Compounds and methods targeting interleukin-34

Abstract

The present invention provides alternative and / or improved anti-IL-34 antibodies, as well as pharmaceutical compositions thereof. [Solution] A drug comprising an anti-IL-34 antibody for use in a method of treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of human subjects, wherein the method comprises administering an anti-N3pG Aβ antibody simultaneously, separately, or sequentially in combination with the anti-IL-34 antibody, wherein the anti-IL-34 antibody comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 each containing a specific sequence, and light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 each containing another specific sequence.

Description

[Technical Field]

[0001] This disclosure 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 therapeutic and / or diagnostic applications related to Alzheimer's disease and other tauopathies.

[0002] Alzheimer's disease (AD), the leading cause of dementia, affects 1% of the population between the ages of 65 and 69, increasing to 40% to 50% in those over 95. Patients with AD exhibit clear clinical symptoms, including cognitive impairment and memory deficits. In these patients, the presence of AD is confirmed by severe senile plaque load and neurofibrillary tangles (NFTs) found in the cerebral cortex on postmortem histopathological examination. Mature senile plaques consist of extracellular β-amyloid peptides derived from enzymatic treatment of amyloid precursor protein and intracellular neurofibrillary tangles (NFTs) derived from filaments of hyperphosphorylated tau protein. Aggregates of hyperphosphorylated tau, such as neurofibrillary tangles, are associated with the degree of cognitive impairment in Alzheimer's disease. In AD and various other tauopathy, tau aggregates appear in specific brain regions and patterns associated with disease risk, onset, and / or progression, 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 conditions. The central nervous system (CNS), where blood-derived immune cells circulate minimally, appears to be particularly vulnerable to dysregulated cytokine networks. In neurodegenerative diseases, CNS resident cells are major producers of pro-inflammatory cytokines and may contribute to dysregulated cytokine networks and neuroinflammation. Damage to the central nervous system may involve the recruitment of circulating immune cells, resulting in an innate immune response consisting of resident microglia, peripheral-derived monocytes, macrophages, and dendritic cells. The activated states of microglia and macrophages are not strictly pro-inflammatory or anti-inflammatory, but rather can exhibit a variety of functional states. Microglia and / or peripheral-derived monocytes and macrophages may acquire an anti-inflammatory phenotype that removes necrotic tissue fragments and promotes regeneration and homeostasis. Neurological dysfunction or injury may also activate microglia to produce pro-inflammatory cytokines and recruit leukocytes from the bloodstream. In neurodegenerative states such as Alzheimer's disease (AD), microglial activation is frequently observed, reflecting the tissue response to the accumulation of extracellular beta-amyloid plaques and hyperphosphorylated tau aggregates. Neuroinflammation is a crucial component 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 be underlying mechanisms in neurodegenerative diseases such as plaque accumulation in Alzheimer's disease and neuronal cell death and dysfunction in Parkinson's disease and Huntington's disease.

[0004] Microgliosis involves the abnormal proliferation and / or hypertrophy of microglia in response to inflammatory signals. Generally, IL-34 acts as a potent and multifaceted cytokine in regulating inflammatory and immune processes and is a key regulatory cytokine for the proliferation of CNS-resident microglia in normal tissue homeostasis. IL-34 is expressed by neurons in the cortex, preolar nucleus, and hippocampus. Although IL-34 shows low sequence homology with CSF-1, it has a similar general structure, and both cytokines bind to a common receptor, CSF-1R, causing receptor autophosphorylation and dimerization, which subsequently activates multiple signaling pathways (A. Freuchet, et al J Leukoc Biol 2021 Oct;110(4):771-796). IL-34 is a secreted homodimeric cytokine that acts as one of the two activating ligands for CSF1R, inducing receptor autophosphorylation and dimerization, which subsequently activates multiple signaling pathways (see, for example, Structural basis for the dual recognition of helical cytokines 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 IL-34 polypeptides are disclosed, for example, in U.S. Patent No. 9,770,486, and consist of 242 amino acids in a leader sequence and 222 amino acids in a mature form (SEQ ID NO: 31).

[0005] Anti-IL-34 antibodies have been described in the art; for example, International Publication No. 2016 / 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] Therefore, there remains an unmet need for alternative and / or improved anti-IL-34 antibodies, their pharmaceutical compositions, and methods of using them for therapeutic and / or diagnostic applications related to immune-mediated diseases involving IL-34, and / or neuroinflammatory disorders, and / or Alzheimer's disease. [Overview of the project]

[0007] Embodiments of this disclosure provide novel anti-human IL-34 antibodies. According to some embodiments, the disclosure provides antibodies 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 a set of CDR combinations provided in Table 1. Sequence identifiers used herein are listed in Table 1 and throughout the specification, and sequences are provided in the amino acid and nucleotide sequence listings provided herein.

[0008] [Table 1]

[0009] Accordingly, embodiments of the present disclosure provide an antibody that binds to human IL-34, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, the VL comprising light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, the HCDR1 comprising SEQ ID NO: 5, the HCDR2 comprising SEQ ID NO: 6, the HCDR3 comprising SEQ ID NO: 7, the LCDR1 comprising SEQ ID NO: 8, the LCDR2 comprising SEQ ID NO: 9, and the LCDR3 comprising SEQ ID NO: 10.

[0010] Accordingly, embodiments of the present disclosure also provide antibodies comprising LCVR having the amino acid sequence of SEQ ID NO: 4 and HCVR having the amino acid sequence of SEQ ID NO: 3.

[0011] Accordingly, embodiments of the present disclosure further provide an antibody that binds to human IL-34, the antibody comprising a heavy chain (HC) containing SEQ ID NO: 1 and a light chain (LC) containing SEQ ID NO: 2.

[0012] In other embodiments, the disclosure also provides antibodies comprising 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, wherein the hinge region and Fc region are selected from SEQ ID NO: 32 and SEQ ID NO: 33.

[0013] 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, and the LC amino acid sequence of SEQ ID NO: 2. Antibody 1 may be encoded by the HC DNA sequence of SEQ ID NO: 11 and the LC DNA sequence of SEQ ID NO: 12. The framework and CDR sequences in each of the antibodies whose sequences are shown herein are annotated using annotation rules consistent with the method of North, et al., J.Mol.Biol.2011:406:228-256, unless otherwise specified.

[0014] In other embodiments, the disclosure also provides an antibody comprising an LC having an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 2, and an HC having an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 1.

[0015] In other embodiments, the disclosure also provides an antibody comprising LC having the amino acid sequence of SEQ ID NO: 2 and HC having the amino acid sequence of SEQ ID NO: 35, which is further referred to herein as antibody 2.

[0016] In other embodiments, the disclosure also provides an antibody comprising LC having the amino acid sequence of SEQ ID NO: 2 and HC having the amino acid sequence of SEQ ID NO: 36, which is further referred to herein as antibody 3.

[0017] In other embodiments, the disclosure also provides an antibody comprising LC having the amino acid sequence of SEQ ID NO: 2 and HC having the amino acid sequence of SEQ ID NO: 37, which is further referred to herein as antibody 4.

[0018] The carboxy-terminal portion of each HC defines a constant region that primarily bears effector function, and in some embodiments of the present disclosure, the antibody has one or more modifications in the constant region of each HC that reduce effector function. Preferably, embodiments of the present disclosure 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.

[0019] 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 include modifications in the constant regions of both HCs that include amino acid alanine at both residues 230 and 231 (each exemplified by the HC of antibody 1 and SEQ ID NO: 33), further modifications in the constant regions of both HCs that include amino acid proline at residue 224 and promote stability (the HC of antibody 1 and exemplified by, for example, SEQ ID NO: 32), and a deletion of amino acid lysine at residue 443 (exemplified by the HC of SEQ ID NO: 1).

[0020] The antibodies of the present disclosure are thought to have a particularly advantageous combination of properties relative to prior art anti-IL-34 antibodies, including, but not limited to, one or more of the following properties: 1) desirable association and dissociation rates, 2) potency in neutralizing human IL-34 to achieve an anti-neuroinflammatory response and in vivo efficacy, 3) sufficient strength as a monotherapy for the treatment and / or prevention of immune-mediated and / or inflammatory disorders, 4) a sustained duration of action, 5) induction of sufficiently limited undesirable cytokine release, 6) acceptably low immunogenicity (i.e., sufficiently non-immunogenic in humans), 7) avoidance of undesirable immune depletion, and / or 8) desirable in vivo stability, physical and chemical stability, including, but not limited to, thermal stability, solubility, low self-association, and pharmacokinetic characteristics, that are acceptable for development and / or use in the treatment of inflammatory or neuroinflammatory disorders, such as AD.

Mode for Carrying Out the Invention

[0021] Embodiments of the present disclosure represent a significant advance over the prior art by providing compositions and methods useful for the prevention, downregulation, or improvement of inflammatory and / or neuroinflammatory disorders via the neutralization of IL-34, using pharmacologically advantageous anti-human IL-34 antibodies, such as those provided in the embodiments described herein. The anti-human IL-34 antibodies of the present disclosure can improve immune and / or inflammatory pathologies or restore immunohomeostasis, preferably by inhibiting the innate immune portion of the immune response and / or suppressing the activation and / or proliferation of microgliosis or other monocyte / macrophage cell lineages, thereby directly altering the pathology of the underlying disease. Clinical use of such antibodies may lead to long-term persistence of the disease being treated.

[0022] Furthermore, there is a need for diagnostic anti-human IL-34 antibodies that are specific to human IL-34, have improved binding affinity, and exhibit improved sensitivity in human IL-34 measurement, as well as for improved enzyme-linked immunosorbent assay (ELISA) assay conditions that result in minimal interference and broad dilution linearity. According to some aspects of this disclosure, anti-human IL-34 antibodies are provided, including human IL-34 neutralizing antibodies that bind to human IL-34 as given by SEQ ID NO: 31. Interleukin 34 (IL-34; also known as the unidentified protein C16 or f77) is secreted as a homodimer consisting of a 39 kDa monomer. It does not belong to any known cytokine family. Human IL-34 is synthesized as a 242-amino acid (AA) precursor containing 20 AA signal sequences, resulting in 222 AA mature chains. As used herein, IL-34 refers to the mature chains. The mature chains contain one site that is potentially N-linked glycosylation. IL-34 is expressed in various tissues, including the heart, brain, liver, kidneys, spleen, thymus, testes, ovaries, small intestine, prostate, and colon, and is most abundant in the spleen. When used herein in relation to the IL-34 polypeptide, "h IL-34" or "human IL-34" refers to wild-type human IL-34 unless otherwise specified, preferably having the amino acid sequence shown in SEQ ID NO: 31, 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).

[0023] Exemplary human IL-34 (SEQ ID NO: 31) has the amino acid sequence: NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP.

[0024] As used herein, "human anti-IL34 antibody" or "anti-human IL-34 antibody" refers to an antibody that binds to human IL-34. Preferably, a "human anti-IL34 antibody" or "anti-human IL-34 antibody" administered in vitro or in vivo results in a desirable decrease in IL-34 signaling, as demonstrated by neutralization of IL-34 activity and / or blockade of responses, e.g., 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 central nervous system is an example of an IL-34-responsive molecule or cellular effect possibility. As used herein, the terms "signaling" and "signal transduction" and "IL-34-mediated" when referring to IL-34 refer to cellular and / or intercellular responses resulting from the activity of IL-34.

[0025] 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 conjugate antibodies. Antibodies may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and 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) crosslinked via interchain disulfide bonds. The LCs are classified as kappa or lambda, each characterized by a specific constant region. Embodiments of the present disclosure may include IgG1, IgG2, or IgG4 antibodies, and may further include kappa or lambda light chains. Preferably, the antibodies of the present disclosure include a light chain constant region which is a κ constant region.

[0026] HC is classified as gamma, mu, alpha, delta, or epsilon, defining 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 approximately 100 to 125 or more amino acids, primarily involved in antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region, primarily involved in effector function. Each heavy chain consists 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 is located after 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 is located after the hinge region and before CH3. CH3 is located after CH2 and at the carboxyl terminus of the heavy chain. The constant region of the light chain contains one domain, CL. CL is located after LCVR, and CL and LCVR form the light chain portion of the Fab.

[0027] The antibodies of this disclosure include IgG HCs, which can be further classified into subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, and embodiments of this disclosure may include one or more modifications to the constant region of each HC, for example, to enhance or reduce effector function. As used herein, the term “Fc region” refers to the region of the antibody containing the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion or the entire hinge region of the antibody heavy chain. IgG1 is known to induce antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and the Fc mutations described herein may reduce aggregation, reduce or enhance ADCC or CDC activity (or other function), and / or modify the pharmacokinetics of the antibody. Embodiments of anti-human IL-34 antibodies described herein reduce binding to FcγR and C1q receptors, thereby reducing or eliminating cytotoxicity that may be induced by antibodies having a wild-type IgG Fc region. Accordingly, according to some embodiments, mutations are introduced into the Fc region at the locations described herein. By sufficiently reducing or eliminating the effector function of such anti-human IL-34 antibodies containing a modified Fc region, patient safety can be improved, and in combination with other properties described herein, therapeutic agents with an improved profile of useful activity while avoiding undesirable activity can be provided.

[0028] When expressed in a specific biological system, antibodies are glycosylated at their Fc region. Typically, glycosylation occurs at the highly conserved N-glycosylation sites in the Fc region of antibodies. The N-glycan typically binds to asparagine. Antibodies can also be glycosylated at other sites. The antibodies of this disclosure are monoclonal antibodies. Monoclonal antibodies are antibodies derived from a single copy or clone (e.g., any eukaryote, prokaryote, or phage clone) and are not defined by the method by which they are produced. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage presentation technology, synthesis technology, e.g., CDR transplantation, or a combination of such technologies or other technologies known in the art. This disclosure intends that the antibodies of this disclosure 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 this disclosure also include antibody fragments or antigen-binding fragments, comprising 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 fragment, scFv antibody fragment, disulfide-linked Fv(sdFv), Fd fragment, and linear antibodies.

[0029] The amino-terminal portions of each LC and HC contain a variable region of approximately 100-120 amino acids, primarily responsible for antigen recognition via the CDR contained within. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which contain scattered, more conserved regions called framework regions (FRs). CDRs are exposed on the surface of the protein and are crucial regions of the antibody for antigen-binding specificity. Each VH and VL consists of three CDRs and four FRs, arranged from the amino-terminal to the carboxyl-terminal 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." CDRs contain the majority 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 six CDRs.The assignment of amino acid residues to CDRs is attributed to 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 This can be done according to well-known schemes, including those described in the database available at www.imgt.org (Lefranc et al., Nucleic Acids Res. 1999;27:209-212).

[0030] For the purposes of this disclosure, unless otherwise specified, North's CDR definition is used for the assignment of amino acids to the CDR domains within the LCVR and HCVR regions of the anti-IL-34 antibodies described herein. Table 2 below provides the CDR sequences of antibody 1 and / or the antibodies of this disclosure, generated using Benchling informatics software, according to the rules of North, Kabat, Chothia, and / or IMGT, respectively.

[0031] [Table 2]

[0032] Embodiments of the antibodies described herein possess a combination of pharmacologically useful and important activities and properties, in some respects being able to bind to human IL-34 with high affinity and high specificity, as well as other useful properties. The term “binding” as used herein is intended to mean the ability of a protein or molecule to form an attractive interaction with another protein or molecule, unless otherwise specified, resulting in proximity of the two proteins or molecules as determined by common methods known in the art. The phrase “specifically binding” as used herein with respect to the affinity of an anti-IL-34 antibody to human IL-34 means, unless otherwise specified, essentially as described herein, preferably about 1 × 10⁻⁶, as determined by common methods known in the art, including by the use of solution equilibrium titration (SET) measured by an SPR (Surface Plasmon Resonance) biosensor and / or an MSD (Meso Scale Discovery) instrument. -10 Less than M, more preferably about 1 × 10 -10 M ~ approx. 1×10 -12 M's K D This is intended to mean that the phrase "specifically binds" also indicates the relative affinity of an anti-IL-34 antibody to human IL-34 compared to other antigens, and this affinity for human IL-34 results in the specific recognition of human IL-34.

[0033] Embodiments of the antibodies of this disclosure can be expressed and produced from constructs containing the sequences of these embodiments 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 this disclosure may also include, for example, DNA or RNA polymerase or substrates incorporated therein by synthetic reactions. The DNA molecules of this disclosure are DNA molecules containing a polynucleotide sequence that does not exist in nature and encodes a polypeptide having at least one amino acid sequence from among the polypeptides (e.g., heavy chain, light chain, variable heavy chain, and variable light chain) in the antibodies of this disclosure.

[0034] Isolated DNA encoding the HCVR or LCVR region can be converted into a full-length heavy-chain gene by operably ligating the HCVR or LCVR-encoding DNA to another DNA molecule encoding the heavy-chain or light-chain constant region, respectively, in order to form the heavy-chain or light-chain. The sequences of heavy-chain constant region genes in humans and other mammals are known in the art. DNA fragments containing these regions can be obtained, for example, by standard PCR amplification.

[0035] The polynucleotides of this disclosure can be expressed in host cells after the sequence has been operably ligated to an expression regulatory sequence. Expression vectors are typically replicable in the host organism, either as episomes or as an integrated portion of host chromosomal DNA. Generally, expression vectors include selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to enable detection of those cells transformed with the desired DNA sequence. Vectors containing the polynucleotide sequence of interest (e.g., a polynucleotide encoding an antibody polypeptide and an expression regulatory sequence) can be introduced into host cells by different well-known methods depending on the type of cell host.

[0036] The antibodies of this disclosure can be readily produced in mammalian cells, non-limiting examples of which include CHO, NS0, HEK293, or COS cells. Host cells are cultured using techniques well known in the art. Mammalian antibody expression typically results in glycosylation. Antibody glycosylation is typically 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 a highly conserved N-glycosylation site (e.g., position 297 in IgG1, according to IMGT or EU index numbering). Glycosylation sites can be modified to alter glycosylation (e.g., by blocking or reducing glycosylation, or by altering the amino acid sequence to generate additional or diverse glycosylation).

