of a recombinant virus having inserted into its genome a nucleic acid sequence encoding the polypeptide.

[0336] The viral vector may preferably be selected from adenovirus, retrovirus, in particular lentivirus and adeno-associated virus (AAV), herpes virus, cytomegalovirus (CMV), vaccinia virus, etc. Lentiviral vectors are described, for example, by Firat et al., (2002) J Gene Med 4:38-45.

[0337] Advantageously, the recombinant virus is a defective virus. The term "defective virus" means a virus that is not able to replicate in a target cell. Typically, the genome of a defective virus lacks at least the sequence required for the virus to replicate in an infected cell. These regions may be eliminated or rendered non-functional, or replaced with other sequences, and especially with nucleic acids encoding a polypeptide of interest. Nevertheless, preferably, the defective virus maintains its genome sequence required for encapsulating viral particles.

[0338] The targeted administration of genes is described, for example, in application WO 95 / 28494.

[0339] Another object of the present invention relates to an in vitro / ex vivo method for obtaining an immunosuppressive macrophage population, comprising culturing a monocyte population with a culture medium comprising an amount of an agonist of the present invention.

[0340] The monocyte colony used as starting material can be separated according to any technology known in the art.For example, human monocyte colony can be obtained from various biological samples containing PBMC.Usually, they are separated from peripheral blood.Positive selection can be carried out to separate them by beads marked with different ligands (for example, CD14).Then the cells of such marks can be separated by multiple techniques such as cell sorting as described below.

[0341] In some embodiments, the human monocyte population is thus CD14 + In some embodiments, the human monocyte population is CD14 + CD16 + In some embodiments, the human monocyte population is CD14 + CD16 - Human monocyte populations.

[0342] Another object of the present invention relates to an in vitro / ex vivo method for obtaining a human Treg cell population, comprising the following steps:

[0343] (a) culturing a monocyte population with a medium comprising an amount of an agonist of the present invention to obtain an immunosuppressive macrophage population (also referred to as "IL34-differentiated macrophages"); and

[0344] (b) co-culturing a human peripheral blood mononuclear cell (PBMC) population and the immunosuppressive macrophage population obtained in step (a).

[0345] In some embodiments, the method of the present invention includes a step of separating a human Treg cell colony from a PBMC colony before step (b). Therefore, according to this embodiment, step (b) includes co-culturing a human Treg cell colony separated from a PBMC colony and an immunosuppressive macrophage colony obtained in step (a).

[0346] In some embodiments, the methods of the present invention include the step of isolating the human Treg cell population after the co-culturing of step (b).

[0347] Typically, the agonist is added to the culture medium at a concentration of 1 to 500 ng / ml, preferably 10 to 100 ng / ml, more preferably 50 ng / ml.

[0348] Typically, the agonist is added to the culture medium at a concentration of 1 to 500 ng / ml, preferably 10 to 100 ng / ml, more preferably 20 ng / ml.

[0349] The step (a) of culturing a human monocyte colony in a culture medium containing a certain amount of an agonist of the present invention is carried out for the necessary time required to obtain an immunosuppressive macrophage (or IL34-differentiated macrophage) colony. Typically, culturing monocytes with the target culture medium should be carried out for at least 3 days or 4 days but not more than 8 days, preferably 6 days. In some embodiments, culturing monocytes with the target culture medium is carried out for 3 days, 4 days, 5 days, 6 days, 7 days or 8 days or longer.

[0350] In step (b), the peripheral blood mononuclear cell (PBMC) population can be separated by methods known to those skilled in the art (e.g., by density centrifugation, such as Ficoll-Paque TM Density gradient centrifugation) to separate.

[0351] In general, isolation of human Treg cell populations can be performed by a variety of methods available to technicians for detecting specific immune cell populations, 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, use of bispecific antibodies specific for IL34 and CD proteins (such as CD4, CD8, CD25, CD127 or CD45RC, PD1, GITR), use of bispecific antibodies specific for IL34, IFNγ, TGFβ and IL10, use of trispecific antibodies and combinations of such methods.

[0352] In some embodiments, the methods of the present invention further comprise the step of expanding the obtained Treg population.

[0353] In some embodiments, the culture medium suitable for expanding Treg includes a certain amount of at least one cytokine. Examples of cytokines that may be present in the culture medium suitable for expanding Treg include, but are not limited to, IL-15, IL-12, IL-4, IL-7, IL-2, IFNγ, IL-34, and proinflammatory cytokines (e.g., IL-1 (especially IL-1β), IL-6, and TNFα). In some embodiments, the culture medium suitable for expanding Treg includes a certain amount of interleukin-2 (IL-2) and / or a certain amount of interleukin-15 (IL-15). In some embodiments, the culture medium suitable for expanding Treg includes a certain amount of interleukin-2 (IL-2) and a certain amount of interleukin-15 (IL-15). Typically, IL-2 is added to the culture medium of the present invention at a concentration 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 of 1 to 1000 U / ml, preferably 10 to 500 U / ml, more preferably 25 U / ml.

[0354] The present invention further relates to a method for expanding human Treg cells, comprising the following steps:

[0355] (a) culturing a human monocyte population with a culture medium containing a certain amount of an agonist of the present invention to obtain an immunosuppressive macrophage population;

[0356] (b) co-culturing the Treg population and the immunosuppressive macrophage population obtained in step (a) using a culture medium suitable for expanding the human Treg cell population; and

[0357] (c) optionally, isolating the human Treg cell population obtained in step (b).

