LILRB4 / ILT3 Antagonist Compositions and Methods of Use Thereof

Abstract

LILRB4 antagonists, such as anti-LILRB4 antibodies or LILRB4-Fc fusion proteins, and pharmaceutical compositions thereof are provided. Methods for treating neurological disorders using the LILRB4 antagonists and pharmaceutical compositions thereof are also provided.

Description

[Technical Field]

[0001] The present disclosure generally relates to compositions and methods for treating diseases, disorders, or conditions associated with microglial dysfunction. The present disclosure relates to leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) antagonists, such as anti-human LILRB4 (also known as ILT3) antibodies (anti-LILRB4), LILRB4-Fc fusions, and compositions comprising them. The present disclosure also relates to compositions and methods for delaying and / or preventing Aβ amyloidosis. The present disclosure also relates to compositions and methods for delaying and / or preventing Aβ plaque-related symptoms and / or cerebral amyloid angiopathy (CAA)-related symptoms, such as Alzheimer's disease (AD) or CAA-related symptoms, in a subject. In particular, the present disclosure relates to regulating amyloid beta (Aβ) levels and / or microglial activation in the brain of a subject.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in WIPO ST26.xml format and is incorporated herein by reference in its entirety. The .xml copy is named 047563_780878(020008).xml and is 22KB in size. [Background technology]

[0003] Microglia are resident macrophages in the central nervous system (CNS) that act as the brain's first line of defense by phagocytosing harmful pathogens and cellular debris. Microglia emerge from early erythroid progenitors in the yolk sac and invade the developing brain before the establishment of a fully mature blood-brain barrier. Under physiological conditions, microglia contribute to CNS homeostasis by supporting the proliferation of neural precursors during brain development. After birth, these cells contribute to maintaining the integrity of neural circuits by forming synapses. After CNS injury, microglia change their morphology and downregulate genes that support homeostatic functions. Compelling evidence suggests that controlling microglial function during pathological progression may be a strategy for developing future therapies aimed at combating brain degeneration in neurological disorders such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

[0004] Alzheimer's disease (AD) is the most common cause of dementia and a growing public health problem. It is currently estimated to affect over 5 million people in the United States, with an estimated increase to 13 million by 2050. Alzheimer's disease leads to loss of memory, cognitive function, and ultimately loss of independence. It imposes a significant personal and economic burden on patients and their families. Due to the severity and increasing prevalence of the disease, there is an urgent need to develop better treatments.

[0005] Cerebral amyloid angiopathy (CAA) occurs in approximately 90% of people who develop AD, as well as in some people who develop it independently of AD. CAA can lead to ischemic and hemorrhagic strokes, causing severe disability and death. There is currently no treatment for CAA.

[0006] Biochemical, genetic, and animal model evidence suggests that amyloid beta (Aβ) is the pathogenic peptide in most cases of AD and CAA. Neuropathological and neurochemical hallmarks of AD include synaptic loss, selective neuronal cell death, reductions in specific neurotransmitters, and abnormal proteinaceous deposits within neuronal cells (neurofibrillary tangles) and extracellular spaces (cerebrovascular plaques, diffuse plaques, and neuritic plaques). A hallmark of CAA is the accumulation of fibrillar forms of Aβ in perforating and pial arterioles on the cerebral cortical surface. CAA can lead to ischemic or hemorrhagic stroke. The main component of plaques found in AD and CAA is Aβ, a 38–43 amino acid peptide cleaved from the amyloid precursor protein (APP).

[0007] Throughout life, soluble Aβ is secreted primarily by neurons, but also by other cell types. Excessive Aβ deposition may result from increased Aβ synthesis, decreased brain Aβ clearance, or increased Aβ fibril formation, as occurs, for example, in some cases of familial early-onset AD and familial early-onset CAA. The lack of compelling evidence for Aβ overproduction in the more common late-onset AD suggests that insufficient Aβ clearance may also lead to Aβ deposition, amyloid plaque formation, and CAA.

[0008] Microglia can either aid in the clearance of age-related amyloid deposits or promote widespread inflammation in response to amyloid or tau, ultimately leading to widespread neurodegeneration. Importantly, this difference may be driven by the temporal stage of the disease: early in AD, microglia may be neuroprotective, but as amyloid and tau pathology progresses, they may become neurotoxic. Summary of the Invention

[0009] Among various aspects of the present disclosure are methods for treating diseases, disorders, and conditions associated with microglial dysfunction. Disclosed herein are anti-human leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) antibodies. The antibodies may comprise a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO:1 (L1), SEQ ID NO:2 (L2), and SEQ ID NO:2 (L3); and a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO:3 (H1), SEQ ID NO:4 (H2), and SEQ ID NO:5 (H3). The light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO:6, or a sequence at least about 90% identical thereto. The heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO:7, or a sequence at least about 90% identical thereto. The framework regions of each variable region may have at least 75% sequence identity with human framework region sequences. The anti-LILRB4 antibodies may further comprise one or more constant regions, or portions of constant regions. The constant region or portion thereof may have at least 90% sequence identity with a human constant region sequence.

[0010] The anti-LILRB4 antibody may be a monoclonal antibody, a humanized antibody, a single domain antibody, a single-chain variable fragment (scFv), an antibody fragment selected from Fv, Fab, Fab', Fab'-SH, and F(ab')2, a bivalent scFv (di-scFv), a trivalent scFv (tri-scFv), a tetravalent scFv (tetra-scFv), a diabody, a triabody, or a tetrabody.

[0011] Disclosed herein are pharmaceutical compositions comprising an anti-LILRB4 antibody and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may further comprise one or more dispersants, buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers, or any combination thereof. The pharmaceutical composition may further comprise one or more dispersants, buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers, or any combination thereof. The carrier may include saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, human serum albumin, buffer solutions, phosphates, glycine, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene polyoxypropylene block polymers, polyethylene glycol, lanolin, or any combination thereof.

[0012] Disclosed herein are methods for treating neurological disorders in a subject in need thereof, comprising administering an anti-LILRB4 antibody or LILRB4-Fc fusion protein to the subject. Disclosed herein are anti-LILRB4 antibodies, LILRB4-Fc fusion proteins, and pharmaceutical compositions thereof for use as pharmaceuticals. Further provided herein are anti-LILRB4 antibodies or LILRB4-Fc fusion proteins for treating neurological disorders, and uses of anti-LILRB4 antibodies or LILRB4-Fc fusion proteins in the manufacture of medicaments for treating neurological disorders. The neurological disorder may be associated with microglial dysfunction. The neurological disorder may be Alzheimer's disease, Parkinson's disease, Nasu-Hakola disease, prion disease, multiple sclerosis, HIV dementia, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, neuropathic pain, or autism spectrum disorder. The disclosed methods may involve systemic or local administration of the antibodies or LILRB4-Fc fusion proteins provided herein.

[0013] Disclosed herein are methods for reducing amyloid plaque load, CAA load, or both in the brain of a subject in need of treatment, comprising administering an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to the subject. Also provided herein are anti-LILRB4 antibodies or LILRB4-Fc fusion proteins for reducing amyloid plaque load, CAA load, or both, as well as uses of anti-LILRB4 antibodies or LILRB4-Fc fusion proteins in the manufacture of a medicament for reducing amyloid plaque load, CAA load, or both. Anti-LILRB4 antibodies can be administered peripherally or locally, and in some cases, can also be administered directly into the central nervous system. Disclosed herein are methods for treating at least one Aβ plaque-associated symptom or at least one CAA-associated symptom in a subject in need of treatment, comprising administering an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to the subject. Also provided herein are anti-LILRB4 antibodies or LILRB4-Fc fusion proteins for treating Aβ plaque-related symptoms or at least one CAA-related symptom, and uses of anti-LILRB4 antibodies or LILRB4-Fc fusion proteins in the manufacture of medicaments for treating at least one Aβ plaque-related symptom or at least one CAA-related symptom. Treatment can include preventing, alleviating, reversing, or ameliorating at least one Aβ plaque-related symptom or sign or at least one CAA-related symptom in a subject. Aβ plaque-related symptoms or CAA-related symptoms can include neurodegeneration, cognitive dysfunction, behavioral changes, language dysfunction, emotional dysregulation, seizures, nervous system structural impairment, nervous system dysfunction, increased risk of developing Alzheimer's disease, and increased risk of developing cerebral amyloid angiopathy. Anti-LILRB4 antibodies or LILRB4-Fc fusion proteins for use in treating at least one Aβ plaque-associated symptom or at least one CAA-associated symptom can be administered systemically or locally, optionally directly into the central nervous system.

[0014] Other aspects and iterations of the present disclosure are detailed below. [Brief explanation of the drawings]

[0015] The patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] [Figure 1] FIG. 1 shows a diagram of human inhibitory leukocyte Ig-like receptors and mouse PIRB orthologues.

[0017] [Figure 2A] FIG. 2A shows the expression of LILR and APOE in the human brain.

[0018] [Figure 2B] Figure 2B shows the expression of LILRB4 and all other LILRs in snRNAseq datasets of human AD versus control (Ctrl).

[0019] [Figure 3A] Figure 3A shows the expression of LILRB4 (ILT3) in microglia in the Seattle Alzheimer's Disease (SEA-AD) dataset.

[0020] [Figure 3B] FIG. 3B shows that LILRB4 expression correlates with Braak score in the SEA-AD dataset.

[0021] [Figure 3C] FIG. 3C shows that LILRB4 expression correlates with brain GuHCl-pTau in the SEA-AD dataset.

[0022] [Figure 3D] Figure 3D shows increased APOE expression in AD (Zhou et al. 2020) and the correlation between APOE expression and Braak score in the SEA-AD dataset.

[0023] [Figure 3E] Figure 3E shows that LILRB4 expression correlates with APOE expression in the snRNAseq dataset from the (SEA-AD) dataset.

[0024] [Figure 4A] FIG. 4A shows the expression of LILRB4 and ApoE by immunofluorescence staining.

[0025] [Figure 4B] Figure 4B provides a comparison of LILRB4 and ApoE expression in brain sections from Ctrl and AD patients.

[0026] [Figure 5A] Figure 5A is a schematic of the bacterial artificial chromosome (BAC) of the telomeric LILR cluster.

[0027] [Figure 5B] FIG. 5B is a schematic diagram of the generation of ILT-TELO-BAC transgenic x 5XFAD mice (ILT-Telo Tg x 5XFAD).

[0028] [Figure 6] Figure 6 shows the gating of microglia, the representative expression of LILRB4, LILRB1, and LILRA2 in microglia, and its quantification in four different genotypes.

[0029] [Figure 7A] Figure 7A shows representative immunofluorescence images of microglial clustering and LILRB4 expression in Telo Tg x 5XFAD and 5XFAD mice. Quantitative data are provided in the accompanying graphs.

[0030] [Figure 7B]Figure 7B is a bar graph showing measurements of microglial density within 15- and 30-μm shells surrounding Aβ plaques, performed by placing IBA1+ PU.1+ microglia and methoxy-X04+ plaques. ILT-Telo Tg x 5XFAD mice showed less microglial coverage of Aβ plaques within the 15-μm shell than 5XFAD mice, suggesting that "reactive microgliosis" near amyloid deposits is preferentially reduced in ILT-Telo Tg x 5XFAD mice.

[0031] [Figure 7C] FIG. 7C is a bar graph showing that the entire cortex of 5XFAD and ILT-Telo Tg x 5XFAD mice exhibits similar microglial densities.

[0032] [Figure 8] Figure 8 shows representative immunofluorescence images showing the volume of microglial bodies in Telo Tg x 5XFAD mice and 5XFAD mice. When the volume of microglial bodies was measured, Telo Tg x 5XFAD mice had smaller microglial body volumes than 5XFAD mice, indicating reduced microglial activation.

[0033] [Figure 9A] FIG. 9A provides images of brain sections stained with the methoxy-X04 probe, showing that Telo Tg x 5XFAD male mice had a higher amyloid burden in the cortex and hippocampus.

[0034] [Figure 9B] FIG. 9B provides a bar graph showing that Aβ accumulation was significantly increased in the cortical and hippocampal regions, but not in the amygdala region, of ILT-Telo Tg x 5XFAD mice.

[0035] [Figure 10]Figure 10 shows neurite dystrophy in Telo Tg x 5XFAD mice and 5XFAD mice. Neuritic dystrophy was examined by staining for LAMP1, which accumulates in dystrophic neurites. LAMP+ voxels were measured within 15- and 30-μm shells surrounding Aβ plaques. Compared to control 5XFAD mice, ILT-Telo Tg x 5XFAD mice showed an overall significant increase in LAMP+ voxels within 15- and 30-μm shells at 6 months of age.