[0037] The expression of antibodies from IgG subclasses in mammals can result in clipping of C-terminal amino acids from one or both heavy chains; for example, in the case of IgG1 antibodies, one or two C-terminal amino acids may be removed. In the case of IgG1 antibodies, if C-terminal lysine is present, it may be truncated or cleaved from the heavy chain during expression. Additionally, the second-to-last glycine may also be truncated or cleaved from the heavy chain.

[0038] Antibody expression in mammals can also lead to modifications of the N-terminal amino acid. For example, if the N-terminal amino acid of the heavy or light chain is glutamine, it can be modified to pyroglutamic acid.

[0039] The antibodies disclosed herein, or pharmaceutical compositions containing them, may be administered by parenteral routes, non-limiting examples of which include subcutaneous and intravenous administration. The antibodies disclosed herein may be administered to a patient in single or multiple doses together with a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions disclosed herein may 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 may include the antibodies disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0040] Use of antibody embodiments of the present invention: According to several embodiments, the anti-IL-34 antibodies of this disclosure are useful for the treatment of immune-mediated diseases. As used herein, the terms “immune-mediated disease” and “inflammatory disease or disorder” are interchangeable and refer to undesirable conditions resulting from an inappropriate or excessive immune response in which IL-34 inhibition elicits a more homeostatic and less pathological response. The terms “immune-mediated disease” and “inflammatory disorder” mean that such conditions include those mediated by a cellular immune response of microglia or macrophages, or by a response of similar tissue-resident cell types such as histiocytes, Kupffer cells, alveolar macrophages, intestinal macrophages, macrophage-like synovial cells, or Langerhans cells. Examples of diseases that are intended to be treated with the antibodies of this disclosure as described herein include Alzheimer's disease; tauopathic diseases; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD); atopic dermatitis; renal disease; sepsis; amyotrophic lateral sclerosis (ALS); and / or non-alcoholic fatty liver disease (NAFLD).

[0041] In some more specific embodiments, the immune-mediated disease is Alzheimer's disease (AD). According to other embodiments of the present disclosure, anti-IL-34 antibodies are useful for diagnostic purposes 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, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0042] The Disclosure further provides a pharmaceutical composition comprising the anti-IL-34 antibody of the Disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients. Furthermore, the Disclosure provides a method for treating immune-mediated diseases such as AD; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD), comprising administering the pharmaceutical composition of the Disclosure to a patient in need thereof.

[0043] In addition, the Disclosure provides a method for treating immune-mediated diseases. More specifically, the Disclosure provides a method for treating immune-mediated diseases, including AD; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD), comprising administering an effective amount of the anti-IL-34 antibody of the Disclosure to a patient in need thereof.

[0044] This disclosure also provides anti-IL-34 antibodies for therapeutic use. More specifically, this disclosure provides anti-IL-34 antibodies for use in the treatment of immune-mediated diseases, including AD; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0045] In certain embodiments, the Disclosure provides the use of the anti-IL-34 antibody of the Disclosure in the manufacture of a pharmaceutical product for the treatment of one or more immune-mediated diseases, including AD; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

[0046] The antibodies of this disclosure are useful for identifying immune-mediated disorders in which IL-34 may contribute to the pathogenesis of the disorder. In further embodiments, this disclosure provides a method for treating an immune-mediated disorder in a patient. Such a method includes the steps of contacting a patient sample with an anti-IL-34 antibody and detecting the binding between human IL-34 in the patient sample and the antibody, and diagnosing that, if the presence of IL-34 in the patient sample is detected above a baseline level observed in an unaffected individual, the patient has, is at risk of having, requires treatment for, and / or is at risk of symptoms associated with an immune-mediated disorder (e.g., Xie, HH, et al. Elevated Serum Interleukin-34 Level in Patients with Systemic Lupus Erythematosus Is Associated with Disease Activity. Sci Rep. See 8,3462 (2018). According to some more specific embodiments of the therapeutic methods provided herein, such methods further include the steps of: determining a reference value, which includes contacting a control standard with a first antibody that binds to the same first epitope region of IL-34 as that used when contacting a patient sample; contacting a control standard with a second antibody having a detectable label and binding to the same second epitope region of IL-34 as that used when contacting a patient sample; and detecting a signal provided by a detectable signal. In some specific embodiments, the anti-IL-34 antibody is LC and HC provided in Table 1. This includes a combination of CDRs. In further embodiments, the second antibody includes a combination of LCVR and HCVR provided in Table 1. According to some embodiments, the reference value is about 10 to 30 pg / mL, for example, from 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, renal 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 includes 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 further embodiments, the second antibody includes a combination of LC and HC CDR provided in Table 1. In further embodiments, the second antibody includes a combination of LCVR and HCVR provided in Table 1. According to certain embodiments, the first and second anti-IL-34 antibodies are not placed in a vial together.

[0047] According to several embodiments, the Disclosure provides a method for detecting IL-34 in a patient sample, comprising the steps of: contacting the patient sample with a first antibody bound to a first epitope region of IL-34; contacting the patient sample with a second antibody bound to a second epitope region of IL-34 and having 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. Furthermore, in some embodiments, the steps of contacting with the first and second antibodies occur simultaneously. In some specific embodiments, the first antibody includes a combination of LC and HC CDR provided in Table 1. In further embodiments, the first antibody includes a combination of LCVR and HCVR provided in Table 1.

[0048] According to some embodiments of this disclosure, a method is provided for quantifying IL-34 in a patient sample. Such a method includes the steps of: contacting the patient sample with a first antibody bound to a first epitope region of IL-34; contacting the patient sample with a second antibody bound to a second epitope region of IL-34 and having a detectable label; detecting a signal provided by the detectable label; contacting a control standard with the first antibody bound to the same first epitope region of IL-34 (as used when contacting the patient sample); contacting the control standard with the second antibody bound to the same second epitope region of IL-34 (as used when contacting the patient sample) and having a detectable label; and detecting a 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. Furthermore, in some embodiments, the step of contacting the first and second antibodies occurs simultaneously. In some specific embodiments, the first antibody includes a combination of LC and HC CDR provided in Table 1. In further embodiments, the first antibody includes a combination of LCVR and HCVR provided in Table 1. In some specific embodiments, the second antibody includes a combination of LC and HC CDR provided in Table 1 or herein. In further embodiments, the second antibody includes a combination of LCVR and HCVR provided in Table 1.

[0049] According to several embodiments, a method for diagnosing immune-mediated diseases is provided. Such a method includes contacting a patient sample with an anti-IL-34 antibody and detecting the binding between IL-34 in the patient sample and the antibody. According to some specific embodiments, the diagnostic method includes diagnosing that, if the presence of IL-34 in the patient sample is detected above a reference value, the patient has, is at risk of having, requires treatment for, and / or is at risk of symptoms associated with an immune-mediated disease. According to some more specific embodiments, such a method further includes a step of determining a reference value, which includes contacting a control standard with a first antibody that binds to the same first epitope region of IL-34 used when contacting the patient sample; a step of contacting the control standard with a second antibody having a detectable label and binding to the same second epitope region of IL-34 used when contacting the patient sample; and a step of detecting a signal provided by a detectable signal. In some embodiments, the first antibody includes a combination of LC and HC CDR provided in Table 1. Some embodiments of methods for diagnosing immune-mediated diseases provided herein further include the steps of contacting a patient sample with a second anti-IL-34 antibody conjugated to a second epitope region of IL-34 and having a detectable label, and detecting a signal provided by a detectable signal. In some specific embodiments, the anti-IL-34 antibody includes combinations of LC and HC CDR provided in Table 1. In further embodiments, the antibody includes combinations of LCVR and HCVR provided in Table 1. According to specific embodiments, the first epitope region of IL-34 partially overlaps with the second epitope region of IL-34. According to certain embodiments, the first and second antibodies are not placed together in a vial. According to further embodiments, the reference range is an approximate range of about 10 to 30 pg / mL from CNS tissue lysate and / or is determined by those skilled in the art for appropriate reference groups and sample sources.In further embodiments, the immune-mediated disease is one of the following: AD; tauopathy; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD); atopic dermatitis; renal disease; sepsis; and / or non-alcoholic fatty liver disease (NAFLD).

[0050] In one embodiment, the present disclosure provides a method for determining the level of human IL-34 in a body fluid sample, comprising: (a) contacting the body fluid with an anti-human IL-34 diagnostic monoclonal antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment includes light chain complementarity determination regions LCDR1, LCDR2, and LCDR3 containing the amino acid sequences (SEQ ID NO: 8), (SEQ ID NO: 9), and (SEQ ID NO: 10), respectively, and heavy chain complementarity determination regions HCDR1, HCDR2, and HCDR3 containing the amino acid sequences (SEQ ID NO: 5), (SEQ ID NO: 6), and (SEQ ID NO: 7), respectively; (b) optionally removing any nonspecifically bound monoclonal antibody or its antigen-binding fragment; and (c) detecting and / or quantifying the amount of monoclonal antibody or its antigen-binding fragment specifically bound to human IL-34. Preferably, the bodily fluid is blood, serum, plasma, or cerebrospinal fluid, and such contact occurs ex vivo.

[0051] Tauopathic diseases 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), Guam parkinsonism-dementia complex, Niemann-Pick disease type C, and myotonic dystrophy. (Li, C., Gotz, J. Tau-based therapies in neurodegeneration: opportunities and challenges. Nat Rev Drug Discov) See 16,863-883 (2017).

[0052] In embodiments of this disclosure, the patient is a human being diagnosed with a medical risk, condition, or disorder, such as one of the diseases or disorders described herein, requiring treatment with the antibodies described herein. Where the disorders that can be treated by the methods of this disclosure are known by established and accepted classifications, such as Alzheimer's disease; tauopathic diseases; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD), these classifications can be found in various well-known medical texts. For example, the Fifth Edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) currently provides a diagnostic tool for identifying the specific disorders described herein. Also, the Tenth Edition of the International Classification of Diseases (ICD-10) provides classifications for the specific disorders described herein. Those skilled in the art will recognize that there are alternative nomenclature, disease classification, and classification systems for the diseases and disorders described herein, including those described in DSM-5 and ICD-10, and that terminology and classification systems evolve with advances in medical science.

[0053] The terms “treating” (or “treat” or “treatment”) refer to delaying, interfering with, preventing, mitigating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease in a subject. The term “subject” refers to a human being. The terms “human subject” and “patient” are used interchangeably in this disclosure.

[0054] As used herein, “method of treatment” is equivalent to the use of a composition for treating a disease or disorder described herein, and / or its use in the manufacture of a medicinal product for treating a disease or disorder described herein, and / or the use of a composition for use.

[0055] The terms "prevent" or "prevention" refer to the prophylactic administration of the antibodies of the present invention to asymptomatic subjects or subjects suffering from preclinical Alzheimer's disease in order to prevent the onset or progression of Alzheimer's disease.

[0056] As used herein, the term “slow progression” means to slow or suppress the progression of a disease or its symptoms in the subject.

[0057] The terms “diseases characterized by Aβ deposition” or “diseases characterized by Aβ deposits” are used interchangeably and refer to diseases pathologically characterized by Aβ deposits in the brain or vascular structures. These include diseases such as Alzheimer’s disease, Down syndrome, and cerebral amyloid vascular disease. The clinical diagnosis, staging, or progression of Alzheimer’s disease can be readily determined by a diagnostician or healthcare professional skilled in the art by using known techniques and observing the results. This generally involves brain plaque imaging, mental or cognitive assessments (e.g., Clinical Dementia Rating-summary of box (CDR-SB), Mini-Mental State Exam (MMSE), or Alzheimer’s Disease Assessment Scale-Cognitive (ADAS-Cog)), or functional assessments (e.g., Alzheimer’s Disease Cooperative Study-Activities of Daily Living). Including Living, ADCS-ADL. Cognitive and functional assessments can be used to determine changes in a patient's cognition (e.g., cognitive decline) and function (e.g., functional decline). Thus, a subject may be determined to have “slowly progressive” cognitive decline according to the techniques described herein. In an exemplary embodiment, “slowly progressive” cognitive decline can be identified by iADRS, where the subject’s iADR has declined by less than approximately 20 over a given period (e.g., 6, 12, 18, or 24 months). Another exemplary embodiment Therefore, “slowly progressive” cognitive decline can be identified by APOE-4 genotyping, in which the subject is APOE-4 homozygous-negative or APOE-4 heterozygous. In another exemplary embodiment, “slowly progressive” cognitive decline can be identified by MMSE, where the subject is determined to have an MMSE score of approximately 27, or an MMSE decline of less than approximately 3 over a given period (e.g., 6, 12, 18, or 24 months). As used herein, “clinical Alzheimer’s disease” refers to the diagnosed stage of Alzheimer’s disease.This includes conditions diagnosed as prodromal Alzheimer's disease, mild Alzheimer's disease, moderate Alzheimer's disease, and severe Alzheimer's disease. The term “preclinical Alzheimer's disease” refers to the stage preceding clinical Alzheimer's disease, where measurable changes in biomarkers (such as CSFAβ42 levels or deposited brain plaques on amyloid PET) indicate the earliest signs of Alzheimer's disease in patients progressing to clinical Alzheimer's disease. This is usually before symptoms such as memory loss and confusion become prominent. Preclinical Alzheimer's disease includes not only patients at high risk of developing AD due to carrying one or two APOE e4 alleles, but also pre-symptomatic autosomal dominant carriers.

[0058] The reduction or slowing of cognitive decline can be measured by cognitive assessments such as the Clinical Dementia Assessment-Box Summary, the Mini-Mental State Examination, or the Alzheimer's Disease Assessment Scale-Cognition. The reduction or slowing of functional decline can also be measured by functional assessments such as the ADCS-ADL.

[0059] As used herein, "mg / kg" means the amount in milligrams of antibody or drug administered to a subject based on their body weight in kilograms. The dose is given in a single dose. For example, a 10 mg / kg dose of antibody for a subject weighing 70 kg is equivalent to a single 700 mg dose of antibody administered in one dose. Similarly, a 20 mg / kg dose of antibody for a subject weighing 70 kg is equivalent to a single 1400 mg dose of antibody administered in one dose.

[0060] When used in this specification, 18Using F-flortaucipir-based quantitative analysis, if the tau load is less than 1.10SUVr (<1.10SUVr), the human subject has a “very low” tau load, and the quantitative analysis refers to the calculation of SUVr, which represents the count in a specific target region of interest in the brain compared to a reference region (see multiblock centroid discriminant analysis or MUBADA, Devous et al, “Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18,” J.Nucl.Med.59:937-943(2018)) (see reference signal intensity or parametric estimate of PERSI, Southekal et al., “Flortaucipir F 18 Quantitation Using Parametric Estimation of Reference Signal Intensity,” J.Nucl.Med.59:944-951(2018)). As used herein, when using 18F-flotaucipir-based quantitative analysis, a human subject has a “very low to moderate” tau loading if the tau loading is less than or equal to 1.46SUVr (i.e., ≤1.46SUVr), and quantitative analysis refers to the calculation of SUVr, which represents the count in a specific target region of interest in the brain compared to a reference region (see MUBADA, Devous et al., “Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18,” J.Nucl.Med.59:937-943(2018)) (see PERSI, Southekal et al., “Flortaucipir F 18 Quantitation Using Parametric Estimation of Reference Signal Intensity,” J.Nucl.Med.59:944-951(2018)).

[0061] When used in this specification, 18Using F-flortaucipir-based quantitative analysis, if the tau load is between 1.10 and 1.46 (i.e., ≤1.10SUVr to ≤1.46SUVr), human subjects have a “low to moderate tau” load, and the quantitative analysis refers to the calculation of SUVr, which represents the count in a specific target region of interest in the brain compared to a reference region (see MUBADA, Devous et al., “Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18,” J.Nucl.Med.59:937-943(2018)) (see PERSI, Southekal et al., “Flortaucipir F 18 Quantitation Using Parametric Estimation of Reference Signal Intensity,” J.Nucl.Med.59:944-951(2018)). Human subjects with a "low to moderate" tau load may be described as having an "intermediate" tau load.

[0062] When used in this specification, 18 Using F-flortaucipir-based quantitative analysis, if the tau load is greater than 1.46SUVr (i.e., >1.46SUVr), a human subject has a “high tau” load, and the quantitative analysis refers to the calculation of SUVr, which represents the count in a specific target region of interest in the brain compared to a reference region (see MUBADA, Devous et al., “Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18,” J.Nucl.Med.59:937-943(2018)) (see PERSI, Southekal et al., “Flortaucipir F 18 Quantitation Using Parametric Estimation of Reference Signal Intensity,” J.Nucl.Med.59:944-951(2018)).

[0063] As used herein, the term "approximately" means a maximum of ±10%.

[0064] As used herein, the term “innate immunity” includes the arm of the immune response required to initiate and maintain the adaptive immune response (antibody and T-cell responses), as opposed to the adaptive arm of the immune response.

[0065] "Effective dose" means the amount of the anti-human IL-34 antibody of this disclosure, or a pharmaceutical composition containing such antibody, that would induce a biological or medical response in a tissue, system, or person, or a desired therapeutic effect, as sought by a therapeutic medical professional. As used herein, the terms "effective response" of a patient, or patient responsiveness to treatment, refer to the clinical or therapeutic benefit conferred to a patient upon administration of the antibody of this disclosure. The effective dose of an antibody may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the antibody's ability to induce a desired response in the individual. The effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the antibody. Such benefits include one or more of the following: a reduction in the level of inflammation or immune activation; stabilization of an immune-mediated disease or disorder; or improvement of the signs or symptoms of an immune-mediated disorder. Alternatively, such benefits include one or more of the following: increased immune tolerance of a transplanted organ; stabilization of an autoimmune disease or disorder; or improvement of the signs or symptoms of an autoimmune disorder.

[0066] The potential benefit of the methods disclosed herein is the possibility of significant and / or long-term relief in patients suffering from immune-mediated disorders or neuroinflammatory disorders with an acceptable safety profile, including acceptable tolerability, toxicity, and / or adverse events, and thus patients benefit from the overall treatment method. The efficacy of the treatments disclosed herein can be measured by a variety of endpoints commonly used when evaluating the treatment of various immune-mediated disorders. For example, other approaches to determining the efficacy of any particular therapy disclosed herein may be optionally used, including the measurement and visualization of immune cell activation markers, measures of inflammation, cell cycle-dependent biomarkers, and / or the measurement of various inflammatory or immune responses, or tissue-specific biomarker evaluation.

