Compounds and Methods Targeting Interleukin-34

Abstract

The present invention relates to IL-34 antibodies, compositions comprising same, and methods of using the antibodies and / or compositions to treat immune-mediated diseases such as neurodegenerative diseases, eg, Alzheimer's disease or tauopathy diseases.

Description

Detailed Description of the Invention

[0001] The present invention relates to compounds, pharmaceutical compositions, and methods comprising antibodies against human interleukin-34 (IL-34), which are expected to be useful in the field of neuroinflammation and acute or chronic inflammatory diseases. In particular, embodiments are expected to be useful for the treatment and / or diagnostic use related to Alzheimer's disease and other tauopathies.

[0002] Alzheimer's disease (AD), the leading cause of dementia, affects 1% of the population aged 65 to 69 and increases to 40% - 50% in those over 95 years old. Patients with AD exhibit distinct clinical symptoms including cognitive impairment and memory deficits. In these patients, the presence of AD is confirmed by the severe senile plaque burden and neurofibrillary changes (NFTs) seen in the cerebral cortex upon postmortem histopathological examination. Mature senile plaques are composed of extracellular β-amyloid peptides derived from enzymatic processing of amyloid precursor protein and intracellular neurofibrillary changes (NFTs) derived from filaments of hyperphosphorylated tau protein. Aggregates of hyperphosphorylated tau, such as neurofibrillary changes, are associated with the degree of cognitive impairment in Alzheimer's disease. In AD and various other tauopathies, tau aggregates appear in specific brain regions and patterns related to the risk, onset, and / or progression of the disease, and these regions and patterns are known to those skilled in the art.

[0003] Cytokines regulate normal homeostatic tissue function, and dysregulation of these cytokine networks is associated with pathological states. The central nervous system (CNS), where blood-derived immune cells rarely circulate, appears to be particularly vulnerable to dysregulated cytokine networks. In neurodegenerative diseases, CNS resident cells are major producers of pro-inflammatory cytokines and can contribute to dysregulated cytokine networks and neuroinflammation. Injury to the CNS can involve the recruitment of circulating immune cells that initiate innate immune responses consisting of resident microglia, peripherally-derived monocytes, macrophages, and dendritic cells. The activation state of microglia and macrophages is not strictly pro-inflammatory or anti-inflammatory, but rather can have a variety of functional states. Microglia and / or peripherally-derived monocytes and macrophages may remove necrotic tissue fragments and acquire an anti-inflammatory phenotype that promotes regeneration and homeostasis. Neurological dysfunction or injury can also activate microglia to produce pro-inflammatory cytokines and recruit leukocytes from the bloodstream. In neurodegenerative conditions such as Alzheimer's disease (AD), activation of microglia is frequently seen, reflecting the tissue response to the accumulation of extracellular beta-amyloid plaques and hyperphosphorylated tau aggregates. Neuroinflammation is a key element of neurodegenerative diseases and is characterized by increased production of pro-inflammatory cytokines by CNS cells (Becher, B., Spath, S. & Goverman, J. Cytokine networks in neuroinflammation. Nat Rev Immunol 17, 49-59 (2017)). Neuroinflammation and microgliosis are thought to underlie the mechanisms of neurodegenerative diseases such as plaque accumulation in Alzheimer's disease, and neuronal death and dysfunction in Parkinson's disease and Huntington's disease.

[0004] Microgliosis involves abnormal proliferation and / or hypertrophy of microglia in response to inflammatory signals. Generally, IL-34 acts as a potent and multifaceted cytokine in the regulation of inflammation and immune processes and is an important regulatory cytokine for the proliferation of CNS resident microglia in the homeostasis of normal tissues. IL-34 is expressed by neurons in the cortex, anterior olfactory nucleus, and hippocampus. IL-34 is closely related to colony-stimulating factor 1 (CSF1; also known as M-CSF), and both cytokines bind to the CSF1 receptor. IL-34 is a secreted homodimeric cytokine and acts as one of the two activating ligands for CSF1R, causing autophosphorylation and dimerization of the receptor, followed by activation of multiple signaling pathways (see, for example, the structural basis for the dual recognition of the helical cytokine IL-34 and CSF-1 by CSF-1R, Structure 20, 676-687, and Felix J, De Munck S, Verstraete K, Meuris L, Callewaert N, Elegheert J. et al.). Human and mouse IL-34 polypeptides are disclosed, for example, in U.S. Patent No. 9,770,486. IL-34 is a protein of 242 amino acids (SEQ ID NO: 41) in humans and 235 amino acids (SEQ ID NO: 42) in mice.

[0005] Anti-IL-34 antibodies have been described in the art. For example, WO2016 / 196679 details various anti-IL-34 antibodies and their potential uses. However, to date, no antibody targeting IL-34 has been approved for therapeutic use.

[0006] Accordingly, there remains an unmet need for alternative and / or improved anti-IL-34 antibodies, pharmaceutical compositions thereof, and methods of using them for the treatment of immune-mediated diseases and / or neuropathological disorders in which IL-34 is involved, and / or diseases treatable with anti-IL-34 antibodies, and / or therapeutic and / or diagnostic applications related to Alzheimer's disease. Further, there remains an unmet need for alternative and / or improved anti-IL-34 antibodies having a combination of properties that are particularly advantageous over prior art anti-IL-34 antibodies, considering at least one or more of the following characteristics: 1) desirable binding and dissociation rates, 2) potency in neutralizing human IL-34 to achieve an anti-neuroinflammatory response and in vivo efficacy, 3) sufficient potency as a monotherapy for the treatment and / or prevention of immune-mediated and / or inflammatory disorders, 4) duration of action, 5) sufficient limitation of the induction of unwanted cytokine release, 6) an acceptably low level of immunogenicity (i.e., sufficient non-immunogenicity in humans), 7) avoidance of harmful immunosuppression, and / or 8) desirable in vivo stability, physical and chemical stability, including but not limited to thermostability, solubility, low self-association, and pharmacokinetic properties acceptable for development and / or use in the treatment of inflammatory or neuropathological disorders such as AD. SUMMARY OF THE INVENTION

[0007] Embodiments of the invention provide novel anti-human IL-34 and anti-mouse IL-34 antibodies. According to some embodiments, the invention provides an antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs, HCDR1, HCDR2, and HCDR3, which are selected from the group of CDR combinations provided in Table 1. The sequence identifiers used herein are listed in Table 1 and the sequences are shown in the amino acid and nucleotide sequence listings provided herein. TABLE 1

[0008] Accordingly, embodiments of the present invention also a. an LCVR having the amino acid sequence of SEQ ID NO: 4 and an HCVR having the amino acid sequence of SEQ ID NO: 3, b. an LCVR having the amino acid sequence of SEQ ID NO: 4 and an HCVR having the amino acid sequence of SEQ ID NO: 14, c. an LCVR having the amino acid sequence of SEQ ID NO: 22 and an HCVR having the amino acid sequence of SEQ ID NO: 21, also provide an antibody comprising an LCVR and an HCVR selected from.

[0009] According to other embodiments, the present invention also provides an antibody comprising a combination of the above LCVR and HCVR having a hinge region and an Fc region selected from SEQ ID NO: 51 and SEQ ID NO: 52.

[0010] According to other embodiments, the present invention also a. an LC having the amino acid sequence of SEQ ID NO: 2 and an HC having the amino acid sequence of SEQ ID NO: 1, b. an LC having the amino acid sequence of SEQ ID NO: 2 and an HC having the amino acid sequence of SEQ ID NO: 13, and c. an LC having the amino acid sequence of SEQ ID NO: 20 and an HC having the amino acid sequence of SEQ ID NO: 19, also provide an antibody comprising an LC and an HC selected from the amino acid sequences of or having an amino acid sequence having at least 95% homology with the amino acid sequences thereof.

[0011] As used herein, "Antibody 1" refers to an antibody having the HCDR1 amino acid sequence of SEQ ID NO: 5, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 7, the LCDR1 amino acid sequence of SEQ ID NO: 8, the LCDR2 amino acid sequence of SEQ ID NO: 9, the LCDR3 amino acid sequence of SEQ ID NO: 10, the HCVR amino acid sequence of SEQ ID NO: 3, the LCVR amino acid sequence of SEQ ID NO: 4, the HC amino acid sequence of SEQ ID NO: 1, the LC amino acid sequence of SEQ ID NO: 2, the HC DNA sequence of SEQ ID NO: 11, and the LC DNA sequence of SEQ ID NO: 12. For each of the antibodies whose sequences are shown herein, the framework and CDR sequences are annotated using annotation rules that are consistent with the method of North, et al., J. Mol. Biol. 2011:406:228-256, unless otherwise specified.

[0012] As used herein, "Antibody 2" refers to an antibody having the HCDR1 amino acid sequence of SEQ ID NO: 15, the HCDR2 amino acid sequence of SEQ ID NO: 16, the HCDR3 amino acid sequence of SEQ ID NO: 17, the LCDR1 amino acid sequence of SEQ ID NO: 8, the LCDR2 amino acid sequence of SEQ ID NO: 9, the LCDR3 amino acid sequence of SEQ ID NO: 10, the HCVR amino acid sequence of SEQ ID NO: 14, the LCVR amino acid sequence of SEQ ID NO: 4, the HC amino acid sequence of SEQ ID NO: 13, the LC amino acid sequence of SEQ ID NO: 2, the HC DNA sequence of SEQ ID NO: 18, and the LC DNA sequence of SEQ ID NO: 12. For each of the antibodies whose sequences are shown herein, the framework and CDR sequences are annotated using annotation rules that are consistent with the method of North, et al., J. Mol. Biol. 2011:406:228-256, unless otherwise specified.