[0036] [Figure 11] Figure 11 shows a schematic diagram illustrating the generation of Fc-mutated anti-LILRB4 mAb (ZM3.1) and Ctrl (27D6) mAbs. To characterize the function of microglial LILRB4 in AD pathology and exclude ADCC effects, a recombinant Fc-mutated mIgG2a anti-LILRB4 mAb (clone: ​​ZM3.1) and an irrelevant Fc-mutated mIgG2a Ctrl mAb (clone: ​​27D6) were generated using the illustrated method.

[0037] [Figure 12] FIG. 12 is a schematic diagram showing the purification of Fc mutant anti-LILRB4 mAb (ZM3.1) and Ctrl (27D6) mAb.

[0038] [Figure 13] FIG. 13 shows the generation of a LILRB4 (ILT3) reporter cell line used for characterization of anti-ILT3 mAbs.

[0039] [Figure 14] Figure 14 shows anti-LILRB4 mAb (also known as anti-ILT3) binding to human LILRB4 ectodomain protein and its activity against a LILRB4 expression reporter (2B4-LILRB4). The activity of anti-LILRB4 mAb against 2B4-LILRB4 was confirmed by reading GFP% in the flow cytometer.

[0040] [Figure 15]Figure 15 is a schematic diagram of the in vivo antibody treatment workflow. ILT-Telo Tg x 5XFAD mice were treated intraperitoneally (ip) with anti-LILRB4 mAb or Ctrl mAb (60 mg / kg) once a week starting at 4 months of age. Relevant values ​​were obtained.

[0041] [Figure 16] Figure 16 shows a study using anti-LILRB4 mAb to measure LILRB4 in plasma and brain, and indicates its concentration. The anti-LILRB4 mAb reached detectable concentrations in both plasma and brain (approximately 10 ng / mg and 100 μg / ml, respectively).

[0042] [Figure 17A] Figure 17A shows a Volcano plot of microglial bulk RNA-seq in mice treated with anti-LILRB4 mAb and Ctrl mAb. One week after the eighth intraperitoneal injection, microglia were isolated from whole brains and bulk RNA-seq was performed (n = 3 anti-LILRB4 mAb-treated mice, 3 Ctrl mAb-treated mice). This screen revealed that 81 transcripts were upregulated and 77 transcripts were downregulated in response to anti-LILRB4 mAb.

[0043] [Figure 17B] Figure 17B shows GO enrichment analysis of anti-LILRB4 mAb-treated and Ctrl mAb-treated mice. Gene sets contributing to microglial motility and phagocytosis were enriched in anti-LILRB4 mAb-treated mice.

[0044] [Figure 18A] Figure 18A shows that among the differentially expressed genes (DEGs) (Padj<0.05, llog2FCl>0.5), gene sets contributing to microglial motility and phagocytosis were enriched in anti-LILRB4 mAb-treated mice.

[0045] [Figure 18B] FIG. 18B shows that the interferon response (IFN-R) and proinflammatory cytokine gene set (Axl, Ifitm3, Usp18, Oas1a, Ifit3) were suppressed in anti-LILRB4 mAb-treated microglia.

[0046] [Figure 19A] FIG. 19A shows string analysis of genes downregulated in anti-LILRB4 mAb-treated mice.

[0047] [Figure 19B] FIG. 19B provides the clusters identified among the genes that were downregulated in anti-LILRB4 mAb-treated mice.

[0048] [Figure 20A] FIG. 20A shows string analysis of genes upregulated in anti-LILRB4 mAb-treated mice.

[0049] [Figure 20B] FIG. 20B provides the clusters identified among the genes upregulated in anti-LILRB4 mAb-treated mice.

[0050] [Figure 21] Figure 21 shows representative immunofluorescence images demonstrating the expression of microglial markers IBA1 (yellow) and CD74 (pink), and amyloid plaque marker X34 (blue) in the cortex, hippocampus (hippo.), and amygdala (amy.) of 6-month-old ILT-Telo Tg x 5XFAD mice after eight injections of anti-ILRB4 or Ctrl IgG. Bar = 30 μm.

[0051] [Figure 22] FIG. 22 shows the quantification of the percentage of IBA1+ voxels within 15 μm surrounding X34+ plaques in different regions.

[0052] [Figure 23] FIG. 23 shows quantification of the percentage of CD74+ / IBA1+ co-localized voxels within 15 μm around X34+ plaques in different regions.

[0053] [Figure 24A] FIG. 24A shows amyloid plaques stained with Methoxy-X04 or 6E10 and brain Aβ load in the cortex, hippocampus, and amygdala in anti-LILRB4 mAb and Ctrl mAb mice.

[0054] [Figure 24B] FIG. 24B provides a bar graph quantifying the percentage of area that is methoxy-X04+ or 6E10+ in the cortex, hippocampus, and amygdala regions.

[0055] [Figure 25] Figure 25 shows levels of PBS-soluble and PBS-insoluble guanidine-soluble fractions of hippocampi assessed by ELISA for Aβ1-40 and Aβ1-42, both from ILT-Telo Tg x 5XFAD mice treated with Ctrl IgG2a mAb.

[0056] [Figure 26] Figure 26 shows representative confocal images of BACE1-labeled (red) dystrophic neurites surrounding methoxy-X04+ (blue) plaques in the cortex of 6-month-old ILT-Telo Tg mice treated with anti-LILRB4 mAb or Ctrl mAb, and quantification of the percentage of BACE1+ voxels within 15 μm around methoxy-X04+ plaques.

[0057] [Figure 27]27 is a schematic diagram showing the elevated plus maze (EMP) test and quantification of behavioral changes in mice after anti-LILRB4 mAb and Ctrl mAb treatment during EMP treatment. During this test, anti-LILRB4-treated mice significantly decreased the time spent in the open arms compared with the Ctrl group, indicating that anti-LILRB4 treatment reversed fear of open spaces and alleviated risk-taking behavior.

[0058] [Figure 28A] FIG. 28A shows a schematic diagram of the steps used to assess contextual memory in the conditioned fear paradigm.

[0059] [Figure 28B] FIG. 28B shows the behavior of anti-LILRB4 mAb-treated and Ctrl mAb-treated mice during tone / shock pairings.

[0060] [Figure 28C] FIG. 28C shows the behavior of anti-LILRB4 mAb-treated and Ctrl mAb-treated mice during tone / shock pairings.

[0061] [Figure 28D] FIG. 28D shows the behavior of anti-LILRB4 mAb- and Ctrl mAb-treated mice during auditory cue conditioning.

[0062] [Figure 29] FIG. 29 shows a binding test between human LILRB4 recombinant protein and recombinant human ApoE2, ApoE3, or ApoE4 using ELISA.

[0063] [Figure 30A] Figure 30A shows the percentage of LILRB4 reporter activation (GFP+ cells) in the presence of the indicated concentrations of rhApoE4 or lipidated rhApoE4 (lrhApoE4). n=3 for each condition.

[0064] [Figure 30B] Figure 30B shows the percentage of LILRB4 reporter activation (GFP+ cells) in the presence of the indicated concentrations of rhApoE3 or ldhApoE3. n=3 for each condition.

[0065] [Figure 31] Figure 31 shows representative flow cytometry plots demonstrating the blocking effect of anti-LILRB4 on rmApoE-induced LILRB4 reporter activation and quantification of GFP+ cells from each treatment. The concentration of anti-LILRB4 or Ctrl IgG is 15 μg / ml. n=3 for each treatment.

[0066] [Figure 32] Figure 32 shows quantification of GFP+LILRB4 reporter cells in the presence of anti-LILRB4 or Ctrl mAb and the indicated concentrations of rhApoE3 or ldhApoE3. n=3 for each condition.

[0067] [Figure 33A] Figure 33A shows the surface and schematic models depicting the top 10 binding predictions for mApoE (depicted in yellow) and hLILRB4 (shown in cyan) via in silico docking modeling (left panel). During the prediction analysis, it was consistently observed that all predicted binding regions of mApoE point to the D1-D2 interdomain site located between the two extracellular Ig-like domains of hLILRB4. Notably, a specific loop region (highlighted in red and dashed oval) containing the amino acid sequence K134ERAAHP140 was found to be consistently involved in the predicted binding sites of mApoE (as shown in the right panel).

[0068] [Figure 33B] Figure 33B shows a surface and schematic model showing the top hits of mApoE (yellow) binding to hLILRB4 (cyan). Key residues T30, P35, and the loop region are labeled. The black dashed ellipse highlights the binding interface between mApoE and hLILRB4 in this predicted complex.

[0069] [Figure 33C] FIG. 33C shows a schematic model indicating the location of various key residues for mApoE binding, T30, P35, Y121, and the loop.

[0070] [Figure 33D] Figure 33D shows a sequence alignment of LILRB4 common variants (CVs) or mutants. The alignment shows the amino acid sequences of the CV, the introduced non-functional loop mutants, the T30A mutant, the P35A mutant, and the Y121A mutant. The mutated residues are shown in dark red to highlight the changes.

[0071] [Figure 33E] Figure 33E shows the purification of the recombinant exodomains of the LILRB4 consensus variant and P35A mutant by size-exclusion chromatography and gel filtration chromatograms of the LILRB4 consensus variant and P35A mutant. For each protein, labeled peak fractions were visualized on SDS-PAGE gels by Coomassie blue staining (left panel) or Western blotting with anti-LILRB4 monoclonal antibody (right panel).

[0072] [Figure 33F] Figure 33F shows the purification of recombinant ectodomains of LILRB4 Y121A, loop, and T30A mutants by size-exclusion chromatography and gel filtration chromatograms of LILRB4 Y121A, loop, and T30A mutants. For each protein, labeled peak fractions were visualized on SDS-PAGE gels via Coomassie blue staining (left panel) or Western blot with anti-LILRB4 monoclonal antibody (right panel).

[0073] [Figure 33G]Figure 33G provides a bar graph quantifying the binding of LILRB4 multimeric or monomeric fractions to rmApoE by ELISA, n=4-6 for each protein.

[0074] Detailed Description Applicants have discovered LILRB4 antagonist compositions useful for treating diseases, disorders, and conditions associated with microglial dysfunction. In one embodiment, Applicants have discovered anti-LILRB4 antibodies and methods of using anti-LILRB4 antibodies to treat Aβ amyloidosis. The methods include effectively administering to a subject a therapeutically effective amount of an anti-LILRB4 antibody that specifically binds to LILRB4. The present disclosure encompasses the discovery that anti-LILRB4 antibodies provide treatment for subjects with Aβ amyloidosis, including, but not limited to, subjects diagnosed with a disease characterized by cerebral Aβ plaques, subjects diagnosed with a disease characterized by vascular Aβ plaques in the brain, subjects diagnosed with an Aβ plaque-associated condition, subjects diagnosed with CAA, subjects with clinical signs of Aβ amyloidosis that may or may not have Aβ plaque-associated and / or CAA-associated symptoms, subjects diagnosed with Alzheimer's disease, and subjects diagnosed with a CAA-associated condition (collectively referred to herein as "subjects in need of treatment"). Methods for identifying clinical signs of Aβ amyloidosis in asymptomatic patients are known in the art and are described below.

[0075] Definition.

[0076] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0077] For the recitation of numerical ranges herein, each intervening number of equal precision is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0078] The term "subject" refers to a human or non-human animal susceptible to Aβ accumulation.

[0079] As used herein, the terms "treat," "treating," or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) undesirable physiological changes or diseases / disorders. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder, as well as those who are prone to the disease, condition, or disorder or in whom the disease, condition, or disorder is to be prevented.

[0080] The term "Aβ" refers to a peptide derived from the carboxy-terminal region of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of Aβ that can have toxic effects. Aβ peptides are typically 37–43 amino acids in length, but their overall size can vary due to truncations and modifications. Aβ can exist intracellularly or extracellularly in soluble and insoluble compartments, as monomers, multimers, and aggregates, and can be complexed with other proteins or molecules. The harmful or toxic effects of Aβ can result from any or all of these forms. For example, these Aβ isoforms include Aβ40 and Aβ42, with the Aβ42 isoform being particularly fibrillogenic or insoluble and associated with disease states.

