EP4652204A2 - Lilrb4/ilt3 antagonist compositions and methods of use thereof - Google Patents

Abstract

Provided are LILRB4 antagonists such as anti-LILRB4 antibody or a LILRB4-Fc fusion proteins and pharmaceutical compositions thereof. Also provided are methods of treating neurological diseases using LILRB4 antagonists and pharmaceutical compositions thereof.

Description

LILRB4/ILT3 ANTAGONIST COMPOSITIONS AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

[0001] The present disclosure generally relates to compositions and methods of treating a microglial dysfunction associated disease, disorder, or condition. The disclosure relates to Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) antagonists, for example anti-human LILRB4 (also known as ILT3) antibodies (anti-LILRB4), LILRB4-Fc fusions and compositions comprising the same. The disclosure also relates to compositions and methods for delaying and/or preventing A amyloidosis. The disclosure also relates to compositions and methods for delaying and/or preventing Ap plaque associated symptoms and/or cerebral amyloid angiopathy (CAA) associated symptoms, such as those associated with Alzheimer’s disease (AD) or CAA in a subject. In particular, the disclosure relates to modulating the concentration of amyloid-P (AP) and/or microglial activation in the brain of a subject.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted in WIPO ST.26 .xml format via EFS-Web and is hereby incorporated by reference in its entirety. The .xml copy is named 047563_780878 (020008). xml and is 22 KB in size.

BACKGROUND OF THE INVENTION

[0003] Microglia are the resident macrophages of the central nervous system (CNS) acting as the first line of defense in the brain by phagocytosing harmful pathogens and cellular debris. Microglia emerge from early erythromyeloid progenitors of the yolk sac and enter the developing brain before the establishment of a fully mature blood-brain barrier. In physiological conditions, during brain development, microglia contribute to CNS homeostasis by supporting cell proliferation of neural precursors. In post-natal life, such cells contribute to preserving the integrity of neuronal circuits by sculpting synapses. After a CNS injury, microglia change their morphology and down-regulate those genes supporting homeostatic functions. There is solid and sound evidence suggesting that regulating microglia functions during disease pathology might represent a strategy to develop future therapies aimed at counteracting brain degeneration in neurological diseases 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 is an increasing public health problem. It is currently estimated to afflict over 5 million people in the United States, with an expected increase to 13 million by the year 2050. Alzheimer’s disease leads to loss of memory, cognitive function, and ultimately loss of independence. It takes a heavy personal and financial toll on the subject and the family. Because of the severity and increasing prevalence of the disease in the population, it is urgent that better treatments be developed.

[0005] Cerebral amyloid angiopathy (CAA) occurs in about 90% of individuals who develop AD, as well as in some individuals independently of AD. CAA can lead to ischemic and hemorrhagic strokes causing severe disability or death. There are no current treatments for CAA.

[0006] Biochemical, genetic, and animal model evidence implicates amyloid-P ( A|3) as a pathogenic peptide in AD and in most cases of CAA. The neuropathologic and neurochemical hallmarks of AD include synaptic loss and selective neuronal death, a decrease in certain neurotransmitters, and the presence of abnormal proteinaceous deposits within neurons (neurofibrillary tangles) and in the extracellular space (cerebrovascular, diffuse, and neuritic plaques). The characteristic features of CAA include the buildup of fibrillar forms of Ap in penetrating and leptomeningeal arterioles on the surface of the cerebral cortex. CAA can lead to ischemic or hemorrhagic stroke. The main constituent of the plaques seen in AD and CAA is Ap, a 38-43 amino acid sequence peptide cleaved from the amyloid precursor protein (APP).

[0007] Throughout life, soluble Ap is secreted primarily by neurons, but also other cell types. Excessive Ap deposition may result from increased Ap synthesis, e.g. as occurs in familial early- onset AD and in some cases of familial early onset CAA, decreased Ap clearance in the brain, or increased Ap fibrillogenesis. The lack of compelling evidence that Ap over-production occurs in the more common late-onset forms of AD suggests that insufficient Ap clearance may drive Ap deposition and amyloid plaque formation and CAA as well. [0008] Microglia can either assist the clearance of age-related amyloid accumulation or can promote extensive inflammation in reaction to amyloid or tau, eventually causing widespread neurodegeneration. Importantly, this difference might be driven by the temporal stage of the disease. In earlier periods of AD, microglia can be neuroprotective; but as either amyloid or tau pathology evolves, they can become neurotoxic.

SUMMARY OF THE INVENTION

[0009] Among the various aspects of the present disclosure is the provision of methods of treating a microglial dysfunction-associated diseases, disorder, and conditions. Disclosed herein is an anti-human Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) antibody. The antibody may comprise a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 1 (LI), 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 (Hl), 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 region of each variable region may have at least 75% sequence identity with a human framework region sequence. The anti-LILRB4 antibody may further comprises one or more constant regions, or a portion of a constant region. 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, singledomain antibody, single chain variant fragment (scFv), an antibody fragment selected from Fv, Fab, Fab', Fab'-SH, and F(ab')2, divalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFv), diabody, triabody or tetrabody

[0011] Disclosed herein is a pharmaceutical composition comprising the anti-LILRB4 antibody and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may further comprise one or more dispersing agent, buffer, surfactant, preservative, solubilizing agent, isotonicity agent, stabilizing agent, or any combination thereof. The carrier may comprise physiological saline, ion exchanger, alumina, aluminum stearate, lecithin, serum protein, human serum albumin, buffer, phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, wool fat, or any combination thereof.

[0012] Disclosed herein is 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. Disclosed herein are the anti-LILRB4 antibodies, LILRB4-Fc fusion proteins and pharmaceutical compositions thereof, for use as a medicament. Further provided herein are the anti-LILRB4 antibody or the LILRB4-Fc fusion protein for treating a neurological disease, and use of the anti-LILRB4 antibody or the LILRB4-Fc fusion protein in the manufacture of a medicament for treating a neurological disease. The neurological disease may be associated with microglial dysfunction. The neurological disease may be Alzheimer's disease, Parkinson's disease, Nasu-Hakola disease, prion diseases, multiple sclerosis, HIV-dementia, amyotrophic lateral sclerosis (ALS), frontal temporal dementia, neuropathic pain, or an autism spectrum disorder. The disclosed method may include systemic or local administration, the antibody or the LILRB4-Fc fusion protein as provided herein.

[0013] Disclosed herein is a method of decreasing an amyloid plaque load, a 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. Also provided herein are the anti-LILRB4 antibody or the LILRB4-Fc fusion protein for decreasing an amyloid plaque load, a CAA load, or both, and use of the anti-LILRB4 antibody or the LILRB4-Fc fusion protein in the manufacture of a medicament for decreasing an amyloid plaque load, a CAA load, or both. The anti-LILRB4 antibody may be peripherally or locally administered, optionally directly within the central nervous system.

Additionally, disclosed herein is a method of treating at least one A0 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. Also provided herein are the anti-LILRB4 antibody or the LILRB4-Fc fusion protein for treating at least one A0 plaque- associated symptom or at least one CAA-associated symptom, and use of the anti-LILRB4 antibody or the LILRB4-Fc fusion protein in the manufacture of a medicament for treating at least one Ap plaque-associated symptom or at least one CAA-associated symptom. The treatment may comprise preventing, attenuating, reversing, or improving at least one symptom or sign of Ap plaque or at least one CAA associated symptom in the subject. The Ap plaque- associated symptom or the CAA-associated symptom may include neuronal degeneration, impaired cognitive function, altered behavior, abnormal language function, emotional dysregulation, seizures, impaired nervous system structure, impaired nervous system function, an increased risk of development of Alzheimer's disease, and an increased risk of development of cerebral amyloid angiopathy. The anti-LILRB4 antibody or the LILRB4-Fc fusion protein for use in treating at least one Ap plaque-associated symptom or at least one CAA-associated symptom may be systemically or locally administered, optionally directly within the central nervous system.

[0014] Other aspects and iterations of the disclosure are detailed below.

BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] FIG. 1 depicts an illustration of the human inhibitory leukocyte Ig-like receptors and the murine PIRB orthologue.

[0017] FIG. 2A depicts LILRs and APOE expression in the human brain.

[0018] FIG. 2B shows expression of LILRB4 and all other LILRs in a human AD snRNAseq dataset versus control (Ctrl).

[0019] FIG. 3A depicts the expression of LILRB4 (ILT3) in microglia in Seattle Alzheimer’s Disease (SEA-AD) dataset. [0020] FIG. 3B shows that the expression of LILRB4 correlates with Braak scores in SEA- AD dataset.

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

[0022] FIG. 3D shows the increased APOE expression in AD (Zhou et al. 2020) and the expression of APOE correlates with Braak scores in SEA-AD dataset.

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

[0024] FIG. 4A depicts LILRB4 and ApoE expression by immunofluorescence staining.

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

[0026] FIG. 5A is a schematic of the Bacterial Artificial Chromosome (BAC) of the telomeric LILR cluster.

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

[0028] FIG. 6 depicts gating of microglia, representative expression of LILRB4, LILRB1 and LILRA2 in microglia, and its quantification in 4 different genotypes.

[0029] FIG. 7A shows representative immunofluorescence images of microglia clustering and expression of LILRB4 in Telo Tg x 5XFAD mice and 5XFAD mice. Quantitative data is provided in the accompanying graph.

[0030] FIG. 7B is a bar graph showing the measurement of microglia density within 15-pm and 30-pm spherical shell surrounding A[3 plaques conducted by positioning IBA1⁺ PU.1⁺ microglia and methoxy -X04+ plaques. ILT-Telo Tg x 5XFAD mice showed less microglia coverage of A|3 plaques than 5XFAD mice within 15pm spherical shell, suggesting a preferential reduction of “reactive microgliosis” near amyloid deposits in ILT-Telo Tg x 5XFAD mice. [0031] FIG. 7C is a bar graph showing that whole cortex of 5XFAD and ILT-Telo Tg x 5XFAD mice exhibits similar overall densities of microglia. .