[0013] As used herein, "Antibody 3" refers to an antibody having the HCDR1 amino acid sequence of SEQ ID NO: 23, the HCDR2 amino acid sequence of SEQ ID NO: 24, the HCDR3 amino acid sequence of SEQ ID NO: 25, the LCDR1 amino acid sequence of SEQ ID NO: 26, the LCDR2 amino acid sequence of SEQ ID NO: 27, the LCDR3 amino acid sequence of SEQ ID NO: 28, the HCVR amino acid sequence of SEQ ID NO: 21, the LCVR amino acid sequence of SEQ ID NO: 22, the HC amino acid sequence of SEQ ID NO: 19, the LC amino acid sequence of SEQ ID NO: 20, the HC DNA sequence of SEQ ID NO: 29, and the LC DNA sequence of SEQ ID NO: 30. The framework and CDR sequences in each of the antibodies whose sequences are shown herein are annotated using annotation rules that are consistent with the method of North, et al., J. Mol. Biol. 2011:406:228-256, unless otherwise specified.

[0014] The carboxy-terminal portion of each HC defines the constant region that is mainly responsible for effector function. In some embodiments of the present invention, the antibody has one or more modifications in the constant region of each HC that reduce effector function. Preferably, embodiments of the present invention are IgG4 antibodies and thus include an IgG4 Fc region, or an Fc region derived from human IgG4, such as a modified IgG4 Fc region.

[0015] According to some embodiments, modifications and amino acid substitutions in the constant regions of both HCs that reduce effector function are introduced into the IgG4 hinge and Fc regions. Thus, some embodiments have modifications in the constant regions of both HCs that include the amino acid alanine at both residues 230 and 231 (exemplified by the HC of Antibody 1, the HC of Antibody 2, and SEQ ID NO: 52, respectively), further modifications in the constant regions of both HCs that include the amino acid proline at residue 224 and promote stability (exemplified by the HC of Antibody 1, the HC of Antibody 2, and SEQ ID NO: 51, for example), and a deletion of the amino acid lysine at residue 443 (exemplified by the HC of SEQ ID NO: 1).

[0016] The antibodies of the present invention are considered to have a combination of properties that are particularly advantageous over prior art anti-IL-34 antibodies, including but not limited to one or more of the following characteristics: 1) desirable binding and dissociation rates, 2) potency in neutralizing human IL-34 to achieve an anti-neuroinflammatory response and in vivo efficacy, 3) sufficient potency as a monotherapy for the treatment and / or prevention of immune-mediated and / or inflammatory disorders, 4) duration of action, 5) sufficient limitation of the induction of undesirable cytokine release, 6) an acceptably low level of immunogenicity (i.e., sufficient non-immunogenicity in humans), 7) avoidance of harmful immunosuppression, and / or 8) heat stability, solubility, low self-association, and desirable in vivo stability, physical and chemical stability, including but not limited to pharmacokinetic properties acceptable for development and / or use in the treatment of inflammatory or neuroinflammatory disorders, such as AD.

[0017] Embodiments of the present invention provide compositions and methods useful for the prevention, downregulation, or improvement of inflammatory and / or neuroinflammatory-related disorders through neutralization of IL-34 using a pharmacologically advantageous anti-human IL-34 antibody as provided in the embodiments described herein, thereby making significant progress over the prior art. The anti-human IL-34 antibodies of the present invention can preferably improve immune and / or inflammatory pathology or restore immune homeostasis, preferably through inhibition of the innate arm of the immune response and / or suppression of microgliosis or other monocytes. Activation and / or proliferation of cells of the macrophage lineage directly alters the underlying disease pathology. Clinical use of such antibodies may lead to long-term persistence of the disease being treated.

[0018] Furthermore, there is a need for diagnostic anti-human IL-34 antibodies that are specific for human IL-34, have improved binding affinity, and show improved sensitivity in the measurement of human IL-34, as well as improved enzyme-linked immunosorbent assay (ELISA) assay conditions that provide minimal interference and broad dilution linearity. According to some aspects of the present invention, anti-human IL-34 antibodies are provided that include human IL-34 neutralizing antibodies that bind to human IL-34 provided by SEQ ID NO: 41. Interleukin 34 (IL-34; also known as uncharacterized protein C16orf77) is secreted as a homodimer consisting of 39 kDa monomers. It does not belong to any known cytokine family. Human IL-34 is synthesized as a 242 amino acid (AA) precursor that includes a 20 AA signal sequence, resulting in a 222 AA mature chain. As used herein, IL-34 refers to the mature chain. The mature chain contains one site that has the potential for N-linked glycosylation. Human IL-34 is 71% identical to mouse IL-34 at the amino acid level. IL-34 is expressed in various tissues such as the heart, brain, liver, kidney, spleen, thymus, testis, ovary, small intestine, prostate, colon, etc., and is most abundant in the spleen. "hIL-34" or "human IL-34", as used herein with respect to the IL-34 polypeptide, refers to wild-type human IL-34, preferably having the amino acid sequence shown in SEQ ID NO: 41, which is mature IL-34 with the leader sequence removed. (See, for example, Lin et.al., Science (2008) Vol. 320, Issue 5877, pp. 807-811).

[0019] Exemplary human IL-34 (including the 20 AA signal peptide sequence removed to obtain the mature polypeptide) is MPRGFTWLRYLGIFLGVALGNEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPI NYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATES VQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP (Refer to SEQ ID NO: 41, NCBI Ref. Seq. No. NP_689669.2 as of March 1, 2018).

[0020] According to some aspects of the present disclosure, provided is an anti-mouse IL-34 antibody comprising a mouse IL-34 neutralizing antibody that binds to human IL-34 provided by SEQ ID NO: 42. Exemplary mouse IL-34 (including a 20 AA signal peptide sequence removed to obtain the mature polypeptide) is MPWGLAWLYCLGILLDVALGNENLEIWTLTQDKECDLTGYLRGKLQYKNRLQYMKHYFPINYRIAVPYEGVLRVANITRLQKAHVSERELRYLWVLVSLNATESVMDVLLEGHPSWKYLQEVQTLLENVQRSLMDVEIGPHVEAVLSLLSTPGLSLKLVRPKALLDNCFRVMELLYCSCCKQSPILKWQDCELPRLHPHSPGSLMQCTATNVYPLSRQTPTSLPGSPSSSHGSLP (Refer to SEQ ID NO: 42, NCBI Ref. Seq. No. NP_00ll28572.l as of March 1, 2018).

[0021] As used herein, "human anti-IL34 antibody" or "anti-human IL-34 antibody" refers to an antibody that binds to human IL-34 and, when administered in vitro or in vivo, results in neutralization of IL-34 activity and / or blockade of responses, for example, a desirable decrease in IL-34 signaling as evidenced by at least one significantly decreased desired activity, e.g., a change in an IL-34-responsive molecule or cellular endpoint. For example, the number, density, or phenotype of microglia in the CNS is an example of an IL-34-responsive molecule or cellular effect. As used herein, the terms "signaling" and "signal transduction" and "IL-34-mediated" when in the context of IL-34 refer to cellular and / or intercellular reactions resulting from the activity of IL-34.

[0022] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, or conjugated antibodies. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and of any subclass (e.g., IgG1, IgG2, IgG3, IgG4). An exemplary antibody is an immunoglobulin G (IgG)-type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The LC is classified as kappa or lambda, each of which is characterized by a specific constant region. Embodiments of the invention can include IgG1 or IgG4 antibodies and can further include a kappa light chain or a lambda light chain. Preferably, the antibody of the invention includes a light chain constant region that is a kappa constant region.

[0023] HC is classified as gamma, mu, alpha, delta, or epsilon, and defines the antibody isotype as IgG, IgM, IgA, IgD, or IgE, respectively. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 or more amino acids that is mainly involved in antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is mainly involved in effector functions. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The constant region of the heavy chain contains CH1, CH2, and CH3 domains. CH1 follows HCVR, and CH1 and HCVR form the heavy chain portion of the antigen-binding (Fab) fragment, which is part of the antibody that binds to the antigen. CH2 follows the hinge region and precedes CH3. CH3 follows CH2 and is at the carboxy terminus of the heavy chain. The constant region of the light chain contains one domain, CL. CL follows LCVR, and CL and LCVR form the light chain portion of Fab.

[0024] The antibodies of the present invention include IgG heavy chains (HCs) that can be further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, and embodiments of the present invention can include one or more modifications in the constant region of each HC that, for example, enhance or reduce effector function. As used herein, the term "Fc region" refers to the region of an antibody that includes the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region can include a portion or the entire hinge region of the antibody heavy chain. IgG1 is known to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and the Fc mutations described herein can reduce aggregation, reduce or enhance ADCC or CDC activity (or other functions), and / or modify the pharmacokinetics of the antibody. Embodiments of the anti-human IL-34 antibodies described herein reduce binding to FcγR and C1q receptors, thereby reducing or eliminating cytotoxicity that can be induced by antibodies having a wild-type IgG Fc region. Thus, according to some embodiments, the mutations are introduced into the Fc region at the positions described herein. By sufficiently reducing or eliminating the effector function of such anti-human IL-34 antibodies that include a modified Fc region, patient safety can be improved, and in combination with other properties described herein, a therapeutic agent can be provided that has an improved profile of useful activity while avoiding undesirable activity.