[0081] "Aβ amyloidosis" is clinically defined as evidence of Aβ deposition in the brain or cerebral blood vessels, typically in the form of amyloid plaques or CAA. Diseases associated with Aβ amyloidosis include, but are not limited to, preclinical Alzheimer's disease, Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), dementia with Lewy bodies, and inclusion body myositis. "Increased risk of developing a disease associated with Aβ amyloidosis" refers to a risk that is higher than expected given the subject's age, family history, genetic status, and other known risk factors.

[0082] " Clinical signs of Aβ amyloidosis " refers to indicators of Aβ deposition known in the art. Clinical signs of Aβ amyloidosis include, but are not limited to, Aβ deposits identified by amyloid imaging (for example, PiB PET, fluorobetapir, or other imaging methods known in the art) or by decreased cerebrospinal fluid (CSF) Aβ42 or Aβ42 / 40 ratio. For example, see Klunk WE et al. Ann Neurol 55(3) 2004, and Fagan AM et al. Ann Neurol 59(3) 2006. Clinical indications of Aβ amyloidosis may also include measurements of Aβ metabolism, particularly Aβ42 metabolism alone or compared to measurements of metabolism of other Aβ variants (e.g., Aβ37, Aβ38, Aβ39, Aβ40, and / or total Aβ), as described in U.S. Patent Serial Nos. 14 / 366,831, 14 / 523,148, and 14 / 747,453, each of which is incorporated herein by reference in its entirety. Additional methods are described in Albert et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 170-179; McKhann et al., Alzheimer's & Dementia 2007 Vol. 7, pp. 263-269; and Sperling et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 280-292, each of which is incorporated herein by reference in its entirety. Importantly, the subject with clinical signs of Aβ amyloidosis may or may not have symptoms associated with Aβ deposition. However, the subject with clinical signs of Aβ amyloidosis has a high risk of developing diseases associated with Aβ amyloidosis.

[0083] "Aβ plaque-associated symptoms" or "CAA-associated symptoms" refer to symptoms resulting from or associated with the formation of amyloid plaques or CAA, respectively, which are composed of ordered, fibrous aggregates called amyloid fibrils. Exemplary Aβ plaque-associated symptoms include, but are not limited to, neurodegeneration, cognitive impairment, memory impairment, behavioral changes, emotional dysregulation, seizures, nervous system structural or functional impairment, and an increased risk of developing or worsening Alzheimer's disease or CAA. Neurodegeneration includes changes in neuronal structure (including molecular-level changes such as intracellular accumulation of toxic proteins and protein aggregates, and macro-level changes such as changes in axon or dendrite shape or length, changes in myelin composition, and loss of myelin), changes in neuronal function, loss of neuronal function, neuronal death, or a combination thereof. Cognitive impairment includes, but is not limited to, impairments in memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and organization. Behavioral alterations include, but are not limited to, physical or verbal aggression, impulsivity, decreased inhibitions, apathy, decreased spontaneity, personality changes, alcohol, tobacco, or drug abuse, and other addiction-related behaviors. Emotional dysregulation disorders include, but are not limited to, depression, anxiety, mania, irritability, and emotional incontinence. Seizures include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures, and non-epileptic and psychogenic seizures. Nervous system structural or functional disorders include, but are not limited to, hydrocephalus, parkinsonism, sleep disorders, psychosis, and balance and coordination disorders. This may include movement disorders such as monoplegia, hemiplegia, quadriplegia, ataxia, ballismus, and tremor. This may also include sensory loss or dysfunction, such as smell, touch, taste, vision, and hearing. Additionally, autonomic nervous system disorders may include bowel or bladder dysfunction, sexual dysfunction, and impaired blood pressure and temperature regulation. Finally, hormonal disorders resulting from hypothalamic and pituitary dysfunction, such as deficiencies or dysregulation of growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, gonadotropin-releasing hormone, prolactin, and many other hormones and regulators, may be included.

[0084] The terms "LILRB4" and "ILT3" are used interchangeably herein. "LILRB4" (NP_001265355.2, UniProtKB identifier Q8NHJ6) is a member of the leukocyte immunoglobulin-like receptor (LILR) family and is present in a gene cluster at chromosome 19q13.4 (e.g., nucleotide sequence identified as HGNC:6608 or NCBI Entrez Gene:11006 or Ensembl:ENSG00000186818 or OMIM:604821 or UniProtKB / Swiss-Prot:Q8NHJ6 or Genecard ID:GC19P054643). The encoded protein belongs to the subfamily B class of LIR receptors, which contains two extracellular immunoglobulin domains, one transmembrane domain, and two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The extracellular Ig-like domain of LILRB4 binds to fibronectin. The receptor is expressed on immune cells and transmits negative signals that inhibit the stimulation of an immune response. The receptor may also function in antigen capture and presentation. It is thought to regulate inflammatory responses and cytotoxicity to help focus the immune response and limit self-reactivity. Multiple transcripts encoding different isoforms of this gene have been found. The location of the locus encoding LILRB4 in humans is very different from that encoding mouse LILRB4. LILRB4 is encoded by a gene complex (called the leukocyte receptor complex, or LRC) on chromosome 19 that contains four inhibitory receptors (ILT2-LILRB1, ILT4-LILRB2, ILT5-LILRB3, and ILT3-LILRB4); the mouse LRC complex is located on mouse chromosome 7 and contains only one inhibitory receptor, called Pirb, that is homologous to the inhibitory ILTs; mouse LILRB4 is encoded outside the LRC complex on mouse chromosome 10 and is therefore a distant paralog of human LILRB4 rather than a direct ortholog (Figure 1). Unless otherwise noted, "LILRB4" refers to "human LILRB4" and includes functional fragments."LILRB4" is also known as immunoglobulin-like transcript 3; leukocyte immunoglobulin-like receptor B4 (LILRB4); leukocyte immunoglobulin-like receptor 5 (LIR-5); leukocyte immunoglobulin-like receptor subfamily B (with TM and ITIM domains) member 4; CD85 antigen-like family member K; monocyte inhibitory receptor HM18; and leukocyte Ig-like receptor B4.

[0085] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibodies and antibody-like structures, including, but not limited to, full-length monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), as well as heavy chain antibodies and antibody fragments that exhibit the desired antigen-binding activity. The domain of an antibody that is responsible for binding to the antigen is referred to as the "variable region" or "variable domain" and is described in more detail below. A single variable domain may be sufficient to confer antigen-binding specificity. Preferably, although not necessarily, antibodies useful for discovery are recombinantly produced. Antibodies may be glycosylated or non-glycosylated, although glycosylated antibodies may be preferred. An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, antibodies are purified to greater than 95% or 99% purity, as determined by methods known in the art.

[0086] In addition to the antibodies described herein, it may be possible to design antibody mimetics or aptamers with substantially the same functionality as the antibodies of the present disclosure using methods known in the art. The term "antibody mimetic" refers to a polypeptide or protein that can specifically bind to an antigen but is structurally unrelated to antibodies. Antibody mimetics have masses ranging from approximately 3 kDa to approximately 20 kDa. Non-limiting examples of antibody mimetics include affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies. Aptamers are a type of small nucleic acid ligand composed of RNA or single-stranded DNA oligonucleotides that exhibit high specificity and affinity for their targets. Aptamers interact with and bind to targets through structural recognition, a process similar to antigen-antibody reactions. Aptamers have a lower molecular weight than antibodies, typically ranging from approximately 8 to 25 kDa.

[0087] The terms "full-length antibody" and "intact antibody" are used interchangeably and refer to antibodies having a structure substantially similar to that of a native antibody or having heavy chains containing an Fc region, as defined herein. The basic structural unit of a native antibody is a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). Light chains are classified as gamma, mu, alpha, and lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. The amino-terminal portions of each light and heavy chain contain a variable region of approximately 100 to 110 or more amino acids (VL and VH, respectively) primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. Intact antibodies are suitably cross-linked through disulfide bonds, as is known in the art.

[0088] The variable domains of antibody heavy and light chains generally have similar structures, with each domain consisting of four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains using a VH or VL domain from an antibody that binds to the antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0089] "Framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains, FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order: FR1-HVR1-FR2-HVR2-FR3-HVR3-FR4. The FR domains of the heavy and light chains may differ, as is known in the art.

[0090] As used herein, the term "hypervariable region" or "HVR" refers to each region of a variable domain that is hypervariable in sequence (commonly also referred to as "complementarity-determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contacts"). Generally, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). As used herein, an "HVR from a variable region" refers to an HVR that contains no more than two amino acid substitutions compared to the corresponding HVR from the original variable region. Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 27-32 (L1: SEQ ID NO: 1), 50-52 (L2: WAS), 89-97 (L3: SEQ ID NO: 2), 26-33 (H1: SEQ ID NO: 3), 51-58 (H2: SEQ ID NO: 4), and 97-101 (H3: SEQ ID NO: 5). Definitions of HVRs (CDRs) may vary based on nomenclature and can be determined by one of skill in the art. The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, comprising at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to Chothia et al. 1987 (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).

[0091] A "variant Fc region" comprises an amino acid sequence that differs from a native Fc region by one or more amino acid substitutions and / or an altered glycosylation pattern, compared to a native Fc region or the Fc region of a parent polypeptide. In one example, a variant Fc region may have about one to about ten amino acid substitutions, or about one to about five amino acid substitutions, in a native-sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region herein may have at least about 80% homology, at least about 90% homology, or at least about 95% homology with a native-sequence Fc region and / or the Fc region of a parent polypeptide. In one exemplary embodiment, the Fc region comprises LALA-PG mutations (i.e., L234A, L235A, and / or P329G) as described in M. Lo et al. J. Biol. Chem., 292 (2017), pp. 3900-3908, which is incorporated herein by reference in its entirety.

[0092] "Antibody fragment" refers to a molecule other than an intact antibody and includes the portion of an intact antibody that binds to the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-chain forms of antibodies and hypervariants thereof, single-domain antibodies, and multispecific antibodies formed from antibody fragments.

[0093] Single-chain forms of antibodies and their higher-order forms can include, but are not limited to, single-domain antibodies, single-chain variable fragments (scFvs), divalent scFvs (di-scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra-scFvs), diabodies, and triabodies and tetrabodies. ScFvs connect the variable regions of the heavy and light chains with a linker. In most, but not all, cases, the linker can be a peptide. The linker peptide is preferably about 5 to 30 amino acids in length, or about 10 to 25 amino acids in length. Typically, the linker stabilizes the variable domains without interfering with proper folding and formation of the active binding site. In a preferred embodiment, the linker peptide is rich in glycine and serine or threonine. ScFvs can be used to facilitate phage display, or can be used in flow cytometry, immunohistochemistry, or as targeting domains. Methods for producing and using scFvs are known in the art. ScFvs may also be conjugated to human constant domains (e.g., heavy chain constant domains derived from IgG domains such as IgG1, IgG2, IgG3, or IgG4, or heavy chain constant domains derived from IgA, IgM, or IgE). Diabodies, triabodies, and tetrabodies and higher variants are typically created by varying the length of the linker peptide from zero to several amino acids. Alternatively, it is well known in the art that multivalent binding antibody variants can be generated using self-assembling units linked to variable domains.

[0094] A "single domain antibody" refers to an antibody fragment that consists of a single monomeric variable antibody domain.

[0095] Multispecific antibodies include bispecific, trispecific, or four or more specific antibodies. Multispecific antibodies can be made by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. The chains can be covalently linked.

[0096] A "monoclonal antibody" refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. A "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology well known in the art, as well as recombinant, phage display, or synthetic techniques, or a combination of such techniques and other techniques readily known in the art. Furthermore, monoclonal antibodies can be labeled with a detectable label, immobilized on a solid phase, or conjugated to a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

[0097] "Heavy chain antibodies" refer to antibodies consisting of two heavy chains. Heavy chain antibodies are IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNAR from cartilaginous fish.