[0032] FIG. 8 shows representative immunofluorescence images depicting microglia body volume in Telo Tg x 5XFAD and 5XFAD mice. Measurement of microglia body volumes showed that Telo Tg x 5XFAD mice had smaller microglia body volume to the 5XFAD mice, indicative of reduced activation in microglia.

[0033] FIG. 9A provides images of methoxy-X04 probe-stained brain sections that show that higher amyloid burden was present in cortex and hippocampus of Telo Tg x 5XFAD male mice.

[0034] FIG. 9B provides bar graphs showing significantly increased AJ3 accumulation in the cortex and hippocampal, but not amygdala regions of ILT-Telo Tg x 5XFAD mice.

[0035] FIG. 10 depicts neurite dystrophy in Telo Tg x 5XFAD and 5XFAD mice. Neurite dystrophy was examined by staining for LAMP 1, which accumulates in dystrophic neurites. LAMP⁺ voxels was measured within 15pm and 30pm spherical shells surrounding A|3 plaques. In comparison to control 5XFAD mice, ILT-Telo Tg x 5XFAD mice showed an overall significant increase of LAMP⁺ voxels within 15pm and 30pm spherical shells at 6 months of age.

[0036] FIG. 11 is a schematic depicting the generation of Fc-mutated anti-LILRB4 mAb (ZM3.1) and Ctrl (27D6) mAb. To characterize the function of microglial LILRB4 in AD pathology and to exclude the ADCC effect, recombinant Fc mutated mIgG2a anti-LILRB4 mAb (clone: ZM3.1) and an irrelevant Fc mutated mIgG2a Ctrl mAb (clone: 27D6) were generated using a method illustrated.

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

[0038] FIG. 13 depicts the generation of an LILRB4 (ILT3) reporter cell line for use in characterizing anti-ILT3 mAb.

[0039] FIG. 14 depicts an anti-LILRB4 mAb (also known as anti-ILT3) binding to human LILRB4 ectodomain protein and its activity on LILRB4 expression reporter (2B4-LILRB4). The activity of anti-LILRB4 mAb with 2B4-LILRB4 was corroborated by reading the GFP% by flow.

[0040] FIG. 15 is a schematic of the workflow of an antibody treatment in vivo. ILT-Telo Tg x 5XFAD mice were treated intraperitoneally (i.p.) with anti-LILRB4 mAb or Ctrl mAb (60mg/kg) once a week, starting at 4-months-old. Relevant readouts were taken.

[0041] FIG. 16 illustrates a test for using anti-LILRB4 mAb to measure LILRB4 in plasma and brain and depicts its concentration in . The anti-LILRB4 mAb reached detectable concentrations in both the plasma and brain (~10 ng/mg in the brain and lOOug/ml in the plasma, respectively).

[0042] FIG. 17A depicts a volcano plot of microbial bulk RNA-seq in anti-LILRB4 mAb and Ctrl mAb treated mice. One week after the 8th i.p. injection, microglia were isolated from whole brains, and bulk RNAseq was conducted (n=3 anti-LILRB4 mAb-treated mice, 3 Ctrl mAb- treated mice). In this screen, 81 transcripts were found upregulated and 77 transcripts were found downregulated in response to anti-LILRB4 mAb.

[0043] FIG. 17B depicts GO enrichment analysis in anti-LILRB4 mAb and Ctrl mAb treated mice. Gene sets that contribute to microglial motility and phagocytosis were enriched in anti- LILRB4 mAb-treated mice.

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

[0045] FIG. 18B shows that interferon response (IFN-R) and inflammatory cytokine gene sets (Axl, Ifitm3, Uspl8, Oasla, Ifit3) were suppressed in anti-LILRB4 mAb-treated microglia.

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

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

[0048] FIG. 20A depicts string analysis of genes upregulated in anti-LILRB4 mAb-treated mice. [0049] FIG. 20B provides clusters identified among genes upregulated in anti-LILRB4 mAb treated mice.

[0050] FIG. 21 are the representative immunofluorescence images revealing the expression of microglial markers IBA1 (yellow) and CD74 (pink), and amyloid plaque marker X34 (blue) in the cortex, hippocamps (hippo.), and amygdala (amy.) of 6-month-old ILT-Telo Tg x 5XFAD mice after 8 injections of anti-LILRB4 or Ctrl IgG. Bar = 30pm.

[0051] FIG. 22 shows the quantification of the percent of IBA1⁺ voxels within 15pm around X34⁺ plaques in different regions.

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

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

[0054] FIG. 24B provides bar graphs with quantitation of the methoxy-X04⁺ or 6E10⁺ percentage of area in cortex, hippocampus, and amygdala regions.

[0055] FIG. 25 depicts levels of PBS-soluble and PBS-insoluble guanidine-soluble fractions of hippocampi were assessed for A 1-40 and A 1-42 by ELISA. Hippocampi dissected either from ILT-Telo Tg x 5XFAD mice treated with Ctrl IgG2a mAb.

[0056] FIG. 26 depicts the representative confocal images of B ACE 1-1 ab eled (red) dystrophic neurites around methoxy-X04+ (blue) plaques in the cortex of 6-month-old ILT-Telo Tg mice treated with anti-LILRB4 or Ctrl mAb and quantification of the percent of BACE1⁺ voxels within 15pm around methoxy-X04⁺ plaques.

[0057] FIG. 27 depicts a schematic illustrating the Elevated Plus Maze (EMP) test and provides quantitation of behavioral changes in mice after administration of anti-LILRB4 mAb and Ctrl mAb during EMP treatment. During this test, the total time spent in open arm significantly reduced in anti-LILRB4-treated mice compared with Ctrl group, which indicated that anti- LILRB4 treatment restored fear of open spaces and mitigated risk-taking behavior. [0058] FIG. 28A shows a schematic of the steps used to assess contextual memory in a conditioned fear paradigm.

[0059] FIG. 28B depicts the behavior of anti-LILRB4 mAh- and Ctrl mAb-treated mice during a tone/ shock pairing.

[0060] FIG. 28C depicts the behavior of anti-LILRB4 mAb- and Ctrl mAb-treated mice during a tone/ shock pairing.

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

[0062] FIG. 29 shows binding test of human LILRB4-recombinant protein with recombinant human ApoE2, ApoE3 or ApoE4 using ELISA.

[0063] FIG. 30A depicts the percentages of LILRB4 reporter activation (GFP⁺ cells) in the presence of indicated rhApoE4 or lipidated rhApoE4 (lrhApoE4) concentrations, n = 3 in each condition..

[0064] FIG. 30B depicts the percentages of LILRB4 reporter activation (GFP⁺ cells) in the presence of indicated rhApoE3 or lrhApoE3 concentrations, n = 3 in each condition.

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

[0066] FIG. 32 depicts the quantification of GFP⁺ LILRB4 reporter cells in the presence of anti- LILRB4 or Ctrl mAb and the indicated rhApoE3 or lrhApoE3 concentrations, n = 3 in each condition.

[0067] FIG. 33A depicts a surface and cartoon model illustrating the top 10 predictions of mApoE (depicted in yellow) binding to hLILRB4 (shown in cyan) through in silico docking modeling (left panel). During the prediction analysis, it was consistently observed that all the predicted binding regions of mApoE pointed towards the D1-D2 interdomain site, which is located between the two extracellular Ig-like domains of hLILRB4. Notably, a specific loop region containing the amino acid sequence K134ERAAHP140 (highlighted in red and dashed oval) was found to be consistently engaged with the predicted binding positions of mApoE (as depicted in the right panel).

[0068] FIG. 33B depicts a surface and cartoon model showing the top hit of mApoE (yellow) binding to hLILRB4 (cyan). Key residues T30, P35 and loop region are labeled. Black dashed ovals highlighted the binding interface between mApoE and 11LILRB4 in this predicted complex.

[0069] FIG. 33C depicts a cartoon model showing the locations of different key residues, T30, P35, Y121 and loop, for mApoE binding.

[0070] FIG. 33D depicts the sequence alignment of the LILRB4 common variant (CV) or mutants. In the alignment, the amino acid sequences of CV, introduced non-functional loop mutant, T30A mutant, P35A mutant, and Y121A mutant are shown. The mutated residues are indicated in dark red color to highlight the changes.

[0071] FIG. 33E depicts the purification of recombinant ectodomain of LILRB4 common variant, P35A mutant by size exclusion chromatography, and the gel filtration chromatograms for the LILRB4 common variant and P35A mutant. For each protein, the labeled peak fractions were visualized on an SDS-PAGE gel through Coomassie blue staining (left panel) or through western blot by anti-LILRB4 monoclonal antibody (right panel).

[0072] FIG. 33F depicts the purification of recombinant ectodomain of LILRB4 Y121A mutant, loop mutant, and T30A mutant by size exclusion chromatography, and the gel filtration chromatograms for the LILRB4 Y121A mutant, loop mutant, and T30A mutant. For each protein, the labeled peak fractions were visualized on an SDS-PAGE gel through Coomassie blue staining (left panel) or through western blot by anti-LILRB4 monoclonal antibody (right panel).

[0073] FIG. 33G provides bar graphs with quantitation of the binding of oligomeric or monomeric fractions of LILRB4 to rmApoE by ELISA, n = 4-6 for each protein. DETAILED DESCRIPTION

[0074] Applicants have discovered LILRB4 antagonist compositions to be useful in the treatment of microglial dysfunction-associated diseases, disorder, and conditions. In one aspect, applicants have discovered anti-LILRB4 antibodies and methods of using the anti-LILRB4 antibodies to treat Ap amyloidosis. The method comprises 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 a treatment for subjects with Ap amyloidosis including, but not limited to, subjects diagnosed with a disease characterized by brain Ap plaques, subjects diagnosed with a disease characterized by vascular Ap plaques in the brain, subjects diagnosed with Ap plaque-associated symptoms, subjects diagnosed with CAA- associated symptoms, subjects with clinical signs of Ap amyloidosis that may or may not have Ap plaque associated symptoms and/or CAA associated symptoms, subjects diagnosed with Alzheimer’s disease, and subjects diagnosed with CAA (collectively referred to, herein, as “subjects in need of treatment”). Methods for identifying clinical signs of Ap amyloidosis in asymptomatic patients are known in the art and discussed below.

[0075] Definitions.