[0067] The effective dose can be readily determined by those skilled in the art by using known techniques and by observing results obtained under similar circumstances. The effective dose of the anti-human IL-34 antibody of this disclosure may be administered as a single dose or in multiple doses. Furthermore, the effective dose of the antibody of this disclosure may be administered in multiple doses, in amounts less than the effective dose if administered only once. When determining the effective dose for a patient, several factors will be considered by the attending physician, including but not limited to the patient's size (e.g., weight or mass), body surface area, age and general health status, any specific disease or disorder associated with the patient, the degree or involvement or severity of the disease or disorder, the individual patient's response, the specific compound administered, the mode of administration, the bioavailability characteristics of the administered preparation, the chosen administration regimen, the use of concomitant medications, and other relevant circumstances known to the attending physician.

[0068] Weekly, bi-weekly, monthly, or quarterly parenteral (including but not limited to subcutaneous, intramuscular, and / or intravenous) doses may range from approximately 0.5 mg / kg to approximately 50 mg / kg. As used herein, “one month” or its derivatives refers to a period of 28 to 31 consecutive days.

[0069] The potential benefit of the methods disclosed herein is the possibility of significant and / or long-term relief in patients suffering from immune-mediated disorders or neuroinflammatory disorders with an acceptable safety profile, including acceptable tolerability, toxicity, and / or adverse events, and thus patients benefit from the overall therapeutic method, more specifically, that the antibodies disclosed herein provide effective treatment while avoiding clinically undesirable immunosuppression and / or immune-related adverse events such as “cytokine storms” or significant cytokine release. The antibodies disclosed herein may be useful in treating cytokine storms or adverse cytokine releases. As used herein, “significant cytokine release” refers to a significant increase in measurable cytokines that can be detected by methods known to those skilled in the art. For example, significant cytokine release may be detected in a human blood sample by ELISA, where cytokine levels from unstimulated blood are compared to cytokine levels with blood incubated with the antibody. In some such tests, significant cytokine release may be detected if, for example, the level of IL-6, or IL-8, or IFN-γ is at least three times higher in the blood incubated with the antibody compared to the level in unstimulated blood. Preferably, the immune-mediated disorder is treated as described in the embodiments herein, in which the patient does not experience significant cytokine release.

[0070] Using antibody combination 1: This disclosure further provides simultaneous, separate, or sequential combinations of the antibodies of this disclosure, particularly antibody 1 and anti-N3pGlu Aβ antibody, and methods of using the combination to treat diseases characterized by amyloid beta (Aβ) deposition, such as AD. Some known anti-Aβ antibodies useful in this combination include donanemab, bapineuzumab, gantenerumab, aducanumab, GSK933776, solanezumab, crenezumab, ponezumab, and lecanemab (BAN2401). This disclosure further provides simultaneous, separate, or sequential combinations of antibody 1 and donanemab (CAS number 1931944-80-7, SEQ ID NOs. 38 and 39), and methods of using the combination to treat diseases characterized by amyloid-beta (Aβ) deposition, such as AD ("Donanemab in early Alzheimer's disease", Mintun, MA et al, New England Journal of Medicine (2021), 384(18), 1691-1704). Preferably, the combination provides sequential use of antibody 1 after a series of treatments with donanemab.

[0071] As used herein, “anti-N3pGlu Aβ antibody,” “anti-N3pG antibody,” or “anti-N3pE antibody” are interchangeable and refer to antibodies that preferentially bind to N3pGlu Aβ over Aβ1-40 or Aβ1-42. Those skilled in the art will understand and recognize that “anti-N3pGlu Aβ antibody,” as well as several specific antibodies including “hE8L,” “B12L,” and “R17L,” are identified and disclosed (along with methods for preparation and use) in U.S. Patent No. 8,679,498(B2) (which is incorporated herein by reference in its entirety). See, for example, Table 1 of U.S. Patent No. 8,679,498(B2). Each of the antibodies disclosed in U.S. Patent No. 8,679,498(B2), including the “hE8L,” “B12L,” and “R17L,” may be used as the anti-N3pGlu Aβ antibody of the present invention or as a substitute for the anti-N3pGlu Aβ antibody described in various embodiments of the present invention. The anti-N3pGlu Aβ antibodies in this combination method are antibodies containing HC and LC of SEQ ID NOs. 40 and 41, respectively. Other representative types of anti-N3pGlu Aβ antibodies include, but are not limited to, those disclosed in U.S. Patents 8,961,972, 10,647,759, 9,944,696, International Publication 2010 / 009987(A2), International Publication 2011 / 151076(A2), International Publication 2012 / 136552(A1) and their equivalents (for example, under Section 112(f) of the U.S. Patent Act).

[0072] Those skilled in the art will understand and recognize that “anti-N3pGluAβ antibodies” and several specific antibodies are specified and disclosed (along with methods for preparation and use) in U.S. Patent No. 8,961,972 (which is incorporated herein by reference in its entirety), U.S. Patent No. 10,647,759 (which is incorporated herein by reference in its entirety), and U.S. Patent No. 9,944,696 (which is incorporated herein by reference in its entirety). Any of the anti-N3pGluAβ antibodies disclosed in U.S. Patent No. 8,961,972, U.S. Patent No. 9,944,696, and U.S. Patent No. 10,647,759 may be used as the anti-N3pGluAβ antibody of the present invention, or in place of the anti-N3pGluAβ antibodies described in various embodiments of the present invention.

[0073] Those skilled in the art will understand and recognize that several specific antibodies, including “anti-N3pGlu Aβ antibody” and “antibody VI,” “antibody VII,” “antibody VIII,” and “antibody IX,” are specified and disclosed in International Publication No. 2010 / 009987(A2) (which is incorporated herein by reference in its entirety) (along with methods for producing and using such antibodies). Each of these four antibodies (e.g., “antibody VI,” “antibody VII,” “antibody VIII,” and “antibody IX”) can be used as the anti-N3pGlu Aβ antibody of the present invention, or in place of the anti-N3pGlu Aβ antibody described in various embodiments of the present invention.

[0074] Those skilled in the art will understand and recognize that several specific antibodies, including “anti-N3pGlu Aβ antibody” and “antibody X” and “antibody XI,” are specified and disclosed in International Publication No. 2011 / 151076(A2) (the entire publication of which is incorporated herein by reference) (along with methods for producing and using such antibodies). Each of these two antibodies (e.g., “antibody X” and “antibody XI”) may be used as the anti-N3pGlu Aβ antibody of the present invention, or as a substitute for the anti-N3pGlu Aβ antibody described in various embodiments of the present invention.

[0075] Those skilled in the art will understand and recognize that several specific antibodies, including “anti-N3pGlu Aβ antibody,” and “antibody XII” and “antibody XIII,” are identified and disclosed in International Publication No. 2012 / 136552(A1) (which is incorporated herein by reference in its entirety) (along with methods for producing and using such antibodies). Each of these two antibodies (e.g., “antibody XII” and “antibody XIII”) may be used as the anti-N3pGlu Aβ antibody of the present invention or as a substitute for the anti-N3pGlu Aβ antibody described in various aspects of this disclosure.

[0076] Aspects of this disclosure provide the use of a combination of the antibodies of this disclosure, particularly antibody 1, and an anti-N3pGlu Aβ antibody, particularly donanemab, for a method of treating diseases characterized by Aβ deposition in a subject, the subject being selected based on i) tau levels / load in the whole brain (global tau), ii) tau levels / load in a region of the brain (e.g., different lobes of the brain), and / or the presence of one or two alleles of APOE e4 in the subject's genome. Diseases that may be treated or prevented using the combination methods disclosed herein include, for example, Alzheimer's disease (AD), Down syndrome, and cerebral amyloid angiopathy (CAA). This disclosure also relates to the use of the combination provided herein to slow disease progression in a subject having early symptomatic Alzheimer's disease (AD) in the presence of intermediate cerebral tau load.

[0077] Antibodies against N3pGlu Aβ are known in the art and are described herein. For example, U.S. Patent No. 8,679,498 (which is incorporated herein by reference in its entirety, including the anti-N3pGlu Aβ antibody disclosed therein) discloses an anti-N3pGlu Aβ antibody and a method of treating diseases such as Alzheimer's disease with antibodies. Passive immunization by long-term chronic administration of antibodies against Aβ, including N3pGlu Aβ, found in deposits, has been shown to disrupt Aβ aggregates in the brain and promote plaque clearance in various animal models. Donanemab (disclosed in U.S. Patent No. 8,679,498; see also CAS number 1931944-80-7) is an antibody against the pyroglutamate modification of the third amino acid of the amyloid beta (N3pGlu Aβ) epitope, which is present only in amyloid plaques in the brain. The mechanism of action of donanemab is the targeting and removal of existing amyloid plaques, a key pathological feature of AD. A second neuropathological feature of AD is the presence of intracellular neurofibrillary tangles containing hyperphosphorylated tau protein. Aβ may trigger tau pathology, and more complex, synergistic interactions between Aβ and tau may emerge in later stages, potentially accelerating disease progression (Busche et al., "Synergy Between Amyloid-β and Tau in Alzheimer's disease," Nature Neuroscience 23:1183-93 (2020)).

[0078] Administration of Aβ antibodies has resulted in adverse events in humans, including amyloid-related imaging abnormalities (ARIA), vasogenic edema and groove exudate (ARIA-E), microbleeds and hemosiderin deposits (ARIA-H), injection site reactions, and immunogenic risks. For example, Piazza and Winblad, “Amyloid-Related Imaging Abnormalities (ARIA) in Immunotherapy Trials for Alzheimer's Disease:Need for Prognostic Biomarkers?” Journal of Alzheimer's Disease, 52:417-420 (2016); Sperling, et al., “Amyloid-related Imaging Abnormalities in Patients with Alzheimer's Disease Treated with Bapineuzumab: A Retrospective Analysis," The Lancet Neurology11.3:241-249(2012); Brashear et al., "Clinical Evaluation of Amyloid-related Imaging Abnormalities in Bapineuzumab Phase III Studies," J.of Alzheimer's Disease66.4:1409-1424(2018); Budd et al., "Clinical Development of Aducanumab, an Anti-Aβ Human Monoclonal See "Antibody Being Investigated for the Treatment of Early Alzheimer's Disease," The Journal of Prevention of Alzheimer's Disease 4.4:255 (2017).

[0079] The combination therapy strategy described herein for donanemab and antibody 1 involves targeting N3pGlu Aβ specifically in amyloid plaques in an existing population of early symptomatic AD patients with cerebral amyloid burden, and targeting neuroinflammation in these patients. This rationale is based on the amyloid hypothesis of AD, which states that Aβ production and deposition are early necessary events in the pathogenesis of AD. See, for example, Selkoe, "The Origins of Alzheimer Disease: A is for Amyloid," JAMA 283:1615-1617 (2000). Clinical support for this hypothesis comes from the demonstration that parenchymal Aβ levels are elevated before the onset of AD symptoms, and that this is supported by genetic variants of AD that overproduce cerebral Aβ and genetic variants that prevent Aβ production. For example, Jonsson et al., "A Mutation in APP Protects Against Alzheimer's Disease and Age-related Cognitive Decline," Nature, Vol. 488 (No. 7409): pp. 96-99 (2012), and Fleisher et al., "Associations Between Biomarkers and Age in the Presenilin 1 E280A Autosomal Dominant Alzheimer Disease Kindred: A Cross-sectional Study," JAMA Neurol, Vol. 72: pp. 316-3124 (2015). Therefore, there is a need for improved combinations of drugs to treat the target without causing or increasing problematic adverse events. Neuroinflammation is a key component of neurodegenerative diseases and is characterized by increased production of pro-inflammatory cytokines by CNS cells. Neuroinflammation and microgliosis are thought to be underlying mechanisms of Alzheimer's disease and / or neuronal cell death and dysfunction. Microgliosis involves the abnormal proliferation and / or hypertrophy of microglia in response to inflammatory signals. IL-34 acts as a potent and multifaceted cytokine in regulating inflammatory and immune processes and is expressed by neurons in the cortex, preolar nucleus, and hippocampus.Treatment with N3pGlu Aβ antibody, particularly donanemab, followed by simultaneous, separate, or preferably sequential treatment with antibody 1, is thought to improve the contribution of neuroinflammation and / or microgliosis to the pathogenesis of AD, thereby slowing or preventing the progression of the neurodegenerative process in these patients.

[0080] One aspect of this disclosure is based on the concept that Alzheimer's disease patients with low or moderate tau, very low to moderate tau, or no tau respond to combination therapy using an anti-N3pGlu Aβ antibody such as donanemab and an antibody of this disclosure such as antibody 1. Another aspect of this disclosure is based on the concept that Alzheimer's disease patients with one or two alleles of APOE e4 respond to treatment with an anti-N3pGlu Aβ antibody. Yet another aspect of this disclosure is based on the concept that Alzheimer's disease patients with one or two alleles of APOE e4 and low or moderate tau, very low to moderate tau, or no tau respond to combination therapy using an anti-N3pGlu Aβ antibody such as donanemab and an antibody of this disclosure such as antibody 1. Some aspects of this disclosure relate to diagnosing and treating patients based on their brain pathology. By selecting patients based on brain pathology, a more homogeneous population can be obtained in clinical trials, as well as the stage and progression of Alzheimer's disease (AD) can be appropriately identified. Appropriate identification of the AD stage allows for, for example, timely referral to a memory clinic, accurate and early diagnosis of AD, initiation of symptomatic treatment, future planning, and initiation of disease modification therapy using combination therapy with anti-N3pGlu Aβ antibodies such as donanemab and antibodies of the disclosure, such as antibody 1.

[0081] Some aspects of this disclosure provide combination embodiments for treating human subjects suffering from a disease characterized by Aβ deposits in the brain, the subjects being administered in two steps, first with an anti-N3pGlu Aβ antibody such as donanemab, in combination with simultaneous, separate, or sequential treatment using an antibody of this disclosure, such as antibody 1. In the first step, the human subject is administered one or more doses of a first dose of anti-N3pGlu Aβ antibody, approximately 100 mg to approximately 700 mg, with each first dose administered approximately every four weeks. Approximately four weeks after the administration of one or more first doses, in the second step, the human subject is administered one or more doses of a second dose, greater than 700 mg to approximately 1400 mg, with each second dose administered approximately every four weeks. Preferably, the anti-N3pGlu Aβ antibody is donanemab. Antibody 1 is administered simultaneously, separately, or sequentially after a series of treatments with donanemab. Preferably, antibody 1 is administered sequentially after a series of treatments with donanemab.

[0082] Some aspects of the treatment combination methods relate to identifying the stage / progression of AD in the patient based on i) the overall or total tau load in the brain of the human subject, or ii) the spread of tau in the brain or a region or part thereof of the subject.

[0083] In some embodiments, patients can be stratified / identified / selected / treated based on the amount of tau present in the brain of the subject (e.g., the whole brain or a part of the brain). In some embodiments, patients can be stratified / identified / selected / treated based on the amount of tau present in the brain of the subject (e.g., the whole brain or a part of the brain) and the presence of one or two alleles of APOE e4.

[0084] In other embodiments, patients are stratified / identified / selected / treated based on the stage of AD progression (e.g., based on the extent of tau in the brain). For example, in some stages, tau overload in AD patients is isolated to areas of the temporal lobe that do not include the frontal lobe or the posterolateral temporal region (PLT). Another stage of AD is when tau overload in AD patients is limited to the posterolateral temporal (PLT) or occipital region. Yet another stage of AD is when tau overload in AD patients is present in the parietal, precuneus, or frontal region along with tau overload in the PLT or occipital region. In some embodiments, patients may be stratified / identified / selected / treated based on the stage of AD progression (e.g., based on the extent of tau in the brain) and based on the presence of one or two alleles of APOE e4.

[0085] Patient stratification based on the amount of tau in the brain, the progression of AD in a part of the brain, and / or the presence of one or two alleles of APOE e4 can be used to determine, for example, whether a patient will respond to combination therapy with an anti-N3pGlu Aβ antibody such as donanemab and an antibody of this disclosure such as antibody 1. Stratification / selection of patient populations based on the amount of tau in the brain, the progression of AD in a part of the brain, and / or the presence of one or two alleles of APOE e4 can help solve the problems of patient heterogeneity and repeatability that are faced during the design and conduct of clinical trials, in addition to therapeutic considerations.

[0086] Other embodiments of this disclosure provide human subjects that respond to combination therapy or prophylaxis of an anti-N3pGlu Aβ antibody, such as donanemab, and an antibody of this disclosure, such as antibody 1, for a disease characterized by amyloid-beta (Aβ) deposits in the brain of human subjects. In some embodiments of this aspect of this disclosure, responding human subjects include human subjects with low to moderate tau loading, very low to moderate tau loading, and / or one or two alleles of APOE e4. In some embodiments of this aspect of this disclosure, responding human subjects are excluded from human subjects with high tau loading. In some embodiments of this aspect of this disclosure, responding human subjects are excluded from human subjects with high tau loading and / or one or two alleles of APOE e4. In some embodiments, the combination of an anti-N3pGlu Aβ antibody, such as donanemab, and an antibody of this disclosure, such as antibody 1, is administered to a responding human subject for the treatment or prophylaxis of a disease characterized by amyloid-beta (Aβ) deposits.

[0087] In one embodiment, the Disclosure relates to a concurrent, separately, or sequential combination therapy or prophylaxis for a disease characterized by Aβ deposition in the brain of a human subject, using an anti-N3pGlu Aβ antibody, in particular donanemab, and the antibody of the Disclosure, in particular antibody 1, comprising: i) administering to a human subject one or more doses of a first dose of anti-N3pGlu Aβ antibody, in an amount of approximately 100 mg to approximately 700 mg, with each first dose administered approximately every four weeks; and ii) administering to a human subject one or more doses of a second dose of anti-N3pGlu Aβ antibody, in an amount of more than 700 mg to approximately 1400 mg, with each second dose administered approximately every four weeks, wherein the anti-N3pGlu Aβ antibody comprises donanemab, and the human subject is administered the antibody of the Disclosure, in particular antibody 1. Preferably, antibody 1 is administered sequentially after a series of treatments with donanemab.