[0098] A "humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of eliciting an immune response in humans after administration, while maintaining the same binding specificity and affinity as the original non-human antibody. Humanized antibodies bind to the same or similar epitope as the non-human antibody. The term "humanized antibody" includes antibodies that are constructed with part or all of the amino acid sequence derived from the germline of a human antibody by altering the sequence of an antibody with non-human hypervariable regions ("HVRs"). The simplest such alteration involves simply replacing mouse constant regions with human antibody constant regions, resulting in a human / mouse chimera that is sufficiently immunogenic for pharmaceutical use. Preferably, the variable regions of the antibody are also humanized using techniques well known in the art. For example, the framework regions of the variable regions can be replaced with corresponding human framework regions while retaining one, some, or all six non-human HVRs. Some framework residues can be replaced with corresponding residues from a non-human VL or VH domain (e.g., the non-human antibody from which the HVR residues are derived) to, for example, restore or improve the specificity or affinity of a humanized antibody. A substantially human framework region has at least about 75% homology with a known human framework sequence (i.e., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity). HVRs can also be randomly mutated to maintain or enhance antigen binding activity and affinity in the context of fully human germline framework regions or substantially human framework regions. As noted above, it is sufficient to employ antibody fragments for use in this discovery method. Furthermore, as used herein, the term "humanized antibody" refers to an antibody comprising substantially human framework regions, at least one HVR from a non-human antibody, and in which any constant regions present are substantially human. A substantially human constant region has at least about 90% (ie, about 90%, about 95%, or about 99% sequence identity) to a known human constant sequence.Hence, all parts of a humanized antibody, except possibly the HVRs, are substantially identical to corresponding pairs of one or more germline human immunoglobulin sequences.

[0099] If desired, humanized immunoglobulin design can be performed as follows or using similar methods known to those skilled in the art (see, for example, Almagro, et al. Front. Biosci. 2008, 13(5):1619-33). The murine antibody variable region is aligned to the most similar human germline sequence (e.g., using BLAST or a similar algorithm). CDR residues from the murine antibody sequence are grafted onto the similar human "acceptor" germline sequence. Subsequently, one or more positions (e.g., vernier positions) near the CDRs or within the framework can be reverted to the original murine amino acid to obtain a humanized antibody with binding affinity similar to that of the original murine antibody. Typically, several versions of the humanized antibody with different backmutations are generated and empirically tested for activity. The humanized antibody variant with properties most similar to the parent murine antibody and with the least reversion of the murine framework is selected as the final humanized antibody candidate.

[0100] One aspect of the present disclosure encompasses LILRB4 (ILT3) antagonist drugs that can alleviate one or more of the pathologies associated with microglia-associated neurological disorders. The term "LILRB4 antagonist" refers to a molecule that inhibits LILRB4 (ILT3) activity; inhibits LILRB4 (ILT3) signaling in cells expressing LILRB4 (ILT3); inhibits the binding of LILRB4 (ILT3) to LILRB4 (ILT3) ligands such as fibronectin; inhibits the binding of LILRB4 (ILT3) to ciliary neurotrophic factor receptors; inhibits the binding of LILRB4 (ILT3) to ApoE; inhibits the binding of LILRB4 (ILT3) to CD166; inhibits LILRB4 (ILT3)-induced suppression of myeloid cells; inhibits LILRB4 (ILT3)-induced suppression of myeloid cell activity; restores FcR activation in myeloid cells expressing LILRB4 (ILT3); and restores chemokine production in myeloid cells expressing LILRB4 (ILT3). In some embodiments, the terms "inhibit," "reduce," "block," "antagonize," "suppress," and "prevent" are relative to the level and / or activity in the absence of treatment with a LILRB4 binding agent. In some embodiments, the terms "inhibit," "reduce," "block," "antagonize," "suppress," and "prevent" are relative to the level and / or activity before treatment with a LILRB4 binding agent.

[0101] 1. Anti-LILRB4 antibody Anti-LILRB4 antibodies are provided herein. The anti-LILRB4 antibodies disclosed herein can be described or specified in terms of the epitopes they recognize or bind. The portion of a target polypeptide that specifically interacts with the antigen-binding domain of an antibody is an "epitope." LILRB4 can contain any number of epitopes, depending on the protein source, isoform, conformational state of the isoform, and location of the isoform. Furthermore, it should be noted that an "epitope" on LILRB4 can be a linear epitope or a conformational epitope, and in either case, can include elements other than the polypeptide; for example, the epitope can include carbohydrate or lipid side chains. The term "affinity" refers to a measure of the strength of binding between the antigen-binding site of an antibody and a particular epitope.

[0102] As used herein, "anti-LILRB4 antibody" refers to an isolated antibody that binds to LILRB4 with an affinity constant or interaction affinity (KD) between about 0.1 pM and about 10 μM, preferably between about 0.1 pM and about 1 μM, and more preferably between about 0.1 pM and about 100 nM. Methods for determining the affinity of an antibody for an antigen are known in the art and are further illustrated in the Examples. Anti-LILRB4 antibodies useful herein include antibodies suitable for administration to a subject in therapeutic amounts.

[0103] The anti-LILRB4 antibodies disclosed herein can also be described or specified in terms of cross-reactivity. "Cross-reactivity" refers to the ability of an antibody specific for one antigen to react with another antigen and is a measure of the relatedness between two different antigenic substances. Thus, an antibody is cross-reactive when it binds to an epitope other than the epitope that induced its formation. A cross-reactive epitope generally contains many of the same complementary structural features as the induced epitope and, in some cases, may actually match better than the original epitope. For example, an antibody may have some degree of cross-reactivity in that it binds to related but non-identical epitopes, such as epitopes that are at least about 85%, at least about 90%, or at least about 95% identical to the reference epitope (as calculated using methods known in the art). An antibody can be said to have little or no cross-reactivity if it does not bind to epitopes that are less than about 95%, less than about 90%, or less than about 85% identical to the reference epitope. An antibody can be considered "highly specific" for an epitope if it does not bind to other analogs, orthologs, or homologs of that epitope.

[0104] Another aspect of the isolated anti-LILRB4 antibodies of the present disclosure is that they may or may not have a mutated Fc region. For example, the Fc region can be modified to increase or decrease affinity for Fc receptors on microglial cells and / or to alter glycosylation patterns.

[0105] Other aspects of the anti-LILRB4 antibodies of the present disclosure are described in more detail below.

[0106] The anti-LILRB4 antibody may comprise one or more of L1 comprising the amino acid sequence set forth in SEQ ID NO: 1, L2 comprising the amino acid sequence WAS, and L3 comprising the amino acid sequence set forth in SEQ ID NO: 2. The anti-LILRB4 antibody may comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 6, or a sequence substantially identical thereto. The anti-LILRB4 antibody may comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 6, or a sequence at least about 90% (about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical thereto.

[0107] An anti-LILRB4 antibody may comprise one or more of H1 comprising the amino acid sequence set forth in SEQ ID NO:3, H2 comprising the amino acid sequence set forth in SEQ ID NO:4, and H3 comprising the amino acid sequence set forth in SEQ ID NO:5. An anti-LILRB4 antibody may comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, or a sequence substantially identical thereto. An anti-LILRB4 antibody may comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, or a sequence at least about 90% (about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical thereto. An anti-LILRB4 antibody may comprise any combination of 2, 3, 4, or 5 of SEQ ID NOs:1-5 and the L2 amino acid sequence WAS. An anti-LILRB4 antibody may comprise all of SEQ ID NOs:1-5 and the L2 amino acid sequence WAS.

[0108] L1-L3 and H1-H3 of anti-LILRB4 antibodies can be defined using nomenclature known in the art. Table 1 provides the corresponding HVRs of the antibodies based on the nomenclature of Chothia and Lesk (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)) and the nomenclature of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). It should be understood that the HVR combinations for each VL and VH may be one or more HVR combinations based on these and other known HVR nomenclature methods. [Table 1]

[0109] Anti-LILRB4 may comprise a light chain variable region comprising one or more or all of L1 comprising the sequence set forth in SEQ ID NO: 15, L2 comprising the sequence set forth in SEQ ID NO: 16, and L3 comprising the sequence set forth in SEQ ID NO: 17; and / or a heavy chain variable region comprising one or more or all of H1 comprising the sequence set forth in SEQ ID NO: 18 or 21, H2 comprising the sequence set forth in SEQ ID NO: 19 or 22, and H3 comprising the sequence set forth in SEQ ID NO: 20.

[0110] In various embodiments described above, the antibody may be a humanized antibody. The humanized anti-hLILRB4 antibody may comprise a light chain variable region comprising one or more of the sequences set forth in SEQ ID NO:1 (L1), amino acid sequence WAS (L2), and SEQ ID NO:2 (L3), and a heavy chain variable region comprising one or more of the sequences set forth in SEQ ID NO:3 (H1), SEQ ID NO:4 (H2), and SEQ ID NO:5 (H3). The humanized antibody may comprise a VL having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the amino acid sequence set forth in SEQ ID NO:6, or a VH having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the amino acid sequence set forth in SEQ ID NO:7. Humanized anti-LILRB4 antibodies may comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity to known human framework sequences).

[0111] The anti-LILRB4 antibody or antigen-binding fragment thereof may comprise the L1, L2, and L3 domains of a light chain variable region having the sequence set forth in SEQ ID NO:6, and the H1, H2, and H3 domains of a light chain variable region having the sequence set forth in SEQ ID NO:7.

[0112] Further modifications of antibody sequences are contemplated, including the substitution of specific amino acid residues containing exposed side chains with other amino acid residues to provide enhanced chemical stability of the final antibody and avoid deamidation and isomerization. Deamidation of asparagine can occur at NG, DG, NG, NS, NA, NT, QG, or QS sequences, resulting in an isoaspartic acid residue (the isoaspartic acid effect), which introduces a kink in the polypeptide chain and reduces stability. Isomerization can occur at DG, DS, DA, or DT sequences. Thus, antibodies of the present disclosure can be engineered to contain no deamidation or asparagine isomerization sites. Furthermore, methionine residues in the CDRs (typically solvent-exposed Met) can be altered to Lys, Leu, Ala, Phe, or other amino acids to reduce the likelihood of methionine sulfur oxidation, which can reduce antigen-binding affinity and contribute to molecular heterogeneity in the final antibody formulation. Additionally, it may be desirable to change the Asn-Pro combinations found in the CDRs to Gln-Pro, Ala-Pro, or Asn-Ala to prevent or minimize potentially dissociable Asn-Pro peptide bonds. Antibodies with such substitutions can be designed and subsequently screened to ensure that the substitutions do not reduce the affinity or specificity of the anti-LILRB4 antibody or unacceptably alter other desired biological activities. Additional discussion of antibody framework modifications suitable for in vivo use is provided at least in U.S. Patent Nos. 1,147,9608 and 9,709,568, which are incorporated herein by reference in their entireties for all purposes.

[0113] Also contemplated are anti-LILRB4 antibodies in different antibody formats, such as Fv, Fab, Fab', Fab'-SH, and F(ab')2; single-chain forms of antibodies and hypermutants thereof; and multispecific antibodies formed from antibody fragments. Methods for producing these antibodies, including one or more of the L1-L3 and H1-H3 disclosed herein, are well known in the art and can be determined by those skilled in the art. One or more of the anti-LILRB4 antibodies, VH, or VL can be expressed with a leader sequence, a targeting moiety, or both. Suitable leader sequences are well known in the art, e.g., the amino acid sequences set forth in SEQ ID NO:8 and SEQ ID NO:9. The targeting moiety may comprise an amino acid sequence capable of directing the antibody (e.g., anti-LILRB4 or a fragment thereof) to which it is bound to a target site.

[0114] The present disclosure also encompasses polynucleotides encoding anti-LILRB4 antibodies, which can be readily determined by one of ordinary skill in the art. The polynucleotides may be RNA or DNA molecules and include nucleic acid sequences encoding one or more of the amino acid sequences set forth in SEQ ID NOS: 1-7 and the L2 amino acid sequence WAS. The polynucleotide sequences can be incorporated into other larger DNA molecules, such as vectors or chromosomes, to express anti-LILRB4 antibodies. The polynucleotide sequences may include one or more modifications for delivery to a subject. The polynucleotide sequence encoding the VL of an anti-LILRB4 antibody may comprise the nucleic acid sequence set forth in SEQ ID NOS: 10 or 12, or a sequence at least 80% identical thereto. The polynucleotide sequence encoding the VH of an anti-LILRB4 antibody may comprise the nucleic acid sequence set forth in SEQ ID NOS: 11 or 13, or a sequence at least 80% identical thereto.

[0115] Also contemplated are vectors encoding anti-LILRB4 antibodies. Vectors encoding anti-LILRB4 antibodies or fragments thereof may comprise one or more of the nucleic acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, or a sequence at least about 80% identical thereto. The vector may be a plasmid vector, a transposon, an isolated nucleic acid sequence, or a viral vector.