[0025] When expressed in certain biological systems, antibodies are glycosylated in the Fc region. Typically, glycosylation occurs in the Fc region of antibodies at highly conserved N-glycosylation sites. N-glycans typically bind to asparagine. Antibodies can also be glycosylated at other positions. The antibodies of the present invention are monoclonal antibodies. Monoclonal antibodies are antibodies derived from a single copy or clone (e.g., including any eukaryotic, prokaryotic, or phage clone), and not by the method by which they are produced. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or a combination of such technologies or other technologies known in the art. The present invention contemplates that the antibodies of the present invention are human or humanized antibodies. In the context of monoclonal antibodies, the terms "human" and "humanized" are well known to those skilled in the art (Weiner LJ, J. Immunother. 2006;29:1-9; Mallbris L, et al., J. Clin. Aesthet. Dermatol. 2016;9:13-15). Exemplary embodiments of the antibodies of the present invention also include antibody fragments or antigen-binding fragments that include at least a portion of an antibody that retains the ability to specifically interact with an antigen, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-bonded Fv (sdFv), Fd fragments, and linear antibodies.

[0026] The amino-terminal portions of each LC and HC contain a variable region of approximately 100 to 120 amino acids that is mainly responsible for antigen recognition via the CDRs contained therein. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this specification, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3", and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3". The CDRs contain most of the residues that form specific interactions with the antigen. The functional ability of an antibody to bind to a specific antigen is greatly influenced by the six CDRs.The assignment of amino acid residues to the CDRs can be performed according to well-known schemes, including those described in Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest”, National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).

[0027] For the purposes of the present invention, unless otherwise specified, the North CDR definition is used for the anti-IL-34 antibodies described herein and the assignment of amino acids to the CDR domains within the LCVR and HCVR regions. The following is a table of the CDR sequences of Antibody 1 based on the North definition. Those skilled in the art can also apply the Kabat, Chothia, and / or IMGT alternative definitions to identify the CDRs according to the convention for the antibodies of the present invention, including Antibodies 1-3 in Table 1. [Table 2]

[0028] Embodiments of the antibodies of the present invention have a combination of several pharmacologically useful and important activities and, in some respects, can bind to human IL-34 with high affinity and high specificity for human IL-34, and have other useful properties. As used herein, the term "bind" means, unless otherwise specified, the ability of a protein or molecule to form an attractive interaction with another protein or molecule, resulting in the proximity of two proteins or molecules, as determined by common methods known in the art. The phrase "specifically binds" as used herein with respect to the affinity of an anti-IL-34 antibody for human IL-34, unless otherwise indicated, preferably by about 1×10 -11 M or less, even more preferably, about 1×10 -11 M to about 1×10 -12 M of K D is intended to mean. The phrase "specifically binds" also indicates the relative affinity of an anti-IL-34 antibody for human IL-34 as compared to other antigens, and the affinity for human IL-34 results in specific recognition of human IL-34.

[0029] Embodiments of the antibodies of the present invention can be expressed and produced from constructs containing the sequences of the present embodiment by various techniques known in the art. The terms "nucleic acid" or "polynucleotide" as used interchangeably herein refer to polymers of nucleotides, including single-stranded and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules incorporating natural nucleotides, modified nucleotides, and / or nucleotide analogs. The polynucleotides of the present disclosure can also include, for example, substrates incorporated therein by DNA or RNA polymerase reactions or synthetic reactions. The DNA molecules of the present invention are DNA molecules comprising non-naturally occurring polynucleotide sequences encoding polypeptides having the amino acid sequence of at least one of the polypeptides (e.g., heavy chain, light chain, variable heavy chain, and variable light chain) in the antibodies of the present invention.

[0030] Isolated DNA encoding an HCVR or LCVR region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the HCVR or LCVR, respectively, to another DNA molecule encoding a heavy chain or light chain constant region to form a heavy chain or light chain, respectively. The sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained, for example, by standard PCR amplification.

[0031] The polynucleotides of the present invention can be expressed in host cells after the sequences are operably linked to expression control sequences. Expression vectors are typically replicable in a host organism, either as episomes or as an integrated part of the host chromosomal DNA. Generally, expression vectors contain selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to enable the detection of those cells transformed with the desired DNA sequences. Vectors containing the polynucleotide sequences of interest (e.g., polynucleotides encoding the polypeptides of the antibodies and expression control sequences) can be introduced into host cells by various well-known methods depending on the type of cell host.

[0032] The antibodies of the present invention can be easily produced in mammalian cells, and non-limiting examples thereof include CHO, NS0, HEK293, or COS cells. The host cells are cultured using techniques well known in the art. Antibody expression in mammals typically results in glycosylation. Antibody glycosylation is usually either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, such as N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid. Typically, glycosylation occurs in the Fc region of the antibody at highly conserved N-glycosylation sites (e.g., position 297 in IgG1 according to the IMGT or EU index numbering). The glycosylation sites can be modified to alter glycosylation (e.g., to block or reduce glycosylation, or to change the amino acid sequence to generate additional or diverse glycosylation).

[0033] Expression of antibodies from IgG subclasses in mammals can result in the cleavage of C-terminal amino acids from one or both of the heavy chains. For example, in the case of IgG1 antibodies, one or two C-terminal amino acids can be removed. In the case of IgG1 antibodies, if a C-terminal lysine is present, it can be trimmed or removed from the heavy chain during expression. Additionally, the penultimate glycine can also be trimmed or removed from the heavy chain in a similar manner.

[0034] Expression of antibodies in mammals can also result in the modification of N-terminal amino acids. For example, if the most N-terminal amino acid of the heavy or light chain is glutamine, it can be modified to pyroglutamic acid.

[0035] The antibody of the present invention, or a pharmaceutical composition containing the same, can be administered by a parenteral route, and non-limiting examples of this are subcutaneous administration and intravenous administration. The antibody of the present invention can be administered to a patient in a single dose or multiple doses together with a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition of the present invention can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and contains the antibody disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0036] Use of the antibody embodiments of the present invention: According to some embodiments, the anti-IL-34 antibody of the present invention is useful for the treatment of immune-mediated diseases. As used herein, the terms "immune-mediated disease" or "inflammatory disease or disorder" are used interchangeably and refer to an undesirable condition resulting from an inappropriate or excessive immune response in which homeostasis is enhanced and pathological responses are reduced by IL-34 inhibition. The terms "immune-mediated disease" or "inflammatory disorder" mean including such conditions, whether mediated by the cellular immune response of microglia or macrophages, or by symptoms of similar tissue-resident cell types such as histiocytes, Kupffer cells, alveolar macrophages, intestinal macrophages, macrophage-like synoviocytes, or Langerhans cells. Exemplary diseases contemplated to be treated by the antibody of the present invention described herein include Alzheimer's disease, tauopathy, Sjogren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0037] In some more specific embodiments, the immune-mediated disease is Alzheimer's disease (AD). According to other embodiments of the present invention, the anti-IL-34 antibody is useful for the diagnostic use of immune-mediated diseases. In some embodiments, the immune-mediated disease is at least one of AD, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0038] The present invention further provides a pharmaceutical composition comprising the anti-IL-34 antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients. Further, the present invention provides a method for treating immune-mediated diseases such as AD, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD), the method comprising administering the pharmaceutical composition of the present invention to a patient in need of treatment.

[0039] In addition, the present invention provides a method for treating immune-mediated diseases. More specifically, the present invention provides a method for treating immune-mediated diseases such as AD, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD), the method comprising administering an effective amount of the anti-IL-34 antibody of the present invention to a patient in need of treatment.

[0040] The present invention also provides the anti-IL-34 antibody of the present invention for use in treatment. More specifically, the present invention provides the anti-IL-34 antibody of the present invention for use in the treatment of immune-mediated diseases including AD, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0041] In certain embodiments, the invention provides for the use of the anti-IL-34 antibody of the invention in the manufacture of a medicament for the treatment of one or more immune-mediated diseases including AD, Sjogren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0042] The antibodies of the present invention are useful for the identification of immune-mediated disorders in which IL-34 may contribute to the etiology of the disorder. In a further embodiment, the present invention provides a method of treating an immune-mediated disease in a patient. Such a method comprises contacting a patient sample with an IL-34 antibody and detecting the binding between human IL-34 and the antibody in the patient sample, and diagnosing the patient as having, at risk of having, in need of treatment for, and / or at risk of symptoms associated with an immune-mediated disease if the presence of IL-34 in the patient sample is detected above a reference value observed in healthy individuals (see, e.g., Xie, H.H., et al. Elevated Serum Interleukin-34 Level in Patients with Systemic Lupus Erythematosus Is Associated with Disease Activity. Sci Rep 8, 3462 (2018)). According to some more specific embodiments of the treatment methods provided herein, such a method further comprises determining a reference value, the steps of contacting a control standard with a first antibody that binds to a first epitope region of the same IL-34 used when contacting the patient sample, contacting the control standard with a second antibody that has a detectable label and binds to a second epitope region of the same IL-34 used when contacting the patient sample, and detecting the signal provided by the detectable signal. In some specific embodiments, the anti-IL-34 antibody comprises a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the second antibody comprises a combination of LCVR and HCVR provided in Table 1. According to some embodiments, the reference value is, for example, from about 10 to 30 pg / mL, such as from a CNS tissue lysate. In certain embodiments, the immune-mediated disease is one of AD, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD). In some embodiments, the patient sample is one of CSF, blood, serum, tissue lysate, or plasma.According to some embodiments, the method further comprises contacting a patient sample with a second anti-IL-34 antibody that binds to a second epitope region of IL-34 and has a detectable label, and detecting a signal provided by the detectable signal. In a further embodiment, the second antibody comprises a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the second antibody comprises a combination of LCVR and HCVR provided in Table 1. According to certain embodiments, the first and second anti-IL-34 antibodies cannot be put together in a vial.