[0088] To date, the clinical focus of donanemab treatment has been limited to early symptomatic AD patients with pre-existing cerebral amyloid burden. However, a second neuropathological feature of AD is the presence of intracellular neurofibrillary tangles containing hyperphosphorylated tau protein. Current disease models suggest that Aβ triggers tau pathology, and that more complex, synergistic interactions between Aβ and tau emerge in later stages, accelerating disease progression (Busche et al., "Synergy Between Amyloid-p and Tau in Alzheimer's disease," Nature Neuroscience 23:1183-93 (2020)).

[0089] Currently, there are no disease-modifying treatments for Alzheimer's disease (AD). Therefore, there is a need for improved methods to treat diseases, including AD, characterized by Aβ deposition in human subjects. Such methods should help identify patients based on whether they may benefit from such treatments. Furthermore, such treatments and methods should not be associated with increased cytotoxicity or other known adverse events. This invention satisfies one or more of these needs.

[0090] Doody et al., "Phase 3 Trials of Solanezumab for Mild-to-Moderate Alzheimer's Disease," NEJM, 370;4, 311-321 (2014), showed that "no clear difference in therapeutic effect on efficacy measures was observed between APOE ε4 carriers and non-carriers." It is thought that administering an anti-N3pGlu Aβ antibody in combination with the antibody of this disclosure to human subjects having one or two alleles of APOE e4 (e.g., APOE e4 carriers) would provide unexpected efficacy compared to non-carriers of one or more of those alleles. Therefore, this embodiment includes administering an anti-N3pGlu Aβ antibody, particularly donanemab, concurrently, separately, or sequentially in combination with the antibody of this disclosure, particularly antibody 1, to patients having one or two APOE e4 alleles as a means of delaying cognitive decline in those patients.

[0091] According to certain embodiments, the present invention provides a method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of a human subject determined to have a high neurological tau load, comprising administering, in simultaneous, separate, or sequential doses, a therapeutically effective amount of an anti-Aβ antibody, particularly donanemab, and a therapeutically effective amount of the antibody of the present disclosure, particularly antibody 1. Additionally, according to certain embodiments, the present invention provides a combined method for treating or preventing a disease characterized by Aβ deposits in the brain of a human subject determined to have a posterolateral temporal lobe tau load, comprising administering, in simultaneous, separate, or sequential doses, a therapeutically effective amount of an anti-Aβ antibody, particularly donanemab, and a therapeutically effective amount of the antibody of the present disclosure, particularly antibody 1.

[0092] In certain embodiments, the present invention provides a combined method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of a human subject determined to have a high neurological tau load and possessing one or two alleles of the epsilon 4 allele of apolipoprotein E (referred to herein as APOE e4 or APOE4), comprising administering, simultaneously, separately, or sequentially, a therapeutically effective amount of an anti-Aβ antibody, particularly donanemab, and a therapeutically effective amount of the antibody of the present disclosure, particularly antibody 1. Additionally, in certain embodiments, the present invention provides a method for treating or preventing a disease characterized by Aβ deposits in the brain of a human subject determined to have a posterolateral temporal lobe tau load, comprising administering, simultaneously, separately, or sequentially, a therapeutically effective amount of an anti-Aβ antibody, particularly donanemab, and a therapeutically effective amount of the antibody of the present disclosure, particularly antibody 1.

[0093] According to several embodiments, the present invention provides an anti-Aβ antibody, particularly donanemab, for use concurrently, separately, or sequentially with the antibody, particularly antibody 1, for the treatment or prevention of a disease characterized by Aβ deposition in the brain of a human subject determined to have a high neurological tau load, comprising administering a therapeutically effective amount of an anti-Aβ antibody, particularly donanemab, and a therapeutically effective amount of the antibody of the present disclosure, particularly antibody 1, in concurrent, separately, or sequential doses. In several embodiments, the human subject is determined to have a high neurological tau load and also to have one or two alleles of APOE e4.

[0094] In some embodiments, the present invention provides anti-Aβ antibodies, particularly donanemab, for use concurrently, separately, or sequentially with the antibodies of the present disclosure, particularly antibody 1, for the treatment or prevention of diseases characterized by Aβ deposits in the brain of human subjects determined to have posterolateral temporal lobe tau overload. In some embodiments, the human subjects are determined to have posterolateral temporal lobe tau overload and also to have one or two alleles of APOEe4.

[0095] In addition, in some embodiments, the present invention provides anti-Aβ antibodies, particularly donanemab, for use concurrently, separately, or sequentially with the antibodies of the present disclosure, particularly antibody 1, to treat, prevent, or slow the progression of Alzheimer's disease (AD). In addition, in some embodiments, the present invention provides anti-Aβ antibodies, particularly donanemab, for use concurrently, separately, or sequentially with the antibodies of the present disclosure, particularly antibody 1, to treat, prevent, or slow the progression of Alzheimer's disease (AD) in human subjects determined to have slowly progressive AD cognitive decline and to have one or two alleles of APOE e4.

[0096] Furthermore, according to some embodiments, the Disclosure provides the use of an anti-Aβ antibody, particularly donanemab, in combination with the antibody of the Disclosure, in particular antibody 1, simultaneously, separately, or sequentially, in the manufacture of a pharmaceutical product for the treatment or prevention of Alzheimer's disease. Furthermore, according to some embodiments, the Disclosure provides the use of an anti-Aβ antibody, particularly donanemab, in combination with the antibody of the Disclosure, in particular antibody 1, simultaneously, separately, or sequentially, in the manufacture of a pharmaceutical product for the treatment or prevention of a disease characterized by Aβ deposits in the brain of a human subject determined to have a high neurological tau load, or ii) a high neurological tau load and one or two alleles of APOE e4.

[0097] In some embodiments, the Disclosure provides the use of an antibody of the Disclosure, particularly an anti-Aβ antibody, particularly donanemab, in combination with antibody 1, concurrently, separately, or sequentially, in the manufacture of a pharmaceutical product for the treatment or prevention of a disease characterized by Aβ deposition in the brain of a human subject determined to have i) posterolateral temporal lobe tau overload, or ii) posterolateral temporal lobe tau overload and one or two alleles of APOE e4. In further embodiments, the Invention provides the use of an antibody of the Disclosure, particularly an anti-Aβ antibody, particularly donanemab, in combination with antibody 1, concurrently, separately, or sequentially, in the manufacture of a pharmaceutical product for the treatment, prevention, or slowing the progression of Alzheimer's disease (AD) in a human subject determined to have i) slowly progressive AD cognitive decline, or ii) one or two alleles of APOE e4 and slowly progressive AD cognitive decline.

[0098] According to some embodiments provided herein, a human subject is determined to have tau loading in the posterolateral temporal lobe and occipital lobe. In some embodiments, a human subject is determined to have tau loading in the posterolateral temporal lobe, occipital lobe, and parietal lobe. In some embodiments, a human subject is determined to have tau loading in the posterolateral temporal lobe, occipital lobe, parietal lobe, and frontal lobe. In some embodiments, neurological PET imaging determines that a human subject has one or more tau loadings in the posterolateral temporal lobe, occipital lobe, parietal lobe, and / or frontal lobe. In some embodiments, one or more tau loadings in the posterolateral temporal lobe, occipital lobe, parietal lobe, and / or frontal lobe correspond to a neurological tau loading greater than 1.46 SUVr.

[0099] According to some embodiments provided herein, a human subject is determined to have one or two alleles of APOE e4 and tau loading in the posterolateral temporal and occipital lobes. In some embodiments, a human subject is determined to have one or two alleles of APOE e4 and tau loading in the posterolateral temporal, occipital, and parietal lobes. In some embodiments, a human subject is determined to have one or two alleles of APOE e4 and tau loading in the posterolateral temporal, occipital, parietal, and frontal lobes. In some embodiments, a human subject is determined to have one or more tau loadings in the posterolateral temporal, occipital, parietal, and / or frontal lobes by neurological PET imaging, and to have one or two alleles of APOE e4. In some embodiments, one or more tau loadings in the posterolateral temporal, occipital, parietal, and / or frontal lobes correspond to a neurological tau loading greater than 1.46 SUVr.

[0100] In additional embodiments, the present invention provides a method for treating, preventing, or slowing the progression of Alzheimer's disease (AD) in a human subject determined to have slowly progressive AD cognitive decline, comprising administering, simultaneously, separately, or sequentially, a therapeutically effective amount of an anti-Aβ antibody, particularly donanemab, and a therapeutically effective amount of the antibody of the present disclosure, particularly antibody 1. In some embodiments, the human subject is determined to have a high neurological tau load. In some embodiments, the human subject is determined to have one or two alleles of APOE e4. In some embodiments, the human subject is determined to have a posterolateral temporal lobe tau load. In some embodiments, the human subject is determined to have a posterolateral temporal lobe and occipital lobe tau load. In some embodiments, the human subject is determined to have a posterolateral temporal lobe, occipital lobe, and parietal lobe tau load. In some embodiments, human subjects are determined to have tau loading in the posterolateral temporal, occipital, parietal, and frontal lobes. In some embodiments, human subjects are determined to have posterolateral temporal lobe tau loading and one or two alleles of APOE e4. In some embodiments, human subjects are determined to have one or two alleles of APOE e4 and tau loading in the posterolateral temporal and occipital lobes. In some embodiments, human subjects are determined to have one or two alleles of APOE e4 and tau loading in the posterolateral temporal, occipital, and parietal lobes. In some embodiments, human subjects are determined to have one or two alleles of APOE e4 and tau loading in the posterolateral temporal, occipital, parietal, and frontal lobes.

[0101] According to embodiments of the present invention provided herein, human subjects are determined to have slowly progressive AD cognitive decline by one or more of the following: ADAS-Cog, iADL, CDR-SB, MMSE, APOE-4 genotyping, and / or iADRS. In some embodiments, human subjects are determined to have slowly progressive AD cognitive decline by iADRS. In some embodiments, the iADRS decreased by less than 20. In some embodiments, the iADRS decreased by less than 20 over a period of 6 months. In some embodiments, the iADRS decreased by less than 20 over a period of 12 months. In some embodiments, the iADRS decreased by less than 20 over a period of 18 months. In some embodiments, the iADRS decreased by less than 20 over a period of 24 months. In some embodiments, human subjects are determined to have slowly progressive AD cognitive decline by APOE-4 genotyping. In some embodiments, human subjects are determined to be APOE-4 heterozygotes. In some embodiments, human subjects were determined to be APOE-4 homozygous negative. In some embodiments, human subjects were determined to have AD cognitive impairment by MMSE. In some embodiments, human subjects were determined to have an MMSE score greater than 27. In some embodiments, the MMSE score decreased by less than 3. In some embodiments, the MMSE score decreased by less than 3 over a period of 6 months. In some embodiments, the MMSE score decreased by less than 3 over a period of 12 months. In some embodiments, the MMSE score decreased by less than 3 over a period of 18 months. In some embodiments, the MMSE score decreased by less than 3 over a period of 24 months.

[0102] According to embodiments of the present invention provided herein, a human subject is determined to have a high neurological tau load by neurological PET imaging. In some embodiments, the human subject is determined to have a high neurological tau load greater than 1.46 SUVr by neurological PET imaging. In some embodiments, the human subject is determined to have a high neurological tau load by quantification of human tau phosphorylated with threonine at residue 217 ("hTau-pT217"). In some embodiments, hTau-pT217 is quantified in a biological sample of the human subject. In some embodiments, the biological sample is cerebrospinal fluid. In some embodiments, the biological sample is one of blood, plasma, or serum.

[0103] For the purposes of the present invention, the tau level or load (as used interchangeably herein) of a human subject can be determined, for example, by using techniques or methods for detecting or quantifying i) neurological or cerebral tau deposition, ii) tau in blood, serum and / or plasma, or iii) tau in cerebrospinal fluid. In some embodiments, subjects can be stratified based on neurological tau load (e.g., low, moderate, or high neurological tau load) using neurological tau load (whether determined via PET or via blood, serum, plasma, or cerebrospinal fluid assays).

[0104] Neurological tau loading is a PET ligand [ 18Tau imaging using a radiolabeled PET compound containing F]-flortaucipir (Leuzy et al., "Diagnostic Performance of RO948 F18 Tau Positron Emission Tomography in the Differentiation of Alzheimer Disease from Other Neurodegenerative Disorders," JAMA Neurology 77.8:955-965(2020); Ossenkoppele et al., "Discriminative Accuracy of 18These can be determined using methods such as those described in "F]-flortaucipir Positron Emission Tomography for Alzheimer Disease vs Other Neurodegenerative Disorders," JAMA 320, 1151-1162, doi:10.1001 / jama.2018.12917 (2018), which are incorporated herein by reference in their entirety. PET tau images can be quantitatively evaluated to estimate the SUVr (Standardized Uptake Ratio) using, for example, published methods (Pontecorvo et al., "A Multicentre Longitudinal Study of Flortaucipir (18F) in Normal Ageing, Mild Cognitive Impairment and Alzheimer's Disease Dementia," Brain 142:1723-35 (2019); Devous et al., "Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18," Journal of Nuclear Medicine 59:937-43 (2018); Southekal et al., "Flortaucipir F18 Quantitation Using Parametric Estimation of Reference Signal"). Intensity, "J.Nucl.Med.59:944-51(2018), these are incorporated herein by reference in their entirety," and / or can be quantitatively assessed to visually evaluate a patient, for example, to determine whether a patient has an AD pattern (Fleisher et al., "Positron Emission Tomography Imaging With[ 18F18-flortaucipir and Postmortem Assessment of Alzheimer Disease Neuropathologic Changes, JAMA Neurology 77:829-39 (2020), which is incorporated herein by reference in its entirety. A lower SUVr value indicates a lower tau load, and a higher SUVr value indicates a higher tau load. In embodiments, quantitative assessment by flortaucipir scan is achieved by an automated image processing pipeline described in Southekal et al., "Flortaucipir F18 Quantitation Using Parametric Estimation of Reference Signal Intensity," J.Nucl.Med. 59:944-951 (2018), which is incorporated herein by reference in its entirety. In some embodiments, counts within a specific target region in the brain (e.g., multiblock centroid discriminant analysis or MUBADA, the whole of which is incorporated herein by reference; see Devous et al., "Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18," J.Nucl.Med. 59:937-943 (2018)) are compared to a reference region, which is, for example, the whole cerebellum (wholeCere), cerebellar GM (cereCrus), atlas-based white matter (atlasWM), or subject-specific WM (ssWM, e.g., parametric estimate of reference signal intensity (PERSI), the whole of which is incorporated herein by reference; see Southekal et al., "Flortaucipir F18 Quantitation Using Parametric Estimation of Reference Signal Intensity," J.Nucl.Med. 59:944-951 (2018)). An exemplary method for determining tau loading is quantitative analysis, which is reported as the standardized intake ratio (SUVr).This represents the count (e.g., MUBADA) within a specific target region of interest in the brain, compared to a reference region (e.g., using PERSI).

[0105] In some embodiments, tau load / load can be measured for the purposes of the present invention using phosphorylated tau (P-tau; phosphorylated with threonine 181 or 217, or any combination thereof) (Barthelemy et al., "Cerebrospinal Fluid Phospho-tau T217 Outperforms T181 as a Biomarker for the Differential Diagnosis of Alzheimer's Disease and PET Amyloid-positive Patient Identification," Alzheimer's Res.Ther. 12,26,doi:10.1186 / s13195-020-00596-4 (2020); Mattsson et al., "Aβ Deposition is Associated with Increases in Soluble and Phosphorylated Tau that Precede a Positive Tau PET in Alzheimer's Disease," Science Advances). 6,eaaz2387(2020), these are incorporated herein by reference in their entirety. In certain embodiments, tau load / load in a subject can be measured using an antibody against human tau phosphorylated with threonine at residue 217 (see International Publication 2020 / 242963, which is incorporated herein by reference in its entirety). In some embodiments, this disclosure includes measuring tau load / load in a subject using an anti-tau antibody disclosed in International Publication 2020 / 242963. The anti-tau antibody disclosed in International Publication 2020 / 242963 is made against isoforms of human tau expressed in the CNS (e.g., recognizes isoforms expressed in the CNS and does not recognize isoforms of human tau expressed exclusively outside the CNS).

[0106] If amyloid is detected in the brain by methods such as amyloid imaging using radiolabeled PET compounds, or by diagnostic methods that detect Aβ or Aβ biomarkers, the subject is positive for amyloid deposits. Exemplary methods that may be used to measure brain amyloid load / burden include, for example, florbetapir (the entire text of which is incorporated herein by reference, Carpenter, et al., "The Use of the Exploratory IND in the Evaluation and Development of 18 F-PET Radiopharmaceuticals for Amyloid Imaging in the Brain: A Review of One Company's Experience, The Quarterly Journal of Nuclear Medicine and Molecular Imaging 53.4:387 (2009), Florbetaben (the whole text is incorporated herein by reference, Syed et al., "[ 18 F]Florbetaben: A Review in β-Amyloid PET Imaging in Cognitive Impairment, CNS Drugs 29, 605-613 (2015), and Flutemetamol (the whole article is incorporated herein by reference, Heurling et al., "Imaging β-amyloid Using[ 18 [F]Flutemetamol Positron Emission Tomography: From Dosimetry to Clinical Diagnosis, European Journal of Nuclear Medicine and Molecular Imaging 43.2:362-373 (2016) is one example. 18[F]-Florbetapil can provide qualitative and quantitative measurements of cerebral plaque load in patients, including those with prodromal AD or mild AD dementia, and can also be used to assess the reduction of amyloid plaques from the brain.

[0107] Additionally, amyloid load / burden can be measured using cerebrospinal fluid or plasma-based analysis of β-amyloid. For example, brain amyloid can be measured using Aβ42 (the entire text is incorporated herein by reference, Palmqvist, S. et al., "Accuracy of Brain Amyloid Detection in Clinical Practice Using Cerebrospinal Fluid Beta-amyloid 42: a Cross-validation Study Against Amyloid Positron Emission Tomography." JAMA Neurol 71, 1282-1289 (2014)). In some embodiments, the Aβ42 / Aβ40 or Aβ42 / Aβ38 ratio can be used as a biomarker for amyloid beta (the entire text is incorporated herein by reference, Janelidze et al., "CSF Abeta42 / Abeta40 and Abeta42 / Abeta38 Ratios: Better Diagnostic Markers of Alzheimer Disease," Ann Clin Transl Neurol 3, 154-165 (2016)). In some embodiments, subjects can be stratified into groups based on amyloid load / burden using CSF or brain amyloid plaques or Aβ deposited in plasma.