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

[0077] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, 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 explicitly contemplated.

[0078] The term “subject” refers to a human, or to a non-human animal susceptible to Ap accumulation. [0079] The terms “treat,” "treating," or "treatment" as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disease/disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether 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 prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented.

[0080] The term “AP” refers to peptides derived from a region in the carboxy terminus of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of Ap that may have toxic effects: Ap peptides are typically 37-43 amino acid sequences long, though they can have truncations and modifications changing their overall size. They can be found in soluble and insoluble compartments, in monomeric, oligomeric and aggregated forms, intracellularly or extracellularly, and may be complexed with other proteins or molecules. The adverse or toxic effects of Ap may be attributable to any or all of the above noted forms, as well as to others not described specifically. For example, two such Ap isoforms include Ap40 and Ap42; with the Ap42 isoform being particularly fibrillogenic or insoluble and associated with disease states.

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

[0082] A “clinical sign of Ap amyloidosis” refers to a measure of Ap deposition known in the art. Clinical signs of Ap amyloidosis may include, but are not limited to, Ap deposition identified by amyloid imaging (e g. PiB PET, fluorbetapir, or other imaging methods known in the art) or by decreased cerebrospinal fluid (CSF) Ap42 or AP42/40 ratio. See, for example, Klunk WE et al. Ann Neurol 55(3) 2004, and Fagan AM et al. Ann Neurol 59(3) 2006, each hereby incorporated by reference in its entirety. Clinical signs of Ap amyloidosis may also include measurements of the metabolism of Ap, in particular measurements of Ap42 metabolism alone or in comparison to measurements of the metabolism of other Ap variants (e.g. AP37, Ap38, Ap39, Ap40, and/or total Ap), as described in U.S. Patent Serial Nos. 14/366,831, 14/523,148 and 14/747,453, each hereby incorporated 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 hereby incorporated by reference in its entirety. Importantly, a subject with clinical signs of Ap amyloidosis may or may not have symptoms associated with Ap deposition. Yet subjects with clinical signs of Ap amyloidosis are at an increased risk of developing a disease associated with Ap amyloidosis.

[0083] An “Ap plaque associated symptom” or a “CAA associated symptom” refers to any symptom caused by or associated with the formation of amyloid plaques or CAA, respectively, being composed of regularly ordered fibrillar aggregates called amyloid fibrils. Exemplary Ap plaque associated symptoms may include, but are not limited to, neuronal degeneration, impaired cognitive function, impaired memory, altered behavior, emotional dysregulation, seizures, impaired nervous system structure or function, and an increased risk of development or worsening of Alzheimer's disease or CAA. Neuronal degeneration may include a change in structure of a neuron (including molecular changes such as intracellular accumulation of toxic proteins, protein aggregates, etc. and macro level changes such as change in shape or length of axons or dendrites, change in myelin sheath composition, loss of myelin sheath, etc.), a change in function of a neuron, a loss of function of a neuron, death of a neuron, or any combination thereof. Impaired cognitive function may include but is not limited to difficulties with memory, attention, concentration, language, abstract thought, creativity, executive function, planning, and organization. Altered behavior may include, but is not limited to, physical or verbal aggression, impulsivity, decreased inhibition, apathy, decreased initiation, changes in personality, abuse of alcohol, tobacco or drugs, and other addiction-related behaviors. Emotional dysregulation may include, but is not limited to, depression, anxiety, mania, irritability, and emotional incontinence. Seizures may include but are not limited to generalized tonic-clonic seizures, complex partial seizures, and non-epileptic, psychogenic seizures. Impaired nervous system structure or function may include, but is not limited to, hydrocephalus, Parkinsonism, sleep disorders, psychosis, impairment of balance and coordination. This may include motor impairments such as monoparesis, hemiparesis, tetraparesis, ataxia, ballismus and tremor. This also may include sensory loss or dysfunction including olfactory, tactile, gustatory, visual and auditory sensation. Furthermore, this may include autonomic nervous system impairments such as bowel and bladder dysfunction, sexual dysfunction, blood pressure and temperature dysregulation. Finally, this may include hormonal impairments attributable to dysfunction of the hypothalamus and pituitary gland such as deficiencies and dysregulation of growth hormone, thyroid stimulating hormone, lutenizing hormone, follicle stimulating hormone, gonadotropin releasing hormone, prolactin, and numerous other hormones and modulators.

[0084] “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, which is found in a gene cluster at chromosomal region 19q 13.4 (for example, the 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 contain two extracellular immunoglobulin domains, a transmembrane domain, and two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The extracellular Ig-like domains of LILRB4 bind to fibronectin. The receptor is expressed on immune cells and transduces a negative signal that inhibits stimulation of an immune response. The receptor can also function in antigen capture and presentation. It is thought to control inflammatory responses and cytotoxicity to help focus the immune response and limit autoreactivity. Multiple transcript variants encoding different isoforms have been found for this gene. The configuration of the locus encoding LILRB4 in humans is quite divergent from that encoding mouse LILRB4.

LILRB4 is encoded in a gene complex (called leukocyte receptor complex-LRC) on chromosome 19 that includes 4 inhibitory recpetors (ILT2-LILRB 1 , ILT4-LILRB2, ILT5- LILRB3, ILT3-LILRB4); the mouse LRC complex, found on mouse chromosome 7, includes only one inhibitory receptor homologous to inhibitory JET, called Pirb; mouse LILRB4 is encoded outside the LRC complex on mouse chromosome 10 and therefore is a distant paralogue of human LILRB4 rather than a direct orthologue of human LILRB4 (FIG. 1). Unless expressly stated otherwise, “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 leucocyte Ig-Like Receptor B4.

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

[0086] In addition to antibodies described herein, it may be possible to design an antibody mimetic or an aptamer using methods known in the art that functions substantially the same as an antibody of the disclosure. An “antibody mimetic” refers to a polypeptide or a protein that can specifically bind to an antigen but is not structurally related to an antibody. Antibody mimetics have a mass of about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies. Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Aptamers interact with and bind to their targets through structural recognition, a process similar to that of an antigen-antibody reaction. Aptamers have a lower molecular weight than antibodies, typically about 8-25 kDa. [0087] The terms “full length antibody” and “intact antibody” may be used interchangeably, and refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein. The basic structural unit of a native antibody comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). Light chains are classified as gamma, mu, alpha, and lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. The amino-terminal portion of each light and heavy chain includes a variable region of about 100 to 110 or more amino acid sequences primarily responsible for antigen recognition (VL and VH, respectively). 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 acid sequences, with the heavy chain also including a "D" region of about 10 more amino acid sequences. Intact antibodies are properly cross-linked via disulfide bonds, as is known in the art.

[0088] The variable domains of the heavy chain and light chain of an antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e g., Kindt et al. Kuby Immunology, 6^th 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 a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., 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 generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence: FR1-HVR1-FR2-HVR2-FR3-HVR3-FR4. The FR domains of a heavy chain and a light chain may differ, as is known in the art. [0090] The term “hypervariable region” or “HVR” as used herein refers to each of the regions of a variable domain which are hypervariable in sequence (also commonly referred to as “complementarity determining regions” or “CDR”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs: three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). As used herein, “an HVR derived from a variable region” refers to an HVR that has no more than two amino acid substitutions, as compared to the corresponding HVR from the original variable region. Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 27-32 (LI : SEQ ID NO: 1), 50-52 (L2: WAS), 89-97 (L3: SEQ ID NO: 2), 26-33 (Hl : SEQ ID NO: 3), 51-58 (H2: SEQ ID NO: 4), and 97-101 (H3: SEQ ID NO: 5). The HVR (CDR) definitions may vary based on nomenclature, and can be determined by a person of skill in the art. The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The 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, 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 can differ from that of a native Fc region by virtue of one or more amino acid substitution(s) and/or by virtue of a modified glycosylation pattern, as compared to a native Fc region or to the Fc region of a parent polypeptide. In an example, a variant Fc region can have from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein may possess at least about 80% homology, at least about 90% homology, or at least about 95% homology with a native sequence Fc region and/or with an 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 by M. Lo et al. J. Biol. Chem., 292 (2017), pp. 3900-3908, which is incorporated herein by reference in its entirety. [0092] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Nonlimiting examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain forms of antibodies and higher order variants thereof; single-domain antibodies, and multispecific antibodies formed from antibody fragments.

[0093] Single-chain forms of antibodies, and their higher order forms, may include, but are not limited to, single-domain antibodies, single chain variant fragments (scFvs), divalent scFvs (di- scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra-scFvs), diabodies, and triabodies and tetrabodies. ScFv’s are comprised of heavy and light chain variable regions connected by a linker. In most instances, but not all, the linker may be a peptide. A linker peptide is preferably from about 5 to 30 amino acids in length, or from about 10 to 25 amino acids in length.

Typically, the linker allows for stabilization of the variable domains without interfering with the proper folding and creation of an active binding site. In preferred embodiments, a linker peptide is rich in glycine, as well as serine or threonine. ScFvs can be used to facilitate phage display or can be used for flow cytometry, immunohistochemistry, or as targeting domains. Methods of making and using scFvs are known in the art. ScFvs may also be conjugated to a human constant domain (e.g. a heavy constant domain is derived from an IgG domain, such as IgGl, lgG2, lgG3, or lgG4, or a heavy chain constant domain derived from IgA, IgM, or IgE).

Diabodies, triabodies, and tetrabodies and higher order variants are typically created by varying the length of the linker peptide from zero to several amino acids. Alternatively, it is also well known in the art that multivalent binding antibody variants can be generated using selfassembling units linked to the variable domain.

[0094] A “single-domain antibody” refers to an antibody fragment consisting of a single, monomeric variable antibody domain.

[0095] Multispecific antibodies include bi-specific antibodies, tri-specific, or antibodies of four or more specificities. Multispecific antibodies may be created by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. These chains can be covalently linked. [0096] "Monoclonal antibody" refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone. "Monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be produced using hybridoma techniques well known in the art, as well as recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies and other technologies readily known in the art. Furthermore, the monoclonal antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

[0097] A “heavy chain antibody” refers to an antibody that consists of two heavy chains. A heavy chain antibody may be an IgG-like antibody from camels, llamas, alpacas, sharks, etc., or an IgNAR from a cartiliaginous fish.