[0358] In some embodiments, Treg cells or monocytes can be obtained from iPSCs (induced pluripotent stem cells).

[0359] In some embodiments, Treg colonies are allogeneic with immunosuppressive macrophages. Therefore, Treg can be separated from transplant donors, and immunosuppressive macrophages can be separated from recipients. Alternatively, Treg can be separated from patients with autoimmune diseases or allergies, or patients who need or wait for organ transplants, or bone marrow donors (for example, for the treatment of GVHD), or healthy individuals. In some embodiments, Treg is syngeneic with immunosuppressive macrophages.

[0360] In some embodiments, the Treg population is CD4 + Foxp3 + Treg and / or CD8+ Foxp3 + In some embodiments, the Treg population is CD4 + CD45RC 低 Treg and / or CD8 + CD45RC 低 Treg population.

[0361] Typically, the culture of Treg should be carried out for at least 12 days, such as 12 days to no more than 6-8 weeks, preferably 15 days. In some embodiments, PBMCs are cultured with the target culture medium for 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days. In some embodiments, PBMCs are cultured with the target culture medium for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10 weeks or longer.

[0362] In some embodiments, cytokines, preferably IL-2 and / or IL-15, are added to the culture medium on day 0 of culturing Treg. 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 day 5, 6, 7 or 8 of culturing Treg. 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 end of the culture.

[0363] In some embodiments of the methods of the present invention, antibody anti-CD3 and / or antibody anti-CD8 is added to the culture medium on day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and / or 20 of culturing PBMCs or Tregs, preferably on day 0 and / or day 11, 12, 13, 14 and / or 15.

[0364] 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.

[0365] Another object of the present invention relates to a pharmaceutical composition comprising a mutant IL-34 polypeptide of the present invention and a pharmaceutically acceptable excipient, and optionally a sustained-release matrix, such as a biodegradable polymer.

[0366] In the pharmaceutical composition for oral, sublingual, subcutaneous, intramuscular, intravenous, percutaneous, local or rectal administration of the present invention, active ingredient, alone or in combination with another active ingredient, can be administered to animals and humans in unit administration form as a mixture supported by conventional drugs. Suitable unit administration forms include oral route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous (subcutaneous), transdermal (transdermal), local, intraperitoneal, intramuscular, intravenous, subdermal (subdermal), transdermal, intrathecal and intranasal administration forms and rectal administration forms. Generally, pharmaceutical compositions contain pharmaceutically acceptable vehicles for preparations that can be injected. These can be especially isotonic and sterile saline solutions (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts), or dry, especially freeze-dried compositions, which can constitute injectable solutions when adding sterile water or saline (according to the situation). Pharmaceutical forms suitable for injection purposes include sterile aqueous solutions or dispersions; preparations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must have fluidity to the extent that it is easy to inject. It must be stable under the conditions of preparation and storage, and must prevent the contamination of microorganisms such as bacteria and fungi. Solutions containing the compounds of the present invention as free bases or pharmacologically acceptable salts can be prepared in water with a surfactant such as hydroxypropylcellulose in appropriate mixing. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0367] The mutant IL-34 polypeptide can be formulated as a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein) and are formed using inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed using free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0368] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a suitable mixture thereof and vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using a surfactant. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. The extended absorption of the injectable composition can be achieved by using a delayed absorption agent in the composition, such as aluminum monostearate and gelatin.

[0369] Sterile injectable solutions are prepared by introducing the required amount of active polypeptide into a suitable solvent together with several other ingredients listed above (as required), followed by filtration sterilization. Typically, dispersions are prepared by introducing various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients (among those listed above). In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce powders of the active ingredient and any other desired ingredients from previously sterile filtered solutions.

[0370] When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, such as the injectable solution type described above, but drug release capsules and the like may also be used.

[0371] For example, for parenteral administration in the aqueous solution, if necessary, the solution should be suitably buffered, and the liquid diluent is first isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, in view of the disclosure, the sterile aqueous medium that can be used is known to those skilled in the art. For example, a dosage can be dissolved in the isotonic NaCl solution of 1ml, and then added to the 1000ml subcutaneous injection or injected into the infusion site of suggestion. According to the situation of the treated subject, some changes in dosage will occur. In any case, the person responsible for using will determine the appropriate dose for individual subjects.

[0372] The mutant IL-34 polypeptide can be formulated in a therapeutic mixture to contain about 0.0001 to 1.0 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 or even about 10 mg per dose. Multiple doses can also be administered.

[0373] The following items are also disclosed in the specification:

[0374] Item 1: A mutant IL-34 polypeptide (P1) having an amino acid sequence ranging from an asparagine (N) residue at position 21 to a proline (P) residue at position 242 in SEQ ID NO: 1, wherein the histidine (H) residue at position 56 and the glycine (G) residue at position 112 are both substituted by a cysteine ​​(C) residue.