[0108] Additional embodiments of combined uses and methods of using the antibodies of this disclosure are described below. A combined embodiment may refer to antibody 1, but the embodiment further includes similar methods, uses, and all limitations described herein for the antibodies of this disclosure described herein. A combined embodiment may refer to “anti-N3pG Aβ antibody” referring to each of the anti-N3pG Aβ antibodies described herein, however, for clarity, these embodiments further include similar methods, uses, and all limitations described herein individually for each of the anti-N3pG Aβ antibodies, and are preferably used in combination with, for example, donanemab. Additional embodiments of this disclosure are provided below, numbered and including internal references to other numbered embodiments. For clarity, these embodiments should be read individually and / or collectively together with the numbered embodiments they reference. The embodiments described below begin at number 26. The term “series of treatments” means a specific patient or subject, listed antibodies, listed doses, listed frequencies and / or durations, listed order, and any other limitations, to the extent described in each case.

[0109] Further combined embodiments of this disclosure include:

[0110] 26. A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of a human subject, comprising administering an effective amount of anti-N3pG Aβ antibody to a human subject in need of such treatment or prevention, simultaneously with, separately from, or sequentially in combination with an effective amount of antibody 1.

[0111] 27. The method according to Embodiment 26, wherein the anti-N3pG Aβ antibody is donanemab.

[0112] 28. The method according to Embodiment 26, wherein the disease is Alzheimer's disease.

[0113] 29. The method according to Embodiment 26, wherein the anti-N3pG Aβ antibody is donanemab and the disease is Alzheimer's disease.

[0114] 30. The method according to Embodiment 29, wherein antibody 1 is administered sequentially after a series of treatments with donanemab.

[0115] 31. A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of human subjects, i) Administering a first dose of anti-N3pG Aβ antibody to human subjects, consisting of one or more doses ranging from approximately 100 mg to approximately 700 mg, with each first dose administered approximately once every four weeks. ii) Approximately four weeks after administering one or more first doses, administer one or more second doses of anti-N3pG Aβ antibody, ranging from over 700 mg to approximately 1400 mg, to human subjects, with each second dose administered approximately once every four weeks. The anti-N3pGlu Aβ antibody is donanemab, and it is administered accordingly. iii) A method comprising administering an effective amount of antibody 1 to a human subject simultaneously, separately, or sequentially.

[0116] 32. The method according to Embodiment 31, wherein a human subject is administered one, two, or three doses of the first dose of donanemab before being administered the second dose.

[0117] 33. The method according to Embodiment 31 or 32, wherein a human subject is administered a first dose of donanemab of approximately 700 mg.

[0118] 34. The method according to any one of Embodiments 31 to 33, wherein a human subject is administered one or more doses of a second dose of donanemab of approximately 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, or 1400 mg.

[0119] 35. The method according to any one of embodiments 31 to 34, wherein a human subject is administered one or more second doses of donanemab of approximately 1400 mg.

[0120] 36. The method according to any one of Embodiments 31 to 35, wherein an anti-N3pGlu Aβ antibody is administered to a human subject over a series of treatment periods up to 72 weeks, or until normal levels of amyloid are achieved.

[0121] 37. The method according to any one of embodiments 31 to 36, wherein an anti-N3pGlu Aβ antibody is administered to a human subject until the patient's amyloid plaque level is approximately 25 centiloids or less.

[0122] 38. The method according to any one of embodiments 31 to 36, wherein an anti-N3pGlu Aβ antibody is administered to a human subject for a series of treatments until the amyloid plaque level in the human subject is reduced to approximately 25 centiloids or less in two consecutive PET imaging scans, or to approximately 11 centiloids or less in one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least 6 months apart.

[0123] 39. The method according to any one of embodiments 31 to 36, wherein a human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks, followed by a second dose of 1400 mg once every four weeks, over a series of treatment periods of up to 72 weeks.

[0124] 40. The method according to any one of embodiments 31 to 36, wherein a human subject is administered a first dose of 700 mg three times, once every four weeks, followed by a second dose of 1400 mg once every four weeks, until the amyloid plaque level of the subject is approximately 25 centiloids or less.

[0125] 41. The method according to any one of embodiments 31 to 36, wherein human subjects are administered three doses of a first dose of 700 mg of donanemab every four weeks, followed by a second dose of 1400 mg every four weeks, until the amyloid plaque level of the subjects is less than or equal to approximately 25 centiloids in two consecutive PET imaging scans, or less than or equal to approximately 11 centiloids in one PET imaging scan, with the two consecutive PET imaging scans optionally spaced at least six months apart.

[0126] 42. The method according to any one of Embodiments 31 to 41, wherein a human subject is administered a second dose of donanemab over a series of treatment periods sufficient to treat or prevent a disease.

[0127] 43. The method according to any one of Embodiments 31 to 42, wherein the treatment or prevention of a disease results in i) a reduction in Aβ deposits in the brain of a human subject, and / or ii) a delay in cognitive or functional decline in a human subject.

[0128] 44. The method according to Embodiment 43, wherein the reduction of Aβ deposits in the brain of a human subject is determined by amyloid PET brain imaging or a diagnostic method for detecting an Aβ biomarker.

[0129] 45. The method according to Embodiment 43 or 44, wherein a second dose is administered to a human subject until the amount of Aβ deposits in the brain of the human subject is reduced by approximately 20-100%.

[0130] 46. ​​The method according to Embodiment 45, which reduces Aβ deposits in the brain of human subjects by approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 100%.

[0131] 47. The method according to any one of Embodiments 31 to 44, wherein a second dose of donanemab is administered to a human subject until the Aβ deposits in the brain of the human subject are reduced by i) approximately 25 centroids to approximately 100 centroids on an average, ii) approximately 50 centroids to approximately 100 centroids on an average, iii) approximately 100 centroids, or iv) approximately 84 centroids.

[0132] 48. The method according to any one of Embodiments 31 to 47, wherein the disease characterized by Aβ deposition in the brain of a human subject is selected from preclinical Alzheimer's disease (AD), clinical AD, prodromal AD, mild AD, moderate AD, severe AD, Down syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy.

[0133] 49. The method according to any one of embodiments 31 to 48, wherein the human subject is a patient with early symptomatic AD.

[0134] 50. The method according to Embodiment 49, wherein the human subject has prodromal AD and mild dementia due to AD.

[0135] 51. The method according to any one of Embodiments 26 to 50, wherein the human subject is i) determined to have a very low to moderate tau load, or is determined to have a very low to moderate tau load, ii) determined to have a low to moderate tau load, or is determined to have a low to moderate tau load, iii) determined to have a very low to moderate tau load, or is determined to have a very low to moderate tau load and one or two alleles of APOE e4, iv) determined to have a low to moderate tau load, or is determined to have a low to moderate tau load and one or two alleles of APOE e4, or v) has one or two alleles of APOE e4.

[0136] 52. The method according to Embodiment 51, wherein a human subject has a very low to moderate tau load if i) the tau load measured by PET brain imaging is 1.46 SUVr or less, or ii) the tau load measured by PET brain imaging is 1.10 SUVr to 1.46 SUVr, and the human subject has a low to moderate tau load.

[0137] 53. The method according to any one of Embodiments 26 to 50, wherein the human subject is i) not having a high tau burden or has been determined not to have a high tau burden, or ii) possessing one or two alleles of APOE e4 and not having a high tau burden or has been determined not to have one.

[0138] 54. The method according to Embodiment 53, in which a human subject has a high tau load if the tau load measured by PET brain imaging exceeds 1.46 SUVr.

[0139] 55. The method according to Embodiment 51 or 53, wherein the tau load in a human subject is determined using PET brain imaging or a diagnostic method for detecting tau biomarkers.

[0140] 56. In the manufacture of pharmaceuticals for the treatment or prevention of diseases characterized by Aβ deposition in the brain of human subjects, the use of an anti-N3pGlu Aβ antibody, either simultaneously, separately, or sequentially combined with antibody 1, A first dose of anti-N3pGlu Aβ antibody, approximately 100 mg to 700 mg, is administered once or twice, with each first dose administered approximately every four weeks. Subsequently, a second dose, greater than 700 mg to approximately 1400 mg, is administered four weeks after the first dose, with each second dose of anti-N3pGlu Aβ antibody administered approximately every four weeks. The anti-N3pGlu Aβ antibody used is donanemab.

[0141] 57. The use according to Embodiment 56, wherein human subjects are administered one, two, or three doses of the first dose of donanemab before being administered the second dose of donanemab.

[0142] 58. The use described in Embodiment 56 or 57, in which human subjects are administered a first dose of approximately 700 mg of donanemab three times.

[0143] 59. The use according to any one of Embodiments 56 to 58, wherein human subjects are administered one or more second doses of donanemab of approximately 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, or 1400 mg.

[0144] 60. The use according to any one of embodiments 56 to 59, wherein the human subject is administered one or more second doses of donanemab of approximately 1400 mg.

[0145] 61. The use according to any one of Embodiments 56 to 60, wherein the anti-N3pGlu Aβ antibody is administered to a human subject over a series of treatment periods up to 72 weeks, or until normal levels of amyloid are achieved.

[0146] 62. The use according to any one of Embodiments 56 to 61, wherein the anti-N3pGlu Aβ antibody is administered to a human subject until the amyloid plaque level in the patient is approximately 25 centiloids or less.

[0147] 63. Use according to any one of Embodiments 56 to 61, wherein the anti-N3pGlu Aβ antibody is administered to a human subject until the amyloid plaque level in the patient is less than or equal to approximately 25 centiloids in two consecutive PET imaging scans, or less than or equal to approximately 11 centiloids in one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least 6 months apart.

[0148] 64. The use according to any one of Embodiments 56 to 61, wherein a human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks, followed by a second dose of 1400 mg of donanemab once every four weeks, for a period of up to 72 weeks.

[0149] 65. The use according to any one of Embodiments 56 to 61, wherein a human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks, followed by a second dose of 1400 mg of donanemab once every four weeks, until the amyloid plaque level in the patient is approximately 25 centiloids or less.

[0150] 66. Use according to any one of Embodiments 56 to 61, wherein a human subject is administered three doses of a first dose of 700 mg donanemab once every four weeks, followed by a second dose of 1400 mg donanemab once every four weeks, until the amyloid plaque level in the patient is less than or equal to approximately 25 centiloids in two consecutive PET imaging scans, or less than or equal to approximately 11 centiloids in one PET imaging scan, with the two consecutive PET imaging scans optionally spaced at least six months apart.

[0151] 67. The use according to any one of Embodiments 56 to 66, wherein a human subject is administered a second dose of donanemab over a series of therapeutic periods sufficient to treat or prevent a disease.

[0152] 68. Use according to any one of Embodiments 56 to 67, wherein the treatment or prevention of a disease results in i) a reduction in Aβ deposits in the brain of a human subject, and / or ii) a delay in cognitive or functional decline in a human subject.

[0153] 69. The use according to Embodiment 68, wherein the reduction of Aβ deposits in the brain of a human subject is determined by amyloid PET brain imaging or a diagnostic method for detecting an Aβ biomarker.

[0154] 70. The use according to Embodiment 68 or 69, wherein a second dose of donanemab is administered to a human subject until the amount of Aβ deposits in the brain of the human subject is reduced by approximately 20–100%.

[0155] 71. The use according to Embodiment 70, which reduces Aβ deposits in the brain of human subjects by approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 100%.

[0156] 72. The use according to Embodiment 70 or 71, which reduces Aβ deposits in the brain of a patient by 100%.

[0157] 73. The use according to any one of Embodiments 56 to 72, wherein a second dose of donanemab is administered to a human subject until the Aβ deposits in the brain of the human subject are reduced by i) approximately 25 centroids to approximately 100 centroids on an approximate average, ii) approximately 50 centroids to approximately 100 centroids on an approximate average, iii) approximately 100 centroids, or iv) approximately 84 centroids.

[0158] 74. Use according to any one of Embodiments 56 to 73, wherein the disease characterized by Aβ deposition in the brain of a human subject is selected from preclinical Alzheimer's disease, clinical AD, prodromal AD, mild AD, moderate AD, severe AD, Down syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy.

[0159] 75. Use according to any one of Embodiments 56 to 74, wherein the human subject is a patient with early symptomatic AD, or the human subject has prodromal AD or mild dementia due to AD.

[0160] 76. Use according to any one of Embodiments 56 to 75, wherein the human subject is i) has a very low to moderate tau load or is determined to have a very low to moderate tau load, ii) has a low to moderate tau load or is determined to have a low to moderate tau load, iii) has a very low to moderate tau load or is determined to have a very low to moderate tau load and one or two alleles of APOE e4, iv) has a low to moderate tau load or is determined to have a low to moderate tau load and one or two alleles of APOE e4, or v) has one or two alleles of APOE e4.

[0161] 77. The use according to Embodiment 76, wherein the human subject has a very low to moderate tau load if i) the tau load measured by PET brain imaging is 1.46 SUVr or less, or ii) the tau load measured by PET brain imaging is 1.10 SUVr to 1.46 SUVr, and the human subject has a low to moderate tau load.

[0162] 78. Use according to any one of Embodiments 56 to 75, wherein the human subject i) does not have a high tau burden or is determined not to have a high tau burden, or ii) possesses one or two alleles of APOE e4 and does not have a high tau burden or is determined not to have a high tau burden.

[0163] 79. If the tau load measured by PET brain imaging exceeds 1.46 SUVr, the human subject has a high tau load, as described in Embodiment 78.

[0164] 80. The use according to Embodiment 76 or 78, wherein the tau load in a human subject is determined using tau PET brain imaging or a diagnostic method for detecting tau biomarkers.

[0165] 81.i) A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of a human subject who is determined to have a very low to moderate tau load, or a low to moderate tau load, or who is determined to have a very low to moderate tau load, or a low to moderate tau load and one or two alleles of APOE e4, i) Administering a first dose of donanemab to human subjects, approximately 100 mg to 700 mg, once or twice, with each first dose of donanemab administered approximately once every four weeks. ii) Administering a second dose of donanemab to human subjects at a dose of more than 700 mg to approximately 1400 mg, four weeks after administering one or more first doses, with each second dose administered approximately once every four weeks. A method comprising simultaneously, separately, or sequentially combining an effective amount of antibody 1.

[0166] 82. A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of human subjects, This includes determining whether a human subject has tau loading in the temporal, occipital, parietal, or frontal lobes of the brain, and if the human subject has tau loading in the temporal, occipital, parietal, or frontal lobes of the brain, i) Administering a first dose of anti-N3pGlu Aβ antibody to human subjects, consisting of one or more doses ranging from approximately 100 mg to 700 mg, with each first dose administered approximately once every four weeks. ii) Approximately four weeks after administering one or more first doses, administer one or more second doses of anti-N3pGlu Aβ antibody, ranging from over 700 mg to approximately 1400 mg, to human subjects, with each second dose administered approximately once every four weeks. A method comprising simultaneously, separately, or sequentially combining an effective amount of antibody 1.

[0167] 83. The method according to Embodiment 82, wherein the human subject has tau loading in the posterolateral temporal lobe or temporal lobe of the brain.

[0168] 84. The method according to Embodiment 82, wherein the human subject has tau loading in the occipital lobe of the brain.

[0169] 85. The method according to Embodiment 82, wherein the human subject has a tau load in the parietal lobe of the brain.

[0170] 86. The method according to Embodiment 82, wherein the human subject has tau loading in the frontal lobe of the brain.

[0171] 87. The method according to Embodiment 82, wherein the human subject has tau loading in the posterolateral temporal lobe (PLT) and / or occipital lobe of the brain.

[0172] 88. The method according to any one of embodiments 82 to 87, wherein the human subject has tau loading in i) the parietal or precuneus region, or ii) the frontal region, along with tau loading in the PLT or occipital region of the brain.

[0173] 89. The method according to any one of embodiments 82 to 86, wherein the human subject has a tau load isolated in the frontal lobe, or a tau load in a region of the temporal lobe that does not include the posterolateral temporal region (PLT) of the brain.

[0174] 90. The method according to any one of embodiments 82 to 88, wherein the human subject has tau loading in the posterolateral temporal lobe, occipital lobe, and parietal lobe of the brain.

[0175] 91. The method according to any one of embodiments 82 to 88, wherein the human subject has tau loading in the posterolateral temporal lobe, occipital lobe, parietal lobe, and frontal lobe of the brain.

[0176] 92. The method according to any one of embodiments 82 to 88, wherein the human subject has tau loading in the posterolateral temporal lobe, occipital lobe, parietal lobe and / or frontal lobe of the brain.

[0177] 93. The method according to any one of Embodiments 82 to 92, wherein a human subject is administered the first dose once, twice, or three times before being administered the second dose.

[0178] 94. The method according to any one of embodiments 82 to 93, wherein a human subject is administered a first dose of approximately 700 mg.

[0179] 95. The method according to any one of Embodiments 82 to 94, wherein a human subject is administered one or more second doses of approximately 800 mg, approximately 900 mg, approximately 1000 mg, approximately 1100 mg, approximately 1200 mg, approximately 1300 mg, or approximately 1400 mg.

[0180] 96. The method according to any one of embodiments 82 to 95, wherein a human subject is administered a second dose of approximately 1400 mg once or more.

[0181] 97. The method according to any one of Embodiments 82 to 96, wherein an anti-N3pGlu Aβ antibody is administered to a human subject for a period of up to 72 weeks, or until a normal level of amyloid is achieved.

[0182] 98. The method according to any one of Embodiments 82 to 97, wherein an anti-N3pGlu Aβ antibody is administered to a human subject until the patient's amyloid plaque level is approximately 25 centiloids or less.