[0116] Disclosed herein are host cells comprising one or more of the nucleic acid sequences provided herein. The host cells can be mammalian cells, bacterial cells, B cells, hybridomas, or cell lines. The host cells comprising one or more of the nucleic acid sequences provided herein can encode antibodies comprising one or more of the amino acid sequences set forth in SEQ ID NOS: 1-5 and the L2 amino acid sequence WAS. The host cells can comprise a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 6, or a sequence at least about 90% identical thereto. The host cells can comprise a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 7, or a sequence at least about 90% identical thereto. The host cells can comprise one or more of the nucleic acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a sequence at least about 80% identical thereto.

[0117] Each of the above antibodies may also comprise a mutant Fc region, including but not limited to, a mutant Fc region that has been modified to alter its natural interaction with microglial FcR.

[0118] An anti-LILRB4 antibody can competitively inhibit the binding of a reference antibody to an epitope. An antibody is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay.

[0119] 2. LILRB4 decoy receptor In one aspect, the present disclosure provides a LILRB4 polypeptide or a fragment thereof. The mature LILRB4 polypeptide comprises two or four extracellular immunoglobulin domains, a transmembrane domain, and one or more of two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Thus, the fragment can be a fragment of the extracellular domain, the transmembrane domain, or the cytoplasmic domain. In one embodiment, the fragment is an extracellular domain fragment. The LILRB4 polypeptide can comprise the amino acid sequence set forth in SEQ ID NO: 14, a fragment thereof, a derivative thereof, or a sequence at least about 80% identical thereto.

[0120] It is understood that the present disclosure is directed to homologs, variants, derivatives, or fragments of LILRB4 in other organisms and is not limited to human LILRB4 (hLILRB4). Homologs, variants, derivatives, or fragments can also be found in other species using methods known in the art. When determining whether LILRB4 has significant homology or shares a certain percentage of sequence identity with the sequences of the present invention, sequence similarity can be determined using conventional algorithms, which usually allow for the introduction of a small number of gaps to achieve the best match. In particular, the "percent identity" of two polypeptide or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). To obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention, BLAST nucleotide searches can be performed using the BLASTN program. Similarly, BLAST protein searches can be performed using the BLASTX program to obtain amino acid sequences homologous to the polypeptides of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) are employed. For more information, see www.ncbi.nlm.nih.gov.

[0121] A homolog, variant, derivative, or fragment of LILRB4 may be at least 80%, 85%, 90%, or 95% homologous to human LILRB4 (SEQ ID NO: 14) or a fragment thereof. In certain embodiments, a homolog, variant, or derivative of LILRB4 may be at least 80%, 85%, 90%, or 95% homologous to a human LILRB4 extracellular domain fragment.

[0122] In one aspect, the present disclosure provides a LILRB4 fusion protein. A LILRB4 fusion protein according to the present disclosure refers to a polypeptide that can be fused to a "targeting moiety" and can direct a bound entity (e.g., LILRB4 or a fragment thereof) to a target site. Targeting moieties can include, but are not limited to, cell surfaces and cell surface proteins. The targeting moiety can include a binding domain derived from a target receptor ligand. A target receptor ligand is a ligand that binds to a target receptor. Suitable target receptors include cell surface receptors found on microglial cells. Non-limiting examples of suitable target receptors include Fc receptors, such as FcRγ, FcRα, FcRε, and FcRμ. FcRγ belongs to the immunoglobulin superfamily and includes FcRγI (CD64), FcRγIIA (CD32), FcRγIIB (CD32), FcRγIIIA (CD16a), and FcRγIIIB (CD16b). In a specific embodiment, the target receptor is FcRγI (CD64). Fc receptors are cell surface receptors that recognize the Fc region of an antibody. Non-limiting examples of target receptor ligands for Fc receptors include the Fc regions of IgG, IgA, IgE, and IgM. In a specific embodiment, the target receptor ligand is the IgG Fc region. In another embodiment, the targeting moiety may comprise an antibody capable of specifically binding to an antigenic determinant on the target site, or a fragment thereof that retains specific binding to the antigenic determinant.

[0123] The targeting moiety may be capable of directing the entity to which it is bound to a target receptor on the surface of a cell capable of expressing LILRB4. The cell capable of expressing LILRB4 may be a microglial cell. The targeting moiety may be capable of directing the entity to which it is bound to a target receptor on the surface of a microglial cell.

[0124] In some embodiments, the targeting moiety can be an antibody or fragment thereof, or a binding domain derived from a target receptor ligand. In certain embodiments, the targeting moiety is an antibody or fragment thereof. For example, the antibody fragment can be a constant region (e.g., hinge, CH2, and / or CH3 domain). In specific embodiments, the targeting moiety is an antibody fragment, such as an Fc fragment. The Fc fragment can include the heavy chain constant region of an antibody. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the antibody isotype as IgG, IgM, IgA, IgD, or IgE, respectively. In specific embodiments, the Fc fragment is an IgG Fc fragment. In humans, there are four IgG subclasses (IgG1, 2, 3, and 4). Each of the four IgG subclasses can be used as a targeting moiety in the present invention. In certain embodiments, the targeting moiety can be a single-chain or linear antibody.

[0125] 3. Treatment method A process for treating a neurological disease, disorder, or condition is provided. In some embodiments, the neurological disease, disorder, or condition is associated with microglia in a subject. In some embodiments, the method for treating a neurological disease, disorder, or condition generally comprises administering a therapeutically effective amount of a LILRB4 antagonist to enhance microglial function, inhibit a neurological disease, disorder, or condition associated with microglial dysfunction, slow the progression of a neurological disease, disorder, or condition associated with microglial dysfunction, or limit the onset of a neurological disease, disorder, or condition associated with microglial dysfunction.

[0126] As used herein, the terms "microglial cells" or "microglia" refer to a class of glial cells involved in mediating immune responses within the central nervous system by acting as macrophages. Microglial cells are capable of producing exosomes and include various morphologies, including amoeboid microglial cells, ramified microglial cells, and reactive microglial cells. Microglial cells include reactive microglia, defined as resting ramified microglia that transform into a reactive macrophage-like state and accumulate at sites of brain injury and inflammation to aid in tissue repair and neuroregeneration.

[0127] One aspect of the present disclosure provides a method for treating a subject with a microglia-related disease or disorder. The microglia-related disease or disorder may be a central nervous system disease or disorder in which disrupted microglial function contributes to the pathology or symptoms. In non-limiting examples, diseases and disorders associated with microglia dysfunction include Alzheimer's disease, Parkinson's disease, Nasu-Hakola disease, prion disease, multiple sclerosis, HIV-1 dementia, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, neuropathic pain, and autism spectrum disorder. For example, diseases and disorders associated with microglia dysfunction include those described in Salter and Stevens, Nature Medicine, volume 23, pages 1018-1027 (2017), which is incorporated herein by reference.

[0128] The present disclosure provides a method for treating Aβ amyloidosis, comprising administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to a subject in need of treatment. The present disclosure also provides a method for treating a subject diagnosed with a disease characterized by cerebral Aβ plaques, comprising administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to the subject. The present disclosure also provides a method for treating a subject diagnosed with a disease characterized by vascular Aβ plaques in the brain, comprising administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to the subject. The present disclosure also provides a method for preventing the progression of a disease characterized by cerebral Aβ plaques, comprising administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to a subject in need of treatment. The present disclosure also provides a method for treating a subject diagnosed with Alzheimer's disease, comprising administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to the subject. The present disclosure also provides a method for treating a subject diagnosed with CAA, the method comprising administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to the subject. Suitable anti-LILRB4 antibodies are described herein. In embodiments where the subject is a human, the anti-LILRB4 antibody is adapted for administration to a human subject (e.g., is humanized).

[0129] In one embodiment, the present disclosure provides a method for preventing or slowing the progression of a disease characterized by Aβ plaques in the brain. The method comprises administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to a subject in need of treatment. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion proteins include those disclosed herein. The progression of a disease characterized by Aβ plaques in the brain can be assessed by methods known in the art and described herein, including the worsening of clinical signs of Aβ amyloidosis, Aβ plaque-related symptoms, or CAA-related symptoms. In an exemplary embodiment, the clinical sign is amyloid plaque load.

[0130] In another embodiment, the present disclosure provides a method for activating microglial cells in a subject. The method comprises administering a therapeutically effective amount of an anti-LILRB4 antibody or LILRB4-Fc fusion protein to a subject in need of treatment. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion proteins include those disclosed herein. Non-limiting examples of activated microglial cells include increased accumulation around Aβ plaques, increased CD68 expression, increased Cst7 expression, increased phagocytic activity, increased CD74 expression, and decreased tyrosine phosphatase activity.

[0131] In another embodiment, the present disclosure provides a method for ameliorating clinical symptoms of Aβ amyloidosis. The method comprises administering a therapeutically effective amount of an anti-LILRB4 antibody or an LILRB4-Fc fusion protein to a subject in need of treatment. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion proteins include those disclosed herein. Non-limiting examples of improved clinical signs of Aβ amyloidosis include a decrease in amyloid plaque load, stabilization of amyloid plaque load (i.e., no further increase), an increase in CSF Aβ42 concentration, an increase in the CSF Aβ42 / Aβ40 ratio, a decrease in the Aβ42 / Aβ40 peak time ratio (e.g., to approach 1) as measured by stable isotope labeling kinetics, a decrease in the Aβ42 / Aβ40 FTR ratio (e.g., to approach 1) as measured by stable isotope labeling kinetics, and a change in the ratio of relative labeled Aβ42 to relative labeling of Aβ40 (or another Aβ peptide) after stable isotope labeling to approach 1. In each of the above embodiments, the improvement (i.e., change) in clinical signs is at least statistically significant. In certain embodiments, the change is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to untreated or negative control-treated subjects. In some embodiments, the change is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to untreated or negative control-treated subjects. In other embodiments, the change may be at least 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or 450% compared to untreated or negative control-treated subjects.

[0132] In another embodiment, the present disclosure provides a method for reducing amyloid plaque load in the brain of a subject. The method comprises administering a therapeutically effective amount of an anti-LILRB4 antibody or LILRB4-Fc fusion protein to the subject. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion proteins include those disclosed herein. The method of the present disclosure may reduce amyloid plaque load in the hippocampus of a subject and / or reduce amyloid plaque load in the brain cortex of a subject. In each of the above embodiments, the amyloid plaque load may be reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to an untreated or negative control-treated subject. In some embodiments, amyloid plaque load may be reduced by at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to untreated or negative control-treated subjects. In other embodiments, amyloid plaque load may be reduced by at least 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or 450% compared to untreated or negative control-treated subjects.

[0133] In another embodiment, the present disclosure provides a method for reducing CAA load in a subject's brain. The method comprises administering a therapeutically effective amount of an anti-LILRB4 antibody or LILRB4-Fc fusion protein to a subject having fibrillar forms of Aβ in the perforating arterioles and / or leptomeningeal arterioles at the cerebral cortical surface. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion proteins include those disclosed herein. The method of the present disclosure can reduce CAA load in the perforating arterioles and / or leptomeningeal arterioles at the cerebral cortical surface of a subject. In each of the above embodiments, the CAA load can be reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to a subject untreated or treated with a negative control. In some embodiments, amyloid plaque load may be reduced by at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to untreated or negative control-treated subjects. In other embodiments, amyloid plaque load may be reduced by at least 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or 450% compared to untreated or negative control-treated subjects.

[0134] In another embodiment, the method comprises administering to a subject in need of treatment, insoluble Aβ in the brain. 42 The present invention also includes a method for reducing the level of Aβ40 in the brain of a subject. The method comprises administering a therapeutically effective amount of an anti-LILRB4 antibody or a LILRB4-Fc fusion protein to a subject. In one example, the method further comprises reducing the insoluble Aβ40 level in the brain of the subject. In another example, the method comprises selectively reducing the insoluble Aβ40 level, reducing the insoluble Aβ42 level, or a combination thereof, in the brain of the subject, compared to the soluble Aβ40 level, Aβ42 level, or a combination thereof.

[0135] The level of Aβ can be assessed by any suitable method known in the art, including, for example, analyzing Aβ by one or more techniques selected from Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectrometry (MS), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), surface-enhanced laser desorption / ionization time-of-flight (SEMI-TOE), high-performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS / MS), and laser densitometry. In vivo imaging of Aβ is particularly suitable for assessing amyloid plaque load. Non-limiting examples of in vivo imaging methods include positron emission tomography (PET), single-photon emission computed tomography (SPECT), near-infrared (NIR) optical imaging, or magnetic resonance imaging (MRI). Suitable imaging agents are also known in the art (eg, PIB).