[0098] A "humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of the non-human antibody eliciting an immune response in humans following administration but retains similar binding specificity and affinity as the starting non-human antibody. A humanized antibody binds to the same or similar epitope as the non-human antibody. The term “humanized antibody” includes an antibody that is composed partially or fully of amino acid sequences derived from a human antibody germline by altering the sequence of an antibody having non-human hypervariable regions (“HVR”). The simplest such alteration may consist simply of substituting the constant region of a human antibody for the murine constant region, thus resulting in a human/murine chimera which may have sufficiently low immunogenicity to be acceptable for pharmaceutical use. Preferably, the variable region of the antibody is also humanized by techniques that are by now well known in the art. For example, the framework regions of a variable region can be substituted by the corresponding human framework regions, while retaining one, several, or all six non-human HVRs. Some framework residues can be substituted with corresponding residues from a non-human VL domain or VH domain (e.g., the non-human antibody from which the HVR residues are derived), e.g., to restore or improve specificity or affinity of the humanized antibody. Substantially human framework regions have 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 may also be randomly mutated such that binding activity and affinity for the antigen is maintained or enhanced in the context of fully human germline framework regions or framework regions that are substantially human. As mentioned above, it is sufficient for use in the methods of this discovery to employ an antibody fragment. Further, as used herein, the term "humanized antibody" refers to an antibody comprising a substantially human framework region, at least one HVR from a nonhuman antibody, and in which any constant region present is substantially human. Substantially human constant regions have at least about 90% with a known human constant sequence (i.e. about 90%, about 95%, or about 99% sequence identity). 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, the design of humanized immunoglobulins may be carried out as follows or using similar methods familiar to those with skill in the art (for example, see Almagro, et al. Front. Biosci. 2008, 13(5): 1619-33). A murine antibody variable region is aligned to the most similar human germline sequences (e.g. by using BLAST or similar algorithm). The CDR residues from the murine antibody sequence are grafted into the similar human “acceptor” germline. Subsequently, one or more positions near the CDRs or within the framework (e.g., Vernier positions) may be reverted to the original murine amino acid in order to achieve a humanized antibody with similar binding affinity to the original murine antibody. Typically, several versions of humanized antibodies with different reversion mutations are generated and empirically tested for activity. The humanized antibody variant with properties most similar to the parent murine antibody and the fewest murine framework reversions is selected as the final humanized antibody candidate.

[0100] One aspect of the present disclosure encompasses an LILRB4 (ILT3) antagonist agent capable of mitigating one or more of the pathologies associated with a microglial-associated neurological disease. The term "LILRB4 antagonist", refers to a molecule that inhibits LILRB4 (ILT3) activity; inhibits LILRB4(ILT3) signaling in cells that express LILRB4(ILT3); inhibits binding of LILRB4(ILT3) to LILRB4(ILT3) ligands such as fibronectin; inhibits binding of LILRB4 (ILT3) to Ciliary Neurotrophic Factor Receptor; inhibits binding of LILRB4(ILT3) to ApoE; inhibits binding of LILRB4(ILT3) to CD 166; inhibits LILRB4(ILT3) -induced suppression of myeloid cells; inhibits LILRB4(ILT3) -induced suppression of myeloid cell activity; restores FcR activation in myeloid cells that express LILRB4(ILT3); and restores chemokine production in myeloid cells that express LILRB4(ILT3). In some embodiments, the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity in the absence of treatment with the LILRB4-binding agent. In some embodiments, the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity prior to treatment with the LILRB4-binding agent.

1. Anti-LILRB4 antibody

[0101] Provided herein is an anti-LILRB4 antibody. Anti-LILRB4 antibodies disclosed herein can be described or specified in terms of the epitope(s) that 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 comprise any number of epitopes, depending on the source of the protein, 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 both instances can include non-polypeptide elements, e.g., an epitope can include a carbohydrate or lipid side chain. The term “affinity” refers to a measure of the strength of the binding of an individual epitope with an antibody’s antigen binding site.

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

[0103] Anti-LILRB4 antibodies disclosed herein can also be described or specified in terms of their cross-reactivity. The term “cross-reactivity” refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original. For example, certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least about 85%, at least about 90%, or at least about 95% identity (as calculated using methods known in the art) to a reference epitope. An antibody can be said to have little or no cross-reactivity if it does not bind epitopes with less than about 95%, less than about 90%, or less than about 85% identity to a reference epitope. An antibody can be deemed “highly specific” for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.

[0104] Another aspect of isolated, anti-LILRB4 antibodies of this disclosure is that they may or may not have a variant Fc region. For example, an Fc region can be modified to have increased or decreased affinity for an Fc receptor on a microglial cell and/or an altered glycosylation pattern.

[0105] Other aspects of anti-LILRB4 antibodies of this disclosure are described more thoroughly below.

[0106] The anti-LILRB4 antibody may comprise one or more of an LI comprising the amino acid sequence set forth in SEQ ID NO: 1, an L2 comprising the amino acid sequenceWAS, and an 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 as 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] The anti-LILRB4 antibody may comprise one or more of an Hl comprising the amino acid sequence set forth in SEQ ID NO: 3, an H2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and an H3 comprising the amino acid sequence set forth in SEQ ID NO: 5. The 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. The 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. The anti-LILRB4 antibody may comprise any combination of any 2, 3, 4, or 5 of SEQ ID NOs: 1-5 and L2 amino acid sequence WAS. The anti-LILRB4 antibody may comprise all of SEQ ID NOs: 1-5 and L2 amino acid sequence WAS.

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

Table 1 :

[0109] The anti-LILRB4 may comprise a light chain variable region comprising one or more, or all of, an LI comprising the sequence set forth in SEQ ID NO: 15, an L2 comprising the sequence set forth in SEQ ID NO: 16, and an 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, an Hl comprising the sequence set forth in SEQ ID NO: 18 or 21, an H2 comprising the sequence set forth in SEQ ID NO: 19 or 22, and an H3 comprising the sequence set forth in SEQ ID NO: 20.

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

[0111] The anti-LILRB4 antibody or antigen binding fragment thereof may comprise LI, L2, and L3 domains of the light chain variable region having the sequence set forth in SEQ ID NO: 6, and Hl, H2, and H3 domains of the light chain variable region having the sequence set forth in SEQ ID NO: 7. [0112] Additional modifications of the antibody sequence are envisaged and may comprise substitutions of certain amino acid residues containing exposed side-chains to other amino acid residues in order to provide for greater chemical stability of the final antibody, which may avoid deamidation or isomerization. The deamidation of asparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect). Isomerization can occur at DG, DS, DA or DT sequences. Thus, the antibodies of the present disclosure may be engineered not to contain deamidation or asparagine isomerism sites. In addition, any methionine residues (typically solvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe or other amino acids in order to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigen-binding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Further, in order to prevent or minimize potential scissile Asn-Pro peptide bonds, it may be desirable to alter any Asn-Pro combinations found in a CDR to Gin-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions may be designed and subsequently screened to ensure that the substitutions do not decrease the affinity or specificity of the anti-LILRB4 antibody, or change other desired biological activity to unacceptable levels. Additional description of suitable antibody framework modifications for in vivo use can be found at least in U.S. Patent No. 11479608 and U. S. Patent No. 9709568, which are incorporated herein by reference in their entirety for all purposes.

[0113] Also envisaged are the anti-LILRB4 antibody in different antibody forms, for example Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain forms of antibodies and higher order variants thereof; and multispecific antibodies formed from antibody fragments. Method for generating these antibodies comprising one or more of the L1-L3 and H1-H3 disclosed herein are well known in the art and can be determined by one of skill in the art. One or more of the anti- LILRB4 antibody, the VH or the VL may be expressed with a leader sequence, or a targeting moiety, or both. Suitable leader sequences are well known in the art, for example, the amino acid sequences as set forth in SEQ ID NO: 8 and SEQ ID NO: 9. The targeting moiety may comprise an amino acid sequence that is able to direct the antibody to which it is attached (e.g., the anti- LILRB4, or a fragment thereof) to a target site. [0114] The present disclosure also encompasses a polynucleotide encoding the anti-LILRB4 antibody, which can readily be determined by one of skill in the art. The polynucleotide may be an RNA molecule or a DNA molecule and comprise a nucleic acid sequence encoding one or more of the amino acid sequences set forth in SEQ ID NOs: 1-7 and L2 amino acid sequence WAS. The polynucleotide sequence may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express the anti-LZLRB4 antibody. The polynucleotide sequence may comprise one or more modifications for delivery into a subject. The polynucleotide sequence encoding the VL of the anti-LILRB4 antibody may comprise the nucleic acid sequence set forth in SEQ ID NO: 10, or SEQ ID NO: 12 or a sequence at least 80% identical thereto. The polynucleotide sequence encoding the VH of the anti-LILRB4 antibody may comprise the nucleic acid sequence as set forth in SEQ ID NO: 11, or SEQ ID NO: 13 or a sequence at least 80% identical thereto.

[0115] Also envisaged are vectors encoding the anti-LILRB4 antibody. The vector encoding the anti-LILRB4 antibody, or a fragment 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 cell may be any mammalian cell, a bacterial cell, a B-cell, a hybridoma or a cell line. The host cell comprising one or more of the nucleic acid sequences provided herein may encode the antibody comprising one or more of the amino acid sequences as set forth in SEQ ID NOs: 1-5 and L2 amino acid sequence WAS . The host cell may comprise the nucleic acid sequence encoding the amino acid sequence as set forth in SEQ ID NO: 6, or a sequence at least about 90% identical thereto. The host cell may comprise the nucleic acid sequence encoding the amino acid sequence as set forth in SEQ ID NO: 7, or a sequence at least about 90% identical thereto. The host cell may comprise one or more of the nucleic acid sequences as set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or the sequence at least about 80% identical thereto. [0117] Each of the antibodies described above may also contain a variant Fc region, including but not limited to a variant Fc region that is modified to alter the natural interaction with the microglia FcR.