[0183] 99. The method according to any one of embodiments 82 to 98, wherein an anti-N3pGlu Aβ antibody is administered to a human subject until the amyloid plaque level in the human subject is less than or equal to approximately 25 centiloids in two consecutive PET imaging scans, or less than or equal to approximately 11 centiloids in one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least 6 months apart.

[0184] 100. The method according to any one of Embodiments 82 to 99, wherein a human subject is administered a first dose of 700 mg three times, once every four weeks, followed by a second dose of 1400 mg once every four weeks for a maximum of 72 weeks.

[0185] 101. The method according to any one of Embodiments 82 to 100, wherein a human subject is administered a first dose of 700 mg three times at 4 weeks until the amyloid plaque level in the subject is approximately 25 centiloids or less, followed by a second dose of 1400 mg at 4 weeks.

[0186] 102. The method according to any one of Embodiments 82 to 101, wherein a human subject is administered a first dose of 700 mg three times at 4 weeks until the amyloid plaque level in the subject is approximately 25 centiloids in two consecutive PET imaging scans or less than approximately 11 centiloids in one PET imaging scan, and then a second dose of 1400 mg at 4 weeks, wherein optionally, two consecutive PET imaging scans are spaced at least 6 months apart.

[0187] 103. The method according to any one of Embodiments 82 to 102, wherein a human subject is administered a second dose for a period of time sufficient to treat or prevent a disease.

[0188] 104. The method according to any one of Embodiments 82 to 103, wherein the treatment or prevention of a disease results in i) a reduction in Aβ deposits in the brain of a human subject, and / or ii) a delay in cognitive or functional decline in a human subject.

[0189] 105. The method according to Embodiment 97, wherein the reduction of Aβ deposits in the brain of a human subject is determined by amyloid PET brain imaging or a diagnostic method for detecting an Aβ biomarker.

[0190] 106. The method according to Embodiment 97 or 98, wherein a second dose is administered to a human subject until the amount of Aβ deposits in the brain of the human subject is reduced by approximately 20-100%.

[0191] 107. The method according to Embodiment 106, which reduces Aβ deposits in the brain of human subjects by approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 100%.

[0192] 108. The method according to any one of Embodiments 82 to 107, wherein a second dose is administered to a human subject until the Aβ deposits in the brain of the human subject are reduced by i) approximately 25 centroids to approximately 100 centroids on an approximate average, ii) approximately 50 centroids to approximately 100 centroids on an approximate average, iii) approximately 100 centroids, or iv) approximately 84 centroids.

[0193] 109. The method according to any one of Embodiments 82 to 108, wherein the disease characterized by Aβ deposition in the brain of a human subject is selected from preclinical Alzheimer's disease (AD), clinical AD, prodromal AD, mild AD, moderate AD, severe AD, Down syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy.

[0194] 110. The method according to any one of Embodiments 82 to 109, wherein the human subject is a patient with early symptomatic AD.

[0195] 111. The method according to Embodiment 109, wherein the human subject has prodromal AD and mild dementia due to AD.

[0196] 112. The method according to any one of Embodiments 82 to 111, wherein the human subject is i) has a very low to moderate tau load, or is determined to have a very low to moderate tau load, or ii) has a low to moderate tau load, or is determined to have a low to moderate tau load.

[0197] 113. The method according to Embodiment 112, wherein a human subject has a very low to moderate tau load if i) the tau load measured by PET brain imaging is 1.46 SUVr or less, or ii) the tau load measured by PET brain imaging is 1.10 SUVr to 1.46 SUVr, and the human subject has a low to moderate tau load.

[0198] 114. The method according to any one of embodiments 82 to 113, wherein the human subject does not have a high tau load, or is determined not to have a high tau load.

[0199] 115. The method according to Embodiment 114, wherein a human subject has a high tau load if the tau load measured by PET brain imaging exceeds 1.46 SUVr.

[0200] 116. The method according to Embodiment 114 or 115, wherein the tau load in a human subject is determined using PET brain imaging or a diagnostic method for detecting tau biomarkers.

[0201] 117. The method according to any one of Embodiments 82 to 116, wherein the anti-N3pGlu Aβ antibody comprises donanemab.

[0202] 118. The method according to any one of embodiments 82 to 117, wherein the patient has one or two alleles of APOE e4.

[0203] 119. A method for reducing / preventing a further increase in tau load, or for slowing the rate of tau accumulation in the temporal, occipital, parietal, or frontal lobes of a human brain, comprising administering an effective amount of anti-N3pGlu Aβ antibody to a human subject simultaneously, separately, or sequentially in combination with antibody 1. [Brief explanation of the drawing]

[0204] [Figure 1] This study demonstrates antibody-1 neutralization of human IL-34-induced luciferase reporter activity in hCSF1R expressing 293SRE cells. [Examples]

[0205] The following examples are provided to illustrate, and not limit, the claimed invention. The results of the following assays demonstrate that exemplary monoclonal antibodies, such as antibody 1 of the Disclosure, bind to and / or neutralize IL-34 and can therefore be used to treat immune-mediated and inflammatory diseases as described herein.

[0206] Example 1: Antibody generation, expression, and purification A panel of human anti-IL-34 antibodies is obtained using a complete human yeast display library and screened to identify reagents that may 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 antigen concentration and / or increasing dissociation time. Individual mutants are sequenced and used to construct a combinatorial library, which is subjected to additional rounds of selection while increasing stringency to identify additive or synergistic mutation pairings between individual CDR regions. Individual combinatorial clones are sequenced and their binding characteristics are determined. To further increase affinity for IL-34, these combinatorial clones may be subjected to additional rounds of single and combinatorial mutagenesis. This screening can be performed against human or cynomolgus monkey IL-34 to increase affinity for selected species. Selected antibodies can also be mutagenicized to repair post-translational modifications such as isomerization while maintaining their binding affinity to IL-34. Additionally, to reduce the potential risk of immunogenicity, framework (FW) or CDR substitutions can be performed on the antibody to return the sequence to its germline state.

[0207] For example, a modified and / or optimized anti-IL-34 antibody, referred to herein as Antibody 1, is obtained, having amino acid sequences of the variable regions of the heavy and light chains, complete heavy and light chain amino acid sequences, and nucleotide sequences that encode the same ones listed in the section below titled “List of Amino Acid and Nucleotide Sequences”. The sequence numbers corresponding to these sequences, as well as the CDR amino acid sequences of the light and heavy chains, are shown in Table 1.

[0208] The anti-IL-34 antibodies illustrated in this disclosure can be expressed and purified in essentially the following ways: Suitable host cells, e.g., HEK293, NS0, or CHO, can be transfected either transiently or stably with an expression system for secreting antibodies using an optimal predetermined HC:LC vector ratio (such as 1:3, 1:2, or 1:1), or with a single vector system encoding both HC and LC.

[0209] The expression plasmid contains, for example, DNA encoding the LC and HC of antibody 1 (the DNA sequence of SEQ ID NO: 11 encoding the exemplified HC of antibody 1, and the DNA sequence of SEQ ID NO: 12 encoding the exemplified LC amino acid sequence of antibody 1), and is expressed from a suitable construct commonly used for this purpose. Clonal cell lines are grown and screened for antibody 1 production, and a selected and established clonal cell line is created without using any animal-containing materials and used for production.

[0210] The purified medium from which the antibody is secreted can be purified by conventional techniques such as mixed-mode ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to a protein A or protein G column and eluted therefrom using conventional methods, and mixed-mode ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and polymers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The illustrated IL-34 antibody in this disclosure is concentrated and / or sterile filtered using common techniques. The purity of the illustrated antibody after these chromatography steps is greater than 95%. The illustrated anti-IL-34 antibody in this disclosure can be immediately frozen at -70°C or stored at 4°C for several months.

[0211] Example 2: Characterization of anti-IL-34 antibodies Binding affinity for human and cynomolgus monkey IL-34 The binding affinity of the anti-IL-34 monoclonal antibody of this disclosure to human and / or cynomolgus monkey (cyno) IL-34 can be determined by methods known in the art. Briefly, the binding affinity and kinetics of the antibody are evaluated by surface plasmon resonance using BIAcore® 8K (Cytiva) at 37°C. Binding affinity is measured by immobilizing the anti-IL-34 antibody on BIAcore® Sensor Chip Protein A (Cytiva) and flowing human or cynomolgus monkey IL-34 starting at 25 nM or 12.5 nM, which is a 2-fold serial dilution with HBS-EP+ buffer (Teknova). In each cycle, 200 μL of IL-34 is flowed over the immobilized antibody at 100 μL / min, followed by dissociation for 20 minutes. The chip surface is regenerated with 50 μL of glycine buffer at pH 1.5 at a flow rate of 100 μL / min. The data are fitted to a 1:1 Langmuir binding mode to derive kon and koff and calculate KD. Table 3 shows the average of at least three experiments for human and cynomolgus monkey IL-34 for the example antibody 1.

[0212] [Table 3]

[0213] Example 3: In vitro functional characterization of an anti-human IL-34 antibody The antibodies of this disclosure are tested for their ability to neutralize IL-34 binding and / or activity. The neutralization of IL-34 binding and / or activity by the antibodies of this disclosure may be evaluated by one or more IL-34 / CSF1R receptor binding assay formats, as well as IL-34 cell-based activity assays, as described below, for example.

[0214] Ability of antibody 1 to replace IL-34 from CSF1R Assays for neutralizing antibodies conjugating IL-34 / CSF1R can be performed using enzyme assays. Such assays can utilize recombinant expressed CSF1R extracellular domain proteins capable of binding to IL-34. These proteins can be conjugated to an ELISA plate to capture soluble IL-34. IL-34 can then be detected by either biotinylation of the antigen and detection by streptavidin / neutraavidin-conjugated peroxidase or phosphatase enzymes. Such neutralization assays involve pre-incubation (e.g., 1 hour) of the antibody to be evaluated with labeled IL-34 (and a control sample not involving an antibody targeting IL-34) before being added to the conjugation assay.

[0215] The CSF1R extracellular domain protein (hCSF1R_Fc, commercially available from R&D, catalog number 329-MR, cynomolgus monkey CSF1R ECD-Fc (AAA is a linker between the CSF1R extracellular domain and Fc) (SEQ ID NO: 34)) can be conjugated to an ELISA plate at a concentration of 30 nM to capture soluble biotinylated IL-34 and allowed to conjugate for 1 hour. After washing and blocking the plate, biotinylated IL-34 can be added and then detected with streptavidin-conjugated peroxidase. To determine the antibody concentration required to replace IL-34 from CSF1R, concentrations of labeled IL-34 close to the 80% binding level (EC80) (3.7 nM) can be used in combination with various antibody concentrations (0-100 nM). After 1 hour incubation, IL-34 bound to CSF1R is detected via streptavidin-conjugated peroxidase. The antibodies were assayed (n=2), and the mean and standard deviation at each concentration were calculated. The potency of the antibody that substituted IL-34 from CSF1R was reported as IC50 (nM), and the calculated confidence intervals (CI) are shown in Tables 4 and 5.

[0216] [Table 4]

[0217] [Table 5]

[0218] 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. The results in Table 4 show that antibody 1 has high affinity for human IL-34 and can replace human CSF1R with IL-34 with an IC50 of 0.1537 nM. The results in Table 4 show that antibody 1 has high affinity for human IL-34, and in particular, antibody 1 shows affinity for human IL-34 comparable to that for hCSF1R, and therefore possesses binding properties that enable them to effectively neutralize IL-34 in vivo. Blocking IL-34 is thought to provide a useful means for disease modification while avoiding safety concerns associated with some existing immunomodulatory therapies. Therefore, 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. (For example, see Lelios, I. et al. Emerging roles of IL-34 in health and disease, J Exp Med (2020) 217(3):e20190290).

[0219] In vitro inhibition of IL-34-induced response The neutralization of IL-34 activity by the antibodies of this disclosure can be evaluated by one or more IL-34 cell-based assays, for example, as described below. The ability of the antibodies of this disclosure to neutralize human IL-34-inducible luciferase reporter activity can be evaluated in 293 hCSF1R SRE cells transfected with cDNA expressing human CSF1R (received: NP_001275634.1). 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-cultured 96-well plates. The following day, the growth medium is removed and the cells are starved in DMEM-F12 (Dulbecco's modified Eagle medium: nutrient mixture F-12) supplemented with heat-inactivated 1% FBS (fetal bovine serum). After 24 hours of starvation, cells were treated with 100 ng / ml of human IL-34 and either multiple concentrations of hCSF1R-Fc or antibody 1 for 6 hours. Following incubation, cells were lysed in 50 μl of Promega® Glo® lysis buffer (Promega® E266A) with gentle agitation for 5 minutes. 50 ml of BrightGlo® luminescence reagent (Promega® E2620) was added, and the cells were incubated on top of the lysed cells for 2 minutes. Luminescence was read using a Perkin Elmer Wallac 1420 Victor2® microplate reader. The reduction in relative fluorescence units (RFU) shown in Table 7 and Figure 1 reflects the ability of antibody 1 to neutralize human IL-34-induced luciferase activity. For the neutralization of hIL-34, the semi-maximal inhibitory concentration (IC50) of antibody 1 was 0.04582 μg / ml. Human CSF1R-Fc was used as a positive control in this assay and inhibited luciferase activity with an IC50 of 0.09603 μg / ml.

[0220] [Table 6]

[0221] Flow cytometry-based inhibition of IL-34-induced expression of CD163 by anti-IL34 antibodies in human monocytes: IL-34 neutralization can also be evaluated by measuring the expression of the cell surface antigen CD163 in human monocytes after treatment with IL-34 using 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 Rep8, 256 (2018)). CD14-positive monocytes were treated with IL-34 for 6 days, and after staining for CD163 with antibodies, CD163 expression was evaluated by flow cytometry. In the experiment, the change in the number of cells expressing CD163 indicated that IL-34 treatment increases the expression of this antigen in monocytes. The addition of antibody 1 inhibited the increase in CD163 expression. In this experiment, an isotype-matched IgG4 antibody was used as a negative control.

[0222] CD14+ human monocytes can differentiate into macrophages upon addition of IL-34 (100 ng / ml). The macrophage marker CD163 can be used to monitor the degree of differentiation. This differentiation into macrophages can be inhibited by the addition of an anti-IL-34 antibody. Seed CD14+ human monocytes in a 6-well plate with or without IL-34. Treat the cells with an anti-IL-34 antibody, e.g., antibody 1, or 15 μg / ml IgG4 PAA for a total of 6 days, refreshing the treatment on day 3. On day 6, collect the cells removed from the plate with non-enzymatic cell dissociation buffer and wash with FACS buffer (PBS + 2% FBS + 0.1% sodium azide + 2% EDTA). Block the cells with TruStain FcX (catalog no. 422302) for 30 minutes according to the manufacturer's recommendations. After blocking, cells were washed with FACS buffer and stained with anti-CD163-PE or IgGk isotype control-PE at 4°C for 1 hour. At the end of incubation, cells were washed and flow analysis was performed using Accuri with a minimum of 10,000 events. Median-PE-A levels were collected for each treatment. The results are shown in Table 10.

[0223] [Table 7]

[0224] Inhibition of CD163 expression in human monocytes in response to IL-34 with antibody 1 demonstrates the ability of the antibody of this disclosure to modulate the monocyte / macrophage number and / or phenotypic differentiation response to IL-34, supporting the use of this antibody for the treatment of 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).

[0225] Example 4: Characterization of the potential immunogenicity of antibody 1 Dendritic cell (DC) internalization assay Monocyte-derived DC culture (MDDC) CD14+ monocytes are isolated from peripheral blood mononuclear cells (PBMCs), cultured, and differentiated into DCs according to a standard protocol. Briefly, PBMCs are isolated from LRS-WBCs using density gradient centrifugation with Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies). CD14+ monocytes are isolated using positive selection with a CD14+ microbead kit (#130-050-201, Miltenyi Biotec) according to the manufacturer's manual. Next, cells are cultured at 1,000,000 / ml for 6 days with 1,000,000 units / ml of GM-CSF and 600 units / ml of IL-4, and then induced into immature dendritic cells (MDDCs) in RPMI medium (hereinafter referred to as complete RPMI medium or medium purchased from Life Technologies) supplemented with 10% FBS, 1 mM sodium pyruvate, 1 × penicillin-streptomycin, 1 × non-essential amino acids, and 55 μM 2-mercaptoethanol. The medium is changed twice, on days 2 and 5. On day 6, cells are gently collected with a cell scraper and used for experiments. MDDCs are visually characterized for dendritic morphology by microscopy and for the expression of CD14, CD11c, and HLA-DR by flow cytometry. The ability to respond to LPS treatment is confirmed by measuring increases in CD80, CD83, and CD86 using flow cytometry.

[0226] Fab-TAMRA-QSY7 fusion F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch) is double-labeled with QSY7-NHS and TAMRA-SE (Molecular Probes) to obtain Fab-TAMRA-QSY7, which is used as a universal probe to track the internalization of the test substance. Each vial of F(ab')2 (approximately 1 ml at 1.3 mg / ml) is concentrated to approximately 2 mg / ml by centrifugation at 14,000 rcf for 2 minutes using an Amico Ultra-0.5 centrifugal filter (#UFC501096, Millipore). The pH is adjusted to basic (>pH8) with 10% (v / v) 1M sodium bicarbonate, and 6.8 μl of 10 mM QSY-NHS preservative solution in DMSO is added and mixed. The reaction vial is kept in the dark at room temperature for 30 minutes. The intermediate product, Fab-QSY7, is purified by centrifugation at a relative centrifugal force (RCF) of 1000 for 2 minutes using a Zeba Spin desalting column (#89890, Thermo Scientific). The concentration and degree of labeling (DOL) are calculated by measuring the absorbance at 280 nm and 560 nm using NanoDrop (ThermoFisher). Next, Fab-QSY7 is concentrated to approximately 2 mg / ml by centrifugation again at 14,000 rcf for 2 minutes using an Amico Ultra-0.5 centrifuge filter. After adjusting the pH with 10% (v / v) 1M sodium bicarbonate, 4.3 μl of 15 mM TAMRA-SE preservative solution in DMSO is added and mixed. After 30 minutes in the dark at room temperature, the final product, Fab-TAMRA-QSY7, is purified and collected using a Zeba Spin desalting column by centrifugation at 1000 rcf for 2 minutes. The concentration and DOL are requantified by reading the absorbance at 280 nm, 555 nm, and 560 nm using a NanoDrop spectrophotometer. Using this protocol, approximately 300 μl of Fab-TAMRA-QSY7 is obtained at a concentration of approximately 1.5 mg / ml, containing approximately 2 QSY7 and 2 TAMRA per F(ab')2.