[0375] Item 2: The mutated IL-34 polypeptide (P1) as described in Item 1, further characterized by at least one mutation selected from the group consisting of:

[0376] - The threonine (T) residue at position 36 is substituted by a tyrosine (Y) residue, a tryptophan (W) residue, a proline (P) residue, a phenylalanine (F) residue, an arginine (R) residue, a histidine (H) residue, an asparagine (N) residue, a cysteine ​​(C) residue, an isoleucine (I) residue, a proline (P) residue, or a leucine (L) residue

[0377] - The serine (S) residue at position 100 is replaced by an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue

[0378] - The threonine (T) residue at position 124 is replaced by a phenylalanine (F) residue or a tryptophan (W) residue

[0379] - The asparagine (N) residue at position 128 is replaced by a tyrosine (Y) or phenylalanine (F) residue

[0380] - The glutamine (Q) residue at position 131 is substituted by 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

[0381] - The serine (S) residue at position 147 is replaced by a glutamic acid (E) residue or an aspartic acid (D) residue

[0382] - the asparagine (N) residue at position 150 is substituted by a glutamic acid (E) residue or an aspartic acid (D) residue, and

[0383] - The leucine (L) residue at position 186 is substituted by an arginine (R) residue or a phenylalanine (F) residue.

[0384] Item 3: A mutant IL34 polypeptide (P2) having an amino acid sequence ranging from an asparagine (N) residue at position 21 to a proline (P) residue at position 242 in SEQ ID NO: 1, wherein the proline (P) residue at position 59 is substituted by 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).

[0385] Item 4: The mutated IL34 polypeptide (P2) as described in Item 3, wherein the proline (P) residue at position 59 is substituted by a lysine (K) residue or an arginine (R) residue.

[0386] Item 5: The mutated IL-34 polypeptide (P2) as described in Item 2, further characterized by at least one mutation selected from the group consisting of:

[0387] - The threonine (T) residue at position 36 is substituted by a tyrosine (Y) residue, a tryptophan (W) residue, a proline (P) residue, a phenylalanine (F) residue, an arginine (R) residue, a histidine (H) residue, an asparagine (N) residue, a cysteine ​​(C) residue, an isoleucine (I) residue, a proline (P) residue, or a leucine (L) residue

[0388] - The serine (S) residue at position 100 is replaced by an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue

[0389] - The threonine (T) residue at position 124 is replaced by a phenylalanine (F) residue or a tryptophan (W) residue

[0390] - The asparagine (N) residue at position 128 is replaced by a tyrosine (Y) or phenylalanine (F) residue

[0391] - The glutamine (Q) residue at position 131 is substituted by 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

[0392] - The serine (S) residue at position 147 is replaced by a glutamic acid (E) residue or an aspartic acid (D) residue

[0393] - the asparagine (N) residue at position 150 is substituted by a glutamic acid (E) residue or an aspartic acid (D) residue, and

[0394] - The leucine (L) residue at position 186 is substituted by an arginine (R) residue or a phenylalanine (F) residue.

[0395] Item 6: A mutant IL34 polypeptide (P3) having an amino acid sequence ranging from an asparagine (N) residue at position 21 to a proline (P) residue at position 242 in SEQ ID NO: 1, comprising at least one mutation selected from the group consisting of:

[0396] - The threonine (T) residue at position 36 is substituted by a tyrosine (Y) residue, a tryptophan (W) residue, a proline (P) residue, a phenylalanine (F) residue, an arginine (R) residue, a histidine (H) residue, an asparagine (N) residue, a cysteine ​​(C) residue, an isoleucine (I) residue, a proline (P) residue, or a leucine (L) residue

[0397] - The serine (S) residue at position 100 is replaced by an aspartic acid (D) residue, a phenylalanine (F) residue, a glutamic acid (E) residue, or a tryptophan (W) residue

[0398] - The threonine (T) residue at position 124 is replaced by a phenylalanine (F) residue or a tryptophan (W) residue

[0399] - The asparagine (N) residue at position 128 is replaced by a tyrosine (Y) or phenylalanine (F) residue

[0400] - The glutamine (Q) residue at position 131 is substituted by 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

[0401] - The serine (S) residue at position 147 is replaced by a glutamic acid (E) residue or an aspartic acid (D) residue

[0402] - the asparagine (N) residue at position 150 is substituted by a glutamic acid (E) residue or an aspartic acid (D) residue, and

[0403] - The leucine (L) residue at position 186 is substituted by an arginine (R) residue or a phenylalanine (F) residue.

[0404] Item 7: The mutant IL34 polypeptide as described in Item 1, 3 or 6, wherein the glutamine (Q) residue at position 81 is deleted.

[0405] Item 8: A fusion protein consisting of the mutated IL-34 polypeptide described in Item 1, 3 or 6 and a heterologous polypeptide fused thereto.

[0406] Item 9: The fusion protein as described in Item 8, wherein the mutant IL-34 polypeptide is fused to the Fc region.

[0407] Item 10: An isolated, synthetic or recombinant nucleic acid encoding the mutant IL-34 polypeptide described in Item 1, 3 or 6.

[0408] Item 11: A vector comprising the nucleic acid described in Item 10.

[0409] Item 12: A host cell that has been transfected, infected or transformed with the nucleic acid described in Item 10 and / or the vector described in Item 11.

[0410] Item 13: Use of the mutated IL-34 polypeptide described in Item 1 or 6 as an agonist.

[0411] Item 14: Use of the mutated IL-34 polypeptide described in Item 3 as an antagonist.