[0043] According to some embodiments, the present invention provides a method for detecting IL-34 in a patient sample, comprising contacting the patient sample with a first antibody that binds to a first epitope region of IL-34, contacting the patient sample with a second antibody that binds to a second epitope region of IL-34 and has a detectable label, and detecting a signal provided by the detectable label. In some embodiments, the patient sample is one of blood, serum, tissue lysate, or plasma. According to some more specific embodiments, the first epitope region of IL-34 partially overlaps with the second epitope region of IL-34. Further, in some embodiments, the steps of contacting with the first and second antibodies occur simultaneously. In some specific embodiments, the first antibody comprises a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the first antibody comprises a combination of LCVR and HCVR provided in Table 1.

[0044] According to some embodiments of the present invention, a method for quantifying IL-34 in a patient sample is provided. Such a method comprises contacting the patient sample with a first antibody that binds to a first epitope region of IL-34, contacting the patient sample with a second antibody that binds to a second epitope region of IL-34 and has the detectable label, detecting a signal provided by the detectable label, contacting the control standard with a first antibody that binds to the same first epitope region of IL-34 as (used when contacting the patient sample); contacting the control standard with a second antibody that has a detectable label and binds to the same second epitope region of IL-34 as (used when contacting the patient sample); and detecting the signal provided by the detectable signal. In some embodiments, the patient sample is one of blood, serum or plasma, or tissue lysate. According to some more specific embodiments, the first epitope region of IL-34 partially overlaps with the second epitope region of IL-34. Further, in some embodiments, the step of contacting with the first and second antibodies occurs simultaneously. In some specific embodiments, the first antibody comprises the combination of LC and HC CDRs provided in Table 1. In a further embodiment, the first antibody comprises the combination of LCVR and HCVR provided in Table 1. In some specific embodiments, the second antibody comprises the combination of LC and HC CDRs provided in Table 1. In a further embodiment, the second antibody comprises the combination of LCVR and HCVR provided in Table 1.

[0045] According to some embodiments, a method for diagnosing an immune-mediated disease is provided. Such a method includes contacting a patient sample with an anti-IL-34 antibody and detecting the binding between IL-34 and the antibody in the patient sample. According to some specific embodiments, the diagnostic method includes diagnosing the patient as having, at risk of having, in need of treatment for, and / or at risk of symptoms associated with, an immune-mediated disease if the presence of IL-34 in the patient sample is detected as exceeding a reference value. According to some more specific embodiments, such a method further includes determining a reference value, the steps of contacting a control standard with a first antibody that binds to a first epitope region of IL-34 that is the same as the one used when contacting the patient sample, contacting the control standard with a second antibody that has a detectable label and binds to a second epitope region of IL-34 that is the same as the one used when contacting the patient sample, and detecting the signal provided by the detectable signal. In some embodiments, the first antibody includes the combination of LC and HC CDRs provided in Table 1. According to some embodiments, the method further includes contacting the patient sample with a second anti-IL-34 antibody that binds to the second epitope region of IL-34 and has a detectable label, and detecting the signal provided by the detectable signal. In some specific embodiments, the anti-IL-34 antibody includes the combination of LC and HC CDRs provided in Table 1. In a further embodiment, the antibody includes the combination of LCVR and HCVR provided in Table 1. According to a more specific embodiment, the first epitope region of IL-34 partially overlaps with the second epitope region of IL-34. According to a particular embodiment, the first and second antibodies cannot be put together in a vial. According to a further embodiment, the reference value is in the approximate range of about 10-30 pg / mL from a CNS tissue lysate and / or is determined by one of ordinary skill in the art for an appropriate reference group and sample source.In a further embodiment, the immune-mediated disease is one of AD, tauopathy, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0046] In one embodiment, the present invention is a method for determining the level of human IL-34 in a body fluid, comprising: (a) contacting the body fluid with an anti-human IL-34 diagnostic monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to human IL-34 and consists of the amino acid sequence of SEQ ID NO: 41, an antibody, or an antigen-binding fragment thereof (including the light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 each containing the amino acid sequences (SEQ ID NO: 8), (SEQ ID NO: 9), and (SEQ ID NO: 10), and the heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 each containing the amino acid sequences (SEQ ID NO: 15), (SEQ ID NO: 16), and (SEQ ID NO: 17)); (b) optionally, removing any non-specifically bound monoclonal antibody or antigen-binding fragment thereof; and (c) detecting and / or quantifying the amount of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human IL-34. Preferably, the body fluid is blood, serum or plasma, or cerebrospinal fluid, and the contacting occurs ex vivo.

[0047] Tauopathies include, but are not limited to, Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain dementia, Down syndrome, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Guamanian parkinsonism-dementia complex, Niemann-Pick disease type C, and myotonic dystrophy (see Li, C., Gotz, J. Tau-based therapies in neurodegeneration: opportunities and challenges. Nat Rev Drug Discov 16, 863-883 (2017)).

[0048] In embodiments of the invention, the patient is a human diagnosed with a medical risk, condition, or disorder, such as one of the diseases or disorders described herein, who is in need of treatment with an antibody described herein. When the disorders treatable by the methods of the invention are established and known by the accepted classifications, such as by Alzheimer's disease, tauopathy, Sjogren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD), those classifications are described in various well-known medical texts. For example, currently, the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) provides diagnostic tools for identifying specific disorders described herein. Also, the 10th edition of the International Classification of Diseases (ICD-10) provides classifications for specific disorders described herein. One of ordinary skill in the art will recognize that there are alternative nomenclatures, taxonomies, and classification systems for the diseases and disorders described herein, including those described in DSM-5 and ICD-10, and that the terminology and classification systems evolve with the advancement of medical science.

[0049] The term "treating" (or "treat" or "treatment") refers to delaying, interfering with, suppressing, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0050] As used herein, the term "innate immunity" includes the arm of the immune response that is required to initiate and maintain an adaptive immune response (antibody and T cell responses), as contrasted with the adaptive arm of the immune response.

[0051] "Effective amount" means the amount of the anti-human IL-34 antibody of the invention, or a pharmaceutical composition comprising such an antibody, that will induce a biological or medical response in a tissue, system, or human that is sought by a treating medical professional, or will induce a desired therapeutic effect. As used herein, the term "effective response" of a patient, or responsiveness of a patient to treatment, refers to the clinical or therapeutic benefit conferred on a patient upon administration of an antibody of the present disclosure. The effective amount of an antibody can vary depending on factors such as the medical condition of the individual, age, gender, and weight of the individual, and the ability of the antibody to induce the desired response in the individual. The effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody. Such benefits include any one or more of a decrease in the level of inflammation or immune activation, stabilization of an immune-mediated disease or disorder, or improvement in the signs or symptoms of an immune-mediated disorder. Alternatively, such benefits include any one or more of an increase in immune tolerance of a transplanted organ, amelioration of a stable autoimmune disease or disorder, or improvement in the signs or symptoms of an autoimmune disorder.

[0052] A potential advantage of the methods disclosed herein is the possibility of providing significant and / or long-term alleviation in patients suffering from an immune-mediated disorder or a neuroinflammatory disorder, with an acceptable safety profile including acceptable tolerability, toxicity, and / or adverse events, such that the patient benefits from the overall treatment method. The efficacy of the treatments of the present disclosure can be measured by various endpoints commonly used when evaluating the treatment of various immune-mediated disorders. For example, any particular therapy of the invention can optionally use other approaches to determining efficacy, including measurement and visualization of immunocyte activation markers, measures of inflammation, cell cycle-dependent biomarkers, and / or measurement of various inflammatory or immune responses, or tissue-specific biomarker evaluation.

[0053] An effective amount can be readily determined by one of ordinary skill in the art by use of known techniques and by observing the results obtained under similar circumstances. The effective amount of the anti-human IL-34 antibody of the present invention can be administered in a single dose or in multiple doses. Further, the effective amount of the antibody of the present invention can be administered in multiple doses of an amount less than the effective amount if administered only once. When determining the effective amount for a patient, a number of factors are considered by the attending diagnostician, including, but not limited to, the size (e.g., weight or mass), body surface area, age, and overall health of the patient; the particular disease or disorder involved; the degree, or involvement, or severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dosing regimen selected; the use of concomitant medications; and other relevant circumstances known to the diagnosing physician.