[0136] In another embodiment, the present disclosure provides a method for improving Aβ plaque-related symptoms and / or CAA-related symptoms in a subject. The method comprises administering a therapeutically effective amount of an anti-LILRB4 antibody that specifically binds to LILRB4 or a LILRB4-Fc fusion protein to a subject with at least one Aβ plaque-related symptom and / or at least one CAA-related symptom. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion proteins include those disclosed herein. Non-limiting examples of the improvement of Aβ plaque-related symptoms are described above. In some embodiments, the improved Aβ plaque-related symptoms include neurodegeneration, cognitive dysfunction, behavioral changes, emotional dysregulation, and / or reduced seizures. In each of the above embodiments, the improvement (i.e., change) in symptoms is at least statistically significant. In certain embodiments, the change is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to untreated or negative control-treated subjects. In some embodiments, the change is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to untreated or negative control-treated subjects. In other embodiments, the change is at least 100, 125, 150, 200, 250, 300, 350, 400, or 450% compared to untreated or negative control-treated subjects.

[0137] The anti-LILRB4 antibodies disclosed herein can also be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, biological response modifier, drug, or PEG. In some embodiments, the therapeutic agent is a drug, radioisotope, lectin, or toxin. Immunotoxin conjugates are widely described in the art. Toxins can be attached to antibodies using conventional coupling techniques, or immunotoxins containing protein toxin moieties can be produced as fusion proteins. When using radioisotope-conjugated anti-LILRB4 antibodies for immunotherapy, the specific isotope can be selected depending on factors such as leukocyte distribution and stability and emission. Depending on the autoimmune response, several emitters can be used. Radioisotopes that emit alpha particles or beta particles are commonly used in immunotherapy. 212 Short-range, high-energy α-emitters such as Bi may also be used. Examples of radioisotopes that can be attached to the anti-LILRB4 antibodies disclosed herein for therapeutic purposes include: 123 I, 124 I, 125 I, 131 I, 89 Zr, 90 Y, 67 Cu, 64 Cu, 111 In, 212 Bi, 212 At, 211 Pb, 47 Sc, 109 Pd, and 188 Other therapeutic agents that can be conjugated to anti-LILRB4 antibodies, as well as ex vivo and in vivo treatment protocols, are known or readily ascertainable by those skilled in the art.

[0138] Administration of anti-LILRB4 antibodies, or compositions containing anti-LILRB4 antibodies, or LILRB4-Fc fusion proteins, is carried out using standard effective techniques, including peripheral (i.e., not into the central nervous system) or local administration to the central nervous system. Peripheral administration includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, including direct administration to the central nervous system (CNS), includes, but is not limited to, administration via a lumbar, intraventricular, or intraparenchymal catheter, or administration using a surgically implanted controlled-release formulation.

[0139] For effective administration, pharmaceutical compositions are purposefully designed to suit the chosen mode of administration and include pharmaceutically acceptable excipients, such as compatible dispersants, buffers, surfactants, preservatives, solubilizers, tonicity agents, stabilizers, and the like, as appropriate. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16th Ed. ISBN: 0-912734-04-3, latest edition, is incorporated herein by reference in its entirety and provides an overview of formulation techniques commonly known to practitioners. Altering the solubility properties of antibodies useful in this discovery may be particularly useful; for example, the antibody may be rendered more lipophilic by encapsulation in liposomes or by blocking polar groups.

[0140] The antibody concentration in the administered formulation is an effective amount, ranging from as low as about 0.1% by weight to as high as about 15% or about 20% by weight, and is selected primarily based on fluid volume, viscosity, etc., depending on the particular mode of administration selected, as desired. A typical composition for injection into a subject can be prepared to contain 1 mL of sterile buffered water such as phosphate-buffered saline and approximately 1-1,000 mg of any one or combination of antibodies disclosed herein. The formulation can be sterile filtered after formulation or rendered microbiologically acceptable. A typical composition for infusion has a fluid volume of 1-250 mL, such as sterile Ringer's solution, and an anti-LILRB4 antibody concentration of 1-100 mg or greater per mL. The anti-LILRB4 antibodies or LILRB4-Fc fusion proteins disclosed herein can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. Lyophilization and reconstitution can result in varying degrees of antibody activity loss (e.g., with conventional immunoglobulins, IgM antibodies tend to lose more activity than IgG antibodies). The dosage is an effective amount and may need to be adjusted to compensate. The pH of the formulation is selected to balance antibody stability (chemical and physical) with the subject's comfort upon administration. Generally, a pH between 4 and 8 is acceptable. The dosage will vary from individual to individual, based on the size, weight, and other physiological characteristics of the individual receiving the appropriate dose.

[0141] As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., an anti-LILRB4 antibody or an LILRB4-Fc fusion protein) that produces a measurable beneficial effect, i.e., significant efficacy, in a subject receiving the substance. The therapeutically effective amount or dosage of a compound administered in accordance with this discovery is determined using standard clinical techniques and may be influenced by the circumstances of the case, such as the antibody administered, the route of administration, and the condition being treated. A typical dosage may comprise about 0.01 mg / kg to about 100 mg / kg of an anti-LILRB4 antibody or an LILRB4-Fc fusion protein described herein. Dosages may range from about 0.05 mg / kg to about 50 mg / kg, more preferably from about 0.1 mg / kg to about 25 mg / kg. Administration frequency may be daily, weekly, twice weekly, three times weekly, or monthly or more, as needed to effectively treat the condition.

[0142] The timing and duration of treatment for the disease itself will be determined by the circumstances of the case, and may vary from a one-time single dose to a lifelong course of therapeutic treatment.

[0143] As long as an appropriate formulation is used herein, other effective administration techniques, such as intracerebroventricular administration, transdermal administration, and oral administration, can be adopted depending on the appropriate application. Furthermore, those skilled in the art can use the polynucleotide of the present disclosure encoding any one of the above antibodies instead of the proteinaceous substance itself. The polynucleotide of the present disclosure can be modified and appropriately formulated for delivery.

[0144] Additionally, it may be desirable to employ controlled release formulations using biodegradable films or materials, or osmotic minipumps, or delivery systems based on dextran beads, alginate, or collagen.

[0145] 4. Pharmaceutical Compositions The present disclosure encompasses pharmaceutical compositions comprising the anti-LILRB4 antibodies disclosed herein to facilitate administration and promote stability of the active agent. For example, the anti-LILRB4 antibodies of the present disclosure can be mixed with at least one pharmaceutically acceptable carrier or excipient, resulting in a (predetermined) pharmaceutical composition that is administered volumetrically and effectively to a subject, such as an appropriate subject (i.e., a "subject in need of treatment" or a "subject in need thereof"). Methods for preparing and administering the anti-LILRB4 antibodies or LILRB4-Fc fusion proteins disclosed herein to a subject in need of treatment are well known to, or can be easily determined by, those skilled in the art. The route of administration of the anti-LILRB4 antibodies or LILRB4-Fc fusion proteins can be, for example, peripheral, oral, parenteral, inhalation, or topical.

[0146] Pharmaceutical compositions for effective administration are purposefully designed to suit the selected mode of administration, and suitable pharmaceutically acceptable excipients such as compatible carriers, dispersants, buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers, etc. are used as appropriate.

[0147] Non-limiting examples of pharmaceutically acceptable carriers include physiological saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, and partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol, lanolin, or combinations thereof.

[0148] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, and the like, are often included in the composition.

[0149] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0150] The compositions disclosed herein can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use.

[0151] Anti-LILRB4 antibodies or LILRB4-Fc fusion proteins can be formulated for parenteral administration. Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. Parenteral formulations may be administered as a single bolus, by infusion, or as a load bolus followed by a maintenance dose. These compositions can be administered at specific fixed or variable intervals, for example, once daily, or on an "as needed" basis.

[0152] Certain pharmaceutical compositions disclosed herein can be orally administered in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions, or solutions. Some pharmaceutical compositions can also be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other conventional solubilizers or dispersants.

[0153] The amount of anti-LILRB4 antibody or LILRB4-Fc fusion protein to be combined with the carrier material to produce a single dosage form can vary depending on the host treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses, or by infusion over a period of time. Dosage regimens can also be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). [Example]

[0154] The following examples are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the examples below represent techniques discovered by the inventors to work well in implementing the present disclosure. However, those skilled in the art should understand in light of the present disclosure that many changes can be made in certain disclosed embodiments and still obtain the same or similar results without departing from the spirit and scope of the present disclosure; therefore, all matter described or shown in the accompanying examples and drawings should be interpreted as illustrative and not limiting.

[0155] Example 1 Generation of ILT-Telo Tg mice (transgenic mice expressing the telomeric region of the human LILR complex) ILT-Telo Tg mice were generated according to a previously described procedure (Song et al., J Exp Med. 2018 Mar 5;215(3):745-760). The CH17-401N15 bacterial artificial chromosome (BAC) was purchased from the BACPAC Resources Center (BPRC). The entire BAC was injected into the nuclei of fertilized eggs of C57BL / 6 x CBA / J (CBA) mice to obtain TELO founders. Offspring from the founders were screened for expression of the ILT encoded by the BAC transgene in various tissues. Strains selected for further study were then backcrossed to the C57BL / 6 background for at least eight generations to ensure that their genetic backgrounds were similar to those of the recipient C57BL / 6 strain. ILT-Telo Tg mice were crossed with 5XFAD mice to generate ILT-Telo Tg x 5XFAD mice. All mice were housed in a specific pathogen-free environment. The Institutional Animal Care and Use Committee at Washington University in St. Louis approved all protocols used in this study.

[0156] Example 2 Generation of LILRB4 reporter cell line A chimeric gene consisting of the extracellular Ig domain and transmembrane domain of LILRB4 fused to the intracellular domain of CD3zeta was cloned into the retroviral vector pMX. Retroviral particles were assembled in the Phoenix-Amphotropic cell line PHA (from ATCC). The supernatant containing the packaged virus was recovered and incubated with the 2B4 cell line. This cell line (provided by Dr. Saito, RIKEN, Japan) expresses GFP under the control of a promoter sequence linked to the transcription factor NFAT. LILRB4 expression was tested 96 hours after transduction. LILRB4-expressing cells were FACS-sorted multiple times until 20% of LILRB4-high expressing cells were purified by FACS, amplified, and cryopreserved in stock batches.

[0157] Activation of the anti-LILRB4 reporter. Activation was assessed by examining GFP expression upon plating of anti-LILRB4 reporter cells bound to plastic with anti-LILRB4 antibody or an irrelevant mouse IgG2a control. Anti-LILRB4 mAb, but not control mAb, engaged and activated the anti-LILRB4 reporter cells.

[0158] Example 3 Anti-LILRB4 mAb testing in the ILT-Telo Tg x 5XFAD model a. In vivo antibody treatment Four-month-old ILT-Telo Tg x 5XFAD male mice were intraperitoneally (ip) injected weekly with either the Fc-mutated anti-LILRB4 antibody (60 mg / kg) or the Fc-mutated control antibody (60 mg / kg). After eight consecutive injections, the animals were deeply anesthetized with ketamine / xylazine (10:1 ratio) and perfused with 0.1% heparin (1:1000 dilution in phosphate-buffered saline). The brains were then removed and immersed in 4% paraformaldehyde (PFA) for 24 hours, followed by immersion in 30% sucrose in phosphate-buffered saline for 24 hours. They were then embedded in a 2:1 mixture of 30% sucrose and optimal cutting compound. 40 μm-thick sections were prepared for immunofluorescence staining.

[0159] b. Immunofluorescence Free-floating mouse brain sections were blocked with 3% BSA and 0.25% Triton X-100 in PBS and stained overnight at 4°C with anti-IBA1 (rabbit monoclonal, 1:500, Cell Signaling Technology), anti-CD74 (Alexa Fluor® 647-conjugated rat IgG2b, 1:200, BioLegend), and anti-6E10 (Alexa Fluor® 488-conjugated anti-Aβ1-16, 1:1000, BioLegend), followed by staining with anti-rabbit IgG Alexa Fluor 555 (1:2,000; Abcam), anti-rat IgG Alexa Fluor 647 (chicken polyclonal, 1:2,000; Invitrogen), and methoxy-X04 (3 μg / ml; Tocris) for 1 hour at room temperature. All antibodies were used in blocking buffer, and between all incubations, sections were washed three times for 10 min in PBS. Images were collected using a Nikon A1Rsi confocal microscope equipped with a 20x 0.95-NA objective. Z-stacks were recorded at 1,024 x 1,024 pixel resolution with 2 µm steps in the z direction. 3D reconstruction and parameter extraction of microglia and Aβ plaques were performed in Imaris (version 9.2.0), and further processing was performed using automated Matlab scripts.