[0118] The anti-LILRB4 antibody may competitively inhibits binding of a reference antibody to its epitope. The antibody is said to competitively inhibit binding of a reference antibody to a given epitope if the antibody preferentially binds to that epitope to the extent that it blocks binding of the reference antibody to the epitope by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays.

2. LILRB4 Decoy Receptor

[0119] In an aspect, the present disclosure provides a LILRB4 polypeptide or a fragment thereof. The mature LILRB4 polypeptide comprises one or more of the following domains; two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Accordingly, the fragment thereof may be an extracellular-domain, transmembrane-domain, or cytoplasmic-domain fragment. In an embodiment, the fragment thereof is an extracellular-domain fragment. The LILRB4 polypeptide may comprise the amino acid sequence as 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 appreciated 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 be found in other species by methods known in the art. In determining whether LILRB4 has significant homology or shares a certain percentage of sequence identity with a sequence of the invention, sequence similarity may be determined by conventional algorithms, which typically allow introduction of a small number of gaps in order to achieve the best fit. In particular, “percent identity” of two polypeptides 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). BLAST nucleotide searches may be performed with the BLASTN program to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. Equally, BLAST protein searches may be performed with the BLASTX program to obtain amino acid sequences that are homologous to a polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) are employed. See www.ncbi.nlm.nih.gov for more details.

[0121] The 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, the homolog, variant or derivative of LILRB4 may be at least 80%, 85%, 90%, or 95% homologous to human LILRB4 extracellular-domain fragment.

[0122] In an aspect, the present disclosure provides a LILRB4-fusion protein. The LILRB4- fusion protein according to the disclosure can be fused to a “targeting moiety,” which refers to a polypeptide that is able to direct the entity to which it is attached (e.g., LILRB4 or a fragment thereof) to a target site. Target sites may include, but are not limited to, the cell surface and a cell-surface protein. The targeting moiety may comprise a binding domain derived from a target receptor ligand. A target receptor ligand is a ligand that binds a target receptor. Suitable target receptors include cell-surface receptors found on microglia cells. Non-limiting examples of suitable target receptors include the Fc receptors: FcRy, FcRoc, FcRs, and FcRp. FcRy belongs to the immunoglobulin superfamily and includes several members, FcRyl (CD64), FcRyllA (CD32), FcRyllB (CD32), FcRylllA (CD16a), and FcRylUB (CD16b). In a specific embodiment, the target receptor is FcRyl (CD64). Fc receptors are cell-surface receptors that recognize the Fc region of an antibody. Non-limiting examples of target receptor ligands for an Fc receptor are IgG, IgA, IgE and IgM Fc regions. In a specific embodiment, the target receptor ligand is an IgG Fc region. In another embodiment, a targeting moiety may comprise an antibody capable of specifically binding to an antigenic determinant on a 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 attached to a target receptor on the surface of cell that is capable of expressing LILRB4. The cell that is capable of expressing LILRB4 may be a microglia cell. The targeting moiety may be capable of directing the entity to which it is attached to a target receptor on the surface of a microglia cell.

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

3. Treatment Methods

[0125] Provided is a process of treating a neurological disease, disorder, or condition. In some embodiments, the neurological disease, disorder, or condition is associated with microglia in a subject. In some embodiments, the methods of treating the neurological disease, disorder, or condition generally comprises administration of a therapeutically effective amount of a LILRB4 antagonist, so as to enhance microglial function, inhibit a neurological disease, disorder, or condition associated with microglial dysfunction, slow the progress of a neurological disease, disorder, or condition associated with microglial dysfunction, or limit the development of a neurological disease, disorder, or condition associated with microglial dysfunction.

[0126] The term “microglial cell” or “microglia”, as used herein, refers to a class of glial cells involved in the mediation of an immune response within the central nervous system by acting as macrophages. Microglial cells are capable of producing exosomes, and further include different forms of microglial cells, including amoeboid microglial cells, ramified microglial cells and reactive microglial cells. Microglial cells include reactive microglia, which are defined as quiescent ramified microglia that transform into a reactive, macrophage-like state and accumulate at sites of brain injury and inflammation to assist in tissue repair and neural regeneration.

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

[0128] The present disclosure provides a method of treating Ap amyloidosis, the method comprising administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to a subject in need thereof. The present disclosure also provides a method of treating a subject diagnosed with a disease characterized by brain Ap plaques, the method comprising administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to the subject. The present disclosure also provides a method of treating a subject diagnosed with a disease characterized by vascular Ap plaques in the brain, the method comprising administering a therapeutically effective amount of the anti- LILRB4 antibody or LILRB4-Fc fusion protein to the subject. The present disclosure also provides a method of preventing the progression of a disease characterized by Ap plaques in the brain, the method comprising administering a therapeutically effective amount of the anti- LILRB4 antibody or LILRB4-Fc fusion protein to a subject in need thereof. The present disclosure also provides a method of treating a subject diagnosed with Alzheimer’s disease, the method comprising administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to the subject. The present disclosure also provides a method of treating a subject diagnosed with CAA, the method comprising administering a therapeutically effective amount of the anti-LILRB4 antibody or 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. humanized).

[0129] In one embodiment, the disclosure provides a method of preventing the progression, or slowing the rate of progression, of a disease characterized by A plaques in the brain. The method comprises administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to a subject in need thereof. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion protein include those disclosed herein. Progression of a disease characterized by Ap plaques in the brain can be evaluated by methods known in the art and described herein, including a worsening of a clinical sign of Ap amyloidosis, an Ap plaque associated symptom, or a CAA associated symptom. In exemplary embodiments, the clinical sign is amyloid plaque load.

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

[0131] In another embodiment, the disclosure provides a method for improving a clinical sign of Ap amyloidosis. The method comprises administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to a subject in need thereof. Suitable anti- LILRB4 antibodies and LILRB4-Fc fusion protein include those disclosed herein. Non-limiting examples of improved clinical signs of Ap amyloidosis may include a decrease in amyloid plaque load, stabilization of amyloid plaque load (i.e. no further increase), an increase in CSF Ap42 concentration, an increase in CSF Ap42/Ap40 ratio, a decreased AP42/AP40 peak time ratio as measured by stable isotope labeling kinetics (e.g. such that is closer to 1), a decreased AP42/AP40 FTR ratio as measured by stable isotope labeling kinetics (e.g. such that is closer to 1), and a change in the ratio of the relative labeling Ap42 to the relative labeling of Ap40 (or another Ap peptide) after stable isotope labeling such that the ratio is closer to 1. In each of the above embodiments, the improvement (i.e. the change) in the clinical sign is at least statistically significant. In certain embodiments, the change may be 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 may be 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 disclosure provides a method for decreasing amyloid plaque load in the brain of a subject. The method comprises administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to a subject. Suitable anti- LILRB4 antibodies and LILRB4-Fc fusion protein include those disclosed herein. A method of the disclosure may decrease the amyloid plaque load in the hippocampus of a subject and/or decrease the amyloid plaque load in the brain cortex of a subject. In each of the above embodiments, the amyloid plaque load may be decreased 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 untreated or negative control treated subjects. In some embodiments, the amyloid plaque load may be decreased 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, the amyloid plaque load may be decreased 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 disclosure provides a method for decreasing CAA load in the brain of a subject. The method comprises administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to a subject with fibrillar forms of A in penetrating and/or leptomeningeal arterioles on the surface of the cerebral cortex. Suitable anti- LILRB4 antibodies and LILRB4-Fc fusion protein include those disclosed herein. A method of the disclosure may decrease CAA load in the penetrating and/or leptomeningeal arterioles on the surface of the cerebral cortex of a subject. In each of the above embodiments, CAA load may be decreased 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 untreated or negative control treated subjects. In some embodiments, the amyloid plaque load may be decreased 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, the amyloid plaque load may be decreased by at least 100, 125, 150, 200, 250, 300, 350, 400, or 450% compared to untreated or negative control treated subjects.

[0134] Another embodiment includes a method of reducing insoluble AP42 levels in the brain of a subject in need thereof. The method comprises administering a therapeutically effective amount of the anti-LILRB4 antibody or LILRB4-Fc fusion protein to the subject. In one example, the method further comprises reducing insoluble AP40 levels in the brain of the subject. In another example, the method comprises selectively reducing insoluble Ap40 levels, reducing insoluble Ap42 levels, or a combination thereof compared to soluble Ap40, Ap42 levels, or a combination thereof in the brain of a subject.

[0135] The level of Ap can be assessed by any suitable method known in the art comprising, e.g., analyzing Ap by one or more techniques chosen from Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix- assisted laser desorption/ionization-time of flight-MS (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 Ap is particularly suited for evaluating amyloid plaque load. Non-limiting examples of in vivo imaging methods include positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI). Suitable imaging agents are also known in the art (e.g. PIB).

[0136] In another embodiment, the disclosure provides a method for improving an Ap plaque associated symptom and/or a CAA associated symptom in a subject. The method comprises administering a therapeutically effective amount of the anti-LILRB4 antibody that specifically binds LILRB4 or LILRB4-Fc fusion protein to a subject with at least one Ap plaque associated symptom and/or at least one CAA associated symptom. Suitable anti-LILRB4 antibodies and LILRB4-Fc fusion protein include those disclosed herein. Non-limiting examples of improved Ap plaque associated symptoms are identified above. In certain embodiments, improved Ap plaque associated symptoms may include reduced neuronal degeneration, impaired cognitive function, altered behavior, emotional dysregulation, and/or seizures. In each of the above embodiments, the improvement (i.e. the change) in the symptom is at least statistically significant. In certain embodiments, the change may be 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 may be 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.