[0412] Item 15: The mutated IL-34 polypeptide described in Item 1, 3 or 6 is used as a medicament.

[0413] Item 16: A method of inducing immune tolerance in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the agonist described in Item 13.

[0414] Item 17: A method for preventing or reducing transplant rejection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the agonist described in Item 13.

[0415] 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 the agonist described in Item 13.

[0416] Item 19: The method of Item 16, 17 or 18, wherein the agonist is administered to the patient in combination with an immunosuppressive drug.

[0417] 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.

[0418] Item 21: A method as described in Item 20, wherein the antagonist is administered to the patient in combination with chemotherapy or immunotherapy, particularly an immune checkpoint inhibitor.

[0419] Item 22: An in vitro / ex vivo method for obtaining an immunosuppressive macrophage population, comprising culturing the monocyte population with a culture medium containing a certain amount of the agonist described in Item 13.

[0420] Item 23: A pharmaceutical composition comprising the mutant IL-34 polypeptide described in Item 1, 3 or 6 and a pharmaceutically acceptable excipient, and optionally a sustained release matrix, such as a biodegradable polymer.

[0421] The present invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way. Sequence Listing <110> French National Institute of Health and Medical Research (INSERM (Institut National de la Santéet de la Recherche Médicale)) University of Nantes <120> Mutated interleukin-34 (IL-34) polypeptides and their use in therapy <130> CV-1370 / PCT <150> EP19305046.5 <151> 2019-01-15 <160> 1 <170> BiSSAP 1.3.6 <210> 1 <211> 242 <212> PRT <213> Homo sapiens <400> 1 Met Pro Arg Gly Phe Thr Trp Leu Arg Tyr Leu Gly Ile Phe Leu Gly 1 5 10 15 Val Ala Leu Gly Asn Glu Pro Leu Glu Met Trp Pro Leu Thr Gln Asn 20 25 30 Glu Glu Cys Thr Val Thr Gly Phe Leu Arg Asp Lys Leu Gln Tyr Arg 35 40 45 Ser Arg Leu Gln Tyr Met Lys His Tyr Phe Pro Ile Asn Tyr Lys Ile 50 55 60 Ser Val Pro Tyr Glu Gly Val Phe Arg Ile Ala Asn Val Thr Arg Leu 65 70 75 80 Gln Arg Ala Gln Val Ser Glu Arg Glu Leu Arg Tyr Leu Trp Val Leu 85 90 95 Val Ser Leu Ser Ala Thr Glu Ser Val Gln Asp Val Leu Leu Glu Gly 100 105 110 His Pro Ser Trp Lys Tyr Leu Gln Glu Val Glu Thr Leu Leu Leu Asn 115 120 125 Val Gln Gln Gly Leu Thr Asp Val Glu Val Ser Pro Lys Val Glu Ser 130 135 140 Val Leu Ser Leu Leu Asn Ala Pro Gly Pro Asn Leu Lys Leu Val Arg 145 150 155 160 Pro Lys Ala Leu Leu Asp Asn Cys Phe Arg Val Met Glu Leu Leu Tyr 165 170 175 Cys Ser Cys Cys Lys Gln Ser Ser Val Leu Asn Trp Gln Asp Cys Glu 180 185 190 Val Pro Ser Pro Gln Ser Cys Ser Pro Glu Pro Ser Leu Gln Tyr Ala 195 200 205 Ala Thr Gln Leu Tyr Pro Pro Pro Pro Trp Ser Pro Ser Ser Pro Pro 210 215 220 His Ser Thr Gly Ser Val Arg Pro Val Arg Ala Gln Gly Glu Gly Leu 225 230 235 240 Leu Pro

[0422] BRIEF DESCRIPTION OF THE DRAWINGS

[0423] Figure 1 : Free energy of mutation. Expected effect of single point mutations of residues H56, G112, P59, L109, I60 and V108 to cysteine ​​at pH 7.5.

[0424] Figure 2 : Free energy of mutation. Expected effect of mutation of residue P59 among all amino acids at pH 7.5.

[0425] Figure 3: Free energy of mutation. Expected effects of mutations of several residues at the IL-34 / CSF-1R interface at pH 7.5 for all amino acids ( 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; Fig. 3I , N128; Figure 3J , Q131; Figure 3K , S147; Figure 3L , N150; Figure 3M , L186 and Figure 3N , N187).

[0426] Figure 4: Interaction and kinetics of IL-34 mutant proteins with CF1R. SPR experiments were performed on Biacore T200 (GE Healthcare) for 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; Fig. 4I , P59K; Figure 4J , N150; Figure 4K , G112C / H56C and Figure 4L , T124F).

[0427] Figure 5: Binding parameters of different IL-34 muteins. The affinity (KD), kinetic parameters (ka and kd) and resonance maximum (Rmax) of IL-34 for CSF-1R were determined using a series of protein dilutions in the "single cycle kinetics" (SCK) model.