[0054] Parenteral (including, but not limited to, subcutaneous, intramuscular, and / or intravenous) doses on a weekly, bi-weekly, monthly, or quarterly basis can be from about 0.5 mg / kg to about 50 mg / kg.

[0055] The parenteral (including, but not limited to, subcutaneous, intramuscular, and / or intravenous) dosage, administered weekly, every two weeks, monthly, or quarterly, may be about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 10 mg / kg, about 4 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 1 mg / kg to about 8 mg / kg, about 2 mg / kg to about 8 mg / kg, about 3 mg / kg to about 8 mg / kg, about 4 mg / kg to about 8 mg / kg, about 5 mg / kg to about 8 mg / kg, about 6 mg / kg to about 8 mg / kg, about 1 mg / kg to about 6 mg / kg, about 2 mg / kg to about 6 mg / kg, about 3 mg / kg to about 6 mg / kg, about 4 mg / kg to about 6 mg / kg, about 5 mg / kg to about 6 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 3 mg / kg to about 5 mg / kg, about 4 mg / kg to about 5 mg / kg, about 1 mg / kg to about 4 mg / kg, about 2 mg / kg to about 4 mg / kg, about 3 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, or about 4 mg / kg to about 5 mg / kg.

[0056] The parenteral (including, but not limited to, subcutaneous, intramuscular, and / or intravenous) dosage, administered weekly, every two weeks, monthly, or quarterly, can be, for example, from about 50 mg to about 500 mg, from about 75 mg to about 500 mg, from about 100 mg to about 500 mg, from about 125 mg to about 500 mg, from about 250 mg to about 500 mg, from about 300 mg to about 500 mg, from about 350 mg to about 500 mg, from about 400 mg to about 500 mg, from about 450 mg to about 500 mg, from about 50 mg to about 400 mg, from about 75 mg to about 400 mg, from about 100 mg to about 400 mg, from about 125 mg to about 400 mg, from about 250 mg to about 400 mg, from about 300 mg to about 400 mg, from about 350 mg to about 400 mg, from about 50 mg to about 300 mg, from about 75 mg to about 300 mg, from about 100 mg to about 300 mg, from about 125 mg to about 300 mg, from about 150 mg to about 300 mg, from about 175 mg to about 300 mg, from about 200 mg to about 300 mg, from about 250 mg to about 300 mg, from about 50 mg to about 250 mg, from about 75 mg to about 250 mg, from about 100 mg to about 250 mg, from about 125 mg to about 250 mg, from about 150 mg to about 250 mg, from about 175 mg to about 250 mg, from about 200 mg to about 250 mg, from about 75 mg to about 250 mg, from about 50 mg to about 200 mg, from about 75 mg to about 200 mg, from about 100 mg to about 200 mg, from about 125 mg to about 200 mg, from about 150 mg to about 200 mg, from about 175 mg to about 200 mg, from about 50 mg to about 175 mg, from about 75 mg to about 175 mg, from about 100 mg to about 175 mg, from about 125 mg to about 175 mg, or from about 150 mg to about 175 mg.

[0057] However, lower or higher dosages than those described herein are also contemplated, particularly in consideration of dosage considerations known to those of ordinary skill in the art and / or described herein. The progress of the patient being treated is monitored by regular evaluation, and the dosage is adjusted accordingly as needed.

[0058] A potential advantage of the methods disclosed herein is that they may provide significant and / or long-term alleviation with an acceptable safety profile, including acceptable tolerability, toxicity, and / or adverse events, in patients suffering from an immune-mediated disorder or a neuroinflammatory disorder, and thus the patient may benefit from the overall treatment method. More specifically, the antibodies of the present invention provide effective treatment while avoiding clinically undesirable immunosuppression and / or immune-related adverse events such as "cytokine storm" or significant cytokine release. The antibodies of the present invention may be useful for the treatment of cytokine storm, or the reverse release of cytokines. As used herein, "significant cytokine release" refers to a significant increase in measurable cytokines that can be detected by methods known to those of skill in the art. For example, significant cytokine release can be detected in human blood samples by ELISA, where cytokine levels from unstimulated blood are compared to cytokine levels from blood incubated with the antibody. In some such tests, significant cytokine release can be detected if, for example, the levels of IL-6, or IL-8, or IFN-γ are at least three-fold higher in blood incubated with the antibody compared to levels in unstimulated blood. Preferably, the treatment of immune-mediated disorders described in the embodiments herein is performed when the patient does not experience significant cytokine release. BRIEF DESCRIPTION OF THE DRAWINGS

[0059]

Figure 1

Figure 2

Figure 3

Figure 4

Example

[0060] The following examples are provided to illustrate the claimed invention and are not intended to limit it. The results of the following assays show that the exemplified monoclonal antibodies and / or antigen-binding fragments thereof of the invention bind to and / or neutralize IL-34 and can thus be used to treat the immune-mediated and inflammatory diseases described herein.

[0061] Example 1: Generation, Expression, and Purification of Antibodies A panel of human anti-IL-34 antibodies is obtained using a human antibody phage display library and screened to identify reagents that can be effective human IL-34 neutralizing antibodies. To isolate clones with improved affinity, mutations are systematically introduced into the individual complementarity determining regions (CDRs) of each antibody, and the resulting library is subjected to multiple rounds of selection while decreasing the antigen concentration and / or increasing the dissociation time. The sequences of the individual variants are determined and used to construct a combinatorial library, which is subjected to additional rounds of selection while increasing the stringency to identify additive or synergistic pairings of mutations between the individual CDR regions. The individual combinatorial clones are sequenced and their binding properties determined. To further increase the affinity for IL-34, these combinatorial clones are subjected to additional rounds of single and combinatorial mutagenesis. This screening can be performed against human or mouse IL-34 (e.g., antibody 1 against human IL-34 and antibody 3 against mouse IL-34) to increase the affinity for the selected species. The selected antibodies can also be mutagenized to repair post-translational modifications such as methionine oxidation while maintaining the binding affinity for IL-34. In addition, to reduce the potential immunogenicity risk, framework (FW) replacements can be made to the antibodies to return these FW1 sequences to their germline state.

[0062] Modified and / or optimized anti-IL-34 antibodies, referred to herein as antibody 1, antibody 2, and / or antibody 3, are obtained and have the amino acid sequences of the variable regions of the heavy and light chains, the complete heavy and light chain amino acid sequences, and the nucleotide sequences encoding the same as those listed in the section entitled "List of Amino Acid and Nucleotide Sequences" below. The sequence numbers corresponding to these fragments, as well as the CDR amino acid sequences of the light and heavy chains, are shown in Table 1 below.

[0063] The exemplary anti-IL-34 antibodies of the present invention can be expressed and purified essentially as follows. Suitable host cells such as HEK293, NS0 or CHO can be transiently or stably transfected in an expression system for secreting the antibody using an optimal predetermined HC:LC vector ratio (such as 1:3 or 1:2) or a single vector system encoding both HC and LC. The clarified culture medium in which the antibody is secreted is purified using any of a number of commonly used techniques. For example, the medium can be applied to a MabSelect® column (GE Healthcare) or a KappaSelect column (GE Healthcare) for Fab fragments, equilibrated with a suitable buffer such as phosphate buffered saline (pH 7.4). The column can be washed to remove non-specific binding components. The bound antibody can be eluted, for example, by a pH gradient (such as 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or phosphate buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0). The antibody fractions can be detected by SDS-PAGE etc. and then pooled. The bound antibody is eluted, for example, by a pH gradient (such as from 0.1 M sodium phosphate buffer (pH 6.8) to 0.1 M sodium citrate buffer (pH 2.5)). The antibody fragments are detected by SDS-PAGE etc. and then pooled. Further purification is optional depending on the intended use. The antibody can be concentrated and / or sterile filtered using common techniques. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, or ion exchange, multimodal, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is greater than 99%. The product can be immediately frozen at -70 °C, or lyophilized, or, if to be used immediately, stored at 4 °C for immediate use. The amino acid sequence numbers of exemplary human antibodies of the present invention are shown in Table 1 below.

[0064] The expression plasmid contains cDNA versions of the LC and HC genes of antibody 1 (e.g., the DNA sequence of SEQ ID NO: 12 encoding the exemplary HC of antibody 1 presented in Table 1, and the DNA sequence encoding the LC amino acid sequence according to Table 1, e.g., the DNA sequence of SEQ ID NO: 13 encoding the exemplary LC of antibody 1 presented in Table 1), and is expressed from commonly used and suitable constructs for this purpose, such as those based on the major immediate-early promoter of human cytomegalovirus. The LC and HC genes are flanked by inverted terminal repeat (ITR) sequences recognized by the transposase enzyme. The parental cell line is co-transfected with the expression plasmid and transposase mRNA that enables transient expression of the transposase enzyme to facilitate stable integration of the antibody 1 gene expression cassette into the genomic DNA.

[0065] The selected bulk culture is subjected to single cell cloning using fluorescence-activated cell sorting (FACS) technology. The cell lines derived from the clones are grown and screened for antibody 1 production. The cell lines derived from the clones are selected and established. This cell line is generated without using any materials containing animal components and is used for production.