[0137] The anti-LILRB4 antibody disclosed herein can also be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, biological response modifiers, pharmaceutical agents, or PEG. In certain embodiments, therapeutic agent may be a drug, a radioisotope, a lectin, or a toxin. Conjugates that are immunotoxins have been widely described in the art. The toxins can be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. In using radioisotopically conjugated anti-LILRB4 antibodies for immunotherapy, certain isotopes can be chosen depending on such factors as leukocyte distribution as well as stability and emission. Depending on the autoimmune response, some emitters can be used. In general, a and 0 particle emitting radioisotopes are utilized in immunotherapy. Short range, high energy a emitters such as ²¹²Bi may be used. Examples of radioisotopes which can be bound to the anti-LILRB4 antibodies disclosed herein for therapeutic purposes include, but are not limited to ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹1, ⁸⁹Zr, ⁹⁰Y, ⁶⁷CU, ⁶⁴CU, ^{n i}In, ²¹²Bi, ²¹²At, ²¹¹Pb, ⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re. Other therapeutic agents which can be coupled to the anti-LILRB4 antibodies, as well as ex vivo and in vivo therapeutic protocols, are known, or can be easily ascertained, by those of ordinary skill in the art.

[0138] Administration of the anti-LILRB4 antibody, or a composition comprising the anti- LILRB4 antibody, or LILRB4-Fc fusion protein is performed using standard effective techniques, include peripherally (i.e. not by administration into the central nervous system) or locally 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 directly into the central nervous system (CNS) includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation.

[0139] Pharmaceutical compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like are used as appropriate. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners. It may be particularly useful to alter the solubility characteristics of the antibodies useful in this discovery, making them more lipophilic, for example, by encapsulating them in liposomes or by blocking polar groups.

[0140] The concentration of the antibody in formulations to be administered is an effective amount and ranges from as low as about 0.1% by weight to as much as about 15 or about 20% by weight and will be selected primarily based on fluid volumes, viscosities, and so forth, in accordance with the particular mode of administration selected if desired. A typical composition for injection to a subject could be made up to contain 1 mL sterile buffered water of phosphate buffered saline and about 1-1000 mg of any one of or a combination of the antibodies disclosed herein. The formulation could be sterile filtered after making the formulation, or otherwise made microbiologically acceptable. A typical composition for intravenous infusion could have volumes between 1-250 mL of fluid, such as sterile Ringer's solution, and 1-100 mg per ml, or more in anti-LILRB4 antibody concentration. The anti-LILRB4 antibodies or LILRB4-Fc fusion protein disclosed herein can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. Lyophilization and reconstitution may lead to varying degrees of antibody activity loss (e.g. with conventional immune globulins, IgM antibodies tend to have greater activity loss than IgG antibodies). Dosages administered are effective dosages and may have to be adjusted to compensate. The pH of the formulations will be selected to balance antibody stability (chemical and physical) and comfort to the subject when administered. Generally, a pH between 4 and 8 is tolerated. Doses will vary from individual to individual based on size, weight, and other physiobiological characteristics of the individual receiving the successful administration. [0141] As used herein, the term "therapeutically effective amount" means an amount of a substance (e.g. the anti-LILRB4 antibody or LILRB4-Fc fusion protein) that leads to measurable and beneficial effects for the subject administered the substance, i.e., significant efficacy. The therapeutically effective amount or dose of compound administered according to this discovery will be determined using standard clinical techniques and may be influenced by the circumstances surrounding the case, including the antibody administered, the route of administration, and the status of the symptoms being treated, among other considerations. A typical dose may contain from about 0.01 mg/kg to about 100 mg/kg of the anti-LILRB4 antibody or LILRB4-Fc fusion protein described herein. Doses can 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. The frequency of dosing may be daily or once, twice, three times or more per week or per month, as needed as to effectively treat the symptoms.

[0142] The timing of administration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments.

[0143] Suitable adaptations, other effective techniques for administration, such as intraventricular administration, transdermal administration and oral administration may be employed provided proper formulation is utilized herein. In addition, a person skilled in the art can use a polynucleotide of the disclosure encoding any one of the above-described antibodies instead of the proteinaceous material itself. The polynucleotide of the disclosure may be modified or suitably formulated for delivery.

[0144] In addition, it may be desirable to employ controlled release formulations using biodegradable films and matrices, or osmotic mini-pumps, or delivery systems based on dextran beads, alginate, or collagen.

4. Pharmaceutical Compositions

[0145] The present disclosure encompasses pharmaceutical compositions comprising the anti-

LILRB4 antibody disclosed herein, to facilitate administration and promote stability of the active agent. For example, the anti-I.n.RB4 antibody of this disclosure may be admixed with at least one pharmaceutically acceptable carrier or excipient resulting in a pharmaceutical composition which is capably and effectively administered (given) to a subject, such as to a suitable subject (i.e. “a subject in need of treatment” or “a subject in need thereof’). Methods of preparing and administering the anti-LILRB4 antibodies or LILRB4-Fc fusion protein disclosed herein to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of the anti-LILRB4 antibody or LILRB4-Fc fusion protein can be, for example, peripheral, oral, parenteral, by inhalation or topical.

[0146] Pharmaceutical compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible carriers, dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like 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, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, poly acrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, wool fat or a combination 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 and the like. In many cases, isotonic agents can be included, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride 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 and gelatin. [0150] Compositions disclosed herein can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use.

[0151] The anti-LILRB4 antibodies or LILRB4-Fc fusion protein may be formulated for parenteral administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are 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, 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 can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, e.g., once a day, or on an “as needed” basis.

[0152] Certain pharmaceutical compositions, as disclosed herein, can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

[0153] The amount of the anti-LILRB4 antibody or LZLRB4-Fc fusion protein to be combined with the carrier materials to produce a single dosage form may vary depending upon the host treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0154] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure, therefore all matter set forth or shown in the accompanying examples and drawings is to be interpreted as illustrative and not in a limiting sense.

Example 1

Generation of ILT-Telo Tg mice (transgenic mice expressing the telomeric region of the human LILR complex

[0155] ILT-Telo Tg mice were generated following 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 BACPAC Resources Center (BPRC). The entire BAC was injected into the nucleus of fertilized eggs from C57BL/6 x CBA/J (CBA) mice, and TELO founders were obtained. Offspring from founders were screened for expression of the ILTs encoded by the BAC transgene in various tissues. The line selected for further work was subsequently backcrossed onto the C57BL/6 background for at least 8 generations to make sure the genetic background is similar to 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 bred and housed in specific pathogen-free conditions. The Institutional Animal Care and Use Committee at Washington University in St. Louis approved all protocols used in this study.

Example 2

Generation of LILRB4 reporter cell line

[0156] Generation of the 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 in the retroviral vector pMX. Retroviral particles were assembled in the Phoenix- Amphotropic cell line PHA (from ATCC). Supernatants containing the packaged virus were ricovered and incubated with the 2B4 cell line. This line (provided by Dr. Saito, Riken, Japan) expresses GFP under a promoter sequence that binds the transcription factor NF AT. Expression of LILRB4 was tested 96 hours after transduction. LILRB4-expressing cells were FACS sorted for multiple rounds until 20% of the cells with the highest LILRB4 expression were

FACS purified, amplified and frozen in stock batches.

[0157] Activation of the anti-LILRB4 reporter. Activation was assessed by surveying GFP expression upon plating anti-LILRB4 reporter cells on plastic bound anti-LILRB4 antibody, or irrelevant mouse IgG2a control. Anti-LILRB4 mAb engages and activates anti-LILRB4 reporter cells, but not the control mAb.

Example 3

Anti-LILRB4 mAb test in ILT-Telo Tg x 5XFAD model a. In vivo antibody treatment

[0158] 4-month-old ILT-Telo Tg x 5XFAD male mice were injected intraperitoneally (i.p.) with Fc-mutated anti-LILRB4 antibody (60mg/kg) or Fc-mutated control antibody (60mg/kg) every week. After 8 consecutive injections, animals were deeply anesthetized with ketamine/xylazine (with the ratio of 10: 1), perfused with 0.1% heparin (1 : 1000 dilution in phosphate-buffered saline). Brains were then removed and were immersed in 4% paraformaldehyde (PF A) for 24 hours, followed by soaking them in 30% sucrose in phosphate-buffered saline for 24 hours prior to embedding in a 2: 1 mixture of 30% sucrose and optimal cutting temperature compound. Sections at 40-pm thickness were prepared for the immunofluorescence staining. b. Immunofluorescence

[0159] Floating mouse brain sections were blocked with 3%BSA and 0.25% Triton X-100 in PBS, and stained with anti-IBAl (rabbit monoclonal, 1 :500, Cell Signaling Technology), anti- CD74 (Alexa Fluor® 647-labeled rat IgG2b, 1 :200, Biolegend), and anti-6E10 (Alexa Fluor® 488-labeled anti A|31— 16, 1 : 1000, BioLegend) overnight at 4 °C, followed by staining with antirabbit IgG Alexa Fluor 555 (1 :2,000; Abeam), anti-rat IgG Alexa Fluor 647 (chicken polyclonal, 1 :2,000; Invitrogen) and Methoxy-X04 (3 pg/ml; Tocris) for 1 h at room temperature. All antibodies were used in blocking buffer, and between all incubations, sections were washed for 10 min in PBS 3 times. Images were collected using a Nikon AlRsi confocal microscope with a 20* 0.95-NA objective. z-Stacks with 2-pm steps in the z direction, 1,024 x 1,024-pixel resolution, were recorded. Three-dimensional reconstruction of microglia and A plaques, and extraction of parameters were performed in Imaris (version 9.2.0), and further processing was performed using automated scripts in Matlab.

Example 4

Determination of LILRB4 expression in human AD and its correlation with APOE expression and AD progression

[0160] Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor of the leukocyte immunoglobulin-like receptor (LILRs) family mainly expressed on human cells of myeloid origin. Currently, the expression and functions of LILRB4 in Alzheimer's disease is unknown.

[0161] In this study, LILRB4 expression was examined in human AD snRNAseq dataset (Zhou, et al, Nat Med. 2020: 11 AD, 11 Ctrl). The UMAP showed that the LILRB4 gene was selectively expressed by microglia in human brain (FIG. 2A), and the LILRB4 was the highest expressed gene in AD among all the leukocyte immunoglobulin-like receptors (LILRs) (FIG. 2B).