[0428] Figure 6: Differential effects of IL-34 mutants on the viability of cultured monocytes isolated from total human PBMCs. Fig. 6A , Percentage of viable cells after 3 days of culture with wild-type IL-34 (IL-34WT), each IL-34 mutant, or medium control. Figure 6B , Absolute number of viable cells, expressed as the percentage of viable cells after 3 days of culture with WT IL-34, each IL-34 mutant, or medium control, relative to the percentage of viable cells obtained under WT IL-34 conditions (set to 100% and indicated by a dotted line). Figure 6C , CD14 in the monocyte population in a dose-response curve (from 1.5 to 200 ng / ml) of IL-34WT, IL-34 mutants, or medium control (no cytokine as negative control). + Cell percentage.

[0429] Figure 7: CD14 isolated from total human PBMCs + Differential phosphorylation of Akt and ERK1 / 2 in response to WT IL-34 or IL-34 mutants in monocytes. Fig. 7A -B, Akt ( Fig. 7A ) and ERK1 / 2( Figure 7B ) Flow cytometric analysis of phosphorylation. The results are shown as the mean fluorescence intensity of pAkt-AF647 and pERK1 / 2-AF647 compared with T0.

[0430] Figure 8: Prediction of free energies (ΔΔGmut) for mutations of certain residues of IL-34 located within the interface with CSF-1R. ΔΔGmut was calculated for substitution of residue Thr36 (T36) of IL-34 by Tyr (Y) and substitution of Ser100 (S100) and Gln131 (Q131) residues of IL-34 by Phe (F) at pH 7.4. The input data are 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 were also predicted.

[0431] Figure 9: Interaction, kinetics and binding parameters of WT IL-34 and mutant S100F with CD138. Fig.9A -B, SPR experiments were performed on Biacore T200 (GE Healthcare) for WT IL-34 ( Fig.9A ) and mutant S100F ( Fig. 9B )conduct. Fig. 9C , the affinity parameters (KD) of WT IL-34 and mutant S100F for CD138 were determined using a series of protein dilutions in a “single cycle kinetic” (SCK) model.

[0432] Figure 10: Interaction, kinetics and binding parameters of WT IL-34 and mutant S100F with PTP-ζ. Fig. 10A -B, SPR experiments were performed on Biacore T200 (GE Healthcare) for WT IL-34 ( Fig. 10A ) and mutant S100F ( Fig. 10B )conduct. Fig. 10C , the affinity parameters (KD) of WT IL-34 and mutant S100F for CD138 were determined using a series of protein dilutions in a “single cycle kinetic” (SCK) model.

[0433] Example

[0434] Example 1

[0435] method

[0436] The mutation energy (the difference between the binding free energy of the mutant and the wild type) was calculated by using the method developed by Spassov and Yan (Spassov and Yan, Proteins 2013; 81: 704-714) in the "Calculate Mutation Energy (Binding)" procedure implemented in Discovery Studio (DS) (Dassault Systèmes BIOVIA software, San Diego, CA) to predict the effect of mutating several IL-34 residues to cysteine ​​on the stability of the dimeric form of IL-34.

[0437] Briefly, the X-ray crystal structure of the dimer form of IL-34 (Protein Data Bank code 4DKC) was used. The structure was first prepared using the Prepare Protein tool in DS2017 and the CHARMm Polar H force field to add hydrogen atoms, remove water molecules, and insert missing atoms or loop regions. The mutation energies of residues located at the IL-34 dimer interface and the IL-34 / CSF-1R interface were then calculated. If the mutation energy was less than -0.5 kcal / mol, the effect of the mutation was defined as stabilizing, if the mutation energy was between -0.5 and 0.5 kcal / mol, it was defined as neutral, and if the mutation energy was greater than 0.5 kcal / mol, it was defined as destabilizing.

[0438] result

[0439] Stabilization of IL-34 dimers

[0440] Mutational energies were calculated for the following mutations: H56C, G112C, P59C, L109C, I60C, and V108C.

[0441] Mutations H56C, G112C, and V108C are predicted to be neutral, whereas mutations P59C, L109C, and I60C are predicted to be destabilizing ( Figure 1 The double mutation H56C / G112C is not expected to adversely affect the stability of IL-34 dimers, but may lead to the formation of disulfide bonds that stabilize the dimer ( Figure 1 ).

[0442] Destabilization of IL-34 dimers

[0443] Calculate the mutation energy of P59. Mutation of P59K and P59R will prevent the formation of IL-34 dimers ( Figure 2 ).

[0444] Stabilization of the IL-34 / CSF-1R interface

[0445] The following mutations would potentially stabilize the complex IL-34 / CSF-1R (Figure 3):

[0446] -T36Y, T36F, or T36W

[0447] -S100D or S100F

[0448] -T124F or T124W

[0449] -N128Y or N128F

[0450] -Q131R, Q131F

[0451] -S147E

[0452] -N150E

[0453] -L186R

[0454] Example 2

[0455] PBMCs from the blood of healthy volunteers (HV) were isolated by Ficoll gradient (CMSMSL01-01; Eurobio), and T, B, and NK cells were depleted by using magnetic beads due to anti-CD3 (clone OKT3), anti-CD19 (clone HIB19; BD Biosciences), and anti-CD56 (clone MY31; BD Biosciences) antibodies. Monocytes were then sorted based on forward scatter (FSC), side scatter (SSC) morphological parameters and positive staining for CD14 (clone M5E2; BD Biosciences). Freshly sorted monocytes were washed and lysed at 1×10 6 / ml was inoculated into complete medium (RPMI1640, 2mM glutamine, 100U / ml penicillin, 0.1mg / ml streptomycin, 10% FCS and supplemented with a certain amount of polypeptides (P1), (P2) and (P3) of the present invention). On the second day, the cells were stimulated with 100ng / mL LPS (L4391; Sigma-Aldrich) for 24 hours or not stimulated. 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 using a FACSCanto II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software. Cells were first gated for cell morphology; dead cells were excluded by selecting DAPI live cells to analyze CD14 + CD16 - and CD14 + CD16 + Expression of different markers in monocytes.