[0160] Example 4 Measurement of LILRB4 expression in human AD and its correlation with APOE expression and AD progression Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor of the leukocyte immunoglobulin-like receptor (LILR) family and is primarily expressed on cells of myeloid origin in humans. Currently, the expression and function of LILRB4 in Alzheimer's disease are unknown.

[0161] In this study, we examined a human AD snRNA-seq dataset (Zhou, et al., Nat Med. 2020: 11 AD, 11 Ctrl). UMAP revealed that the LILRB4 gene is selectively expressed by microglia in the human brain (Figure 2A), and LILRB4 was the most highly expressed gene in AD among all leukocyte immunoglobulin-like receptors (LILRs) (Figure 2B).

[0162] Next, we reanalyzed human microglial snRNA-seq data from the Seattle Alzheimer's Disease dataset (SEA-AD, 84 patients: 2 Braak 0; 10 Braak II and III; 23 Braak IV; 34 Braak V; 15 Braak VI). UMAP revealed LILIB4 expression in microglia (Figure 3A). Furthermore, we observed that patients with higher Braak stages had higher microglial LILRB4 expression compared with Braak 0 patients (Figure 3B). Furthermore, analysis of the SEA-AD dataset also showed a positive correlation between microglial LILRB4 expression and phospho-tau (pTau) content in AD brain lysates (Figure 3C). ApoE expression was also examined in the SEA-AD snRNA-seq dataset. Microglial APOE expression was higher in AD patients than in controls, reflecting disease severity (Figure 3D). Notably, LILRB4 expression strongly correlated with APOE, indicating its upregulation during microglial activation in response to AD pathology ( Fig. 3E ).

[0163] Next, we examined microglial LILRB4 expression by immunofluorescence in brain sections from AD and Ctrl patients (n = 4 AD, 4 Ctrl). Representative confocal images stained with IBA1 (green), LILRB4 (red), ApoE (white), and Methoxy-X04 (blue) (Figure 4A) show that LILRB4 and APOE expression were highly upregulated in Aβ plaque-associated microglia from AD patients. There was a positive correlation between LILRB4 and APOE expression, and patients with high LILRB4 expression showed elevated APOE expression (Figure 4B).

[0164] Example 5 Characteristics of Aβ pathology in ILT-Telo Tg x 5XFAD mice We generated ILT-TELO-BAC transgenic mice (Telo) and crossed them with the 5XFAD model of AD, as shown in Figures 5A and 5B. Four genotypes were generated for analysis: wild-type; ILT-Telo Tg; 5XFAD; and ILT-Telo Tg x 5XFAD. Expression of LILRB4, LILRB1, and LILRA2 was confirmed in ILT-Telo Tg microglia by FACS (Figure 6). LILRB4 expression was significantly upregulated in 6-month-old ILT-Telo Tg x 5XFAD male mice compared with sex- and age-matched ILT-Telo Tg mice (Figure 6).

[0165] Furthermore, we performed immunofluorescence analysis of microglial LILRB4 expression by co-staining for the microglial markers IBA1 (red), LILRB4 (green), ApoE (white), and the Aβ plaque marker Methoxy-X04 (blue) (Figure 7A). Quantification of microglial LILRB4 expression in the entire cortex of ILT-Telo Tg x 5XFAD mice and control 5XFAD mice revealed that LILRB4 expression was highly specific and elevated in microglia associated with Aβ plaques in ILT-Telo Tg x 5XFAD mice (6 months old, male) (Figure 7A). + PU1 +Microglia and Methoxy-X04 + We measured microglia density within 15- and 30-μm shells surrounding Aβ plaques by placing plaques. ILT-Telo Tg x 5XFAD mice had fewer microglia surrounding Aβ plaques within 15-μm shells compared to 5XFAD mice, suggesting preferential reduction of "reactive microgliosis" near amyloid deposits in ILT-Telo Tg x 5XFAD mice (Figure 7B). Microglia showed similar densities throughout the cortex in 5XFAD and ILT-Telo Tg x 5XFAD mice (Figure 7C).

[0166] In amyloid pathology, "reactive microgliosis" is characterized by hypertrophic or amoeboid microglial proliferation surrounding Aβ plaques. Three-dimensional reconstructions of immunofluorescence for IBA1 (red), LILRB4 (cyan), ApoE (green), and Aβ plaques (blue) showed that plaque-associated microglia in ILT-Telo Tg x 5xFAD mice had smaller cell body size than those in 5xFAD mice, as quantified by the total volume of microglial cell bodies and processes (Figure 8).

[0167] To investigate whether the loss of microglial activity in ILT-Telo Tg x 5XFAD mice impacts Aβ pathology, we stained matched coronal brain sections from ILT-Telo Tg x 5XFAD mice and control 5XFAD mice at 6 months of age with 6E10 to examine Aβ aggregate deposition. Significant increases in Aβ accumulation were observed in the cortex, hippocampus, and amygdala of ILT-Telo Tg x 5XFAD mice (Figures 9A and 9B).

[0168] Since the microglial barrier promotes amyloid compaction and shielding, preventing neurite dystrophy, we investigated neurite dystrophy by staining for LAMP1, which accumulates in dystrophic neurites. +Voxels were measured within 15 μm and 30 μm shells surrounding Aβ plaques (Figure 10). Compared to control 5XFAD mice, ILT-Telo Tg x 5XFAD mice had significantly more LAMP within 15 μm and 30 μm shells at 6 months of age. + Voxels showed an overall significant increase (Fig. 10).

[0169] In summary, we demonstrate that LILRB4 is expressed in microglia of ILT-Telo Tg mice, its expression increases with amyloid pathology, and that LILRB4 expression is elevated in microglia associated with Aβ plaques in 6-month-old ILT-Telo Tg x 5XFAD mice. Furthermore, Telo transgenic mice exhibited defects in microglial activity, enlarged Aβ aggregates, and extensive neuritic dystrophy.

[0170] Example 6 Generation of anti-LILRB4 antibodies and in vivo testing in ILT Telo Tg x 5xFAD mice Binding of Fc to Fc receptors has been shown to induce cytotoxicity via the antibody-dependent cell-mediated cytotoxicity (ADCC) pathway. To characterize the function of microglial LILRB4 in AD pathology and exclude ADCC effects, recombinant Fc-mutated mIgG2a anti-LILRB4 mAb (clone: ​​ZM3.1) and an irrelevant Fc-mutated mIgG2a control mAb (clone: ​​27D6) were generated using the method shown in Figure 11. Further purification of the Fc-mutated anti-LILRB4 mAb (ZM3.1) and Ctrl (27D6) mAb was performed (Figure 12). Next, to characterize the anti-LILRB4 mAb, a calcium-driven reporter cell line was generated as described in Example 2 (Figure 13). The 2B4-LILRB4 reporter was stably transfected with a chimeric gene consisting of the extracellular portion of LILRB4 and the intracellular domain of CD3ζ. The involvement of hLILRB4 led to the Ca2 +This promotes signal transduction, leading to the nuclear translocation of NFAT and the synthesis of enhanced GFP (EGFP) driven by NFAT. The specificity of anti-LILRB4 was first demonstrated by binding to the recombinant ectodomain of LILRB4 by ELISA (Figure 14). To measure the activity of anti-LILRB4 mAb, anti-LILRB4 mAb or Ctrl mAb was immobilized on a plate at different concentrations, and 2B4-LILRB4 reporter cells were added to the plate overnight. Anti-LILRB4 mAb induced EGFP in the 2B4-LILRB4 reporter cells in a dose-dependent manner, suggesting that the immobilized anti-LILRB4 mAb specifically binds to hLILRB4 (Figure 14).

[0171] The anti-LILRB4 mAb and control mAb were further tested in vivo in ILT-Telo Tg x 5XFAD male mice. Administration of anti-LILRB4 mAb and control mAb and sample collection were performed as described in Example 3. Briefly, ILT-Telo Tg x 5XFAD mice were treated intraperitoneally (ip) with anti-LILRB4 mAb or CTRL mAb (60 mg / kg) once a week starting from 4 months of age (Figure 15). Mouse behavior was evaluated after six consecutive injections, and mAb treatment was terminated after two additional injections. After eight consecutive injections, plasma and brain homogenate samples were collected and antibody levels were measured. Plasma and brain samples from male mice were tested as described in Figure 16. The anti-LILRB4 mAb reached detectable concentrations in both plasma and brain (approximately 10 ng / mg in brain and 100 μg / ml in plasma, respectively) (Figure 16). Furthermore, bulk microglial RNA-seq, Aβ burden, and assessment of microglial activity were performed in mAb-treated male mice.

[0172] a. Bulk RNA-seq of microglia To clarify the in vivo effects of anti-LILRB4 mAb on microglial activity, we performed transcriptome analysis of microglia in anti-LILRB4 mAb-treated and CTRL mAb-treated mice. One week after the eighth injection, microglia were isolated from whole brains and bulk RNA-seq was performed (n = 3 anti-LILRB4 mAb-treated mice, 3 CTRL mAb-treated mice). In this screen, 81 transcripts were upregulated and 77 transcripts were downregulated in response to anti-LILRB4 mAb (Figure 17A). Among these differentially expressed genes (DEGs), (Padj < 0.05, llog2FCl > 0.5) (Figure 17B), gene sets contributing to microglial motility and phagocytosis were enriched in anti-LILRB4 mAb-treated mice (Figure 17B, Figure 18A). Conversely, interferon response (IFN-R) and proinflammatory cytokine gene sets (Axl, Ifitm3, Usp18, Oas1a, Ifit3) were suppressed in microglia treated with anti-LILRB4 mAb (Figure 18B). Furthermore, string analysis was performed to identify protein-protein interaction networks enriched in the down-regulated gene set (Figure 19A) and the up-regulated gene set (Figure 20A). Significant protein interaction clusters were observed in both the down-regulated gene set (Figure 19B) and the up-regulated gene set (Figure 20B).

[0173] b. Assessment of Aβ burden and microglial activity Increased CD74 expression was verified by co-staining of the microglial marker IBA1 (yellow), the MHC class II-associated marker CD74 (pink), and the Aβ plaque marker X34 (blue) in microglia treated with anti-LILRB4 mAb (Figure 21). Immunofluorescence showed an increase in the size of microglial bodies after anti-LILRB4 mAb treatment, with X34 expression in different regions. + 15 μm of IBA1 around the plaque + Quantified by voxel (Figure 22) and X34 in different regions +CD74 within 15 μm of the plaque + / IBA1 + The percentage of colocalized voxels increased (Figure 23).

[0174] To measure the effect of anti-LILRB4 mAb on total Aβ plaque load, matched coronal brain sections were stained with methoxy-X04 and 6E10, and total Aβ plaque area was measured in the cortex, hippocampus, and amygdala (Figures 24A and 24B). Compared with Ctrl mAb treatment, anti-LILRB4 mAb administration induced an overall significant reduction in Aβ plaques in different brain regions (Figures 24A and 24B). Aβ40 (Aβ42) and Aβ42 (Aβ42) levels were also quantified by ELISA in the soluble and insoluble fractions of the cortex. Snap-frozen cortical tissue was homogenized sequentially in PBS and guanidine solution to obtain PBS-soluble and insoluble fractions. Although the levels of soluble and insoluble Aβ40 were similar, significant decreases in soluble and insoluble Aβ42 were detected in the cortical regions of anti-LILRB4 mAb-treated mice compared with Ctrl mice (Figure 25). Aβ42 is neurotoxic, inducing tau phosphorylation and microtubule destabilization. Microglial plaque containment was inversely correlated with Aβ42 "hot spots" on the plaques. Neuritic damage was then assessed by BACE1 staining, which accumulates in swollen presynaptic dystrophic neurites adjacent to amyloid plaques. The cortical area showing BACE1 staining and the volume within a 15-μm spherical shell surrounding Aβ plaques were significantly reduced in anti-LILRB4-treated mice compared with Ctrl IgG2 (Figure 26), indicating suppression of neuronal dystrophy. Taken together, these data suggest that anti-LILRB4 mAb enhances microglial activation, promotes microglial clearance of Aβ, and attenuates the release of microglial cytotoxic mediators (IFN-R and proinflammatory cytokines).