[0162] Next, the human microglia snRNAseq data from the Seattle Alzheimer’s Disease dataset (SEA-AD, 84 patients: 2 Braak 0; 10 Braak II&III; 23 Braak IV; 34 Braak V, 15 Braak VI) was re-analyzed. The UMAP showed LILIB4 expression in microglia (FIG. 3A). Further, the patients with higher Braak stages were observed to have more microglia LILRB4 expression, compared with Braak 0 patients (FIG. 3B). Moreover, analysis of the SEA-AD dataset also showed a positive correlation of microglial LILRB4 expression with phospho-Tau (pTau) content in AD brain lysates (FIG. 3C). The ApoE expression was also examined in the SEA-AD snRNA-seq datasets. Microglial APOE was more expressed in AD than controls and reflected the severity of disease (FIG. 3D). Notably, LILRB4 expression showed a strong correlation with APOE, indicating that its upregulation occurs during microglia activation in response to AD pathologies (FIG. 3E).

[0163] Microglia LILRB4 expression was then validated in AD and Ctrl brain sections by immunofluorescence (n=4 AD, 4 Ctrl). Representative confocal images stained with IBA1 (green), LILRB4 (red), ApoE (white) and Methoxy-X04 (blue) (FIG. 4A), show highly up- regulated LILRB4 and APOE expression in Ap plaque-associated microglia in AD patients. The expression of LILBR4 and APOE positively correlated-patients with higher LILRB4 expression showed elevated APOE expression (FIG. 4B).

Example 5

Characterization of Ap pathology in ILT-Telo Tg x 5XFAD mice

[0164] ILT-TELO-BAC transgenic mice (Telo) was generated and crossed with the 5XFAD model of AD as shown in FIG. 5A and FIG. 5B. Four genotypes were generated for analysis: Wild type; ILT-Telo Tg; 5XFAD; and ILT-Telo Tg x 5XFAD. The LILRB4, LILRB1 and LILRA2 expression were corroborated in ILT-Telo Tg microglia by FACS (FIG. 6) which indicated that the LILRB4 expression was significantly upregulated in the 6-month-old ILT-Telo Tg x 5XFAD male mice, compared with the gender and age-matched ILT-Telo Tg mice (FIG. 6).

[0165] Further, immunofluorescence analysis of microglial LILRB4 expression was performed by co-staining of microglial marker IB Al (red), LILRB4 (green), ApoE (white), and A plaque marker Methoxy -X04 (blue) (FIG. 7A). Quantification of microglia LILRB4 expression throughout the cortex of ILT-Telo Tg x 5XFAD mice and control 5XFAD mice revealed highly specific and elevated expression of LILRB4 in Ap plaque-associated microglia in the ILT-Telo Tg x 5XFAD mice (6-month-old, males) (FIG. 7A). Measurement of microglia density within 15pm and 30pm spherical shells surrounding Ap plaques was conducted by positioning IBA1⁺ PU.1⁺ microglia and Methoxy -X04⁺ plaques. ILT-Telo Tg x 5XFAD mice showed less microglia coverage of Ap plaques than 5XFAD mice within 15pm spherical shell, suggesting a preferential reduction of “reactive microgliosis” near amyloid deposits in ILT-Telo Tg x 5XFAD mice (FIG. 7B). Microglia exhibited similar overall densities in the whole cortex of 5XFAD and ILT-Telo Tg x 5XFAD mice (FIG. 7C).

[0166] In amyloid pathology, “reactive microgliosis” is characterized by microglia proliferation with a hypertrophic or ameboid shape surrounding Ap plaques. Three-dimensional reconstruction of IB Al (red), LILRB4 (cyan), ApoE (green), and Ap plaque (blue) immunofluorescence demonstrated that plaque-associated microglia in ILT-Telo Tg x 5XFAD mice show smaller cell body size than those in the 5xFAD mice, as quantified by microglial soma and total volume of processes (FIG. 8).

[0167] To investigate whether the defect of microglia activity in ILT-Telo Tg x 5XFAD mice would impact the A pathology, the deposition of Ap aggregates was examined by staining of matched coronal brain sections from ILT-Telo Tg x 5XFAD mice and control 5XFAD mice at 6 months of age with 6E10. Significantly increased A accumulation was observed in the cortex, hippocampal and amygdala regions of ILT-Telo Tg x 5XFAD mice (FIG. 9A and FIG. 9B).

[0168] Because microglia barrier promotes amyloid compaction and insulation and prevents neurite dystrophy, the neurite dystrophy was examined by staining for LAMP1, which accumulates in dystrophic neurites. Further, LAMP⁺ voxels was measured within 15-pm and 30- pm spherical shells surrounding Ap plaques (FIG. 10). In comparison to control 5XFAD mice, ILT-Telo Tg x 5XFAD mice showed an overall significant increase of LAMP⁺ voxels within 15- pm and 30-pm spherical shell at 6 months of age (FIG. 10).

[0169] In summary, the results indicated that LILRB4 was expressed in microglia of ILT-Telo Tg mice and that expression increased with amyloid pathology, with A 3 plaque associated microglia showing higher expression of LILRB4 in in 6-month-old ILT-Telo Tg x 5XFAD mice. Further, Telo transgenic mice showed defective microglial activity, enlargement of Ap aggregates, as well as extensive neurite dystrophy.

Example 6

Generation of anti-LILRB4 antibody and in vivo testing ILT Telo Tg x 5xFAD mice

[0170] The binding of Fc to Fc receptor has been found to induce cytotoxic potential through antibody dependent cell-mediated cytotoxicity (ADCC) pathways. To characterize the function of microglial LILRB4 in AD pathology and to exclude the ADCC effect, recombinant Fc mutated mIgG2a anti-LILRB4 mAb (clone: ZM3.1) and an irrelevant Fc mutated mIgG2a control mAb (clone: 27D6) were generated using a method illustrated in FIG. 11. Purification of Fc-mutated anti-LILRB4 mAb (ZM3.1) and Ctrl (27D6) mAb was further performed (FIG. 12). A calcium-driven reporter cell line was then generated (FIG. 13), as described in Example 2, to characterize anti-LILRB4 mAb. The 2B4-LILRB4 reporter is stably transfected with a chimeric gene consisting of the extracellular portion of LILRB4 and the intracellular domain of CD3^. Engagement of hLILRB4 promotes Ca2⁺ signals that leads to nuclear translocation of NF AT and NFAT-driven synthesis of enhanced GFP (EGFP). The specificity of anti-LILRB4 was first demonstrated by binding to the recombinant ectodomain of LILRB4 by ELISA (FIG. 14). To determine the activity of anti-LILRB4 mAb, the anti-LILRB4 or Ctrl mAb was immobilized on a plate at different concentrations, to which the 2B4-LILRB4 reporter cell was added overnight to the plate. The anti-LILRB4 mAb induced EGFP in 2B4-LILRB4 reporter cell in a dosedependent fashion, suggesting that the immobilized anti-LILRB4 mAb specifically binds hLILRB4 (FIG. 14).

[0171] Anti-LILRB4 mAb and control mAb was 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 (i.p.) with anti-LILRB4 mAb or CTRL mAb (60mg/kg) once a week, starting at 4-month-old (FIG. 15). Mouse behavior was evaluated after six consecutive injections, and the mAb treatment was concluded after two additional doses. After eight consecutive injections, plasma and brain homogenate samples were collected for measurement of antibody levels. The plasma and brain samples from the male mice were tested as illustrated in FIG. 16. The anti- LILRB4 mAb reached detectable concentrations in both the plasma and brain (~10ng/mg in the brain and lOOug/ml in the plasma, respectively) (FIG. 16). Microglia bulk RNA-seq, A burden and microglia activity evaluations were further conducted in mAb treated male mice. a. Microglia bulk RNA-seq

[0172] To characterize the impact of anti-LILRB4 mAb on microglial activity in vivo, microglial transcriptome analysis was performed among the anti-LILRB4 mAb and CTRL mAb treated groups. One week after the 8th i.p. injection, microglia were isolated from whole brains, and bulk RNAseq was conducted (n=3 anti-LILRB4 mAb-treated mice, 3 CTRL mAb-treated mice). In this screen, 81 transcripts were found upregulated and 77 transcripts were found downregulated in response to anti-LILRB4 mAb (FIG. 17A). Among these differentially expressed genes (DEGs) (Padj < 0.05, llog2FCl>0.5) (FIG. 17B), gene sets that contribute to microglial motility and phagocytosis were enriched in anti-LILRB4 mAb-treated mice (FIG. 17B, FIG. 18A). Conversely, interferon response (IFN-R) and inflammatory cytokine gene sets (Axl, IfitrnS. Uspl8, Oasla, were suppressed in anti-LILRB4 mAb-treated microglia (FIG. 18B). Additionally, string analysis was conducted to identify Protein-Protein interaction networks enriched in the down regulated (FIG. 19A) and the upregulated (FIG. 20A) gene sets. Significant protein interaction clusters were observed in both down regulated (FIG. 19B) and upregulated (FIG. 20B) gene sets. b. A 0 burden and microglia activity evaluations

[0173] The increase of CD74 expression was validated in anti-LILRB4 mAb-treated microglia by co-staining for the microglia marker IBA1 (yellow), the MHC Class Il-related marker CD74 (pink), and Ap plaque marker X34 (blue) (FIG. 21). Immunofluorescence demonstrated the increased microglial body size after the anti-LILRB4 mAb treatment, as quantified by IBA1⁺ voxels with 15pm around X34⁺ plaques in different regions (FiG. 22), and the increased percent of co-localized CD74⁺/IBA1⁺ voxels within 15pm around X34⁺ plaques in different regions (FIG. 23)