[0456] Example 3

[0457] For cell signaling experiments, the cell lines were serum starved (0.1% FBS) for 48 hours and treated with polypeptides (P1), (P2) and (P3) (10 ng / ml) of the present invention for 20 minutes, and samples were harvested at this time point. The samples were then analyzed by Western blotting using Tris / glycine buffer and transferred to hybond-P membranes (Amersham, GE, Fairfield, CT, USA). All protein samples were quantified using the BCA assay to ensure similar protein amounts in all lanes of the western gel (Thermo Scientific Inc.). The antibodies used in the Western blot experiments were CSF-1R (sc692, 1:1000, Santa Cruz, Santa Cruz, CA, USA), phosphotyrosine (sc-508, 1:1000, Santa Cruz,), phosphorylated 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 were incubated with 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 for 1 hour at room temperature in 5% milk TTBS. The signal was detected using Super Signal West Pico chemiluminescent substrate (Thermo Scientific Inc., Waltham, MA, USA).

[0458] Example 4

[0459] Materials and Methods

[0460] Reagents and Materials

[0461] S series CM5 sensor chips and HBS-P buffer (10 mM HEPES buffer containing 2.7 mM KCl, 150 mM NaCl and 0.05% surfactant P20, pH 7.4), 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, for analysis) were purchased from GE Healthcare Life Science (Uppsala, Sweden).

[0462] Surface plasmon resonance (SPR) analysis

[0463] SPR experiments were performed on a Biacore T200 (GE Healthcare) at 25°C. The HBS-P buffer was filtered through a 0.45 μm membrane filter and degassed before use. First, the recombinant human CSF-1 receptor (CSF-1R, reference number K10161-H08H) from Sino Biological was immobilized on the chip surface by amine coupling. In brief, the recombinant CSF-1 receptor was diluted to 20 μg / mL in a pH 5.0, 10 mM sodium acetate solution. Using a standard amine coupling kit (Amine Coupling Kit), the diluted CSF-1R was quickly covalently fixed to the flow cell of a CM5 sensor chip through primary amine groups. The IL-34 mutant protein was then analyzed in a "single cycle kinetics" (SCK) model on the chip with fixed CSF-1R.

[0464] Interaction and dynamics of IL-34 mutant proteins with CSF-1R

[0465] The affinity (KD), kinetic parameters (ka and kd) and maximum resonance (Rmax) of IL-34 for CSF-1R were determined by using a series of protein dilutions in the "single cycle kinetics" (SCK) model. The IL-34 mutant protein as analyte was diluted in HBS-P buffer to a concentration range of 25nM to 400nM, which was passed over the immobilized CSF-1R and the response units (RU) obtained were recorded. The flow rate was 30μL / min, with 120s for binding and 600s for dissociation. The sensor chip surface was then regenerated with 10mM NaOH for 30 seconds. The dissociation equilibrium constant KD, the kinetic parameters kd and ka and Rmax were determined by direct curve fitting of the sensorgrams to the Langmuir 1:1 interaction model.

[0466] result

[0467] Bicore analysis showed that multiple mutants of IL-34 had higher affinity for CSF-1R compared with WT IL-34, especially mutants S100F, T36Y, Q131F, T36F, T36W, Q131R, and S100D, suggesting that these mutants would be more effective than WT IL-34 ( Figure 4A -H and Figure 5 In contrast, the P59K, N150E, G112 / H56C, and T124F mutants of IL-34 had reduced or comparable affinity for CSF-1R compared with WT IL-34 ( Fig. 4I -L and Figure 5 ).

[0468] Example 5

[0469] Materials and Methods

[0470] Buffy coat was obtained from Etablissement du Sang (EFS, Nantes, France). PBMCs were obtained by Ficoll gradient and then monocytes were isolated by magnetic bead separation (Classical Monocyte Isolation Kit, Miltenyi Biotec). CD14 + Monocytes are 10 6 Cells / ml were cultured in flat-bottom 96-well plates in complete medium (RPMI1640, 10% FBS, 1% penicillin-streptomycin, 1% glutamine, 1% AANE, 1% Hepes, 1% sodium pyruvate) containing WT IL-34 or mutants: S100F, S100D, T36W, T36Y, T36F, Q131F, Q131R, N150E, C112 / C56, P59K, T124F and IL-34Fc ( Fig. 6A and Figure 6B ) and ranged from 1.5 to 200 ng / mL ( Figure 6C ). Medium without added cytokines (no cytokine) was used as negative control. On day 3, cells were collected by washing in PBS 2% FBS 2mM EDTA and further used for viability staining and phenotypic analysis by flow cytometry. Counting beads (123counteBeads TM Counting Beads, ThermoFisher Scientific) to analyze the absolute number of cells.