[0227] Standardization and internalization testing using FACS Individual test molecules are normalized to 1 mg / ml in PBS, then further diluted to 8 μg / ml in complete RPMI medium. Fab-TAMRA-QSY7 is diluted to 5.33 μg / ml in complete RPMI medium. Equal volumes of antibody and Fab-TAMRA-QSY7 are mixed and incubated in the dark at 4°C for 30 minutes for complex formation. MDDC is resuspended in complete RPMI medium at 4 million / ml and seeded at 50 μl per well in a 96-well round-bottom plate to which 50 μl of antibody / probe conjugate is added. Cells are incubated in a CO2 incubator at 37°C for 24 hours. Cells are washed in 2% FBS PBS and resuspended in 100 μl of 2% FBS PBS containing Cytox Green live / dead dye. Data are collected with a BD LSR Fortessa X-20 and analyzed with FlowJo. Live single cells are gated and the percentage of TAMRA-fluorescent cells is recorded as readout.

[0228] Data display and statistical analysis The molecules are tested in double or triple denominations against three or more donors. For each donor, the proportion of the TAMRA-positive population is considered. A normalized internalization index (NII) is used to allow comparison of molecules with data generated from different donors. The internalization signal is normalized against IgG1 isotype (NII=0) and internally positive control PC (NII=100) using the following formula.

[0229]

number

[0230] [Table 8] (For example, see Wen, Y., Cahya, S., Zeng, W. et al. Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment. AAPS J22, 68 (2020).)

[0231] MAPP assay (MHC-associated peptide proteomics) method Primary human dendritic cells from 10 normal human donors were prepared from buffy coat by isolation of CD-14-positive cells as described, and differentiated into immature dendritic cells by incubation for 3 days at 37°C and 5% CO2 in complete RPMI medium containing 5% serum substitute (Thermo Fisher Scientific, catalog no. A2596101) with 20 ng / ml IL-4 and 40 ng / ml GM-CSF (Knierman et al., "The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein", Cell Reports, 33, 108454 (2020)). On day 4, approximately 5 × 10⁶ micromoles of test antibody were added. 6 After adding the LPS to the cells and incubating for 5 hours, replace the medium with fresh medium containing 5 μg / ml LPS to transform the cells into mature dendritic cells. The following day, lyse the mature cells in 1 ml of RIPA buffer containing a protease inhibitor and DNAse. Store the lysate at -80°C until sample analysis.

[0232] Using an automated liquid processing system, HLA-II molecules were isolated from thawed lysates using a biotinylated anti-pan-HLA class II antibody (clone Tu39). The bound receptor-peptide complexes were eluted with 5% acetic acid and 0.1% TFA. The eluted MHC-II peptides were passed through a pre-washed 10k MWCO filter to remove high molecular weight proteins. The isolated MHC-II peptides were analyzed by nanoLC / MS using a Thermo easy 1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. For this separation, a 75 μm × 7 cm YMC-ODS C18 column was used, with a flow rate of 250 nL / min and a gradient of 65 minutes with 0.1% formic acid aqueous solution as solvent A and 80% acetonitrile with 0.1% formic acid as solvent B. Mass spectrometry is performed in a full-scan manner with a resolution of 240,000, followed by a 3-second data-dependent MS / MS cycle consisting of an ion-trap rapid scan with HCD and EThcD fragmentation.

[0233] Peptide identification is performed by an internal proteomics pipeline (Higgs et al., "Label-free LC-MS method for the identification of biomarkers", Methods in Molecular Biology, 428, 209-230 (2008)) using multiple search algorithms without enzyme search parameters against a bovine / human database containing the test antibody sequence. The sample identification file is processed using the KNIME workflow. Peptides identified from the test material are aligned relative to the parent sequence. A summary is created for all donors, annotating the percentage of donors presenting non-germline residues, the number of different regions presenting peptides with non-germline residues, and the depth of peptide presentation in each region with non-germline residues. Increased degree of non-germline peptide presentation is associated with an increased risk of immunogenicity. The results for antibody 1 are shown in Table 12.

[0234] [Table 9]

[0235] T-cell proliferation assay This assay evaluates the ability of a candidate or candidate MAPP-derived peptide cluster to activate CD4+ T cells by inducing cell proliferation as described below (Walsh et al., "Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms", mAbs, 12, 1764829 (2020)). Cryopreserved PBMCs from 10 healthy donors were used, and CD8+ T cells were depleted from the PBMCs and labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). The PBMCs were placed in AIM-V medium (Life Technologies, catalog no. 12055-083) containing 5% CTS® Immune Cell SR (Gibco, catalog no. A2596101) in 4 × 10⁶ cells. 6 Cells were seeded at / ml / well and tested in triplicates with 2.0 mL of various test substances, DMSO control, medium control, and keyhole limpet hemocyanin (KLH; positive control). Cells were cultured and incubated at 37°C in 5% CO2 for 7 days. On day 7, samples were stained with the following cell surface markers for viability detection by flow cytometry using a BD LSRFortessa® equipped with a High Throughput Sampler (HTS): anti-CD3, anti-CD4, anti-CD14, anti-CD19, and DAPI. Data were analyzed using FlowJo® software (FlowJo, LLC, TreeStar) and the Cellular Division Index (CDI) was calculated. Briefly, the CDI of each test molecule was compared to the CFSE cells growing in the stimulated wells. dim Percent of CD4+ T cells are growing in unstimulated wells of CFSEdim The calculation was performed by dividing by the percentage of CD4+ T cells. A CDI of 2.5 or higher was considered to indicate a positive reaction. The proportion of donor frequencies across all donors was evaluated. The results for antibody 1 are shown in Table 13.

[0236] [Table 10]

[0237] Example 5: Antibody pharmacokinetics in cynomolgus monkeys Cynomolgus monkeys are administered a single intravenous (IV) dose of 3 mg / kg of antibody 1 in 1 mL / kg of PBS (pH 7.4). For pharmacokinetic characterization, blood is collected from two animals / time points 1, 3, 6, 24, 48, 72, 96, 120, 168, 240, 336, 408, 504, and 672 hours after administration and processed into serum. Serum concentrations of antibody 1 are determined by qualified immunoaffinity liquid chromatography-mass spectrometry. Antibody 1 and a human antibody internal standard (stable isotope-labeled human IgG) are extracted from 100% cynomolgus monkey serum using biotinylated goat anti-human IgG antibody, followed by quantification of trypsin surrogate peptides using a Q-Exactive® Orbitrap® mass spectrometer. Pharmacokinetic parameters are calculated for each animal (N=2) using non-compartmental analysis (NCA), and parameters are summarized by their mean values. NCA and summary statistical calculations were performed using Phoenix. As shown in Table 14, antibody I showed an extension of the pharmacokinetic profile in cynomolgus monkeys.

[0238] [Table 11]

[0239] Sequence listings of amino acids and nucleotide sequences Heavy chain of antibody 1 (SEQ ID NO: 1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGYLWHAFDHWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0240] Light chain of antibody 1; LC of antibody 2 (SEQ ID NO: 2) EIVLTQSPGTLSLSPGERATLSCRASQSVSSLYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQVVGSSPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0241] Antibody 1's HCVR (SEQ ID NO: 3) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGYLWHAFDH

[0242] LCVR of antibody 1; LCVR of antibody 2 (SEQ ID NO: 4) EIVLTQSPGTLSLSPGERATLSCRASQSVSSLYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQVVGSSPPFT

[0243] Antibody 1's HCDR1 (SEQ ID NO: 5) AASGFTFSSYAMS

[0244] Antibody 1's HCDR2 (SEQ ID NO: 6) AISGSGGKTY

[0245] Antibody 1's HCDR3 (SEQ ID NO: 7) AKRGYLWHAFDH

[0246] LCDR1 of antibody 1 and antibody 2 (SEQ ID NO: 8) RASQSVSSLYLA

[0247] LCDR2 of antibody 1 and antibody 2 (SEQ ID NO: 9) YGASSRAT

[0248] LCDR3 of antibody 1 and antibody 2 (SEQ ID NO: 10) QVVGSSPPFT

[0249] DNA encoding the heavy chain of antibody 1 (SEQ ID NO: 11)

[0250] DNA encoding the light chain of antibody 1 (SEQ ID NO: 12) gaaatagttctcactcagtcccctgggacactctccctgagtccaggagaacgtgcaacactcagttgccgtgcaagccagtccgtctcatccttgtatcttgcttggtaccaacaaaaacctggacaggccccccgtcttcttatctatggtgcctccagt cgcgcaactggtattcccgaccggttcagcggcagtgggtccggcactgacttcaccctgactataagtcggttggagccagaggactttgccgtgtactattgccaagtggtgggaagctcccctcccttcactttcggcggagggaccaaggtagaaatc aaaagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttcttatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccag gagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggagagtgc

[0251] Antibody 1: HCDR1 (Kabat) (SEQ ID NO: 13) SYAMS

[0252] Antibody 1: HCDR2 (Kabat) (SEQ ID NO: 14) AISGSGGKTYYADSVKG

[0253] Antibody 1: HCDR3 (Kabat) (SEQ ID NO: 15) RGYLWHAFDH

[0254] Antibody 1: LCDR1 (Kabat) (SEQ ID NO: 16) RASQSVSSLYLA

[0255] Antibody 1 LCDR2 (Kabat) (SEQ ID NO: 17) GASSRAT

[0256] Antibody 1 LCDR3 (Kabat) (SEQ ID NO: 18) QVVGSSPPFT

[0257] Antibody 1: HCDR1 (Chothia) (SEQ ID NO: 19) GFTFSSY

[0258] Antibody 1: HCDR2 (Chothia) (SEQ ID NO: 20) SGSGGK

[0259] Antibody 1: HCDR3 (Chothia) (SEQ ID NO: 21) RGYLWHAFDH

[0260] Antibody 1: LCDR1(Chothia) (SEQ ID NO: 22) RASQSVSSLYLA

[0261] Antibody 1 LCDR2(Chothia) (SEQ ID NO: 23) GASSRAT

[0262] Antibody 1: LCDR3 (Chothia) (SEQ ID NO: 24) QVVGSSPPFT

[0263] Antibody 1: HCDR1(IMGT) (SEQ ID NO: 25) GFTFSSYA

[0264] Antibody 1's HCDR2(IMGT) (SEQ ID NO: 26) ISGSGGKT

[0265] Antibody 1's HCDR3(IMGT) (SEQ ID NO: 27) AKRGYLWHAFDH

[0266] Antibody 1: LCDR1(IMGT) (SEQ ID NO: 28) QSVSSLY

[0267] Antibody 1's LCDR2(IMGT) (SEQ ID NO: 29) GAS

[0268] Antibody 1's LCDR3(IMGT) (SEQ ID NO: 30) QVVGSSPPFT

[0269] Human IL-34 (SEQ ID NO: 31) NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP

[0270] IgG4PAA hinge region (SEQ ID NO: 32) ESKYGPPCPPCP

[0271] IgG4PAA Fc region (SEQ ID NO: 33) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0272] Sequence of Cnidinomolgus macaque CSF1R ECD-Fc (SEQ ID NO: 34) IPVIEPSGPELVVKPGETVTLRCVGNGSVEWDGPISPHWTLYSDGPSSVLTTNNATFQNTRTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAKEVVVFEDQDALLPCLLTDPVLEAGVSLVRLRGRPLLRHTNYSFSPWHGFIIHRAKFIQGQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASNIDVDFDVFLQHNTTKLAIPQRSDFHDNRYQKVLTLSLGQVDFQHAGNYSCVASNVQGKHSTSMFFRVVESAYLDLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTSINGSGTLLCAASGYPQPNVTWLQCAGHTDRCDEAQVLQVWVDPHPEVLSQEPFQKVTVQSLLTAETLEHNQTYECRAHNSVGSGSWAFIPISAGARTHPPDEAAAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0273] Heavy chain of antibody 2 (SEQ ID NO: 35) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGYLWHAFDHWGRGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] Heavy chain of antibody 3 (SEQ ID NO: 36) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGYLWHAFDHWGRGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0275] Antibody 4 heavy chain (SEQ ID NO: 37) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGYLWHAFDHWGRGTLVTSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0276] Donanemab heavy chain (SEQ ID NO: 38) QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0277] Donanemab light chain (SEQ ID NO: 39) DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0278] Heavy chain of anti-N3pG antibody (SEQ ID NO: 40) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0279] Light chain of anti-N3pG antibody (SEQ ID NO: 41) DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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), wherein the VH comprises heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3. The HCDR1 includes sequence number 5, The HCDR2 includes sequence number 6, The HCDR3 includes sequence number 7, The aforementioned LCDR1 includes sequence number 8, The LCDR2 includes sequence number 9, The aforementioned LCDR3 is an antibody containing SEQ ID NO:

10.

2. The antibody according to claim 1, wherein VH comprises SEQ ID NO: 3 and VL comprises SEQ ID NO:

4.

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

2.

4. A nucleic acid comprising a sequence encoding a sequence number selected from sequence number 11 or 12.

5. A vector comprising the nucleic acid described in claim 4.

6. The vector according to claim 5, wherein the vector comprises a first nucleic acid sequence encoding sequence number 11 and a second nucleic acid sequence encoding sequence number 12.

7. A composition comprising a first vector containing a nucleic acid sequence encoding sequence number 11, and a second vector containing a nucleic acid sequence encoding sequence number 12.

8. A cell comprising the vector according to claim 5 or 6.

9. A cell comprising a first vector containing a nucleic acid sequence encoding sequence number 11, and a second vector containing a nucleic acid sequence encoding sequence number 12.

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

11. A process for producing an antibody, comprising culturing the cells described in any one of claims 8 to 10 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

12. An antibody produced by the process described in claim 11.

13. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 3 or 12, and a pharmaceutically acceptable excipient, diluent, or carrier.

14. A method for treating an immune-mediated disease in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 3 or 12, or a pharmaceutical composition according to claim 13.

15. The method according to claim 14, wherein the immune-mediated disease is selected from the group consisting of Alzheimer's disease; tauopathic diseases; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

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

17. An antibody according to any one of claims 1 to 3 or 12, for use in therapy.

18. An antibody according to any one of claims 1 to 3 or 12, or a pharmaceutical composition according to claim 13, for use in the treatment of immune-mediated diseases.

19. The antibody or pharmaceutical composition according to claim 18, wherein the immune-mediated disease is selected from the group consisting of Alzheimer's disease; tauopathic diseases; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, kidney disease, sepsis, amyotrophic lateral sclerosis (ALS), and / or non-alcoholic fatty liver disease (NAFLD).

20. The antibody or pharmaceutical composition according to claim 18, wherein the immune-mediated disease is Alzheimer's disease.

21. Use of the antibody according to any one of claims 1 to 3 or 12 in the manufacture of a pharmaceutical product for the treatment of an immune-mediated disease.

22. The use according to claim 21, wherein the immune-mediated disease is selected from the group consisting of Alzheimer's disease; tauopathic diseases; Sjögren's syndrome (SS); rheumatoid arthritis (RA); inflammatory bowel disease (IBD), atopic dermatitis, renal disease, sepsis, and / or non-alcoholic fatty liver disease (NAFLD).

23. The use according to claim 21, wherein the immune-mediated disease is Alzheimer's disease.

24. A method for determining human IL-34 levels in body fluids, (a) Contacting the body fluid with an anti-human IL-34 diagnostic monoclonal antibody or its antigen-binding fragment that specifically binds to human IL-34 consisting of the amino acid sequence of SEQ ID NO: 49, wherein the antibody or its antigen-binding fragment comprises, respectively, light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 containing the amino acid sequences (SEQ ID NO: 8), (SEQ ID NO: 9), and (SEQ ID NO: 10), and heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 containing the amino acid sequences (SEQ ID NO: 5), (SEQ ID NO: 6), and (SEQ ID NO: 7), (b) Optionally, remove any nonspecifically bound monoclonal antibody or its antigen-binding fragment, (c) A method comprising detecting and / or quantifying the amount of a monoclonal antibody or its antigen-binding fragment that is specifically bound to human IL-34.

25. The method according to claim 24, wherein the bodily fluid is blood, serum, plasma, or cerebrospinal fluid, and the contact occurs ex vivo.

26. A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of a human subject, comprising administering an effective amount of anti-N3pG Aβ antibody to the human subject in need of such treatment or prevention, simultaneously, separately, or sequentially in combination with an effective amount of the antibody described in any one of claims 1 to 3 or 12.

27. The method according to claim 26, wherein the anti-N3pGAβ antibody is donanemab, and the antibody described in any one of claims 1 to 3 or 12 is antibody 1.

28. The method according to claim 26, wherein the disease is Alzheimer's disease.

29. The method according to claim 26, wherein the anti-N3pG Aβ antibody is donanemab and the disease is Alzheimer's disease.

30. The method according to claim 29, wherein antibody 1 is administered sequentially after a series of treatments with donanemab.

31. A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of human subjects, i) Administering the aforementioned human subjects one or more doses of a first dose of anti-N3pGAβ antibody ranging from approximately 100 mg to approximately 700 mg, wherein each first dose is administered approximately once every four weeks. ii) Approximately four weeks after administering one or more first doses, administer one or more second doses of the anti-N3pGAβ antibody, ranging from more than 700 mg to approximately 1400 mg, to the human subject, wherein each second dose is administered approximately once every four weeks. The aforementioned anti-N3pGlu Aβ antibody is donanemab, and it is administered as follows: iii) A method comprising administering an effective amount of antibody 1 to the human subject simultaneously, separately, or sequentially.

32. The method according to claim 31, wherein the human subject is administered the first dose of donanemab once, twice, or three times before being administered the second dose.

33. The method according to claim 31 or 32, wherein the human subject is administered a first dose of donanemab of about 700 mg.