[0066] The clarified culture medium in which the antibody has been secreted is applied to a protein A affinity column equilibrated with a compatible buffer such as phosphate-buffered saline (pH 7.4) or 20 mM Tris (pH 8.0) containing 150 mM sodium chloride. The column is washed with 1 M NaCl to remove non-specific binding components. The bound antibody is eluted, for example, with sodium citrate at pH (about) 3.5, and the fractions are neutralized with 1 M Tris buffer. The antibody fractions are detected by, for example, SDS-PAGE or analytical size exclusion and then pooled. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The exemplified anti-IL-34 antibody of the present invention is concentrated and / or sterile filtered using common techniques. The purity of the exemplified antibody after these chromatography steps is greater than 95%. The exemplified anti-IL-34 antibody of the present invention can be immediately frozen at -70°C or stored at 4°C for several months.

[0067] Example 2: Characterization of Anti-IL-34 Antibody Binding Affinity for Human IL-34 The binding affinity of the anti-IL-34 monoclonal antibody of the present invention for human IL-34 containing the leader sequence or mature human IL-34 can be determined by methods known in the art. Unless otherwise specified, all reagents and materials can be purchased from Meso Scale Discovery (MSD®), and the measurements can be performed at 37°C. Human and mouse IL-34 can be prepared and purified using IMAC and size exclusion chromatography or purchased from commercial suppliers. The human CSF1R Fc fusion protein (exemplified compound D) is also prepared, purified by MabSelect® SuRe® (GE Healthcare), and further purified by size exclusion chromatography.

[0068] Briefly, generally, two-fold or three-fold dilution series of human, cynomolgus monkey (cyno), or mouse IL-34 are prepared from starting concentrations of 40, or 50, or 60, or 80, or 100, or 120 nM down to 100 fM, and each series includes an IL-34 blank control. Samples are prepared in 3% (w / v) Blocker A solution (MSD®, number R93AA-1), and each antibody exemplified in Table 1 at a final immobilized concentration of 5 - 50 pM is added to each sample. An antibody-only control is included. Each protein-antibody sample with a volume of 50 μl is added to individual wells of a 96-well microtiter plate (Greiner, EK-20101). The plate is sealed with an optical adhesive film (Thermo Fisher Scientific, number 4311971) and incubated at 37 °C for 1 - 3 days to allow equilibration of the samples. The day before analysis, each column of a 96-well MSD® standard plate (MSD®, number L15XA) is coated with 30 μl of the corresponding IL-34 (used in the titration series) at a concentration of 1 μg / ml in phosphate-buffered saline (PBS). On the day of the experiment, the MSD® standard plate is washed three times with 150 μl of PBST (PBS containing 0.05% Tween) and blocked with 150 μl of 3% Blocker A solution for 60 minutes at room temperature while shaking at 300 rpm on a MaxQ 4450 benchtop shaker (Thermo Fisher Scientific). After washing three times with PBST, 50 μl of each protein-antibody sample (prepared and incubated as described above) is added to the MSD® standard plate and incubated at 37 °C for 150 seconds while shaking at 300 rpm. After washing three times with PBST, 50 μl of a 1 μg / ml SULFO-TAG-labeled detection antibody prepared in 1% (w / v) Blocker A solution is added to the MSD® standard plate, and the plate is incubated at 37 °C for 150 seconds while shaking at 300 rpm. The plate is washed three more times with PBST, followed by the addition of 150 μl / well of 1-fold read buffer (MSD®, number R92TC-2). The MSD® standard plate is MESO Quickplex

[0069] Read using the SQ 120 / 1300 device (Meso Scale Discovery). Data analysis was performed using GraphPad Prism 8 (version 8.0.0 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com), and the binding affinity (K D ) was determined using the integrated four-parameter logistic curve model of GraphPad Prism 8. The results are shown in Tables 2, 3, and 4.

Table 3

Table 4

Table 5

[0070] Since IL-34 binds to human CSF1R with an affinity of approximately 50 - 100 pM, a high-affinity antibody is required to effectively neutralize this cytokine in the CNS. Blocking IL-34 is thought to provide a useful means for disease modification while avoiding safety concerns associated with some existing immunomodulatory therapies. Thus, neutralization of IL-34-mediated signaling is a therapeutic approach for the management of neuroinflammation, microgliosis, and neurodegenerative diseases such as Alzheimer's disease and other tauopathies and inflammatory diseases.

[0071] The results in Tables 2, 3, and 4 show that antibodies 1, 2, and 3 have high affinity for human IL-34, and in particular, antibodies 1 and 2 show affinity comparable to hCSF1R-Fc for human IL-34. Thus, the antibodies of the present invention have binding properties that enable effective neutralization of IL-34 in vivo.

[0072] Physicochemical properties: Chemical stability, Regarding the product attributes of the therapeutic antibody, including photo-stability, triple slow freeze / thaw, and solubility, Antibody 1 exhibits a desirable combination of physicochemical attributes for use as a human therapeutic agent.

[0073] Preparation of Stability Samples To prepare stability samples, the aqueous antibody solution is dialyzed overnight at 4°C against PBS, or 10 mM histidine / 280 mM mannitol, pH 6 (H6M), or 10 mM histidine / 150 mM NaCl, pH 6 (H6N), or 10 mM histidine / 17% sucrose, pH 6.0 (H6S). If necessary, polysorbate-80 is added to the final stability samples at 0.05% (「T」). The formulated Antibody 1 is further concentrated to the desired concentration using an Amicon spin concentrator. Abbreviations: 4wk = 4 weeks, H6MT = 10 mM histidine / 280 mM mannitol / 0.05% polysorbate, pH 6.0, H6ST = 10 mM histidine / 17% sucrose, 0.05% polysorbate, pH 6.0.

[0074] Chemical Stability Antibody 1 shows an acceptable evaluation in a 4-week (4wk) chemical stability hot-spot check at 5 mg / ml in PBS. After storage in PBS at 35°C for 4 weeks, compared to the time 0 control, Antibody 1 shows a 0.26% aggregate growth as measured by analytical size exclusion chromatography (aSEC). The hot-spot is confirmed by LC-MS (liquid chromatography - mass spectrometry) peptide mapping. When compared to the 4-week / 4°C sample, the following degradation / hot-spot changes are reported for PBS at 35°C: isomerization of D104 (0.1%), cleavage of G93 / D94 / S95 (0.8%). The total change rate of CDR degradation was 0.9%.

[0075] High-Concentration Stability Temperature Holding Antibody 1 shows an acceptable evaluation for high-concentration stability at 100 mg / ml in H6MT for 4 weeks. After storage at 35°C for 4 weeks, compared to the time 0 control, as measured by aSEC, there is a growth of 1.0% aggregates, and as measured by CE-SDS (capillary electrophoresis sodium dodecyl sulfate), there is an increase of 0.9% of low molecular weight fragments under non-reducing conditions. Hot spots are confirmed by LC-MS peptide mapping. The following degradation / hot spot changes are reported after incubation at 35°C for 4 weeks and compared to the control incubated at 5°C for 4 weeks: deamidation of H6MT:Q91 (0.1%), isomerization of D104 (0.1%), cleavage of G93 / D94 / S95 (0.5%). The total change rate of CDR degradation was 0.7%.

[0076] Solubility Antibody 1 shows an acceptable evaluation based on solubility assessment. The antibody achieves 150 mg / ml in both H6M and H6N formulations and after storage at 5°C for 3 days followed by storage at -5°C for 1 week, no visible precipitation or phase separation is observed.

[0077] Photo stability Antibody 1 shows an acceptable evaluation for photo stability. The antibody in 100 mg / ml H6MT is exposed to UV / VIS light (20% ICH guideline) and compared to a dark control sample. Chemical degradation is evaluated by LC-MS peptide mapping. The total change rate of CDR hot spot analysis is 0.1%. Growth of cleavage is observed at residues G93 / D94 / S95 of the light chain, which was 0.1%. Upon exposure to UV / VIS, as measured by aSEC, the growth of aggregates is 3.6%.

[0078] High-concentration freeze / thaw stability The physical stability of Antibody 1 was evaluated by slow freeze-thaw and showed an acceptable evaluation when formulated with H6ST. After three slow freeze-thaw cycles in a shelf freeze dryer (VirTis SP Scientific), Antibody 1 showed a 0.1% increase in aggregates of H6ST determined by aSEC when compared to the time 0 control.

[0079] Example 3: In Vitro Functional Characterization of Anti-Human IL-34 Antibodies Neutralization of IL-34 In Vitro The antibodies of the present invention are tested for their ability to neutralize the binding and / or activity of IL-34. Neutralization of the binding and / or activity of IL-34 by the antibodies of the present invention can be evaluated, for example, by one or more IL-34 / CSF1R receptor binding assay formats, as well as IL-34 cell-based activity assays, as described below.

[0080] Screening for neutralizing antibodies of IL-34 / CSF1R binding can be performed initially through an enzyme assay. Such an assay can use a recombinant expressed CSF1R extracellular domain protein that can bind to IL-34. These proteins can be bound to an ELISA plate to capture soluble IL-34. IL-34 can then be detected either by biotinylation of the antigen and detection by streptavidin / neutravidin-conjugated peroxidase or phosphatase enzymes, or alternatively, another detection technique can be used, for example, an antibody that detects the his-tagged version of IL-34. Such a neutralization assay includes pre-incubation (e.g., for 1 hour) of the antibody being evaluated with labeled IL-34 (as well as control samples not involving antibodies targeting IL-34) before addition to the binding assay. Concentrations of labeled IL-34 close to the 50% binding level (EC 50 ), and various concentrations (e.g., when evaluating the dose response of the antibody from about 100 micromolar to about 1 picomolar, etc.) can be used. Determination of the potency (IC 50 ) is possible by evaluating antibody inhibition over a range.