[0471] result

[0472] Mutants T36F, S100F, Q131F, T36W, T36Y, N150E, and IL-34-Fc were as effective as WT IL34 in inducing monocyte / macrophage survival ( Fig. 6A ). S100F, T36Y, Q131F and IL-34-Fc showed stronger ability to maintain cell survival ( Figure 6B ), and is particularly noteworthy.

[0473] Interestingly, Q131R and S100D (which have stronger binding affinity for CSF-1R compared to WT IL34) inhibited macrophage survival ( Fig. 6A In addition, analysis of monocyte CD14 expression in the presence of decreasing concentrations of IL-34 mutants showed that the S100F, Q131F, and T36Y mutants were more effective at differentiating CD14+ monocytes at lower concentrations compared to WT IL-34 at the same concentration ( Figure 6C ).

[0474] Example 6

[0475] Materials and Methods

[0476] To analyze Akt and ERK1 / 2 phosphorylation (pAkt and pERK1 / 2, respectively), freshly sorted CD14 + Monocytes were cultured in FBS-free medium (RPMI 1640, 1% penicillin-streptomycin, 1% glutamine, 1% AANE, 1% Hepes, 1% sodium pyruvate) containing 100 ng / ml of WT IL-34 or different S100F, T36W, T36Y, T36F, Q131F, Q131R, N150E and IL-34-Fc mutants in low attachment 96-well plates for 1, 3 and 5 minutes. Analysis was performed by flow cytometry using phospho-Akt (Ser473) and phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) primary antibodies (reference numbers 4060 and 4370, Cell Signaling), and goat anti-rabbit IgG (H+L)-AF647 (reference number A21245, Life Technologies) secondary antibodies, following the BD Biosciences Phosflow protocol (using BD Cytofix Fixation buffer and BD Phosflow Perm Buffer III, BD Biosciences).

[0477] result

[0478] Flow cytometric analysis of pAkt and pERK1 / 2 was then performed to functionally assess the signaling induced by the binding of each IL-34 mutant to CSF-1R. Flow cytometric analysis of pAkt showed increased phosphorylation of the T36F, IL-34-Fc, S100F, Q131F, T36W, and T36Y mutants compared to WT IL-34 after 3 minutes, and this was still the case for most mutants after 5 minutes ( Fig. 7A ). In contrast, the N150E mutant was less effective than WT IL-34 in inducing Akt phosphorylation after 3 minutes and equal to WT IL-34 after 5 minutes. Finally, the Q131R mutant of IL-34 did not induce any Akt phosphorylation after 3 or 5 minutes. Flow cytometric analysis of pERK1 / 2 showed increased phosphorylation of IL-34-Fc, S100F, Q131F, and T36Y mutants compared to wt IL-34 after 3 minutes, and Q131F and T36Y after 5 minutes ( Figure 7B ). Moreover, the T36W and T36F mutants showed the same kinetics as WT IL-34. In contrast, the N150E mutant was again less potent than WT IL-34 after 3 and 5 minutes. Furthermore, as already observed for pAkt, the Q131R mutant did not induce any ERK1 / 2 phosphorylation ( Figure 7B ).

[0479] Example 7

[0480] Materials and Methods

[0481] The ΔΔGmut at pH 7.4 was calculated for the substitution of residue Thr36 (T36) of IL-34 by Tyr (Y) and the substitution of residues Ser100 (S100) and Gln131 of IL-34 by Phe (F) using the Spassov and Yan method (Spassov and Yann, 2013) implemented in Discovery Studio (Dassault Systèmes BIOVIA Release 2017, San Diego) in the "Calculate Mutation Energy (Binding)" protocol. The input data were the atomic coordinates of the IL-34 / CSF-1R complex (pdb code 4DKD).

[0482] result

[0483] Consistent with the results obtained in the previous example, mutations T36Y, S100F and Q131F were stabilizing by computer simulation (ΔΔGmut<-0.5) ( Figure 8). It was therefore hypothesized that combining two or three of these mutations might be even more stabilizing than any of them alone. Interestingly, the stabilizing effect of the double mutations was predicted to be the same as or even slightly higher than that of the simple mutations. The stabilizing effect of the combination of triple mutations was predicted to be even higher.

[0484] Example 8

[0485] Materials and Methods

[0486] Reagents and Materials

[0487] S series CM5 sensor chips and HBS-P buffer (10 mM HEPES buffer containing 2.7 mM KCl, 150 mM NaCl and 0.05% surfactant P20, pH 7.4), 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, for analysis) were purchased from GE Healthcare Life Science (Uppsala, Sweden).

[0488] Surface plasmon resonance (SPR) analysis

[0489] SPR experiments were performed on Biacore T200 (GE Healthcare) at 25 ° C. HBS-P buffer was filtered through a 0.45 μm membrane filter and degassed before use. First, recombinant human CD138 (SDC1, syndecan-1, reference number 11429-H08H) from Sino Biological and PTPz (reference number OPCA02772) from Aviva System Biologic were fixed on the chip surface by amine coupling. In brief, recombinant CD138 or PTP-ζ were diluted to 20 μg / mL in 10 mM sodium acetate solution, pH 5.0. Using a standard amine coupling kit (Amine Coupling Kit), diluted CD138 or PTP-ζ were quickly covalently fixed to the flow cell of a CM5 sensor chip by primary amine groups. Then IL-34 mutant proteins were analyzed on a chip fixed with CD138 or PTP-ζ in a "single cycle kinetics" (SCK) model.