34. The method according to any one of claims 31 to 33, wherein the human subject is administered one or more doses of donanemab in a second dose of approximately 800 mg, approximately 900 mg, approximately 1000 mg, approximately 1100 mg, approximately 1200 mg, approximately 1300 mg, or approximately 1400 mg.

35. The method according to any one of claims 31 to 34, wherein the human subject is administered one or more doses of a second dose of approximately 1400 mg of donanemab.

36. The method according to any one of claims 31 to 35, wherein the anti-N3pGlu Aβ antibody is administered to the human subject over a series of treatment periods up to 72 weeks, or until a normal level of amyloid is achieved.

37. The method according to any one of claims 31 to 36, wherein the anti-N3pGlu Aβ antibody is administered to the human subject until the amyloid plaque level in the patient is about 25 centiloids or less.

38. The method according to any one of claims 31 to 36, wherein the anti-N3pGlu Aβ antibody is administered to the human subject for a series of treatments until the amyloid plaque level in the human subject is reduced to about 25 centiloids or less in two consecutive PET imaging scans, or to about 11 centiloids or less in one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least six months apart.

39. The method according to any one of claims 31 to 36, wherein the human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks, followed by a second dose of 1400 mg once every four weeks, over a series of treatment periods of up to 72 weeks.

40. The method according to any one of claims 31 to 36, wherein the human subject is administered a first dose of 700 mg three times, once every four weeks, until the amyloid plaque level in the subject is about 25 centiloids or less, and then a second dose of 1400 mg is administered once every four weeks.

41. The method according to any one of claims 31 to 36, wherein the human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks, followed by a second dose of 1400 mg once every four weeks, until the amyloid plaque level in the subject is less than or equal to about 25 centiloids in two consecutive PET imaging scans, or less than or equal to about 11 centiloids in one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least six months apart.

42. The method according to any one of claims 31 to 41, wherein the human subject is administered the second dose of donanemab over a series of treatment periods sufficient to treat or prevent the disease.

43. The method according to any one of claims 31 to 42, wherein the treatment or prevention of the disease causes i) a reduction in Aβ deposits in the brain of the human subject, and / or ii) a delay in cognitive or functional decline in the human subject.

44. The method according to claim 43, wherein the reduction of Aβ deposits in the brain of the human subject is determined by amyloid PET brain imaging or a diagnostic method for detecting a biomarker of Aβ.

45. The method according to claim 43 or 44, wherein the second dose is administered to the human subject until the amount of Aβ deposits in the brain of the human subject is reduced by approximately 20 to 100%.

46. The method according to claim 45, wherein the Aβ deposits in the brain of the human subject are reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 75%, or about 100%.

47. The method according to any one of claims 31 to 44, wherein the second dose of donanemab is administered to the human subject until the Aβ deposits in the brain of the human subject are reduced by i) approximately 25 centroids to approximately 100 centroids on an approximate average, ii) approximately 50 centroids to approximately 100 centroids on an approximate average, iii) approximately 100 centroids, or iv) approximately 84 centroids.

48. The method according to any one of claims 31 to 47, wherein the disease characterized by the Aβ deposits in the brain of the human subject is selected from preclinical Alzheimer's disease (AD), clinical AD, prodromal AD, mild AD, moderate AD, severe AD, Down syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy.

49. The method according to any one of claims 31 to 48, wherein the human subject is a patient with early symptomatic AD.

50. The method according to claim 49, wherein the human subject has prodromal AD and mild dementia due to AD.

51. The method according to any one of claims 26 to 50, wherein the human subject is i) determined to have a very low to moderate tau load, or is determined to have a very low to moderate tau load, ii) determined to have a low to moderate tau load, or is determined to have a low to moderate tau load, iii) determined to have a very low to moderate tau load, or has a very low to moderate tau load and one or two alleles of APOE e4, iv) determined to have a low to moderate tau load, or has a low to moderate tau load and one or two alleles of APOE e4, or v) has one or two alleles of APOE e4.

52. The method according to claim 51, wherein the human subject has a very low to moderate tau load if the tau load measured by PET brain imaging is 1.46 SUVr or less, or ii) has a low to moderate tau load if the tau load measured by PET brain imaging is between 1.10 SUVr and 1.46 SUVr.

53. The method according to any one of claims 26 to 50, wherein the human subject i) does not have a high tau load or is determined not to have a high tau load, or ii) possesses one or two alleles of APOE e4 and does not have a high tau load or is determined not to have a high tau load.

54. The method according to claim 53, wherein the human subject has a high tau load if the tau load measured by PET brain imaging exceeds 1.46 SUVr.

55. The method according to claim 51 or 53, wherein the tau load in the human subject is determined using PET brain imaging or a diagnostic method for detecting a tau biomarker.

56. In the manufacture of pharmaceuticals for the treatment or prevention of diseases characterized by Aβ deposition in the brain of human subjects, the use of anti-N3pGlu Aβ antibodies, simultaneously, separately, or sequentially combined with antibody 1, wherein one or more first doses of anti-N3pGlu Aβ antibodies of approximately 100 mg to approximately 700 mg are administered, each first dose administered approximately every four weeks, followed by one or more second doses of more than 700 mg to approximately 1400 mg administered four weeks after the administration of the first doses, each second dose of anti-N3pGlu Aβ antibodies administered approximately every four weeks. The aforementioned anti-N3pGlu Aβ antibody is donanemab.

57. The use according to claim 56, wherein the human subject is administered the first dose of donanemab once, twice, or three times before being administered the second dose of donanemab.

58. The use according to claim 56 or 57, wherein the human subject is administered three times a first dose of donanemab of approximately 700 mg.

59. The use according to any one of claims 56 to 58, wherein the human subject is administered one or more doses of a second dose of donanemab, approximately 800 mg, approximately 900 mg, approximately 1000 mg, approximately 1100 mg, approximately 1200 mg, approximately 1300 mg, or approximately 1400 mg.

60. The use according to any one of claims 56 to 59, wherein the human subject is administered one or more doses of a second dose of approximately 1400 mg of donanemab.

61. The use according to any one of claims 56 to 60, wherein the anti-N3pGlu Aβ antibody is administered to the human subject over a series of treatment periods up to 72 weeks, or until a normal level of amyloid is achieved.

62. The use according to any one of claims 56 to 61, wherein the anti-N3pGlu Aβ antibody is administered to the human subject until the amyloid plaque level in the patient is about 25 centiloids or less.

63. The use according to any one of claims 56 to 61, wherein the anti-N3pGlu Aβ antibody is administered to the human subject until the amyloid plaque level in the patient is less than or equal to about 25 centroids in two consecutive PET imaging scans, or less than or equal to about 11 centroids in one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least six months apart.

64. The use according to any one of claims 56 to 61, wherein the human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks, followed by a second dose of 1400 mg of donanemab once every four weeks, over a period of up to 72 weeks.

65. The use according to any one of claims 56 to 61, wherein the human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks until the amyloid plaque level in the patient is less than or equal to about 25 centiloids, and then a second dose of 1400 mg of donanemab is administered once every four weeks.

66. The use according to any one of claims 56 to 61, wherein the human subject is administered three doses of a first dose of 700 mg of donanemab once every four weeks, followed by a second dose of 1400 mg of donanemab once every four weeks, until the amyloid plaque level in the patient is less than or equal to about 25 centiloids in two consecutive PET imaging scans, or less than or equal to about 11 centiloids in one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least six months apart.

67. The use according to any one of claims 56 to 66, wherein the human subject is administered the second dose of donanemab over a series of treatment periods sufficient to treat or prevent the disease.

68. The use according to any one of claims 56 to 67, wherein the treatment or prevention of the disease causes i) a reduction in Aβ deposits in the brain of the human subject, and / or ii) a delay in cognitive or functional decline in the human subject.

69. The use according to claim 68, wherein the reduction of Aβ deposits in the brain of the human subject is determined by amyloid PET brain imaging or a diagnostic method for detecting a biomarker of Aβ.

70. The use according to claim 68 or 69, wherein the second dose of donanemab is administered to the human subject until the amount of Aβ deposits in the brain of the human subject is reduced by approximately 20 to 100%.

71. The use according to claim 70, wherein the Aβ deposits in the brain of the human subject are reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 75%, or about 100%.

72. The use according to claim 70 or 71, wherein the Aβ deposits in the brain of the patient are reduced by 100%.

73. The use according to any one of claims 56 to 72, wherein the second dose of donanemab is administered to the human subject until the Aβ deposits in the brain of the human subject are reduced by i) approximately 25 centroids to approximately 100 centroids on an approximate average, ii) approximately 50 centroids to approximately 100 centroids on an approximate average, iii) approximately 100 centroids, or iv) approximately 84 centroids.

74. The use according to any one of claims 56 to 73, wherein the disease characterized by the Aβ deposits in the brain of the human subject is selected from preclinical Alzheimer's disease, clinical AD, prodromal AD, mild AD, moderate AD, severe AD, Down syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy.

75. The use according to any one of claims 56 to 74, wherein the human subject is a patient with early symptomatic AD, or the human subject has prodromal AD or mild dementia due to AD.

76. The use according to any one of claims 56 to 75, wherein the human subject is i) determined to have a very low to moderate tau load, or is determined to have a very low to moderate tau load, ii) determined to have a low to moderate tau load, or is determined to have a low to moderate tau load, iii) determined to have a very low to moderate tau load, or has a very low to moderate tau load and one or two alleles of APOE e4, iv) determined to have a low to moderate tau load, or has a low to moderate tau load and one or two alleles of APOE e4, or v) has one or two alleles of APOE e4.

77. The use according to claim 76, wherein the human subject has a very low to moderate tau load if the tau load measured by PET brain imaging is 1.46 SUVr or less, or ii) has a low to moderate tau load if the tau load measured by PET brain imaging is between 1.10 SUVr and 1.46 SUVr.

78. The use according to any one of claims 56 to 75, wherein the human subject i) does not have a high tau load or is determined not to have a high tau load, or ii) possesses one or two alleles of APOE e4 and does not have a high tau load or is determined not to have a high tau load.

79. The use according to claim 78, wherein the human subject has a high tau load if the tau load measured by PET brain imaging exceeds 1.46 SUVr.

80. The use according to claim 76 or 78, wherein the tau load in the human subject is determined using tau PET brain imaging or a diagnostic method for detecting tau biomarkers.

81. i) A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of a human subject who is determined to have a very low to moderate tau load, or a low to moderate tau load, or who is determined to have a very low to moderate tau load, or a low to moderate tau load and one or two alleles of APOE e4, i) Administering a first dose of donanemab, approximately 100 mg to approximately 700 mg, to the human subject, wherein each first dose of donanemab is administered approximately once every four weeks. ii) Administering to the human subject one or more times a second dose of donanemab, ranging from more than 700 mg to approximately 1400 mg, four weeks after administering the first dose, wherein each second dose is administered approximately once every four weeks. A method comprising simultaneously, separately, or sequentially combining an effective amount of antibody 1.

82. A method for treating or preventing a disease characterized by amyloid-beta (Aβ) deposits in the brain of human subjects, The process includes determining whether the human subject has tau loading in the temporal lobe, occipital lobe, parietal lobe, or frontal lobe of the brain, and if the human subject has tau loading in the temporal lobe, occipital lobe, parietal lobe, or frontal lobe of the brain, i) Administering the aforementioned human subjects one or more doses of a first dose of anti-N3pGlu Aβ antibody ranging from approximately 100 mg to approximately 700 mg, wherein each first dose is administered approximately once every four weeks. ii) Approximately four weeks after administering the first dose, administer to the human subject one or more times a second dose of anti-N3pGlu Aβ antibody ranging from more than 700 mg to approximately 1400 mg, wherein each second dose is administered approximately once every four weeks. A method comprising simultaneously, separately, or sequentially combining an effective amount of antibody 1.

83. The method according to claim 82, wherein the human subject has a tau load in the posterolateral temporal lobe or temporal lobe of the brain.

84. The method according to any one of claims 82, wherein the human subject has a tau load in the occipital lobe of the brain.

85. The method according to claim 82, wherein the human subject has a tau load in the parietal lobe of the brain.

86. The method according to claim 82, wherein the human subject has a tau load in the frontal lobe of the brain.

87. The method according to claim 82, wherein the human subject has tau loading in the posterolateral temporal lobe (PLT) and / or occipital lobe of the brain.

88. The method according to any one of claims 82 to 87, wherein the human subject has tau loading in i) the parietal or precuneus region, or ii) the frontal region, along with tau loading in the PLT or occipital region of the brain.

89. The method according to any one of claims 82 to 86, wherein the human subject has a tau load isolated in the frontal lobe of the brain, or a tau load in a region of the temporal lobe that does not include the posterolateral temporal region (PLT).

90. The method according to any one of claims 82 to 88, wherein the human subject has tau loading in the posterolateral temporal lobe, occipital lobe, and parietal lobe of the brain.

91. The method according to any one of claims 82 to 88, wherein the human subject has tau loading in the posterolateral temporal lobe, occipital lobe, parietal lobe, and frontal lobe of the brain.

92. The method according to any one of claims 82 to 88, wherein the human subject has tau loading in the posterolateral temporal lobe, occipital lobe, parietal lobe, and / or frontal lobe of the brain.

93. The method according to any one of claims 82 to 92, wherein the human subject is administered the first dose once, twice, or three times before being administered the second dose.

94. The method according to any one of claims 82 to 93, wherein the human subject is administered a first dose of approximately 700 mg.

95. The method according to any one of claims 82 to 94, wherein the human subject is administered a second dose of approximately 800 mg, approximately 900 mg, approximately 1000 mg, approximately 1100 mg, approximately 1200 mg, approximately 1300 mg, or approximately 1400 mg once or more.

96. The method according to any one of claims 82 to 95, wherein the human subject is administered a second dose of approximately 1400 mg once or more.

97. The method according to any one of claims 82 to 96, wherein the anti-N3pGlu Aβ antibody is administered to the human subject for a period of up to 72 weeks, or until a normal level of amyloid is achieved.

98. The method according to any one of claims 82 to 97, wherein the anti-N3pGlu Aβ antibody is administered to the human subject until the amyloid plaque level in the patient is about 25 centiloids or less.

99. The method according to any one of claims 82 to 98, wherein the anti-N3pGlu Aβ antibody is administered to the human subject until the amyloid plaque level in the human subject is less than or equal to about 25 centiloids in two consecutive PET imaging scans, or less than or equal to about 11 centiloids in one PET imaging scan, wherein optionally, the two consecutive PET imaging scans are spaced at least six months apart.

100. The method according to any one of claims 82 to 99, wherein the human subject is administered a first dose of 700 mg three times, once every four weeks, and then a second dose of 1400 mg once every four weeks for a maximum of 72 weeks.

101. The method according to any one of claims 82 to 100, wherein the human subject is administered a first dose of 700 mg three times, once every four weeks, until the amyloid plaque level in the subject is about 25 centiloids or less, and then a second dose of 1400 mg once every four weeks.

102. The method according to any one of claims 82 to 101, wherein the human subject is administered a first dose of 700 mg three times at 4 weeks, followed by a second dose of 1400 mg at 4 weeks, until the amyloid plaque level in the subject is less than or equal to about 25 centiloids after two consecutive PET imaging scans, or less than or equal to about 11 centiloids after one PET imaging scan, and optionally, the two consecutive PET imaging scans are spaced at least 6 months apart.

103. The method according to any one of claims 82 to 102, wherein the human subject is administered the second dose for a period of time sufficient to treat or prevent the disease.

104. The method according to any one of claims 82 to 103, wherein the treatment or prevention of the disease causes i) a reduction in Aβ deposits in the brain of the human subject, and / or ii) a delay in cognitive or functional decline in the human subject.

105. The method according to claim 97, wherein the reduction of Aβ deposits in the brain of the human subject is determined by amyloid PET brain imaging or a diagnostic method for detecting a biomarker of Aβ.

106. The method according to claim 97 or 98, wherein the second dose is administered to the human subject until the amount of Aβ deposits in the brain of the human subject is reduced by approximately 20 to 100%.

107. The method according to claim 106, wherein the Aβ deposits in the brain of the human subject are reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 75%, or about 100%.

108. The method according to any one of claims 82 to 107, wherein the second dose is administered to the human subject until the Aβ deposits in the brain of the human subject are reduced by i) approximately 25 centroids to approximately 100 centroids on an approximate average, ii) approximately 50 centroids to approximately 100 centroids on an approximate average, iii) approximately 100 centroids, or iv) approximately 84 centroids.

109. The method according to any one of claims 82 to 108, wherein the disease characterized by the Aβ deposits in the brain of the human subject is selected from preclinical Alzheimer's disease (AD), clinical AD, prodromal AD, mild AD, moderate AD, severe AD, Down syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy.

110. The method according to any one of claims 82 to 109, wherein the human subject is a patient with early symptomatic AD.

111. The method according to claim 109, wherein the human subject has prodromal AD and mild dementia due to AD.

112. The method according to any one of claims 82 to 111, wherein the human subject is i) determined to have a very low to moderate tau load, or is determined to have a very low to moderate tau load, or ii) has a low to moderate tau load, or is determined to have a low to moderate tau load.

113. The method according to claim 112, wherein the human subject has a very low to moderate tau load when the tau load measured by PET brain imaging is 1.46 SUVr or less, or ii) has a low to moderate tau load when the tau load measured by PET brain imaging is between 1.10 SUVr and 1.46 SUVr.

114. The method according to any one of claims 82 to 113, wherein the human subject does not have a high tau load, or is determined not to have a high tau load.

115. The method according to claim 114, wherein the human subject has a high tau load if the tau load measured by PET brain imaging exceeds 1.46 SUVr.

116. The method according to claim 114 or 115, wherein the tau load in the human subject is determined using PET brain imaging or a diagnostic method for detecting a tau biomarker.

117. The method according to any one of claims 82 to 116, wherein the anti-N3pGlu Aβ antibody comprises donanemab.

118. The method according to any one of claims 82 to 117, wherein the patient has one or two alleles of APOE e4.

119. A method for reducing / preventing a further increase in tau load, or for slowing the rate of tau accumulation in the temporal, occipital, parietal, or frontal lobes of a human brain, comprising administering an effective amount of anti-N3pGlu Aβ antibody to a human subject simultaneously, separately, or sequentially in combination with antibody 1.

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