[0081] Inhibition of IL-34 induction response in vitro Neutralization of IL-34 activity by the antibodies of the present invention can be evaluated by, for example, one or more IL-34 cell-based assays as described below. The ability of the antibodies of the present invention to neutralize human IL-34-inducible luciferase reporter activity can be evaluated in NIH-3T3 hCSF1R AP1 or 293 hCSF1R SRE cells transfected with cDNA expressing human CSF1R, mouse CSF1R, or cynomolgus monkey CSF1R (human CSF1R (accession: NP_001275634.1), mouse CSF1R (accession: NM_001037859.2), and cynomolgus monkey CSF1R (accession: NC_022277)). (The terms "cynomolgus", "cynomolgus monkey", or "cynomolgus macaque" are used interchangeably herein). For example, 293 / SRE cells stably overexpressing human CSF1R (hCSF1R) are dissociated in 0.05% trypsin-PBS and seeded at 70,000 cells per 100 μl in tissue culture-treated 96-well plates. The next day, the growth medium is removed and the cells are starved in DMEM-F12 (Dulbecco's modified Eagle's medium: nutrient mixture F-12) supplemented with heat-inactivated 1% FBS (fetal bovine serum). After 24 hours of starvation, the cells are treated for 6 hours with either 100 ng / ml of human IL-34 or cynomolgus monkey IL-34 and various concentrations of hCSF1R-Fc or antibody 1. After incubation, the cells are lysed by gentle agitation for 5 minutes with 50 μl of Promega® Glo® lysis buffer (Promega® E266A). 50 μl of BrightGlo® luciferase reagent (Promega® E2620) is added and incubated on the lysed cells for 2 minutes. Luminescence is read on a Perkin Elmer Wallac 1420 Victor2® microplate reader. The reduction in relative fluorescence units (RFU) shown in Table 5 and Figure 1 reflects the ability of antibody 1 to neutralize IL-34 and reduce luciferase activity. The half-maximal inhibitory concentration (IC 50)The value is 385 pM for the neutralization of hIL-34 (Figure 1) and 654 pM for cynomolgus monkey IL-34. Human CSF1R-Fc was used as a positive control in this assay and inhibited luciferase activity at an IC 50 of 560 pM.

Table 6

[0082] Table 5 and Figure 1 show that Antibody 1 effectively neutralizes human IL-34-induced luciferase reporter activity in the above-described 293 hCSF1R cell-based assay (IC 50 0.3852 nM) and exhibits at least equivalent or greater potency than hCSF1R-Fc in this assay. These data support the ability of Antibody 1 to neutralize human IL-34-mediated signal transduction and treat diseases in which IL-34-mediated signal transduction contributes to the pathogenesis.

[0083] The ability of Antibody 1 to neutralize CSF1R dimerization in the PathHunter® eXpress Dimerization Assay Human IL-34 neutralization can be further evaluated by seeding U2OS CSF1R / CSF1R cells (PathHunter® eXpress Dimerization Assay, DiscoverX) in 96-well plates and assessing the ability of anti-IL-34 antibodies to inhibit CSF1R dimerization. These assays utilize Enzyme Fragment Complementation (EFC) technology in which the β-galactosidase (β-gal) enzyme is split into two fragments, ProLink (PK) and Enzyme Acceptor (EA). Independently, these fragments have no β-gal activity, but when complementation is forced by protein-protein interaction, they form an active β-gal enzyme. The PathHunter® eXpress Dimerization assay detects ligand-induced dimerization of two subunits of the CSF1R receptor-dimer pair. Cells are designed to co-express one CSF1R receptor subunit fused to the Enzyme Donor (ED) and a second CSF1R dimer partner fused to the Enzyme Acceptor (EA). When human IL-34 binds to one of the receptor subunits, its interaction with its dimer partner is induced, forcing complementation of the two enzyme fragments. This forms a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal. The reduction in relative fluorescence units (RFU) shown in Table 6 and / or Figure 2 reflects the ability of Antibody 1 to neutralize human IL-34 and reduce chemiluminescence. The half-maximal inhibitory concentration (IC 50 ) value of Antibody 1 is 226 pM. Human CSF1R-Fc is used as a positive control in this assay and inhibits luciferase activity with an IC 50 of 353 pM. The data in Table 6 and Figure 2 support the ability of Antibody 1 to block the interaction between human IL-34 and CSF1R, and by that ability, inhibit CSF1R dimerization in this assay. This data supports the use of the antibodies of the present invention to neutralize human IL-34.

Table 7

[0084] The ability of an anti-IL-34 antibody to inhibit ERK phosphorylation in NIH-3T3 / CSF1R cells. Alternatively, neutralization of IL-34 can be determined by seeding NIH-3T3 / CSF1R cells in 96-well plates and evaluating the ability of an anti-IL-34 antibody to inhibit extracellular signal-regulated kinase (ERK) phosphorylation. In this assay, cells are seeded in DMEM supplemented with 10% FBS on day 1 and incubated overnight at 37 °C. On day 2, the medium is removed, the cells are washed with serum-free DMEM, and incubated for an additional 24 hours in serum-free DMEM. On day 3, the medium is removed and 1 μg / ml of IL-34 is added to the cells for 5 minutes in the presence or absence of an anti-IL-34 antibody. Phospho / total ERK1 / 2 is evaluated by the Whole Cell Lysate Kit (Meso Scale Discovery, catalog number K15107D-3). The reduction in relative light units (RLU) shown in Table 7 and / or Figure 3 reflects the ability of antibody 3 to neutralize IL-34 and reduce chemiluminescence. The half-maximal inhibitory concentration (IC 50 ) value of antibody 3 is 16 nM. Human CSF1R-Fc was used as a positive control in this assay and inhibited luciferase activity with an IC 50 of 63 nM.

Table 8

[0085] The ability of an anti-IL34 antibody to inhibit IL-34-induced expression of CD163 in human monocytes by flow cytometry: Neutralization of IL-34 can also be evaluated by measuring the expression of cell surface antigen CD163 in human monocytes after treatment with IL-34 by flow cytometry (see, for example, Boulakirba, S., et al. IL-34 and CSF-1 display an equivalent macrophage differentiation ability but a different polarization potential. Sci Rep 8, 256 (2018)). CD14-positive monocytes are treated with IL-34 for 72 days, stained with an antibody against CD163, and then the expression of CD163 is evaluated by flow cytometry. In the experiment described in FIG. 4, the rightward shift in the number of cells expressing CD163 indicates that IL-34 treatment increases the expression of this antigen in monocytes. The addition of antibody 3 inhibits the increase in CD163 expression. In this experiment, an isotype-matched IgG4 antibody is used as a negative control.

[0086] Inhibition of CD163 expression in human monocytes by antibody 3 in response to IL-34 demonstrates the ability of the antibodies of the present invention to modulate the monocyte / macrophage number and / or phenotypic differentiation response to IL-34 and supports the use of such antibodies for treating immune-mediated diseases such as neuroinflammation and other inflammatory conditions (see, for example, Lelios, I. et al. Emerging roles of IL-34 in health and disease, J Exp Med (2020) 217(3):e20190290).

[0087] Example 4: In Vivo Functional Characterization of Anti-IL-34 Antibodies Ability of antibody 3 to reduce the number of microglial cells in the dorsal cortex and hippocampus of mice The ability of the anti-IL-34 antibody of the present invention to reduce microglial cell numbers is tested using a mouse model. Briefly, FBV female mice (Envigo, 6-8 weeks old) are administered subcutaneously with 50 mg / kg of antibody 3 or an isotype-matched IgG control antibody. At different time points (1, 3, 7, and 14 days) after antibody administration, the animals are euthanized with CO2 and the brains are perfused with saline. After perfusion, brain tissues are harvested, and the dorsal cortex and hippocampus are dissected under a microscope and snap-frozen in liquid nitrogen. mRNA is prepared from the dorsal cortex and hippocampus and used to evaluate the effect of anti-IL34 antibody or control antibody treatment on the expression of the microglial markers Iba-1 and CD11b by TaqMan. Changes in mRNA expression are normalized to an internal GAPDH mRNA control and presented as fold change. The expression of Iba-1 and CD11b mRNA is routinely used as surrogate markers for microglial cell numbers, and a reduction in the expression of these markers is generally recognized to reflect a reduction in brain microglia. The expression of two of the receptors for IL-34, CSF1R and PTP zeta, and the mRNA of both CSF1R ligands (IL-34 and CSF-1) are also evaluated after administration of antibody 3. The results are shown in Table 8 (A - hippocampus, and B - cortex).