[0490] Interaction and kinetics of IL-34 mutant proteins with CD138 or PTP-ζ

[0491] The maximum resonance (Rmax) of IL-34 relative to CD138 or PTP-ζ was determined using a series of protein dilutions in the "single cycle kinetics" (SCK) model. The IL-34 mutant protein as the analyte was diluted in HBS-P buffer to a concentration of 25nM to 400nM, which flowed through the fixed CD138 or PTP-ζ, and the response unit (RU) obtained was recorded. The flow rate was 30μL / min, with 120s of binding and 600s of dissociation. The sensor chip surface was then regenerated with 10mM NaOH for 30 seconds. Rmax was determined by directly curve fitting the sensorgram to the Langmuir 1:1 interaction model.

[0492] result

[0493] Bicore analysis showed that the S100F mutant of IL-34 had a stronger effect on CD138 ( Fig.9A -C) and PTP-ζ( Fig. 10A -C) has a higher affinity, indicating that this mutant is more potent than WT IL-34.

[0494] References

[0495] In this application, several references describe prior art related to the present invention. The disclosures of these references are hereby incorporated into the present disclosure by reference.

Claims

1. A mutant IL34 polypeptide consisting of an amino acid sequence selected from the group consisting 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, into which a mutation selected from the group consisting of: - the serine residue at position 100 is substituted by a phenylalanine residue (S100F), - the threonine residue at position 36 is substituted by a tyrosine residue (T36Y), - the glutamine residue at position 131 is substituted by a phenylalanine residue (Q131F), - the threonine residue at position 36 is substituted by a tryptophan residue (T36W), or - The threonine residue at position 36 is substituted by a phenylalanine residue (T36F).

2. A mutant IL34 polypeptide consisting of an amino acid sequence selected from the group consisting 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, into which a mutation selected from the group consisting of: - the serine residue at position 100 is substituted by an aspartic acid residue (S100D), or - The glutamine residue at position 131 is substituted by an arginine residue (Q131R).

3. A fusion protein consisting of the mutated IL-34 polypeptide according to any one of claims 1 to 2 and a heterologous polypeptide fused thereto.

4. The fusion protein of claim 3, wherein the mutant IL-34 polypeptide is fused to an Fc region.

5. An isolated, synthetic or recombinant nucleic acid encoding the mutant IL-34 polypeptide according to any one of claims 1 to 2 or encoding a fusion protein consisting of the mutant IL-34 polypeptide and a heterologous polypeptide fused thereto. A vector comprising the nucleic acid according to claim 5.

7. A host cell comprising the nucleic acid according to claim 5 and / or a vector comprising the nucleic acid.

8. The mutant IL-34 polypeptide according to claim 1, a fusion protein consisting of the mutant IL-34 polypeptide and a heterologous polypeptide fused thereto, a nucleic acid encoding the mutant IL-34 polypeptide or the fusion protein, or a vector comprising the nucleic acid, 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 in vitro.

9. Use of the mutant IL-34 polypeptide according to claim 1, a fusion protein consisting of the mutant IL-34 polypeptide and a heterologous polypeptide fused thereto, a nucleic acid encoding the mutant IL-34 polypeptide or the fusion protein, or a vector comprising the nucleic acid in the preparation of a medicament for preventing or reducing transplant rejection in a patient in need thereof.

10. Use of the mutant IL-34 polypeptide according to claim 1, a fusion protein consisting of the mutant IL-34 polypeptide and a heterologous polypeptide fused thereto, a nucleic acid encoding the mutant IL-34 polypeptide or the fusion protein, or a vector comprising the nucleic acid in the preparation of a medicament for preventing or treating a neurodegenerative disease, an autoimmune disease or an allergy in a patient in need thereof.

11. Use of the mutant IL-34 polypeptide according to claim 2, a fusion protein consisting of the mutant IL-34 polypeptide and a heterologous polypeptide fused thereto, a nucleic acid encoding the mutant IL-34 polypeptide or the fusion protein, or a vector comprising the nucleic acid as an antagonist of a ligand of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138 in vitro.

12. Use of the mutant IL-34 polypeptide according to claim 2, a fusion protein consisting of the mutant IL-34 polypeptide and a heterologous polypeptide fused thereto, a nucleic acid encoding the mutant IL-34 polypeptide or the fusion protein, or a vector comprising the nucleic acid in the preparation of a medicament for treating cancer or a bone disease involving abnormal proliferation in a patient in need thereof.

13. An in vitro / ex vivo method for obtaining an immunosuppressive macrophage population, comprising culturing a monocyte population using a culture medium comprising a mutant IL-34 polypeptide according to claim 1.

14. A pharmaceutical composition or drug comprising the mutant IL-34 polypeptide according to any one of claims 1 to 2, a fusion protein consisting of the mutant IL-34 polypeptide and a heterologous polypeptide fused thereto, a nucleic acid encoding the mutant IL-34 polypeptide or the fusion protein or a vector comprising the nucleic acid, and at least one pharmaceutically acceptable excipient.

15. The pharmaceutical composition or medicament of claim 14, further comprising at least one sustained release matrix.

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