[0175] c. Behavioral testing Furthermore, to understand whether the reduction in Aβ pathology and IFN response induced by anti-LILRB4 mAb treatment were associated with behavioral changes, an elevated plus maze (EPM) test was first performed to assess risk-taking behavior and anxiety-like traits in mice. As illustrated in Figure 27, the EPM was designed to assess risk-taking behavior, exploratory impulses, and anxiety-like traits by allowing mice free access to a maze containing both unprotected open arms and protected closed arms, approximately 1 m above the floor. During this test, anti-LILRB4-treated mice significantly reduced the total time spent in the open arms compared to the control group (Figure 27), indicating that anti-LILRB4 administration restores fear of open spaces and attenuates risk-taking behavior. Furthermore, contextual memory was assessed in a conditioned fear paradigm (Figure 28A-D). In this test, there were no significant differences between the two groups in baseline freezing behavior on Day 1 and freezing during tone / shock presentation (Figure 28B). Assessment of contextual memory on day 2 also showed comparable freezing times between the two groups (Figure 28C). However, analysis of freezing behavior during tone presentation on day 3 showed a trend toward a treatment effect on freezing in response to tone presentation, with post-hoc tests showing that anti-LILRB4 mice froze more than ctrl mice (p = 0.0896) (Figure 28D). This suggests that anti-LILRB4 mAb-treated mice have stronger amygdala-dependent memory for the cue compared with control mice.

[0176] In summary, anti-LILRB4 antibody enhanced microglial phagocytic function and reduced microglial IFN-R and proinflammatory cytokine release, thereby reducing Aβ plaques and suppressing risk-taking behavior in mice. Furthermore, anti-LILRB4 antibody ameliorated Aβ plaque-induced pathological and behavioral changes.

[0177] Example 7 Validation of LILRB4-ApoE interaction ApoE secreted by microglia is an important component of amyloid plaques and promotes their compaction. In turn, plaque-associated ApoE may influence microglial activation by Aβ plaques. Recent studies have shown that APOE variants are recognized and bound by LILRB4. Here, we tested the binding of ApoE2 / 3 / 4 to LILRB4 by ELISA and observed that ApoE3 / 4 has a higher LILRB4 binding affinity than ApoE2 (Figure 29). Furthermore, 2B4-LILRB4 reporter cells were incubated overnight with lipidated or nonlipidated human ApoE3 / 4 or mouse ApoE at different concentrations. In the human ApoE incubation group, reporter cells incubated with ApoE4 had a stronger EGFP signal than the ApoE3 group, and non-lipidated ApoE3 / 4 had a stronger effect on reporter cell activation than lipidated ApoE3 / 4 (Figures 30A and 30B); meanwhile, mouse ApoE showed a stronger effect on reporter cell activation than human ApoE3 / 4 (Figure 14). Anti-LILRB4 reporter cells were further used to test the binding of mouse recombinant ApoE to human recombinant LILRB4. Human ApoE3 / 4- and mouse ApoE-induced reporter cell activation was completely blocked by anti-LILRB4 mAb, demonstrating the specificity of LILRB4-ApoE binding (Figures 31 and 32).

[0178] Three amino acid residues, T30, P35, and Y121, were identified as important for the interaction between human LILRB4 and human ApoE. In another study, the structure of the LILRB4 ectodomain was elucidated and binding sites for various ligands were predicted using SPPIDER (Accessibility-Based Identification and Recognition of Protein-Protein Interfaces). To further develop this model, in silico docking modeling of the LILRB4 ectodomain (PDB: 3P2T) with the N-terminal four-helix bundle of mouse ApoE (mApoE) (PDB: 1YA9) was performed using the public server ClusPro 2.0. All of the top 10 predicted binding sites for mApoE to LILRB4 point to the D1-D2 interdomain site between the two Ig-like domains of LILRB4, where the K 134 ERAAHP 140 The loops in the predicted mApoE region were always involved in the predicted position of mApoE (Figure 33A). The key interacting residues on the top predicted model are T30, P35, and Y121. T30 and P35 are located at the binding interface between mApoE and LILRB4, confirming their high likelihood of interacting with mApoE (Figure 33B). T30 is close to the helical bundle of mApoE, while P35 may be involved in mApoE access to the binding site (Figure 33C). On the other hand, residue Y121 is located in the loop K. 134 ERAAHP 140 , T30, and P35 were located on opposite sides of the predicted binding interface (Figure 33D). 134 ERAAHP 140 , T30, and P35 interact directly with mApoE, while Y121 may play an indirect role.

[0179] To test this hypothesis, we investigated the K 134 ERAAHP 140We performed a nonfunctional loop swap by converting the entire sequence to VGGVGGP, mutating the loop but minimizing the effect on protein folding (Figure 33D). Recombinant LILRB4 ectodomain proteins with T30A, P35A, or Y121A mutations were also generated. After purification of the original and mutant LILRB4 ectodomains by size-exclusion chromatography, the consensus variants of LILRB4 were confirmed, as were the P35A and Y121A variants, which were variably glycosylated in both monomeric and multimeric forms (Figures 33E and 33F). Meanwhile, the loop-swap and T30A variants significantly shifted LILRB4 multimerization (Figure 33F), suggesting that the loop swap and T30 promote LILRB4 multimerization. The binding of the consensus variants and mutant multimers of LILRB4 to recombinant mApoE was compared by ELISA, as not all variants were monomeric. The loop-swapped and P35A mutants weakened the binding of LILRB4 to mApoE, whereas the T30A and Y121A mutants only showed a tendency to decrease (Fig. 33G). The binding of P35A and Y121A to mApoE was reduced compared to that of the prototypical LILRB4 mutants and was independent of the monomeric / multimeric state (Fig. 33G). Importantly, analysis of purified LILRB4 by immunoblotting with anti-LILRB4 revealed that the loop-swapped mutations inhibited the recognition of LILRB4 by ZM3.1 mAb (Fig. 33E). The K of the D1-D2 interdomain of LILRB4 134 ERAAHP 140 The loop is important for binding both mApoE and anti-LILRB4 antibodies, so the latter directly interferes with the region of LILRB4 that binds to mApoE.

[0180] Example 8 overview These examples demonstrate that LILRB4-ApoE signaling regulates microglial responses around Aβ deposits and inhibits Aβ clearance in an ApoE isoform-dependent manner. Therefore, disrupting the LILRB4-ApoE interaction with anti-LILRB4 mAb reverses microglial immunosuppression and delays the onset of AD.

[0181] Sequence Listing (Table 1) [Table 2-1] [Table 2-2]

Claims

1. An anti-human leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) antibody, a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 1 (L1), WAS (L2), and SEQ ID NO: 2 (L3); and a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO:3 (H1), SEQ ID NO:4 (H2), and SEQ ID NO:5 (H3); An anti-LILRB4 antibody comprising:

2. The anti-LILRB4 antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6 or a sequence at least about 90% identical thereto.

3. The anti-LILRB4 antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 or a sequence at least about 90% identical thereto.

4. The anti-LILRB4 antibody of any one of claims 1 to 3, wherein the framework regions of each variable region have at least 75% sequence identity with human framework region sequences.

5. The anti-LILRB4 antibody of any one of claims 1 to 4, further comprising one or more constant regions, or portions of constant regions, having at least 90% sequence identity with a human constant region sequence.

6. The anti-LILRB4 antibody of any one of claims 1 to 5, which is an antibody fragment selected from a monoclonal antibody, a humanized antibody, a single domain antibody, a single chain variant fragment (scFv), an Fv, a Fab, a Fab', a Fab'-SH, and an F(ab')2, a divalent scFv (di-scFv), a trivalent scFv (tri-scFv), a tetravalent scFv (tetra-scFv), a diabody, a triabody, or a tetrabody.

7. The anti-LILRB4 antibody of claim 6, which is a monoclonal antibody or an antibody fragment thereof.

8. A pharmaceutical composition comprising the anti-LILRB4 antibody of any one of claims 1 to 7 and a pharmaceutically acceptable carrier or excipient.

9. 9. The pharmaceutical composition of claim 8, further comprising a dispersing agent, a buffering agent, a surfactant, a preservative, a solubilizing agent, a tonicity agent, a stabilizer, or any combination thereof.

10. 10. The pharmaceutical composition of claim 9, wherein the carrier comprises saline, an ion exchanger, alumina, aluminum stearate, lecithin, serum proteins, human serum albumin, a buffer solution, phosphate, glycine, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, a salt or electrolyte, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, a zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, a wax, a polyethylene polyoxypropylene block polymer, polyethylene glycol, lanolin, or a combination thereof.

11. A method of treating a neurological disease in a subject in need thereof, comprising administering to the subject an anti-LILRB4 antibody or a LILRB4-Fc fusion protein.

12. 12. The method of claim 11, wherein the neurological disease is associated with microglial dysfunction.

13. 13. The method of claim 11 or 12, wherein the neurological disease is Alzheimer's disease, Parkinson's disease, Nasu-Hakola disease, prion disease, multiple sclerosis, HIV dementia, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, neuropathic pain, or autism spectrum disorder.

14. The method of any of claims 11 to 13, wherein the antibody or LILRB4-Fc fusion protein is administered systemically.

15. The method of any of claims 11 to 14, wherein the antibody or LILRB4-Fc fusion protein is administered locally, optionally directly into the central nervous system.

16. The method of any one of claims 11 to 15, wherein the antibody is an anti-LILRB4 antibody of any one of claims 1 to 7.

17. A method for reducing amyloid plaque load, CAA load, or both in the brain of a subject in need thereof, comprising administering to the subject an anti-LILRB4 antibody or a LILRB4-Fc fusion protein.

18. 18. The method of claim 17, wherein the anti-LILRB4 antibody is administered peripherally.

19. 19. The method of claim 18, wherein the anti-LILRB4 antibody is administered locally, optionally directly into the central nervous system.

20. The method of any of claims 17 to 19, wherein amyloid plaque load is reduced.

21. The method according to any of claims 17 to 20, wherein the CAA load is reduced.

22. The method according to any one of claims 17 to 21, wherein the anti-LILRB4 antibody is the anti-LILRB4 antibody of any one of claims 1 to 7.

23. A method of treating at least one Aβ plaque-associated symptom or at least one CAA-associated symptom in a subject in need thereof, comprising administering to the subject an anti-LILRB4 antibody or a LILRB4-Fc fusion protein.

24. 25. The method of claim 24, wherein the treatment comprises preventing, alleviating, ameliorating, or ameliorating at least one symptom or sign of Aβ plaque or at least one CAA-associated symptom in the subject.

25. 25. The method of claim 23 or 24, wherein the Aβ plaque-associated condition or CAA-associated condition is selected from the group consisting of neurodegeneration, cognitive dysfunction, behavioral changes, language dysfunction, emotional dysregulation, seizures, nervous system structural impairment, nervous system dysfunction, increased risk of developing Alzheimer's disease, and increased risk of developing cerebral amyloid angiopathy.

26. The method of any of claims 23 to 25, wherein the anti-LILRB4 antibody or LILRB4-Fc fusion protein is administered systemically.

27. The method of any of claims 23 to 26, wherein the anti-LILRB4 antibody or LILRB4-Fc fusion protein is administered locally, optionally directly into the central nervous system.

28. The method of any one of claims 23 to 27, wherein the anti-LILRB4 antibody is the anti-LILRB4 antibody of any one of claims 1 to 7.

29. The anti-LILRB4 antibody according to any one of claims 1 to 7 or the pharmaceutical composition according to any one of claims 7 to 10, for use as a pharmaceutical.

30. An anti-LILRB4 antibody according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 7 to 10 for use in reducing amyloid plaque load and / or CAA load in the CNS.

31. The anti-LILRB4 antibody of any of claims 1 to 7 or the pharmaceutical composition of any of claims 7 to 10 for use in treating at least one Aβ plaque-associated symptom or at least one CAA-associated symptom.

32. An anti-LILRB4 antibody according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 7 to 10 for use in the treatment of Alzheimer's disease.

33. Use of an anti-LILRB4 antibody according to any one of claims 1 to 7 in the manufacture of a medicament for reducing amyloid plaque load and / or CAA load in the CNS.

34. Use of an anti-LILRB4 antibody according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment of at least one Aβ plaque-associated symptom or at least one CAA-associated symptom.

35. Use of the anti-LILRB4 antibody of any of claims 1 to 7 in the manufacture of a medicament for the treatment of Alzheimer's disease.

36. A LILRB4-Fc fusion protein for use as a pharmaceutical.

37. A LILRB4-Fc fusion protein for use in the treatment of neurological disorders.

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