[0174] To determine the impact of anti-LILRB4 mAb on total Ap plaque load, matched coronal brain sections were stained with methoxy-X04 and 6E10, and total Ap plaque area in the cortices, hippocampi, and amygdala were measured (FIG. 24A and FIG. 24B). In comparison to Ctrl mAb treatment, administration of anti-LILRB4 mAb induced an overall significant reduction of Ap plaques in the different brain regions (FIG. 24A and FIG. 24B). Levels of amyloid-P40 (AP42) and amyloid-P42 (AP42) were also quantified in the soluble and insoluble fractions of cortex by ELISA. Flash-frozen cortices tissue was homogenized sequentially in PBS and guanidine solutions to obtain PBS-soluble and -insoluble fractions. While levels of soluble AP40 and insoluble AP40 were similar, a significant decrease in soluble and insoluble AP42 in the cortex regions of anti-LILRB4 mAb-treated mice was detected compared to CTRL mice (FIG. 25). Ap42 is neurotoxic and can induce tau phosphorylation and microtubule instability. Encapsulation of plaques by microglia was inversely correlated with AP42 ‘hot-spots’ on plaques. The neurite damage was then evaluated by BACE1 staining, which accumulates in swollen presynaptic dystrophic neurites in close proximity to amyloid plaques. The cortical area exhibiting BACE1 staining and its volume within 15pm spherical shells surrounding A0 plaques were significantly reduced in mice treated with anti-LILRB4 versus Ctrl IgG2 (FIG. 26), indicating reduced neuronal dystrophy. Together, these data suggested that anti-LILRB4 mAh enhanced microglial activation, promoted microglial clearance of A0, and alleviated microglial cytotoxic mediators release (IFN-R and inflammatory cytokines). c. Behavioral testing

[0175] Assessment was further done to understand whether the reduction of A(3 pathology and IFN response induced by anti-LILRB4 mAb treatment were associated with behavioral changes. Elevated Plus Maze (EPM) was first conducted to assess mice risk-taking behavior and anxietylike traits. EPM was designed to assess risk-taking behavior, exploratory drive, and anxiety -like traits by giving mice free access to a maze that contains both open unprotected arms and enclosed protected arms, all elevated ~lm from the floor, as illustrated in FIG. 27. During this test, the total time spent in open arm significantly reduced in anti-LILRB4-treated mice compared with control group (FIG. 27), which indicated that anti-LILRB4 treatment restored fear of open spaces and mitigated risk-taking behavior. Moreover, contextual memory was as assessed in a conditioned fear paradigm (FIG. 28A-D). In this test, there was no significant difference on day 1 baseline freezing behavior and freezing during the tone/ shock presentation in the two groups (FIG. 28B). Assessment of contextual memory on day 2 also indicated a comparable time spending freezing in the two groups (FIG. 28C). While analysis of freezing behavior during presentation of the tone on day 3 indicated a trend towards a treatment effect in freezing in response to the tone presentation, with post hoc tests showing that anti-LILRB4 mice froze more than Ctrl mice (p=0.0896) (FIG. 28D). This indicated a stronger amygdala dependent memory for the cue in anti-LILRB4 mAb treated mice compared to control treated mice.

[0176] In summary, the results showed anti-LILRB4 reduced AJ3 plaques and mitigates mice risk-taking behavior by enhancing microglial phagocytosis function and reducing the microglial IFN-R and inflammatory cytokine release. Further, anti-LILRB4 antibody ameliorated pathology and behavioral alterations induced by A plaques. Example 7

Validation of LILRB4-ApoE interaction

[0177] ApoE secreted by microglia is an important constituent of amyloid plaques that promotes their compaction. Plaque-associated ApoE may in turn impact microglial activation by A plaques. Recent findings showed that APOE variants were recognized and bound by LILRB4. Here, the binding of ApoE2/3/4 to LILRB4 was tested by ELISA, and it was observed that ApoE3/4 had higher LILRB4 binding affinity than ApoE2 (FIG. 29). Furthermore, 2B4-LILRB4 reporter cell was incubated with immobilized lipidated or non-lipidated human ApoE3/4 or mouse ApoE overnight at different concentrations. In the human ApoE incubation groups, the reporter cells incubated with ApoE4 had stronger EGFP signal than the ApoE3 group, and non- lipidated ApoE3/4 had a stronger effect than the lipidated ApoE3/4 on the reporter cell activation (FIG. 30A and FIG. 30B); whereas mouse ApoE showed stronger effect on the reporter cell activation than human ApoE3/4 (FIG. 14). Anti-LILRB4 reporter cells were further used to test binding of mouse recombinant ApoE with human recombinant LILRB4.The human ApoE3/4 and mouse ApoE-induced reporter cell activations were totally blocked by the anti-LILRB4 mAb, demonstrating specificity of LILB4-ApoE binding (FIG. 31 and FIG. 32).

[0178] Three amino acid residues crucial for the interaction between human LILRB4 and human ApoE were identified to be: T30 and P35 and Y121. Another study solved the structure of LILRB4 ectodomain and predicted binding site for any ligand by the SPPIDER (accessibilitybased protein-protein interface identification and recognition). To further advance this model, the in silica docking modeling of LILRB4 ectodomain (PDB: 3P2T) together with the N-terminus four-helix bundle of mouse ApoE (mApoE) (PDB: 1YA9) was applied using the public server ClusPro 2.0. The top 10 predictions of mApoE binding to LILRB4 all pointed to the D1-D2 interdomain site between two Ig-like domains of LILRB4, where a loop of K134ERAAHP140 was constantly engaged with predicted mApoE positions (FIG. 33A). The key interaction residues T30, P35 and Y121 on the top predicted model. T30 and P35 were located at the binding interface between mApoE and LILRB4, corroborating their likely interaction with mApoE (FIG. 33B) While T30 approached the helical bundle of mApoE, the P35 might be relevant for the mApoE to access the binding site (FIG. 33C). On the other hand, residue Y121 sat on the opposite site of the predicted binding interface where loop K134ERAAHP140, T30 and P35 are (FIG. 33D). Therefore, the loop K134ERAAHP140, T30 and P35 may directly interact with mApoE while Y121 may play an indirect role.

[0179] To test this hypothesis, a non-functional loop swap was performed, converting the entire K134ERAAHP140 sequence of LILRB4 into VGGVGGP, which mutates the loop but minimizes the impact of protein folding (FIG. 33D). The recombinant LILRB4 ectodomain proteins with T30A, P35A, or Y121A mutations were also produced. After purification of the original and mutated LILRB4 ectodomains by size-exclusion chromatography, the common variant of LILRB4 was noted, as well as its P35A and Y121A mutants yielded both monomeric and oligomeric forms variably glycosylated (FIG. 33E and 33F), whereas the loop swap and T30A mutants markedly shifted LILRB4 towards the oligomeric form (FIG. 33F), suggesting that loop swap and T30 facilitate LILRB4 oligomerization. The binding of the common variant of LILRB4 and mutated oligomers to recombinant mApoE were compared by ELISA as not all mutants yielded monomers. The loop swap and the P35A mutant attenuated binding of LILRB4 to mApoE, while T30A and Y121A mutants only showed a trend of reduction (FIG. 33G), corroborating the predicted model. Reduced binding of P35A and Y121A to mApoE compared to that of the prototypic LILRB4 variant was independent of their monomeric/oligomeric states (FIG. 33G). Importantly, analysis of purified LILRB4 by immunoblot with anti-LILRB4 revealed that the loop swap mutation abrogated recognition of LILRB4 by the ZM3.1 mAb (FIG. 33E). Since the K134ERAAHP140 loop at the D1-D2 interdomain of LILRB4 is crucial for binding both mApoE and the anti-LILRB4 antibody, the latter directly interferes with the region of LILRB4 that binds mApoE.

Example 8

Summary

[0180] Together, examples disclosed demonstrated that LILRB4-ApoE mediated signaling regulated microglia response around Ap deposits, which inhibited A clearance depending on the ApoE isoform. Therefore, disrupting LILRB4-ApoE interaction by anti- LILRB4 mAb could reverse microglia immune-suppression and slow down AD development. List of Sequences (Table 1)

Table 1

Claims

CLAIMS What is claimed is:

1. A anti-human Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) antibody, comprising a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 1 (LI), 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 (Hl), SEQ ID NO: 4 (H2), and SEQ ID NO: 5 (H3).

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 1-3, wherein the framework region of each variable region has at least 75% sequence identity with a human framework region sequence.

5. The anti-LILRB4 antibody of any one of claims 1-4, wherein the antibody further comprises one or more constant regions, or a portion of a constant region, that has at least 90% sequence identity with a human constant region sequence.

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

7. The anti-LILRB4 antibody of claim 6, wherein the antibody 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. The pharmaceutical composition of claim 8, further comprising a dispersing agent, buffer, surfactant, preservative, solubilizing agent, isotonicity agent, stabilizing agent, or any combination thereof.

10. The pharmaceutical composition of claim 9, wherein said carrier comprises physiological saline, ion exchanger, alumina, aluminum stearate, lecithin, serum protein, human serum albumin, buffer, phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, wool fat, 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. The method of claim 11, wherein the neurological disease is associated with microglial dysfunction.

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

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

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

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

17. A method of decreasing an amyloid plaque load, a 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. The method of claim 17, wherein the anti-LILRB4 antibody is peripherally administered.

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

20. The method of any one of claims 17-19, wherein the amyloid plaque load is decreased.

21. The method of any of claims 17-20, wherein the CAA load is decreased.

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

23. A method of treating at least one A0 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. The method of claim 24, wherein the treating comprises preventing, attenuating, reversing, or improving at least one symptom or sign of A0 plaque or at least one CAA associated symptom in the subject.

25. The method of claim 23 or claim 24, wherein the A0 plaque-associated symptom or the CAA-associated symptom is selected from the group consisting of neuronal degeneration, impaired cognitive function, altered behavior, abnormal language function, emotional dysregulation, seizures, impaired nervous system structure, impaired nervous system function, an increased risk of development of Alzheimer's disease, and an increased risk of development of cerebral amyloid angiopathy.

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

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

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

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

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

31. The anti-LILRB4 antibody of any one of claims 1 to 7 or the pharmaceutical composition of any one of claims 7-10, for use in the treatment of at least one A0 plaque- associated symptom or at least one CAA-associated symptom.

32. The anti-LILRB4 antibody of any one of claims 1 to 7 or the pharmaceutical composition of any one of claims 7-10, for use in the treatment of Alzheimer’s disease.

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

34. Use of the anti-LILRB4 antibody of any one of claims 1 to 7 in the manufacture of a medicament for the treatment of at least one A|3 plaque-associated symptom or at least one CAA-associated symptom.

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

36. An LILRB4-fc fusion protein for use as a medicament.

37. An LILRB4-fc fusion for use in the treatment of a neurological disease.