Table 9

Table 10

[0088] Ability of antibody 3 to reduce microglial cell numbers in a mouse model of Alzheimer's disease (Tg4510 mice): The ability of the anti-IL-34 antibody of the present invention to reduce microglial cell numbers in a mouse model of Alzheimer's disease (Tg4510 mice) is also tested. Tg4510 mice are a model of tauopathy characterized by overexpression of the P301L mutant form of human tau in the forebrain. These mice are used to study the formation of neurofibrillary tangles as a model of Alzheimer's disease, neurodegenerative tauopathy, and frontotemporal dementia, and show age-dependent and region-specific progression of neuropathology associated with cognitive impairment. Briefly, 8-week-old female Tg4510 mice are administered 5, 15, and 50 mg / kg of the anti-IL-34 antibody or an isotype-matched IgG control biweekly for 9 consecutive weeks. At the end of the administration period, the animals are euthanized and perfused with PBS. After perfusion, brain tissue is collected, and both cerebral hemispheres are separated from each cerebral hemisphere through a mid-sagittal section, and the dorsal cortex and hippocampus are dissected microscopically and snap-frozen in liquid nitrogen. To evaluate the effect of the anti-IL-34 antibody on the expression of microglial markers, mRNA is prepared from the hippocampus, and the expression of CD11b, Iba-1, and CSF1R is analyzed by real-time PCR as shown in Table 9 for the hippocampus.

Table 11

[0089] Ability of the anti-IL34 antibody to reduce phosphorylated tau in Tg4510 mice: To evaluate whether an anti-IL-34 antibody can reduce phospho-tau, one of the pathological forms of tau associated with Alzheimer's disease, protein lysates are prepared from the cortex of animals treated with the anti-IL-34 antibody or an IgG control antibody. Briefly, wells of a 96-well plate are coated with an anti-phospho-tau antibody (AT8) and incubated at 4°C for 24 hours. The next day, the plate is washed with 0.05% Tween / PBS, blocked with SynBlock™ buffer (ImmunoChemistry Technology), incubated with the cortical lysates at 4°C for 24 hours, followed by addition of a biotinylated tau detection antibody (CP27) and streptavidin-horseradish peroxidase conjugate. As shown in Table 9, treatment with the anti-IL-34 antibody reduced phosphorylated tau in the cortex of Tg4510 mice.

Table 12

[0090] Sequence Listing of Amino Acid and Nucleotide Sequences Heavy Chain of Antibody 1 (SEQ ID NO: 1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYATSWVRQAPGKGLEWVSAISHSGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRSSLDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Light chain of Antibody 1, LC of Antibody 2 (SEQ ID NO: 2) EIVLTQSPGTLSLSPGERATLSCRASQSISSAYLAWYQQKPGQAPRLLIYASSIRPTGIPDRFSGSGSGTDFTLTISPLEPEDFAVYYCSQYGDSLSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HCVR of Antibody 1 (SEQ ID NO: 3) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYATSWVRQAPGKGLEWVSAISHSGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRSSLDTWGQGTLVTVSS LCVR of Antibody 1, LCVR of Antibody 2 (SEQ ID NO: 4) EIVLTQSPGTLSLSPGERATLSCRASQSISSAYLAWYQQKPGQAPRLLIYASSIRPTGIPDRFSGSGSGTDFTLTISPLEPEDFAVYYCSQYGDSLSFGGGTKVEIK HCDR1 of Antibody 1 (SEQ ID NO: 5) AASGFTFSSYATS HCDR2 of Antibody 1 (SEQ ID NO: 6) AISHSGRSTYYADSVKG HCDR3 of Antibody 1 (SEQ ID NO: 7) ARGRSSLDT LCDR1 of Antibody 1 and Antibody 2 (SEQ ID NO: 8) RASQSISSAYLA LCDR2 of Antibody 1 and Antibody 2 (SEQ ID NO: 9) YASSIRPT LCDR3 of Antibody 1 and Antibody 2 (SEQ ID NO: 10) SQYGDSLS DNA encoding the heavy chain of Antibody 1 (SEQ ID NO: 11) DNA encoding the light chain of antibody 1 (SEQ ID NO: 12) gaaatcgtactgacccaaagcccagggactttgagtttgtcccctggggaaagagcaaccctctcatgtcgtgcaagtcaaagtatatccagtgcatatcttgcttggtatcagcaaaagcctggtcaagcaccaaggctgcttatttatgcctcatctattagacctacaggtatccctgaccgattctccggaagtggcagtgggactgatttcacacttacaatttcccccctggaacctgaagactttgccgtatattattgttcacagtatggcgactcacttagtttcgggggcggcacaaaggttgaaataaagcggactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc Heavy chain of antibody 2 (SEQ ID NO: 13) EVQLLESGGGLVQPGGSLRLSCAASGFTFFSYAHSWVRQAPGKGLEWVSAISHSGRSTYYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRSSLDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG HCVR of Antibody 2 (SEQ ID NO: 14) EVQLLESGGGLVQPGGSLRLSCAASGFTFFSYAHSWVRQAPGKGLEWVSAISHSGRSTYYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRSSLDTWGQGTLVTVSS HCDR1 of Antibody 2 (SEQ ID NO: 15) AASGFTFFSYAHS HCDR2 of Antibody 2 (SEQ ID NO: 16) AISHSGRSTYYADAVKG HCDR3 of Antibody 2 (SEQ ID NO: 17) ARGRSSLDT DNA encoding the heavy chain of Antibody 2 (SEQ ID NO: 18) Heavy chain of antibody 3 (SEQ ID NO: 19) EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLEWVSAISHRGGSTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRSSLDGWGQGTMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light chain of antibody 3 (SEQ ID NO: 20) EIVLTQSPGILSLSPGERASLSCRASTSVSSAYLAWYQQKPGQAPRLLIYASSHRPLGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGDSLSFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC HCVR of antibody 3 (SEQ ID NO: 21) EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLEWVSAISHRGGSTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRSSLDGWGQGTMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK LCVR of Antibody 3 (SEQ ID NO: 22) EIVLTQSPGILSLSPGERASLSCRASTSVSSAYLAWYQQKPGQAPRLLIYASSHRPLGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGDSLSFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC HCDR1 of Antibody 3 (SEQ ID NO: 23) AASGFTFSYYAMS HCDR2 of Antibody 3 (SEQ ID NO: 24) AISHRGGSTLYADSVKG HCDR3 of Antibody 3 (SEQ ID NO: 25) ARGRSSLDG LCDR1 of Antibody 3 (SEQ ID NO: 26) RASTSVSSAYLA LCDR2 of Antibody 3 (SEQ ID NO: 27) YASSHRPL LCDR3 of Antibody 3 (SEQ ID NO: 28) QQYGDSLS DNA encoding the heavy chain of antibody 3 (SEQ ID NO: 29) DNA encoding the light chain of antibody 3 (SEQ ID NO: 30) gagatagtactgacccaaagtccgggtatattgtccctcagccctggcgaacgagccagccttagctgtcgggcctccaccagtgtgtcaagcgcgtacttggcctggtaccaacaaaaaccaggccaagcgcctcgactcctgatatatgcctcctcccaccggccccttggaatcccggacagattttccggttcaggctcaggtacagattttaccctcactatatcccgactggagcccgaggacttcgcagtatattactgccagcagtatggtgactccctctcctttggcgggggtactaaggttgaaatcaagcgggctgatgcggcgcccactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgctagcgtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt Human IL-34 (SEQ ID NO: 41) NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP Mouse IL-34 (SEQ ID NO: 42) NENLEIWTLTQDKECDLTGYLRGKLQYKNRLQYMKHYFPINYRIAVPYEGVLRVANITRLQKAHVSERELRYLWVLVSLNATESVMDVLLEGHPSWKYLQEVQTLLENVQRSLMDVEIGPHVEAVLSLLSTPGLSLKLVRPKALLDNCFRVMELLYCSCCKQSPILKWQDCELPRLHPHSPGSLMQCTATNVYPLSRQTPTSLPGSPSSSHGSLP IgG4 PAA hinge region (SEQ ID NO: 51) ESKYGPPCPPCP IgG4 PAA Fc region (SEQ ID NO: 52) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

Claims

1. An antibody that binds to human IL-34, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

10. An antibody.

2. The antibody according to claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO:

4.

3. The antibody according to claim 1 or 2, wherein the antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 1 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:

2.

4. A nucleic acid comprising the HC DNA sequence of SEQ ID NO: 11 and the LC DNA sequence of SEQ ID NO:

12.

5. A vector comprising the nucleic acid according to claim 4.

6. A composition comprising a first vector comprising the HC DNA sequence of SEQ ID NO: 11 and a second vector comprising the LC DNA sequence of SEQ ID NO:

12.

7. A cell comprising the vector according to claim 5.

8. A cell comprising a first vector comprising the HC DNA sequence of SEQ ID NO: 11 and a second vector comprising the LC DNA sequence of SEQ ID NO:

12.

9. The cell according to claim 7 or 8, wherein the cell is a mammalian cell.

10. A process for producing an antibody, comprising culturing the cell according to any one of claims 7 to 9 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

11. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient, diluent, or carrier.

12. A therapeutic agent for an immune-mediated disease in a subject in need of treatment, comprising the antibody according to any one of claims 1 to 3.

13. The therapeutic agent according to claim 12, wherein the immune-mediated disease is selected from the group consisting of Alzheimer's disease, tauopathy, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

14. The therapeutic agent according to claim 12, wherein the immune-mediated disease is Alzheimer's disease.

15. Use of the antibody according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of an immune-mediated disease.

16. The use according to claim 15, wherein the immune-mediated disease is selected from the group consisting of Alzheimer's disease, tauopathy, Sjögren's syndrome (SS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

17. The use according to claim 15, wherein the immune-mediated disease is Alzheimer's disease.

Images